[go: up one dir, main page]

WO2019210080A1 - Methods and compositions for skeletal and neurological disorders - Google Patents

Methods and compositions for skeletal and neurological disorders Download PDF

Info

Publication number
WO2019210080A1
WO2019210080A1 PCT/US2019/029161 US2019029161W WO2019210080A1 WO 2019210080 A1 WO2019210080 A1 WO 2019210080A1 US 2019029161 W US2019029161 W US 2019029161W WO 2019210080 A1 WO2019210080 A1 WO 2019210080A1
Authority
WO
WIPO (PCT)
Prior art keywords
nell
cntnap4
antibody
disorder
antigen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2019/029161
Other languages
French (fr)
Inventor
B. Chia Soo
Kang Ting
Zhong Zheng
Chenshuang LI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California Berkeley
University of California San Diego UCSD
Original Assignee
University of California Berkeley
University of California San Diego UCSD
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of California Berkeley, University of California San Diego UCSD filed Critical University of California Berkeley
Publication of WO2019210080A1 publication Critical patent/WO2019210080A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2842—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta1-subunit-containing molecules, e.g. CD29, CD49
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A01K2217/077—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out heterozygous knock out animals displaying phenotype
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention generally relates a methods and compositions for a disorder such as a neurological disorder or a bone disorder such as osteoporosis.
  • compositions for treating a disorder which composition comprises an effective amount of an agent effective for potentiating an effective binding of blood NELL-1 to blood Cntnap4 in a mammalian subject in need thereof to achieve a normal level of blood NELL-1 binding to blood Cntnap4 in the mammalian subject, wherein the mammalian subject has an abnormal level of blood NELL-1 binding to blood Cntnap4.
  • the agent comprises a Wnt/b -eaten in signaling activator.
  • the agent comprises NELL-1, Cntnap4, and a combination of NELL- 1 and Cntnap4.
  • the agent comprises a GSK3[> inhibitor.
  • the agent comprises an agonist of GABAergic activity and/or an inhibitor of Dopaminergic activity in the subject.
  • the agent further comprises integrin-b ⁇ .
  • composition optionally in combination with any of the various embodiments disclosed herein, the agonist of GABAergic activity is Indiplon.
  • the inhibitor of Dopaminergic activity is any of the various embodiments disclosed herein.
  • the disorder is a neurological disorder or a skeletal disorder.
  • the disorder is osteoporosis.
  • the disorder is autism.
  • the agent is an enhancer or inhibitor of NELL- 1 or Cntnap4.
  • the agent is an exogenous gene construct expressing NELL-1 or Cntnap4.
  • composition optionally in combination with any of the various embodiments disclosed herein, the composition further comprises a
  • composition optionally in combination with any of the various embodiments disclosed herein, the subject is a human being.
  • a method of diagnosing a neurological disorder or skeletal disorder in a mammalian subject which method comprises:
  • the disorder is a neurological disorder.
  • the disorder is autism or osteoporosis.
  • the subject is a human being.
  • a method of treating or ameliorating a disorder in a mammalian subject comprises administering to the mammalian subject in need thereof a composition comprising an effective amount of an agent effective for potentiating an effective binding of blood NELL- 1 to blood Cntnap4 in the mammalian subject to achieve a normal level of blood NELL-1 binding to blood Cntnap4 in the mammalian subject,
  • the mammalian subject has an abnormal level of blood NELL-1 binding to blood Cntnap4 (e.g., compared to a control subject or a subject without a disorder, such as a bone disorder or a neurological disorder), and
  • the disorder is a neurological disorder or a bone disorder.
  • the agent comprises a Wnt/
  • the agent comprises NELL-1, Cntnap4, and a combination of NELL- 1 and Cntnap4.
  • the agent comprises a GSK3 inhibitor.
  • the agent comprises an agonist of GABAergic activity and/or an inhibitor of Dopaminergic activity in the subject.
  • the agent further comprises integrin-b ⁇ .
  • the agonist of GABAergic activity is Indiplon.
  • the inhibitor of Dopaminergic activity is Risperidone.
  • the disorder is a neurological disorder or a skeletal disorder.
  • the disorder is osteoporosis.
  • the disorder is autism.
  • the agent is an enhancer or inhibitor of NELL- 1 or Cntnap4.
  • the agent is a gene construct expressing NELL-1 or Cntnap4.
  • the composition further comprises a pharmaceutically acceptable carrier for local or systemic delivery.
  • the subject is a human being.
  • a method of fabricating a composition which method comprises:
  • an agent which is capable of potentiating an effective binding of blood NELL-1 to blood Cntnap4 in a mammalian subject in need thereof to achieve a normal level of blood NELL-1 binding to blood Cntnap4 in the mammalian subject (e.g., compared to a control subject or a subject without a disorder, such as a bone disorder or a neurological disorder), and
  • the disorder is a neurological disorder or a bone disorder.
  • the agent comprises a Wnt/
  • the agent comprises NELL-1, Cntnap4, and a combination of NELL- 1 and Cntnap4.
  • the agent comprises a GSK3 inhibitor.
  • the agent comprises an agonist of GABAergic activity and/or an inhibitor of Dopaminergic activity in the subject.
  • the agent further comprises integrin-b ⁇ .
  • the agonist of GABAergic activity is Indiplon.
  • the inhibitor of Dopaminergic activity is Risperidone.
  • the disorder is a neurological disorder or a skeletal disorder.
  • the disorder is osteoporosis.
  • the disorder is autism.
  • the agent is an enhancer or inhibitor of NELL- 1 or Cntnap4.
  • the agent is a gene construct expressing NELL-1 or Cntnap4.
  • the composition further comprises a pharmaceutically acceptable carrier for local or systemic delivery.
  • the subject is a human being.
  • an isolated antibody or antigen-binding fragment thereof that specifically binds an epitope present on Cntnap4.
  • the antibody or antigen-binding fragment thereof may be an agonistic antibody.
  • the epitope may be within a Laminin G domain of Cntnap4.
  • the antibody or antigen-binding fragment thereof may be a humanized antibody, a human antibody, or a monoclonal antibody.
  • the antibody or antigen-binding fragment thereof may include human constant regions.
  • the antigen-binding fragment may be an antibody that lacks the Fc portion or is a F(ab’)2, a Fab, an Fv, or an scFv structure.
  • fusion protein containing the antibody or antigen-binding fragment thereof of any of the above embodiments fused to Nell-1 or a fragment thereof.
  • compositions including the antibody or antigen binding fragment thereof of any of the above embodiments in combination with, e.g., a pharmaceutically acceptable carrier, excipient, and/or diluent.
  • bone formation e.g., osteogenesis
  • the subject has a bone disorder, such as osteoporosis.
  • the subject has a neurological disorder, such as Autism spectrum disorder.
  • the antibody or antigen-binding fragment thereof may be administered at a dosage of about 0.001 mg/kg/day to about 10 mg/kg/day (e.g., 0.001 to about 0.01 mg/kg/day, about 0.01 to about 0.1 mg/kg/day, about 0.1 to about 1 mg/kg/day, or about 1 to about 10 mg/kg/day).
  • the antibody or antigen-binding fragment thereof may be formulated at a concentration of 0.5-300 mg/mL, e.g., in a volume of 0.1-2 mL (such as a volume of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0 mg/mL).
  • Figures 1A-1C show Nell-1 structure and function.
  • Figure 1A shows distinct Nell-1 domains.
  • Figure IB shows known functions of Nell-1.
  • Figure 1C shows a nonsense mutation in the mouse Nell-1 locus induced by N-ethyl-N-nitrosourea (ENU) resulted in neonatal lethality with skeletal abnormalities.
  • ENU N-ethyl-N-nitrosourea
  • Figures 2A-2E show generation of floxed Nell-1 and Cntnap4 mice.
  • Figure 2 A shows exon 1 was targeted for floxed Nell-1;
  • Figure 2B shows X-gal stained and microCT scanned newborn mice of floxed Nell-1 bred with Wntl-Cre showing defect in frontal bones (*);
  • Figure 1C shows quantitative measurements of defect area in frontal bones;
  • Figure 2D shows exon3 was targeted for floxed Cntnap4;
  • Figure 2E shows similar fontal bone defect was seen in Cntnap4;Wntl-Cre knockout mice (25).
  • Figures 3A-3F show co-localization of Nell- 1 and Cntnap4 in mouse brain.
  • Figure 3 A shows hippocampal and thalamic areas of 2-month-old WT mouse brain.
  • Figures 3B-3E show immuno fluorescent staining of Nell- 1 (red) and Cntnap4(green) in different areas of the mouse brain in A showing co-localization in pyramidal cells and interneurons.
  • Figure 3F shows complete overlapping of Nell- 1 and Cntnap4 in Purkinje cells in the cerebellum.
  • Insets enlarged images of single or double positively stained neurons.
  • Figures 4 A and 4B show immunohistochemistry of Nell- 1 and Cntnap4 in the human hippocampus.
  • Figure 4A shows double positive neurons in CA1.
  • Figure 4B shows co -localization of Nell- 1 and Cntnap4 in pyramidal cells and interneurons of CA4.
  • Figures 5A-5C show risperidone decreases hyperactivity and repetitive behavior in Nell- 1 1 mice.
  • Figure 5A shows Nell-1+/- mice exhibit overgrooming behavior.
  • Figure 5B shows risperidone, an antagonist to Dopamine, improves the marble burying score of Nell-1+/- mice.
  • Figure 5C shows risperidone improves Nell-1+/- mice’s behavior in the three-chamber social interaction test.
  • n 12 mice/genotype and treatment condition. *p ⁇ 0.05 by Mann-Whitney U test.
  • Figure 6 is a summary of studies in Example 3. Notes: Nl : Nell-1; C4:Cntnap4; WT: wild type; KO: knockout; CB: craniofacial bones; ASD: autism spectrum disorders; NPC: neural progenitor cells; TEM: transmission electronic microscopy.
  • Figures 7A and 7B demonstrate immunohistochemistry of the mouse trigeminal ganglion (TG).
  • Figure 7 A shows Cntnap4 positive ganglion neurons (green) and Nell-1 positive satellite cells (red).
  • Figure 7B shows that there are more parvalbumin (PV) positive (red) than tyrosine hydroxylase (TH) positive (green) ganglion neurons in TG.
  • PV parvalbumin
  • TH tyrosine hydroxylase
  • Figures 8A-8F show postnatal changes of CB by microCT in Nell-1 and Cntnap4 mutant mice.
  • Figure 8A shows abnormal CB with hydrocephalus in KO mice at P35;
  • Figure 8B shows similar CB changes with severely shortened frontonasal axis (red brackets) in KO mice at P20 and P50;
  • Figures 8C-8F show premature fusion (red arrows) of ISS in KO mice at P20.
  • Nl Nell-1;
  • C4 Cntnap4; ISS: intersphenoid synchondrosis; SOS: spheno -occipital synchondrosis. Scale bar in B: lmrn.
  • Figures 9A-9C show double calvarial defect model in juvenile mouse.
  • Figure 9A shows an illustration of calvarial defects and their relation to bones and sutures;
  • Figure 9B shows two 2mm defects were created on the right frontal and left parietal bones without affecting sutures;
  • Figure 9C shows two different tissue origins of the calvarial vault.
  • Dotted circles in red represent bone defects;
  • Dotted line in black represents Jugum limitants by which separates anterior and posterior frontal sutures.
  • Scale bar 1mm.
  • Figures 10A and 10B show Cntnap4 is indispensable for Nell-l’s activation of Wnt/b- catenin signaling pathways in osteogenesis.
  • Figure 10A shows a diagram of Wnt/ -catenin signaling pathway, and its antagonists DKK1 and PNU74654 and agonist CHIR99021;
  • Figure 10B shows expression of Wnt/ -catenin signaling molecules in the whole cell lysate of Control (WT) and Cntnap4-KD MC3T3-E1 cells treated with rhNell-1.
  • Figures l lA and 11B show Wnt inhibitors block Nell- 1 stimulation of Wnt/[i-catenin signaling and osteoblastic differentiation in CNCCs.
  • Figures 11 A and 1 IB show gene expression of Wnt downstream and osteogenic markers at day 7 post-treatment with CNCCs. * p ⁇ 0.05;
  • Figures 12A-12C show distribution of Parvalbumin (PV) and Tyrosine Hydroxylase (TH) positive neurons in the mouse brain.
  • Figure 12A shows PV positive pyramidal cells in hippocampal CA1;
  • Figure 12B shows most interneurons were positively stained with PV in the hypothalamus (HTH);
  • Figure 12C shows PV positive Purkinje cells in the cerebellum (CBL); Arrows pointing to PV+ cells.
  • Figure 13A and 13B show expression of active b-catenin (ABC) in neurons of the mouse brain.
  • Figures 13A and 13B show differential intensities of ABC positive neurons (red staining pointed by arrowheads) were readily detectable in the neocortex. Some ABC positive neurons also stained strongly positive for Nell-1 (yellow staining pointed by arrows).
  • Figures 14A-14C demonstrate the establishment of primary mouse neuronal cell culture.
  • Figure 14A shows primary mouse hippocampal neurons were cultured on poly-D-lysine coated coverslip at Day 5;
  • Figure 14B shows identification of neuronal cells by MAP2 (red) and astrocytes by GFAP (green) using immunocytochemistry at Day 7 primary culture;
  • Figure 14C shows co-localization/expression of Nell- 1 and Cntnap4 in mouse primary neuronal cells at Day 7 culture.
  • Figure 15 show phage biopanning diagram using His-tagged Nell- 1 -coated magnetic beads.
  • a cDNA library was constructed from human brain mRNA and packaged with T7 phages. The library was probed with His-tagged Nell- 1 -coated magnetic beads. After four rounds of biopanning, most of the non-specific binding phages were washed off, and the remaining phages were considered binding candidates. Among the phage candidate cDNA inserts, PCR products over 500 bp in length were sequenced and analyzed.
  • Figures 16A-16D show confirmation of binding affinity between Cntnap4 phages and Nell-1 using a binding dissociation constant ELISA assay.
  • Figure 16A shows the Basic Local Alignment Search Tool (BLAST) result of the amino acid sequence displayed by the T7 phage constructed with human brain cDNA matches the partial protein sequence of human Cntnap4.
  • Query the amino acid sequence enclosed by the T7 phage DNA;
  • Sbjct the matched amino acid sequence Cntnap4.
  • the LamG domains are highlighted in pink.
  • FIG 16B shows increasing the number of phages incubated with Nell-1 pre-coated ELISA plates, the Cntnap4 phage demonstrated significantly higher binding affinity than the control phage.
  • Figure 16C shows Cntnap4 phage revealed high binding affinity only to full length Nell-1, and not to LamG domain- deleted Nell-1.
  • Figure 16D shows structures of Nell- 1 and Cntnap4 and their potential interaction domains.
  • Nell-1 is a secreted protein comprised of 810 amino acids with a molecular weight of ⁇ 90 kDa before N-glycosylation and oligomerization.
  • Cntnap4 contactin associated protein-like 4, also known as Caspr4
  • Caspr4 contactin associated protein-like 4, also known as Caspr4
  • DISC discoidin-like domain
  • FreD fibrinogen-related domain
  • EGF epidermal growth factor
  • the cytoplasmic region contains a binding site for PDZ domains.
  • the potential binding domain of Nell-1 and Cntnap4 is highlighted by the blue dashed line.
  • TM transmembrane.
  • Mean ⁇ S.E. of six independent experiments performed in triplicate are shown. *: P ⁇ 0.05 when compared to control phage.
  • Figures 17A and 17B show mRNA expression levels of Cntnap4 in 12 types of non neuron and glial cells used in various Nell-1 studies.
  • Figure 17A shows of the 8 types of tested cell lines, the MC3T3-E1 cell line expressed the highest levels of Cntnap4.
  • Figure 17B shows of the 4 types of tested primary cells, NMCC exhibited the highest expression levels of Cntnap4.
  • NMCC newborn mouse calvarial cells.
  • mRC mouse rib chondrocytes.
  • hBMSC human bone marrow stem cells.
  • hARC human articular chondrocytes. Mean + S.E. of six independent experiments performed in triplicate are shown.
  • Figures 18A-18C show Co-localization of Cntnap4 and Nell-1 in pre-osteoblastic cells and calvarial bones.
  • Figure 18A shows confocal laser scanning microscopy (CFSM) revealed the co-localization of Nell-1 and Cntnap4 in MC3T3-E1 pre-osteoblasts after 30 minutes of incubation with exogenous recombinant human Nell-1. Co-localization can be found
  • FIG. 18A shows similar co-localization and protein interaction of Nell- 1 and Cntnap4 were also observed in the plasma membrane of NMCC with 30 minutes of Nell- 1 treatment.
  • Figure 18C shows calvarial bone of P60 mice showed high- intensity double staining of Nell- 1 and Cntnap4 in the bone marrow cavity.
  • White arrows marrow cavity cells with both Nell-1/Cntnap4 co-localization staining and PLA signaling; yellow arrow: bone lining cells with Nell-1/Cntnap4 co-localization staining.
  • Scale bar 50 mhi (yellow), 500 mhi (black), mhi 100 (blue), and 20 mhi (white).
  • Figures 19A-19E show physical interaction between Nell-1 and Cntnap4.
  • Figures 19A and 19B show pull-down assays were performed with ( Figure 19A) MC3T3-E1 pre-osteoblasts and ( Figure 19B) neonatal mouse calvarial cells (NMCC). Increased Cntnap4 was detected when beads were coated with His-tagged Nell-1.
  • Figure 19C and 19D show co-Immunoprecipitation assay with ( Figure 19C) MC3T3-E1 pre-osteoblasts and ( Figure 19D) NMCC demonstrated an increase in Cntnap4 when cells were incubated with Nell-1.
  • Figures 20A-20C show that Cntnap4 is indispensable for Nell-1 osteogenic bioactivity in vitro.
  • Figure 20A shows alkaline phosphatase (AFP) staining on day 9 and Alizarin Red staining on day 14 revealed increased staining in the Nell-1 and BMP2 groups of control shRNA transfected MC3T3-E1 cells.
  • AFP alkaline phosphatase
  • Alizarin Red staining on day 14 revealed increased staining in the Nell-1 and BMP2 groups of control shRNA transfected MC3T3-E1 cells.
  • high staining intensities of AFP and Alizarin Red were only present in the BMP2 group.
  • Figure 20B shows a time-dependent, steady increase in Ocn and Opn staining was observed in both PBS and recombinant human Nell- 1 -treated control MC3T3-E1 cells. At each individual time point, the Nell- 1 -treated group demonstrated increased staining intensity when compared to the PBS-treated group. In Cntnap4- KD MC3T3 cells, neither PBS nor Nell-1 treatment resulted in detectable positive staining of Ocn or Opn.
  • Figure 20C shows in control MC3T3-E1 cells, Alp, Collagen Ial, and Collagen Ia2 reached peak expression levels 9 days after stimulation, while Ocn, Opn, and Bsp displayed time- dependent patterns of increased expression.
  • Figures 21A-21C show Cntnap4- knockdown blocks the osteogenic effects of Nell- 1 ex vivo.
  • Figure 21 A shows the mineral deposition in the mouse calvarial explants during the culture period was revealed by Alizarin Complexone. Fentiviral overexpression of Nell-1 increased the density of Alizarin Complexone, however when Nell-1 was overexpressed in Cntnap4-KD samples, the Alizarin Complexone staining was comparable to the control (without Cntnap4- KD or Nell-1 overexpression).
  • Figure 2 IB shows quantification of the maximal width of the frontal and parietal bones overlapping area.
  • Nell-1 overexpression alone increased the overlapping area, while Cntnap4- KD alone slightly reduced the overlapping area.
  • the maximal width of the overlapping area remained unchanged (similar to that of the control group).
  • Figure 21C shows quantification of the unclosed anterior fontanel area.
  • the calvarial explants in the Nell-1 overexpression group demonstrated a completely closed fontanel, while the anterior fontanel in the control group remained open.
  • Cntnap4- KD alone slightly inhibited the closure of the anterior fontanel.
  • the Cntnap4-KO + Nell-1 overexpression group showed a largely open fontanel area.
  • each calvarial bone is outlined by a white dotted line; yellow arrows represent the maximal width of the frontal and parietal bones overlapping area (in the coronal suture).
  • P Parietal
  • F Frontal. 8 calvaria explants were used for each group.
  • B and C the means were used as center values.
  • Figures 22A-22C show Cntnap4 is indispensable for Nell- l’s bioactivity on the activation of MAPK and WNT signaling pathways in vitro.
  • Figure 22 A shows the activation of MAPK signaling in Control and Cntnap4- KD MC3T3-E1 pre-osteoblasts stimulated with Nell-1.
  • control MC3T3-E1 cells significantly higher levels of pERK and pJNK were detected 10 min and 30 min after Nell-1 stimulation, respectively. There was no change detected in the
  • FIGS. 22B and 22C show expression of Wnt signaling molecules in the ( Figure 22B) whole cell lysate and ( Figure 22C) cell nuclear lysate of Control and Cntnap4-KD MC3T3-E1 cells treated with Nell-1.
  • Nell-1 significantly increased the expression levels of Axin2 and active b-catenin, while no effect was observed on these markers in Cntnap4- KD cells treated with Nell-1.
  • Charts demonstrate mean relative band intensity (normalized to control MC3T3-E1 at 0 min) + S.E. for three individual experiments. *, P ⁇ 0.05 when compared with Control at 0 min; #, P ⁇ 0.05 when compared with Cntnap4- KD at 0 min.
  • FIG 23 is a schematic diagram of Nell- 1 signaling pathways in osteogenesis.
  • Nell-1 As a secreted molecule, Nell-1 initiates cellular signaling through binding to its specific receptor, Cntnap4, on the cell surface.
  • the MAPK and Wnt signaling pathways play critical roles in Nell-1- mediated osteogenesis.
  • Nell-1 preferentially activates ERK and JNK in MAPK signaling, and also promotes the phosphorylation/activation of Runx2, which in turn stimulates the expression of Nell-1 and Ocn by directly binding to the OSE2 region of their promoters.
  • Nell-1 promotes the expression of Axin2 and acti vc-()-catcnin, and increases the nuclear translocation of acti ve-[i-catenin.
  • Figures 24A-24F show expression of Cntnap4, Cntnap3, and Cntnap2 in vitro and in vivo.
  • Figures 24A and 24B show Nell-1 significantly increased the levels of Cntnap4 in both ( Figure 24A) MC3T3-E1 pre -osteoblasts and ( Figure 24B) NMCC.
  • expression of Cntnap2, which was markedly lower than that of Cntnap4 was not responsive to Nell-1 simulation. More importantly, transcription of Cntnap3 was not detectable in both MC3T3-E1 pre-osteoblasts and NMCC, regardless of Nell- 1 treatment.
  • Figure 24C shows in accordance with the gene expression analysis, Cntnap3 protein was not detected in MC3T3-E1 pre-osteoblasts and NMCC after 30 minutes of Nell- 1 treatment. Thus, neither co-localization nor direct binding of Cntnap3 and Nell-1 was detected in vitro.
  • Figure 24D shows neither co-localization nor direct binding of Cntnap3 and Nell-1 was detected in P60 mouse calvarial bone.
  • Figure 24E shows minimal Cntnap2 staining was detected in vitro.
  • Figure 25 shows mRNA expression levels of Cntnap4 in control and Cntnap4 shRNA transfected MC3T3-E1 cells. Expression of Cntnap4 in the stable Cntnap4 knockdown MC3T3- E1 cell line was about 85% lower than the expression in control cells. Mean + S.E. of six independent experiments performed in triplicate are shown. *: P ⁇ 0.05 when compared to Control group.
  • Figures 26 A and 26B show Cntnap4 knockdown does not alter Nell- 1 -induced Nfatc2 expression in ATDC5 cells.
  • Figure 26A shows Cntnap4 knockdown reduced the expression level of Nfatc2 in ATDC5 cells.
  • Figure 26B shows Nell-1 stimulated similar levels of Nfatc2 increase in both control and Cntnap4- KD ATDC5 cells in the same dose-dependent manner. Mean + S.E. of three independent experiments performed in duplicate are shown.
  • Figures 28A and 28B show expression and purification of the extracellular portion of human Cntnap4 (Cntnap4 extra ).
  • the purified extracellular portion of recombinant human Cntnap4 protein analyzed by Western blot with Rabbit-anti-Cntnap4 polyclonal antibodies:
  • Figure 28 A shows HPA031859, Sigma-Aldrich, and
  • Figure 28B shows HPA053742, Sigma-Aldrich.
  • Figure 29 demonstrates the effectiveness of the invention composition for autism.
  • the term“effective amount”, as used herein, is an amount of an agent that is sufficient to produce a statistically significant, measurable change of a condition in repaired tissue using the agent disclosed herein as compared with the condition in the repaired tissue without using the agent. Such effective amounts can be gauged in clinical trials as well as animal studies. Such a statistically significant, measurable, and positive change of a condition in repaired tissue using the agent disclosed herein as compared with the condition in the repaired tissue without using the agent is referred to as being an“improved condition”.
  • the term“significantly” or“significant” shall mean statistically significant.
  • the term“normal level of blood NELL-1 binding to blood Cntnap4” refers to the state of binding of blood NELL-1 to blood Cntnap4 in a normal mammalian subject.
  • This term, in the context of“in reference to a disorder selected from a neurological disorder or a bone disorder” shall mean a mammalian subject who does not suffer from or who is not disposed to develop a disorder selected from a neurological disorder or a bone disorder.
  • agent refers to a substance(s) effective for potentiating an effective binding of blood NELL-1 to blood Cntnap4 in a mammalian subject.
  • An agent can be a single substance or a combination of substances.
  • the term“enhancer” refers to a substance which enhances the activity of a particular reactant, catalyst, or a biologic substance (e.g., a protein or enzyme).
  • the term“agonist” refers to a chemical that binds to a receptor and activates the receptor to produce a biological response. Whereas an agonist causes an action, an antagonist blocks the action of the agonist, and an inverse agonist causes an action opposite to that of the agonist.
  • activator refers to a chemical that triggers or causes to occur a chemical or biological process.
  • inhibitor refers a substance which slows down or prevents a particular chemical reaction or other process or which reduces the activity of a particular reactant, catalyst, or a biologic substance (e.g., a protein or enzyme)
  • an inhibitor of glycogen synthase kinase 3 beta is one that slows down or prevents a particular chemical reaction or other process or which reduces the activity of GSK3 ;
  • an inhibitor of dopamine is one that slows down or prevents a particular chemical reaction or other process or which reduces the activity of dopamine, and
  • an activator of Wnt/b- catenin signaling pathway is one that that triggers the Wnt/
  • GSK3 inhibitor examples include, but are not limited to: 1) GSK2a/ inhibitors: SB 216763, SB 415286, CHIR 98014, CHIR 99021, Bios-7- indolylmaleimidem (www.nature.com/articles/nrdl415), BIO (6-bromioindi rub in-3 -oxime ), AZD2858, and AZD1080
  • GSK3 inhibitors AR A014418, 1 -Azakenpaulone, TWS119, Tideglusib, IM-12, Indiruhlin,
  • dopamine inhibitor examples include, but are not limited to:
  • dopamine receptor inhibitors Benztropine mesylate, Alizapride HC1, and Amfebutamone HC1;
  • dopamine receptor antagonists Quetiapine fumarate, Chlorpromazine HC1, Domperidone, Metoclopramide HC1, Olanzapine, Paliperidone, Amisulpride, Rotundine, Chlorprothixene, Lurasidone HC1, and Lcvosulpiridc;
  • dopamine reuptake inhibitor 4-Hydroxy- l-methyl-4-(4-methylphenyl)-3-piperidyl 4- methylphenyl ketone, Altropane, Amfonelic acid, Amineptine, BTCP, 3C-PEP, DBL-583, Difluoropine, GBR-12783, GBR-12935, GBR-13069, GBR-13098, GYKI-52895, Iometopane, Methylphenidate, Ethylphenidate, Modafinil, Armodafmil, RTI-229, Vanoxerine, Adrafinil, Benztropine, Bupropion, Fluorenol, Medifoxamine, Metaphit, Rimcazole, Venlafaxine, Oroxylin A, Dexmethylphenidate, Difemetorex, Fencamfamine, Lefetamine, Levophacetoperane,
  • Wnt/b -catenin signaling pathway activator examples include, but are not limited to: IM-12, AZD2858, Methyl vanillate, Sotrastaurin, Cpdl, Cpd2, Norrin, R-spondins, 2-amino-4- [3,4-(methlyenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine, SKL2001, Wnt agonist 1, and CP21.
  • the term“optional” shall mean having the choice to add or not to add a technical element or feature to an embodiment of invention. As such, the term“optional” can also be construed to mean“with” or“without” a technical element or feature in an embodiment of invention.
  • compositions, methods, and respective component(s) thereof that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.
  • the term“consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • the term“desirable property” refers to any attributes of a biologies that is significant with respect to the biologies’ action as a therapeutics or biologically active agent.
  • Such desirable properties include, for example, blood circulation life, shelf-life, hydrophobicity or hydrophilicity, biological activity, bioavailability, cytotoxicity, non-immunogenicity, or conformational properties, etc.
  • references to“the method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
  • compositions for treating a disorder which composition comprises an effective amount of an agent effective for potentiating an effective binding of blood NELL-1 to blood Cntnap4 in a mammalian subject in need thereof to achieve a normal level of blood NELL-1 binding to blood Cntnap4 in the mammalian subject, wherein the mammalian subject has an abnormal level of blood NELL-1 binding to blood Cntnap4 (e.g., relative to a control subject).
  • the agent comprises a Wnt/b -eaten in signaling activator.
  • the agent comprises NELL-1, Cntnap4, and a combination of NELL- 1 and Cntnap4.
  • the agent comprises a GSK3 inhibitor.
  • the agent comprises an agonist of GABAergic activity and/or an inhibitor of Dopaminergic activity in the subject.
  • the agent further comprises integrin-b ⁇ .
  • composition optionally in combination with any of the various embodiments disclosed herein, the agonist of GABAergic activity is Indiplon.
  • the inhibitor of Dopaminergic activity is any of the various embodiments disclosed herein.
  • the disorder is a neurological disorder or a skeletal disorder.
  • the disorder is osteoporosis.
  • the disorder is autism.
  • the agent is an enhancer or inhibitor of NELL- 1 or Cntnap4.
  • the agent is an exogenous gene construct expressing NELL-1 or Cntnap4.
  • the composition further comprises a
  • composition optionally in combination with any of the various embodiments disclosed herein, the subject is a human being.
  • the composition is a formulation for systemic or local delivery.
  • the at least one desirable property is selected from the group consisting of blood circulation life, shelf-life, hydrophobicity or hydrophilicity, biological activity, bioavailability, cytotoxicity, non-immunogenicity, or conformational properties, etc.
  • the invention composition can include a carrier. Further, the composition can be formulated into various formulations for a desired mode of delivery.
  • Neurological disorders such as Autism spectrum disorder (ASD) are lifelong neurodevelopmental disability conditions that affects a great portion of general population.
  • ASD Autism spectrum disorder
  • this invention provides the basis for more definitive clinical diagnosis in post-natal individuals as well as fetal screening for
  • Nell- 1 is a soluble extracellular matrix protein
  • Nell-based therapies exhibit higher potential and feasibility than other membrane protein-based therapies to treat neurological disorders.
  • Nell-1 also exhibits significant pro-osteogenic bioactivities. Therefore, Nell- 1 -based treatment can replace the complex combinatory treatment strategy of neural related and osteoporosis drugs for the patients suffering from neurodevelopmental disability accompanied by bone related diseases.
  • the agent that binds to Cntnap4 to activate osteogenesis and/or neurogenesis may be an antibody or an antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof may be agonistic.
  • the antibody may bind may specifically bind to Cntnap4 and activate the receptor, e.g., to promote osteogenesis and/or neurogenesis.
  • the antibody or antigen-binding fragment thereof may bind to any suitable epitope on Cntnap4 in order initiate downstream signaling, e.g., via Wnt and MAPK signaling pathways.
  • the antibody may bind in the same location as Nell-1 and mimic Nell-1 binding.
  • the antibody may bind allosterically, e.g., outside of the Nell-1 binding pocket, but cause Cntnap4 to be more likely to bind Nell-1.
  • Cntnap4 is a transmembrane protein of 1310 amino acids consisting of a large extracellular domain, a single membrane-spanning domain, and a short cytoplasmic region at the carboxy-terminus.
  • the extracellular region is composed of a discoidin-like domain (DISC), a fibrinogen-related domain (FreD), two EGF repeats, and four Laminin G domains.
  • DISC discoidin-like domain
  • FreD fibrinogen-related domain
  • two EGF repeats two EGF repeats
  • Laminin G domains Laminin G domains
  • the epitope bound by the antibody or antigen-binding fragment thereof may include a region within Cntnap4, such as within residues 1-100, 100-200, 200-300, 300-400, 400-500, 500- 600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, or 1300-1310.
  • the epitope may include a portion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more residues of Cntnap4.
  • fusion proteins including an antibody or antigen-binding fragment thereof fused to Nell-1 or a fragment thereof.
  • the fusion proteins may be used to promote osteogenesis and/or neurogenesis.
  • Nell-1 may be fused at the N-terminus or the C-terminus of the antibody.
  • the antibody may target Nell-1 to a Cntnap4 receptor, e.g., to direct Nell-1 to a specific tissue type or the enhance binding by Nell-1 to the Cntnap4 receptor.
  • Antibodies can be optimized to target a preferred epitope on Cntnap4 in order to exhibit desirable functional properties.
  • methods for identifying the particular epitope to which an antibody binds are known to those skilled in the art.
  • Standard techniques include peptide scanning, in which overlapping, short peptides (for example, 10-30 amino acids, e.g., 20, in length) derived from the full length protein to which the antibody binds are individually tested for their ability to bind the antibody. From such experiments, the region of the protein to which the antibody binds can then be determined.
  • Site-directed mutagenesis can also be used to identify the antigenic region(s) of Cntnap4.
  • point mutations are systematically introduced into the target polypeptide and the ability of the antibody to bind the peptide with mutations at various positions is used to determine whether a particular region of that protein contains the epitope to which the antibody binds.
  • Antibody epitopes can also be identified using high-throughput mutagenesis techniques, such as Shotgun Mutagenesis (Integral Molecular, Inc., Philadelphia, Pa.), which can be used to generate large numbers of mutations within the target protein. Such methodologies permit efficient identification of epitopes within the protein.
  • an in vitro competitive blockade assay can be performed.
  • Additional antibodies against Cntnap4 can be made, e.g., using any of the numerous methods for making antibodies known in the art.
  • a coding sequence for a fragment of Cntnap4 is expressed as a C-terminal fusion with glutathione S-transferase (GST) (Smith et al., Gene 67:31- 40, 1988).
  • GST glutathione S-transferase
  • the fusion protein is purified on glutathione-Sepharose beads, eluted with glutathione, cleaved with thrombin (at an engineered cleavage site), and purified for immunization of rabbits.
  • Antiserum specificity can be determined using a panel of unrelated GST proteins.
  • peptides corresponding to relatively unique immunogenic regions of a polypeptide described herein can be generated and coupled to keyhole limpet hemocyanin (KLH) through an introduced C-terminal lysine.
  • KLH keyhole limpet hemocyanin
  • Antiserum to each of these peptides is similarly affinity purified on peptides conjugated to BSA, and specificity is tested by ELISA or Western blot analysis using peptide conjugates, or by Western blot or immunoprecipitation using the polypeptide expressed as a GST fusion protein.
  • monoclonal antibodies that specifically bind Cntnap4 can be prepared using standard hybridoma technology (see, e.g., Kohler et al., Nature 256:495-7, 1975; Kohler et al., Eur. J. Immunol. 6:511-9, 1976; Kohler et al., Eur. J. Immunol. 6:292-5, 1976; Hammerling et al., Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981). Once produced, monoclonal antibodies can also be tested for specific recognition by Western blot or
  • monoclonal antibodies can be prepared using a polypeptide described hereinc and a phage display library (Vaughan et al., Nat. Biotechnol. 14:309-14, 1996).
  • Cntnap4 epitopic fragments can be generated by standard techniques, e.g., using PCR and cloning the fragment into a pGEX expression vector. These fragments can be used to produce anti-Cntnap4 antibodies that target a particular epitope. Fusion proteins are expressed in E. coli and purified using a glutathione agarose affinity matrix. To minimize potential problems of low affinity or specificity of antisera, two or three such fusions can be generated for each protein, and each fusion can be injected into at least two rabbits. Antisera are raised by injections in a series, and can include, for example, at least three booster injections.
  • Useful antibodies for binding Cntnap4 in order to promote osteogenesis and/or neurogenesis can be identified in several different screening assays.
  • antibodies can be assayed by ELISA to determine whether they are specific for the immunizing antigen (i.e., a Cntnap4 epitope described herein).
  • immunizing antigen i.e., a Cntnap4 epitope described herein.
  • ELISA plates can be coated with immunogen, the antibody can be added to the plate, washed, and the presence of bound antibody can be detected by using a second antibody specific for the Ig of the species in which the antibody was generated.
  • a functional in vitro assay can also be used to screen the antibodies, e.g., using an osteogenesis assay as described herein.
  • the invention also features humanized antibodies that bind to Cntnap4 in order to activate osteogenesis and/or neurogenesis.
  • Various methods for humanizing non-human antibodies are known in the art.
  • a humanized antibody can have one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as“import” residues, which are typically taken from an“import” variable domain. Humanization can be essentially performed following the method of Winter and co workers (Jones et ah, Nature 321 :522-5, 1986; Riechmann et al., Nature 332:323-7, 1988;
  • such“humanized” antibodies can be chimeric antibodies (U.S. Patent No. 4,816,567), in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which at least some hypervariable region residues as well as other variable region residues are substituted by residues from analogous sites in, e.g., rodent antibodies.
  • variable domains both light and heavy
  • the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies can be important to reduce antigenicity.
  • sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences.
  • the human sequence which is closest to that of the rodent is then accepted as the human framework for the humanized antibody.
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.
  • the same framework may be used for several different humanized antibodies. See, e.g., Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-9, 1992; Presta et al ., J. Immunol. 151 :2623-32, 1993.
  • Humanized antibodies can also be produced that retain high affinity for the antigen (e.g., Cntnap4) and other favorable biological properties, such as potentiating osteogenesis and/or neurogenesis.
  • humanized antibodies can be prepared by analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences.
  • Human antibodies that bind Cntnap4 can be constructed by combining Fv clone variable domain sequence(s) selected from human-derived phage display libraries with known human constant domain sequences(s) (Hoogenboom et al., J. Mol. Biol. 227:381-8, 1992; Marks et al., J. Mol. Biol. 222:581-97, 1991). These antibodies can be used to bind Cntnap4 to activate osteogenesis and/or neurogenesis. Alternatively, human monoclonal antibodies can be made by the hybridoma method.
  • antibody fragments that comprise a portion of an intact antibody that binds Cntnap4, in particular, the antigen binding region thereof.
  • antibody fragments include Fab, Fab’, F(ab’)2 , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. These fragments may be used to bind Cntnap4 in order to active osteogenesis and/or neurogenesis.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual“Fc” fragment, whose name reflects its ability to crystallize readily.
  • Pepsin treatment yields an F(ab’)2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
  • Fv is the minimum antibody fragment which contains a complete antigen-binding site.
  • a two -chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association.
  • one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a“dimeric” structure analogous to that in a two-chain Fv species. It is in this configuration that the three hypervariable regions (HVRs) of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer.
  • HVRs hypervariable regions
  • HVRs i.e. complementarity determine regions (CDRs) confer antigen-binding specificity to the antibody.
  • CDRs complementarity determine regions
  • the Fab fragment contains the heavy- and light-chain variable domains and also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
  • Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.
  • Fab’-SH is the designation herein for Fab’ in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab’) 2 antibody fragments originally were produced as pairs of Fab’ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • Single-chain Fv or scFv antibody fragments comprise the VH and VL domains of antibody, where these domains are present in a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the scFv to form the desired structure for antigen binding.
  • scFv see, e.g., Pluckthiin, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer- Verlag, New York, 1994), pp. 269-315.
  • F(ab’) 2 fragments are isolated directly from recombinant host cell culture.
  • Fab and F(ab’) 2 fragment with increased in vivo half-life comprising salvage receptor binding epitope residues are described in U.S. Patent No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.
  • compositions useful for practicing the therapeutic methods described herein contain a physiologically tolerable carrier together with an active agent as described herein, dissolved or dispersed therein as an active ingredient.
  • the therapeutic composition is not immunogenic when administered to a mammal or human patient for therapeutic purposes.
  • pharmaceutically acceptable “physiologically tolerable” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
  • a pharmaceutically acceptable carrier will not promote the raising of an immune response to an agent with which it is admixed, unless so desired.
  • the preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectable either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified or presented as a liposome composition.
  • the active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein.
  • Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.
  • the therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein.
  • Pharmaceutically acceptable salts include the acid addition salts that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.
  • Physiologically tolerable carriers are well known in the art.
  • Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline.
  • aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.
  • Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
  • the amount of an active agent used in the methods described herein that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
  • compositions are well known in the art.
  • examples of such carrier includes, e.g., salient, for liquid or suspension formulations, natural or synthetic polymeric materials for burst or sustained release formulations or targeted delivery formulations. Some examples of the carriers are further described in detail below.
  • the carrier disclosed herein can be a polymeric material
  • Exemplary polymeric material that can be used here include but are not limited to a biocompatible or bioabsorbable polymer that is one or more of poly(DL-lactide), poly(L-lactide), poly(L-lactide), poly(L-lactide-co-DL-lactide), polymandelide, polyglycolide, poly(lactide-co-glycolide), poly(D,L-lactide-co-glycolide), poly(L-lactide-co-glycolide), poly(ester amide), poly(ortho esters), poly(glycolic acid-co-trimethylene carbonate), poly(D,L-lactide-co-trimethylene carbonate), poly(trimethylene carbonate), poly(lactide-co-caprolactone), poly(glycolide-co- caprolactone), poly(tyrosine ester), polyanhydride, derivatives thereof.
  • the polymeric material comprises poly(D,L-lactide- co-glycolide). In some embodiments, the polymeric material comprises poly(D,L-lactide). In some
  • the polymeric material comprises poly(L-lactide).
  • Additional exemplary polymers include but are not limited to poly(D-lactide) (PDLA), polymandelide (PM), polyglycolide (PGA), poly(L-lactide-co-D,L-lactide) (PLDLA), poly(D,L-lactide) (PDLLA), poly(D,L-lactide-co-glycolide) (PLGA) and poly(L-lactide-co-glycolide) (PLLGA).
  • the stent scaffolding can be made from PLLGA with a 25 mole% of GA between 5-15 mol%.
  • the PLLGA can have a mole% of (LA:GA) of 85: 15 (or a range of 82:18 to 88:12), 95:5 (or a range of 93:7 to 97:3), or commercially available PLLGA products identified as being 85: 15 or 95 :5 PLLGA.
  • LA:GA mole% of (LA:GA) of 85: 15 (or a range of 82:18 to 88:12), 95:5 (or a range of 93:7 to 97:3), or commercially available PLLGA products identified as being 85: 15 or 95 :5 PLLGA.
  • the examples provided above are not the only polymers that may be used. Many other examples can be provided, such as those found in Polymeric Biomaterials, second edition, edited by Severian Dumitriu; chapter 4.
  • polymers that are more flexible or that have a lower modulus that those mentioned above may also be used.
  • exemplary lower modulus bioabsorbable polymers include, polycaprolactone (PCL), poly(trimethylene carbonate) (PTMC), polydioxanone (PDO), poly(3-hydrobutyrate) (PHB), poly(4-hydroxybutyrate) (P4HB), poly(hydroxyalkanoate) (PHA), and poly(butylene succinate), and blends and copolymers thereof.
  • higher modulus polymers such as PLLA or PLLGA may be blended with lower modulus polymers or copolymers with PLLA or PLGA.
  • the blended lower modulus polymers result in a blend that has a higher fracture toughness than the high modulus polymer.
  • Exemplary low modulus copolymers include poly(L-lactide)-b-polycaprolactone (PLLA-b-PCL) or poly(L-lactide)-co-polycaprolactone (PLLA-co-PCL).
  • the composition of a blend can include 1-5 wt% of low modulus polymer.
  • More exemplary polymers include but are not limited to at least partially alkylated polyethyleneimine (PEI); at least partially alkylated poly(lysine); at least partially alkylated polyornithine; at least partially alkylated poly(amido amine), at least partially alkylated homo- and co-polymers of vinylamine; at least partially alkylated acrylate containing aminogroups, copolymers of vinylamine containing aminogroups with hydrophobic monomers, copolymers of acrylate containing aminogroups with hydrophobic monomers, and amino containing natural and modified polysaccharides, polyacrylates, polymethacryates, polyureas, polyurethanes, polyolefins, polyvinylhalides, polyvinylidenehalides, polyvinylethers, polyvinylaromatics, polyvinylesters, polyacrylonitriles, alkyd resins, polysiloxanes and epoxy resins, and mixtures thereof.
  • PEI
  • biocompatible biodegradable polymers include, without limitation, polycaprolactone, poly(L-lactide), poly(D,L-lactide), poly(D,L-lactide-co- PEG) block copolymers, poly(D,L-lactide-co-trimethylene carbonate), poly(lactide-co- glycolide), polydioxanone (PDS), polyorthoester, polyanhydride, poly(glycolic acid-co-trimethylene carbonate), polyphosphoester, polyphosphoester urethane, poly(amino acids), polycyanoacrylates, poly (trimethylene carbonate), poly(iminocarbonate), polycarbonates, polyurethanes, polyalkylene oxalates, polyphosphazenes, PHA-PEG, and combinations thereof.
  • polycaprolactone poly(L-lactide), poly(D,L-lactide), poly(D,L-lactide-co- PEG) block copolymers,
  • the PHA may include poly(a- hydroxy acids), poly(P-hydroxyacid) such as poly(3-hydroxybutyrate) (PHB), poly(3- hydroxybutyrate-co-valerate) (PHBV), poly(3-hydroxyproprionate) (PHP), poly(3- hydroxyhexanoate) (PHH), or poly (4 -hydroxy acid) such as poly poly(4-hydroxybutyrate), poly(4- hydroxy valerate), poly(4-hydroxyhexanoate), poly(hydroxyvalerate), poly(tyrosine carbonates), poly(tyrosine arylates), poly(ester amide), polyhydroxyalkanoates (PHA), poly(3- hydroxyalkanoates) such as poly(3-hydroxypropanoate), poly(3-hydroxybutyrate), poly(3- hydroxy valerate), poly(3-hydroxyhexanoate), poly(3-hydroxyheptanoate) and poly(3- hydroxyoctanoate), poly(4-hydroxyal
  • poly(ethylene oxide-co-lactic acid) PEO/PLA)
  • polyalkylene oxides such as poly(ethylene oxide), polypropylene oxide), polypther ester
  • polyalkylene oxalates phosphoryl choline containing polymer, choline, poly(aspirin)
  • polymers and co-polymers of hydroxyl bearing monomers such as 2-hydroxyethyl methacrylate (HEMA), hydroxypropyl methacrylate (HPMA), hydroxypropylmethacrylamide, PEG acrylate (PEGA), PEG methacrylate, methacrylate polymers containing 2-methacryloyloxyethyl- phosphorylcholine (MPC) and n-vinyl pyrrolidone (VP), carboxylic acid bearing monomers such as methacrylic acid (MA), acrylic acid (AA), alkoxymethacrylate, alkoxyacrylate, and 3- trimethylsilylpropyl methacrylate (TMSPMA), poly(sty
  • polyethylene is used to construct at least a portion of the device.
  • polyethylene can be used in an orthopedic implant on a surface that is designed to contact another implant, as such in a joint or hip replacement.
  • Polyethylene is very durable when it comes into contact with other materials.
  • a metal implant moves on a polyethylene surface, as it does in most joint replacements, the contact is very smooth and the amount of wear is minimal. Patients who are younger or more active may benefit from polyethylene with even more resistance to wear. This can be accomplished through a process called crosslinking, which creates stronger bonds between the elements that make up the polyethylene.
  • the appropriate amount of crosslinking depends on the type of implant. For example, the surface of a hip implant may require a different degree of crosslinking than the surface of a knee implant.
  • a method of diagnosing a neurological disorder or skeletal disorder in a mammalian subject which method comprises:
  • the disorder is a neurological disorder.
  • the disorder is autism or osteoporosis.
  • the subject is a human being.
  • a method of treating or ameliorating a disorder in a mammalian subject comprises administering to the mammalian subject in need thereof a composition comprising an effective amount of an agent effective for potentiating an effective binding of blood NELL- 1 to blood Cntnap4 in the mammalian subject to achieve a normal level of blood NELL-1 binding to blood Cntnap4 in the mammalian subject,
  • the mammalian subject has an abnormal level of blood NELL-1 binding to blood Cntnap4, and
  • the disorder is a neurological disorder or a bone disorder.
  • the agent comprises a Wnt/(i-catcnin signaling activator.
  • the agent comprises NELL-1, Cntnap4, and a combination of NELL- 1 and Cntnap4.
  • the agent comprises a GSK3 inhibitor.
  • the agent comprises an agonist of GABAergic activity and/or an inhibitor of Dopaminergic activity in the subject.
  • the agent further comprises integrin-b ⁇ .
  • the agonist of GABAergic activity is Indiplon.
  • the inhibitor of Dopaminergic activity is Risperidone.
  • the agent is an antibody or antigen-binding fragment thereof that binds to Cntnap4, as described herein.
  • the disorder is a neurological disorder or a skeletal disorder.
  • the disorder is osteoporosis.
  • the disorder is autism.
  • the agent is an enhancer or inhibitor of NELL- 1 or Cntnap4.
  • the agent is a gene construct expressing NELL-1 or Cntnap4.
  • the composition further comprises a pharmaceutically acceptable carrier for local or systemic delivery.
  • the subject is a human being.
  • ASD Autism spectrum disorder
  • this invention provides the basis for more definitive clinical diagnosis in post natal individuals as well as fetal screening for neurodevelopmental abnormalities.
  • Nell-1 is a soluble extracellular matrix protein
  • Nell-based therapies exhibit higher potential and feasibility than other membrane protein-based therapies to treat neurological disorders.
  • Nell-1 also exhibits significant pro-osteogenic bioactivities. Therefore, Nell-1- based treatment can replace the complex combinatory treatment strategy of neural related and osteoporosis dmgs for the patients suffering from neurodevelopmental disability accompanied by bone related diseases
  • a method of fabricating a composition which method comprises:
  • an agent which is capable of potentiating an effective binding of blood NELL- 1 to blood Cntnap4 in a mammalian subject in need thereof to achieve a normal level of blood NELL-1 binding to blood Cntnap4 in the mammalian subject, and
  • composition wherein the disorder is a neurological disorder or a bone disorder.
  • the agent comprises a Wnt/(i-catcnin signaling activator.
  • the agent comprises NELL-1, Cntnap4, and a combination of NELL- 1 and Cntnap4.
  • the agent comprises a GSK3 inhibitor.
  • the agent comprises an agonist of GABAergic activity and/or an inhibitor of Dopaminergic activity in the subject.
  • the agent further comprises integrin-b ⁇ .
  • the agonist of GABAergic activity is Indiplon.
  • the inhibitor of Dopaminergic activity is Risperidone.
  • the disorder is a neurological disorder or a skeletal disorder.
  • the disorder is osteoporosis.
  • the disorder is autism.
  • the agent is an enhancer or inhibitor of NELL- 1 or Cntnap4.
  • the agent is a gene construct expressing NELL-1 or Cntnap4.
  • the agent is an antibody or antigen-binding fragment thereof that binds to Cntnap4, as described herein.
  • the composition further comprises a pharmaceutically acceptable carrier for local or systemic delivery.
  • the subject is a human being.
  • Dosages of an agent for treating a disorder as described herein varies according to different disorders, gender types, and age groups.
  • the dosage of an agent as described herein ranges from 0.0005 mg/kg body weight to 1 g/kg body weight. In some embodiments, the dosage range is from 0.001 mg/kg body weight to 0.5 g/kg body weight, from 0.0005 mg/kg body weight to 0.1 g/kg body weight, from 0.001 mg/kg body weight to 0.05 g/kg body weight.
  • the agent is an antibody or antigen-binding fragment thereof, which may be administered at a dosage of about 0.001 mg/kg/day to about 10 mg/kg/day (e.g., 0.001 to about 0.01 mg/kg/day, about 0.01 to about 0.1 mg/kg/day, about 0.1 to about 1 mg/kg/day, or about 1 to about 10 mg/kg/day).
  • a dosage form of an agent described herein is selected for localized delivery and is not necessarily selected with regard to body weight or to achieve a certain serum level, but to achieve a localized effect, e.g., as for a localized injection, implantation or other localized administration to the eye.
  • the doses recited above can be repeated for a limited period of time.
  • the doses are given once a day, multiple times a day, for example but not limited to three times a day, once every other day, once a week, once a month, once every 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months, or once a year.
  • the doses recited above are administered daily for several days, weeks, or months. The duration of treatment depends upon the subject’s clinical progress and responsiveness to therapy. Continuous, relatively low maintenance doses are contemplated after an initial higher therapeutic dose.
  • Agents useful in the methods and compositions described herein can be administered topically, intravenously (by bolus or continuous infusion), orally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, intracavity, intrathecally, or parenterally, or can be delivered by peristaltic means, if desired, or by other means known by those skilled in the art. It is preferred that the agents for the methods described herein are administered topically to the eye.
  • the agent can be administered systemically, or alternatively, can be administered directly to the tumor e.g., by intratumor injection or by injection into the tumor’s primary blood supply.
  • compositions containing at least one agent disclosed herein can be conventionally administered in a unit dose.
  • unit dose when used in reference to a therapeutic composition refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e., carrier, or vehicle.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • the quantity to be administered and timing depends on the subject to be treated, capacity of the subject’s system to utilize the active ingredient, and degree of therapeutic effect desired.
  • An agent can be targeted by means of a targeting moiety, such as e.g., an antibody or targeted liposome technology.
  • Antibody-based or non-antibody-based targeting moieties can be employed to deliver a ligand or the inhibitor to a target site.
  • a natural binding agent for an unregulated or disease associated antigen is used for this purpose.
  • Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are particular to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for administration are also variable, but are typified by an initial administration followed by repeated doses at one or more intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.
  • An agent may be adapted for catheter-based delivery systems including coated balloons, slow-release drug-eluting stents or other drug-eluting formats, microencapsulated PEG liposomes, or nanobeads for delivery using direct mechanical intervention with or without adjunctive techniques such as ultrasound.
  • Nell-1 overexpression mice We first identified high Nell-1 expression in the prematurely fused sutures of craniosynostosis patients, and later verified Nell-l’s osteogenic property in transgenic Nell-1 overexpression mice. Recently, we identified Cntnap4, a member of the neurexin superfamily of transmembrane molecules, to exert a novel ligand/receptor interaction with Nell-1 in osteoblasts. Specifically, Cntnap4 binding is specific and necessary for Nell-1 mediated Wnt signaling during osteogenesis in calvarial cells.
  • Cntnap4 is a known interneuron presynaptic membrane protein.
  • Nell- 1 is highly expressed in the brain, and co-localizes with Cntnap4 in both mouse and human brains. Moreover, we observed Nell-1 haploinsufficient mice to also exhibit ASD-like behaviors.
  • Nell-1 has dual roles in the brain and craniofacial bones (CB) via a novel interaction between Nell-1 and Cntnap4, and that disruption of the Nell- 1/Cntnap4 functional axis will not only induce deficits in CB, but also interfere with neural transmission in the brain. Studies on such are summarized as follows.
  • Nell- 1/Cntnap4 Functional interaction of Nell- 1/Cntnap4 in craniofacial skeletal and brain tissues.
  • Nell- 1 and Cntnap4 we previously demonstrated the major roles of Nell- 1 and Cntnap4 in normal CB development.
  • the functional contributions of Nell-1/Cntnap4 in the postnatal CB and brain are specified using tissue specific knockout Nell-1 and Cntnap4 mice.
  • Wntl-Cre mice targeting CNNCs-derived CB and trigeminal ganglions (TG), and PV-2A-Cre mice targeting parvalbumin positive (PV+) GABAergic neurons in the brain and TG are bred with the existing Nell-lfl/fl and Cntnap4fl/fl mice to obtain Nell-lWntlKO, Cntnap4WntlKO, Nell- 1PV-2AKO, and Cntnap4PV-2AKO mice.
  • Nell-1 osteogenic effects in craniofacial skeletal tissues within the context of its interaction with Cntnap4
  • double calvarial defects in the frontal bones (CNCC -derived) and parietal bones (non-CNCC-derived) of Cntnap4WntlKO mice are created.
  • the healing process of these defects are evaluated using Nell-1 protein by live microCT and histomorphometry analyses.
  • the major molecular events of the Nell-1/Cntnap4 interaction during intramembranous osteogenesis are dissected with CNCCs.
  • Nell-l In contrast to Cntnap4, Nell-l’s function in the brain, despite its high expression, is completely unknown.
  • Nell-1 is not only effective for patients with skeletal conditions, but also for patients with cognitive and/or degenerative neural conditions.
  • Nell-1 is a unique osteogenic factor with multifaceted advantageous features.
  • Neural EGFL like 1 (Nell-1; also known as Nel-like molecule, type 1) is a unique multimeric secretory protein (1) ( Figure 1A). Watanabe et al. first sequenced Nell-1 from a human fetal brain cDNA library (2) while our group first identified Nell-l’s osteogenic function from its high expression in active bone formation sites in human craniosynostosis (CS) patients (3). Through gain- and loss-of-function models, we have demonstrated the essential role of Nell-1 in craniofacial and appendicular skeletogenesis (4-6).
  • Nell-1 loss-of-function newborn mice demonstrated reduced calvarial bone growth, enlarged sagittal sutures, and short body lengths (1, 4, 7) ( Figure 1C).
  • Figure 1C Reaffirming the importance of Nell-1 in human development, a similar phenotype of delayed cranial fontanelle and suture closure with short body stature was recently reported in a 3-year-old Japanese girl that had a de novo hemizygous interstitial deletion of chromosome I lpl4.1-pl5.3, the gene locus for Nell-1 (11).
  • Nell-1 has excellent safety profile with documented tumor-suppressive properties and provides significant benefits in preventing osteoporotic bone loss, and its anti-inflammatory and adipogenic inhibition properties (5, 18, 20, 21, 24) (Figure IB).
  • 1.2 Cntnav4 a novel susceptibility factor of Autism Spectrum Disorders (ASPs), is a specific cell surface receiver of Nell-1 in yromotine osteogenesis.
  • Nell-1 As a novel ligand for Cntnap4 (25).
  • Contactin-associated protein-like 4 (Cntnap4, also known as Caspr4) is a transmembrane neurexin superfamily member with vital functions in neurodevelopment, neurocognition, and the pathogenesis of
  • Cntnap4 critically regulates brain interneuron synaptic transmission by increasing GABAergic, while decreasing dopaminergic activity (27).
  • Global Cntnap4 knockout mice exhibit relatively decreased GABAergic, and increased dopaminergic activity that associates with repetitive, ASD-like behaviors that is rescued by pharmacological dampening of dopaminergic signaling or augmentation of GABAergic signaling (27).
  • GABA is the major inhibitory neurotransmitter in the brain and ASD has been increasingly linked to brain inhibitory circuit dysfunction (30).
  • Cntnap4 is functionally required for Nell-1 stimulation of Wnt/b -eaten in signaling pathway in vitro, and for Nell-1 mediated-osteogenesis in vivo (25).
  • Specific inactivation of either Cntnap4 or Nell-1 in cranial neural crest cells (CNCCs) using Wntl-Cre resulted in remarkably similar calvarial bone defects ( Figures 2A-2E) (25).
  • Nell- 1 protein has long been known to be highly expressed in the brain with unknown neural functions.
  • the identification of a neuronally expressed molecule, Cntnap4, as Nell-l’s receptor in mediating osteogenesis led us to further postulate that there may be potential Nell-1 functions or Nell-1/Cntnap4 interactions in the neural system and at the craniofacial neuroskeletal interface where the innervation of nerve fibers meet the craniofacial bones (25, 31).
  • Nell- 1 or Cntnap4 expression in the hippocampus, inferior olivary nucleus, and spinal cord (3, 26, 27, 32, 33) we are the first to perform co-localization studies of Nell- 1 and Cntnap4 in the central nervous system (Figure 3).
  • Our detailed in situ Nell-1 and Cntnap4 co localization studies in mouse brain revealed for the first time significant overlap of Nell- 1 and Cntnap4 protein in human hippocampal pyramidal cells and interneurons that might be critical for regulating excitatory (e.g., Dopamine) and inhibitory (e.g., GABA) neurotransmitters in the brain ( Figures 4A and 4B).
  • transcriptome analyses have implicated both Nell-1 and Cntnap4 involvement in several neurodegenerative and neuropsychiatric disorders such as ASD (34, 35) and Alzheimer’s disease (36).
  • ASD Alzheimer’s disease
  • our preliminary study also revealed that, like global Cntnap4 knockout mice, which exhibit diminished GABAergic and increased
  • Nell- 1 is recognized to be critical for mouse development, particularly in the craniofacial skeletal system (4, 5, 7, 47). However, it remains unclear how Nell-1 affects postnatal growth of craniofacial bones and the brain due to lack of appropriate mouse models (4). Significantly, we have successfully generated and characterized two new mouse lines of floxed Nell-1 and floxed Cntnap4 ( Figures 2A-2E). These mice when bred with Wntl-Cre allow us to target the CNCC derived craniofacial bones (CB) (e.g., frontal bone) and trigeminal ganglions (TG) for studies described below (48, 49).
  • CB CNCC derived craniofacial bones
  • TG trigeminal ganglions
  • PV+ parvalbumin positive GABAergic inhibitory neurons are critical for ASD pathogenesis (27) and comprise a significant portion of the TG (see Figures 7A and 7B), we use PV-2A-Cre to determine the effect of Nell- 1 or Cntnap4 deficiency on PV+ neurons in the brain as well as on TG innervation of the CB at the neuroskeletal interface.
  • the craniofacial neuroskeletal interface is where sensory fibers, a large majority of which are from the TG, innervate the CB (50).
  • Nell-1 gene was first cloned in a rat brain cDNA library and identified as a nuclear protein binding partner (1-3). Since then, there have been minimal studies on Nell-l’s role in the nervous system (31). Our technological finding of presynaptic membrane protein, Cntnap4 (27), as Nell-1’s specific receptor combined with the distinct co-localization of Nell- 1 and Cntnap4 in neural tissues ( Figures.
  • GABAergic neurons As outlined below due to the fact that GABA is the major inhibitory neurotransmitter in the brain and ASD has been increasingly linked to brain inhibitory circuit dysfunction (30) (see again section A2) and due to the broader distribution of PV+ inhibitory neurons compared with tyrosine hydroxylase positive (TH+) dopaminergic neurons in brain areas where Nell-1 and Cntnap4 extensively co-express and co-localize (refer to Figures 12A-12C).
  • TH+ tyrosine hydroxylase positive
  • Cntnap4Wntlknockout mice are used to evaluate the requirement for Cntnap4 during Nell- 1 -mediated calvarial bone defect repair as well as investigate Wnt/()-catcnin signaling activation by Nell-1/Cntnap4 interaction during craniofacial osteogenesis. Effects of Nell- 1 and Nell-1/Cntnap4 interaction in neural tissues are assessed through Cntnap4 pv 2A knockout and pharmacological GABAergic augmentation approaches. Influences and signaling pathways of the Nell-1/Cntnap4 interaction in modulating the release of the key inhibitory neurotransmitter, GABA, are defined.
  • Nell-1/Cntnap4 in craniofacial skeletal and neural tissues, we propose foundational studies to reveal the spatiotemporal tissue distribution and co-localization of Nell- 1 and Cntnap4 in the CB, brain, and at craniofacial neuroskeletal interfaces such as between the CB and the TG, whereby the peripheral nervous tissues of sensory and sympathetic fibers coming from TG innervate the CB during development and postnatal growth.
  • Nell-1 and Cntnap4 exhibit similar spatiotemporal expression patterns with areas of co-localization in the CB, brain, and craniofacial neuroskeletal interfaces during development and growth.
  • Nell- 1 and Cntnap4 positive neural cells the antibodies against parvalbumin (R&D), tyrosine hydroxylase (Abeam), GABA (Sigma), Dopamine, MAP2 and GFAP (ThermoFisher) are also used for neural tissues IHC.
  • the antibodies against active b-catenin (clone 8E7 of anti-ABC from Millipore), osteocalcin and cathepsin K (Santa Cruz) are also included for neural tissue and/or CB IHC.
  • Nell-1 is known to significantly impact craniofacial bone development via osteogenic cells (4, 5, 7, 47). However, it remains unknown how Nell-1 and Cntnap4 may interact to modulate postnatal growth of the craniofacial skeletal and brain tissues. For studies here we have created separate, cell lineage specific Nell-1 and Cntnap4 knockout mice (see again preceding descriptions). We use the tissue spatiotemporal distribution pattern of Nell-1/Cntnap4 from above and Cntnap4 knockout mice to specifically focus on Nell- 1/Cntnap4 interactions in the craniofacial skeleton and brain during postnatal growth and function.
  • Live microCT is utilized on postnatal mice to obtain whole skull scanning data for longitudinal measurements following published standard protocols by Jackson Laboratory (56) and our previous report (18).
  • We conduct relevant behavioral tests see list of tests in Vertebrate Animals to examine the functional involvement of Nell- 1 and/or Cntnap4 on obsessive/repetitive and abnormal social behaviors with the precision of cell-specific inactivation.
  • Nell-lfl/fl; Wntl-Cre and Cntnap4fl/fl; Wntl-Cre mice are used to evaluate the effects of Nell- 1 and Cntnap4 on postnatal growth of the CB:
  • We have successfully generated CNCCs specific Nell-1 and Cntnap4 knockout mouse lines (Nell- lWntlKO and Cntnap4WntlKO) by breeding existing floxed Nell-1 (6, 13, 18) and floxed Cntnap4 mice (25) with Wntl-Cre (JAX Stock# 022501), respectively ( Figures 2A-2E).
  • mice from each group (8 males and 8 females for each genotype) undergo live microCT scanning (Skyscan 1176, Bruker-microCT, Kontich, Belgium) of the whole skull at P7 (juvenile), P14 (youth), and P60 (adult) when the changes of the growing calvarial vault and cranial base are at the highest peak (56) (Table 1).
  • 3D reconstructions of the skull is used for measurement of selected parameters (Table 2) (6, 18).
  • PV-2A-Cre knock-in mice express the Cre protein in PV+ interneurons of the neocortex, hippocampus, and cerebellum where Cntnap4 also expresses highly (59, 60).
  • the Nell-lPV-2AKO and Cntnap4PV-2AKO mice are generated by crossing existing floxed Nell-1 and floxed Cntnap4 mouse lines with PV-2A-Cre line (JAX Stock# 008069).
  • EdU is pulsed at 72 hours prior to sacrifice, and then chased for cell proliferation until sacrifice.
  • We performed a power analysis for the sample size estimation of CB measurements with significant differences being detected using the Students' t-test (two-tailed, equal variance) between corresponding measurements at the compared ages with 80% power (alpha 0.05).
  • Figure 9A and 9B shows our successful optimization of the model; we noted that it requires 6 full weeks to completely regenerate the 2mm bone defects without therapeutic intervention.
  • the different embryological origins of the frontal and parietal bones ( Figure 9C) will allow selective Wntl -mediated Cntnap4 knockout of CNCCs in the frontal bone, but not the parietal bone, in each Cntnapt4Wnt-lKO mouse as an intra-animal control.
  • the interaction and signaling of Nell-1/Cntnap4 is required for postnatal CB defect repair and regeneration.
  • Double calvarial defects in juvenile Cntnap4 knockout mice are created and then recombinant human NELL-1 protein (rhNELL-1) delivered thereto (Aragen Bioscience Inc., see Authentication of key biological or chemical resources) using an established protein delivery protocol.
  • rhNELL-1 protein recombinant human NELL-1 protein delivered thereto (Aragen Bioscience Inc., see Authentication of key biological or chemical resources) using an established protein delivery protocol.
  • rhNELL-1 protein Aragen Bioscience Inc., see Authentication of key biological or chemical resources
  • This enables us to compare the crucial role of Nell-1/Cntnap4 interactions in frontal bone defects that have CNCC sources of Cntnap4 knocked out and in parietal bone defects with normal Cntnap4 expression.
  • MicroCT with morphological analyses are used to monitor and evaluate the healing progress with strong scientific rigor.
  • Immunohistochemistry is performed using antibodies against bone markers including osteopontin (OPN) and osteocalcin (OCN), Wnt signaling markers including active b-catenin (ABC) and Axin2, as well as Nell-1 (R&D), Cntnap4 (Sigma) and VEGL (Santa Cruz Biotechnology) using the ABC colorimetric method (Vector Laboratories, Burlingame, CA).
  • Cntnap4 knockout CNCCs during calvarial bone regeneration by fluorescent imaging: To trace the involvement of mutant CNCCs to the Nell- 1 -stimulated defect regeneration process, Cntnap4WntlKO and/or Cntnap4Wntl-R26RtdTomatoKO mice are used for tracking CNCCs in the healing frontal bone defect similar to what we performed in a previous study(6). We also assess cell proliferation at 2 weeks post-operation by EdU pulse labeling 4 hours prior to sacrifice and correlate with labeled Cntnap4 KO CNCCs.
  • Nell-1 activates Wnt/b -eaten in during osteogenesis (18, 47, 67, 68) with Integrin b ⁇ as a key Nell-1 binding partner for Wnt/b- catenin signaling (18, 69-71).
  • Cntnap4 is functionally required for Nell- 1 stimulation of Wnt ⁇ -catenin signaling in osteogenic-committed cells ( Figures 10A and 10B).
  • CNCCs both intracellular (i.e., PNU74654) and surface (i.e.,
  • DKK1 Wnt inhibitors can block Nell-1 mediated osteogenic effects, with PNU74654 providing more complete blockage of Nell-l’s effects ( Figures 11A and 1 IB) (also see Figure 10A).
  • glycogen synthase kinase ⁇ ( ⁇ dK3b ) inhibitors are recognized as potent activators of the Wnt ⁇ -catenin signaling pathway as CSK3[l mediated b-catenin phosphorylation causes its destabilization (72, 73) (see Figure 10A).
  • GSK-3 inhibitors are thought to regulate many cognitive-related processes such as neurogenesis, synaptic plasticity and neural cell survival and are increasingly studied as therapeutic agents for degenerative, cognitive, and psychiatric neural conditions (74, 75).
  • the Wnt canonical pathway downstream molecules, c-myc and cyclin Dl, along with key osteogenic marker genes, Ocn and Runx2, are also analyzed to assess Wnt signaling activation.
  • Wnt3a (at 50-100ng/ml) is used as a positive control for the in vitro experiments described herein. Thirty newborns of each genotype of mutant mice, inclusive of both sexes, are used for primary CNCCs isolation and the in vitro experiments (see Vertebrate Animals).
  • Cntnap4 critically regulates brain interneuron synaptic transmission by increasing GABAergic activity while decreasing dopaminergic activity (27).
  • Global Cntnap4 knockout mice exhibit relatively decreased GABAergic activity and increased dopaminergic activity that is associated with repetitive, ASD-like behaviors that are rescued by pharmacological dampening of dopaminergic signaling or augmentation of GABAergic signaling (27).
  • Nell-1 was identified as the first ligand capable of binding to Cntnap4 protein in the hippocampal pyramidal cells and intemeurons of mouse and human brains ( Figures 3 and, 4) (25).
  • Nell-1 haploinsufficient adult mice exhibit increased repetitive behaviors and social interaction deficits similar to those seen in Cntnap4 global knockout mice that are repressed by Risperidone ( Figures 5A-5C).
  • Nell- 1/Cntnap4 interaction might serve as a new therapeutic target of drugs known for augmenting GABA (e.g., Indiplon) and/or repressing Dopamine (e.g., Risperidone) to reverse the ASD-like phenotypes seen in both global Cntnap4 (27) and Nell-1 knockout mice ( Figures 5A-5C).
  • GABA e.g., Indiplon
  • Dopamine e.g., Risperidone
  • Ligures 14A-14C demonstrates establishment of primary mouse neuronal cell culture.
  • Ligure 14A shows the primary mouse hippocampal neurons were cultured on poly-D-lysine coated coverslip at Day 5;
  • Ligure 14B shows identification of neuronal cells by MAP2 (red) and astrocytes by GLAP (green) using immunocytochemistry at Day 7 primary culture;
  • Ligure 14C shows co-localization/expression of Nell- 1 and Cntnap4 in mouse primary neuronal cells at Day 7 culture.
  • Nell-1/Cntnap4 interaction in neuronal cells and neural progenitor cells NPCs: To better understand the role of Nell- 1 in neural cells, the effects of rhNELL-1 on the growth and maturation of neuronal cell neurites, on cell proliferation of NPCs, and on neuronal differentiation of NPCs and the neuroblastoma cell line, SH-SY5Y, are studied using standard protocols (92, 93).
  • Cntnap4 KO cells and the Wnt inhibitors are utilized to validate their necessity in Nell-1 mediated neural effects as we previously described for osteogenesis studies (18, 21) (also see Ligures 10A and 10B); (4) Assessing how GSK3 inhibitors affect Nell-1/Cntnap4 activation of Wnt/
  • Nell-1 may have significant positive impacts on neurite growth in neuronal cells, on NPC proliferation and on neuronal differentiation of neuroblastoma cell line, SH-SY5Y, by activating Wnt/
  • GSK3 inhibitors may significantly enhance the neurogenic effects of Nell- 1.
  • the studies provide (1) novel insight into Nell-l’s role in the nervous system and in the pathogenesis of ASD-like conditions, and (2) mechanistic insight of the Nell-1/Cntnap4 functional axis in neuroskeletal development, growth, and disease that will significantly impact growth and regeneration of postnatal craniofacial bones in cases of disease or injury.
  • PubMed PMID 12235118; PMCID: 151127.
  • Baig DN et al., Brain Research Bulletin. 2017;129:82-90.
  • Nell-1 Secretory protein Neural EGFL Like 1
  • Nell-1 is known to regulate osteogenesis via Wnt and MAPK signaling
  • the lack of Nell- 1 specific cell surface receptor(s) identification has impeded mechanistic studies.
  • Cntnap4 is a transmembrane neurexin superfamily member located on the presynaptic membrane of interneurons, has vital neurogenesis and central nervous system functions, and is associated with multiple neurodegenerative and neuropsychiatric disorders; nevertheless, it has no known osteogenic function.
  • Cntnap4 knockdown blocks Nell- 1 -responsive Wnt and MAPK signaling, and effectively abolishes Nell-l’s osteogenic effects.
  • the identification of this new interaction between a neurogenesis-associated receptor Cntnap4 and osteogenic ligand Nell-1 facilitates basic and translational studies on osteogenic roles of Nell-1/Cntnap4 signaling, and also has broader impacts on the growing research field of neuroskeletal interplay in homeostasis and diseases of the nervous and skeletal systems.
  • Neural EGFL Like 1 (Nell-1; also known as Nel-like molecule, type 1) is a unique multimeric secretory protein 1 .
  • Watanabe et al. first identified Nell-1 from a human fetal brain cDNA library 2 , while our group recognized its high expression in actively fusing human calvarial sutures from craniosynostosis (CS) patients 3 .
  • Nell-l essential role in craniofacial and appendicular skeletogenesis 4 15 , as well as its interplay with runt-related transcription factor 2 (Runx2, also known as Cbfal) 1, 7 , Wnt 16 18 , and MAPK 19, 20 signaling during osteogenesis.
  • Linx2 also known as Cbfal
  • Wnt 16 18 Wnt 16 18
  • MAPK 19 20 signaling during osteogenesis.
  • Nell-1 is highly conserved across species with 92.6% homology between human and rat 1 . Devoid of a transmembrane domain, Nell-1 is a secretory extracellular protein 1, 33 . Nell-1 contains multiple defined structural motifs, including a secretory signal peptide, an N-terminal thrombospondin- 1 -like (TSPN) module [which overlaps with a laminin G (LamG) domain], a coiled-coil region, several von Willebrand factor-like (vWF) repeats with five cysteine residues [also known as chordin-like, cysteine-rich (CR) domains], and six epidermal growth factor (EGF)-like repeats 1 3, 33, 34 .
  • TSPN N-terminal thrombospondin- 1 -like
  • vWF von Willebrand factor-like
  • Nell-1 requires Wnt and MAPK signaling during osteogenesis 16 20 , multiple attempts to search for a Nell- 1 specific cell surface receptor, including the use of yeast two-hybrid systems 4 , were unsuccessful until now.
  • Cntnap4 contactin associated protein-like 4
  • Caspr4 contactin associated protein-like 4
  • Nell-1 and Cntnap4 were co-localized in osteogenic-committed cells through confocal laser scanning microscopy (CLSM) and in situ proximity ligation assay (PLA). Physical binding between Nell-1 and Cntnap4 was further confirmed by pull-down and co-immunoprecipitation (Co-IP) assays, as well as by surface plasmon resonance (SPR) analysis that revealed a classical ligand/receptor, high-binding affinity between Nell-1 and Cntnap4.
  • CLSM confocal laser scanning microscopy
  • PDA in situ proximity ligation assay
  • Cntnap4 knock-down we demonstrated that Cntnap4 is functionally required for Nell-1 stimulation of Wnt and MAPK signaling in vitro, and Nell-1 mediated osteogenesis of cranial suture explants ex vivo.
  • our data reveal the critical neurogenesis-associated protein 36 38 , Cntnap4, as a specific cell surface receptor for Nell-1 during osteogenesis. More importantly, we have identified Nell-1, which itself is also highly expressed in neural tissues, as the first extracellular ligand to Cntnap4.
  • Cntnap4 belongs to a larger category of synaptic cell adhesion molecules (SCAMs) 39 that have been implicated in a wide variety of neurodevelopmental and neuropsychiatric disorders.
  • SCAMs synaptic cell adhesion molecules
  • Nell-1 The mammalian neural EGFL Like 1 ( Nell-1 ; also known as Nel-like molecule, type 1) gene and its related gene Nell-2 were originally cloned from a human brain cDNA library based on their similarity to the chicken neural epidermal growth factor (EGF)-like molecule ( Nel ) gene 2 , with Nell-2 being the mammalian ortholog of chicken Nel 40 .
  • EGF brain epidermal growth factor
  • Nel human brain cDNA library to construct a T7 phage display cDNA library for biopanning.
  • Amyloid beta A4 protein Appendix
  • Nel protein family (Nel-described in chicken and fish; Nell-1 and Nell- 2 -described in human and mammals, with Nell-2 being the ortholog to chicken and fish Nel 40 ) exhibits similar domain organization 34 .
  • Nell-1 and Nel can form homo- or hetero-complexes with each other through their N-terminal LamG/TSPN domains prior to secretion 40 , but do not form hetero complexes 40 with thrombopondin-1 (TSP-1), despite the Nel family and TSP-1 sharing similar structure and heparin-binding capability in their respective LamG/TSPN regions 33, 40, 41 .
  • the N-terminus LamG/TSPN domain of Nell- 1 is also required for the Nell-1/Cntnap4 interaction, as the deletion of the LamG/TSPN domain from Nell-1 eliminated nearly all binding of the Cntnap4 phage to Nell-1 ( Figure 16C).
  • the LamG/TSPN domains of Nel proteins appear to be important for homo/hetero-complex formation with other Nel family members, as well as for binding to Cntnap4.
  • Nell-1 is known to regulate osteogenic and chondrogenic differentiation 42 46 , we first examined Cntnap4 gene expression in twelve Nell- 1 -responsive cell lines capable of
  • Cntnap4 expression was highest in the highly osteogenic-committed MC3T3-E1 cell line 51, 52 ( Figure 17A), relative to less differentiated cell lines (such as C3H10T1/2 53, 54 , ST-2 55, 56 , 143B 57, 58 , MG63 57, 59 , and SaoS2 57, 58 ), predominantly adipo genic-committed cell lines (such as M2-10B4 60 ) or chondrogenic-committed cell lines (such as ATDC5 61 ).
  • the greater Cntnap4 expression pattern found in cells with greater osteogenic capability was also observed in primary cells.
  • NMCC 7 Primary newborn mouse calvarial cells (NMCC 7 ) exhibited significantly higher levels of Cntnap4 expression than primary mouse rib chondrocytes, human articular chondrocytes, or human bone marrow stem cells (Figure 17B).
  • C3H10T1/2 (Clone 8: ATCC ® CCL-226TM) mouse embryo/sarcoma M2-10B4 (ATCC ® CRL- 1972TM) mouse bone marrow/stroma cell line
  • MC3T3-E1 (Subclone 4: ATCC ® CRL-2593TM) mouse calvaria
  • Nell-1 significantly upregulated Cntnap4 expression in both MC3T3-E1 pre osteoblasts and primary NMCC ( Figures 24A and 24B).
  • CLSM confirmed the co-localization of Nell- 1 and Cntnap4 on the plasma membrane of MC3T3-E1 and primary NMCC, while PLA confirmed a direct Nell- 1/Cntnap4 interaction ( Figures 18A andl8B).
  • Cntnap proteins are known to share intracellular ligands.
  • amyloid beta precursor protein binding family A member 1 (APBA1, also known as Mintl) is an intracellular ligand for both Cntnap3 and Cntnap4, while calcium/calmodulin dependent serine protein kinase (CASK) intracellularly binds both Cntnap2 and Cntnap4 36 .
  • APBA1 amyloid beta precursor protein binding family A member 1
  • Mintl calcium/calmodulin dependent serine protein kinase
  • Nell-1 as the first extracellular ligand of Cntnap4 to be identified, may also bind Cntnap2 and Cntnap3 in osteogenic-committed cells.
  • control MC3T3-E1 cells and Cntnap4- KD MC3T3-E1 cells exhibited similar robust osteogenic responses to bone morphogenetic protein 2 (BMP2), an osteogenic protein with a signaling pathway distinct from Nell- 1 1 ( Figure 20A).
  • BMP2 bone morphogenetic protein 2
  • Figure 20A an osteogenic protein with a signaling pathway distinct from Nell- 1 1
  • Nell-1 protein significantly enhanced osteocalcin (Ocn) and osteopontin (Opn) expression in control MC3T3-E1 pre-osteoblasts, while no positive staining for either of these two osteogenic markers was observed in Nell-1 treated Cntnap4- KD MC3T3-E1 cells ( Figure 20B).
  • Concomitant gene expression profiling of osteogenic markers Alp, Collagen led, Collagen led, Ocn, Opn, and bone sialoprotein (Bsp) Figure 20C revealed inhibited osteogenic differentiation in Cntnap4-KD MC3T3-E1 cells, and consequent lack of response to Nell-1.
  • Nell-1 is also a major regulator of chondrogenic differentiation and maturation 43, 46, 63 , chondrogenic committed ATDC5 cells exhibited notably lower Cntnap4 expression ( Figures 20A-20B).
  • expression of nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 2 (Nfatc2), a known Nell-1 primary response gene in ATDC5 cells 44 was decreased by Cntnap4- KD alone (without Nell-1) in ATDC5 cells ( Figure 26A).
  • Cntnap4-KD did not affect dose-dependent Nell-1 stimulated increases of Nfatc2 expression in Cntnap4-KD ATDC5 cells ( Figure 26B).
  • Nell-1 can regulate Nfatc2 expression, Nell-1 can induce Nfatc2 in chondrogenic-committed cells independently of Cntnap4.
  • Nell- 1 -responsive osteogenic-committed cells e.g., MC3T3-E1 cells and NMCC
  • ADTC5 cells 44 highly Nell- 1 -responsive cells, such as ADTC5 cells 44
  • Cntnap4 is not likely to be the only cell surface receptor for Nell-1.
  • Nell-1 may interact with different receptors in a cell-type and differentiation stage specific manner.
  • Cntnap4 Mediates Nell- 1 -Stimulated MAPK and Wnt Signaling in Osteogenic-Committed Cells
  • Nell- 1 -responsive ERK or JNK activation was markedly diminished in Cntnap4-KD MC3T3-E1 cells ( Figure 22A).
  • Cntnap4- KD completely abolished Nell- 1 -responsive Wnt signaling ( Figures 22B and 22C).
  • Nell-1 signaling pathways ( Figure 23) are distinct from BMP2 16, 19 21, 65 .
  • Nell-1 may hold great promise for future clinical development as a safer therapeutic for bone repair and regeneration 1, 18 .
  • ATRAID all-trans retinoic acid induced differentiation factor
  • APR3 apoptosis related protein 3
  • Nell-1 in osteosarcoma- and kidney-derived cell lines 68 .
  • ATRAID was a protein mainly present on the lysosomal membrane 69 , it was not considered a suitable candidate for extracellular Nell- 1 1 binding and signal transduction. Instead, ATRAID, due to its similarities with lysosomal-associated membrane protein 1/2 69 , may play a role in endocytosis and/or transportation of Nell-1 68 .
  • ATRAID can bind with multiple truncated Nell-1 constructs with or without the N-terminal LamG domain 68 .
  • Previously we also reported the binding of Nell- 1 C-terminal sequence with integrin-b ⁇ 49, 70 , and that integrin-b ⁇ knock-down inhibited Nell- 1 -responsive Wnt- signal transduction 18 .
  • Integrin-b ⁇ demonstrates relatively promiscuous binding with a broad range of molecules in various cell types (including osteoblasts and chondrocytes) 70 72 , and is less likely to be a Nell- 1 -specific receptor.
  • integrin-b ⁇ binds to the C-terminus of Nell-1 70
  • Cntnap4 binds to the Nell-l’s N-terminus.
  • Nell-l-integrin-b ⁇ binding facilitates Nell-1 ligand-receptor complex formation with Cntnap4 to stimulate downstream signal transduction remains for future investigation.
  • Nell-1 was first identified in fetal and adult human brain 2 , and abundant Nell-Fs expression is detected in developing and adult nervous systems 33 .
  • Transmembrane receptor Cntnap4 first identified from a fetal mouse brain cDNA library, is abundantly expressed in the central nervous system and during neurogenesis 36 .
  • the spatial expression patterns of Nell- 1 and Cntnap4 are largely overlapped in the central nervous system hippocampus, inferior olive nucleus, and spinal cord 33, 36, 40 , we successfully detected the binding between Nell-1 and Cntnap4 by T7 phage display from a human brain cDNA library.
  • Cntnap4 is a member of the neurexin superfamily of SCAMs 39 , which play important roles in connecting pre- and post-synapses that are critical to synapse development and cortical interneuron function 36, 37, 39 , recognizing Cntnap4 as a specific, functional receptor for Nell-1 formation in osteogenic-committed cells is unexpected, but highly significant, to elucidate the mechanisms mediating the neural-bone interactions required for the regeneration of skeletal system. This is particularly important in the dental and craniofacial skeletal system for which development and restoration is largely orchestrated by the nervous system 73 .
  • Cntnap4 and other neurexin superfamily members have been identified as susceptible molecules associated with neuropsychiatric disorders such as ASD 74 76 and neurodegenerative disorders including age-related disease, cognitive impairment, late onset Alzheimer’s disease, Alzheimer’s disease, and Parkinson’s disease 77 .
  • Murine Cntnap4- null mice demonstrate significant interneuron dysfunction with abnormally diminished GABAergic and increased dopaminergic synaptic transmission, and display perseverative behaviors that mimic human ASD patients 37 .
  • human genetic linkage studies and murine models strongly implicate Cntnap4 in neurobiological diseases.
  • Cntnap4 may represent a novel set of receptors that play essential roles in both nervous and skeletal tissues. While previous studies revealed CASK and/or APBA1 36 interactions with the cytoplasmic domain of Cntnap members, no extracellular binding proteins or ligands to Cntnap4 have been identified so far. We are the first to describe Nell-1, which itself is also highly expressed in neural tissues 1, 2, 25, 33 , as the first extracellular ligand to Cntnap4.
  • Nell-1 overexpression in mice results in craniofacial anomalies and neural tube defects during the late embryonic development stage, which represent the phenomena observed in acrania 78 , a rare human congenital disease that is often associated with exencephaly 79 .
  • our current ex vivo results showed that interfering with the Nell- 1/Cntnap4 signaling pathway significantly delayed suture closure (Figures 21A-21C), which is in accordance to our previous finding that Nell- 1 -deficiency leads to postponed suture closure resembling cleidocranial dysplasia (CCD) 8 .
  • CCD cleidocranial dysplasia
  • G protein- coupled receptor family C group 6 member A (Gprc6a) has been recognized as the receptor of Ocn in the reproductive system 89, 90 and glucose metabolism 91 , because Gprc6a deficiency in mice does not replicate the neurotransmitter and behavior abnormalities seen in the Ocn-deficient animals 82, 88 , the receptor transducing Ocn signals in the brain is still unknown. Therefore, in order to develop effective and specific therapeutics for the unique skeletal-neural associated conditions, such as CCD and ASD, the nature of interactions between brain and skeletal tissues requires further investigation.
  • Nell-1/Cntnap4 mediated the Nell-1- responsive Ocn expression in osteogenic-committed cells ( Figures 20B and 20C), which suggests that there is a potential Nell-1/Cntnap4 pathway in regulating Ocn-mediated endocrinal modulation among different tissues and organs, including bone and the brain 80 .
  • our ongoing studies have found co-localized Nell-1 and Cntnap4 in the human hippocampus ( Figure 27) as well as in osteogenic-committed cells. It is highly possible that, in addition to the well- known BMP-mediated signal transduction 92 , Nell-1/Cntnap4 represents an alternative pathway that orchestrates the skeletal-neural functional axis.
  • Nell-1 and Cntnap4 are potential loci of several neurodegenerative and neuropsychiatric disorders, such as ASD 31, 32, 74 76 and Alzheimer’s disease 28, 11 .
  • Our preliminary studies revealed that, like Cntnap4-mutant mice 37 , Nell-1 -mutant mice also have aberrant behaviors which can be rescued by specific neuropsychiatric pharmaceutical intervention (unpublished data). Further investigations are currently ongoing to demonstrate the functional involvement of Nell-1/Cntnap4 axis in the brain, as we have already shown in bone tissue.
  • Recombinant full-length human Nell-1 with polyhistidine-tag was provided by Katayama Inc. (Amagasaki-city, Hyogo, Japan) with a purity of 98%, and used as bait in the phage display study.
  • Recombinant full-length human Nell- 1 without tags was provided by Aragen Bioscience Inc. (Morgan Hill, CA, USA) with a purity of 92% 17 for all functional studies.
  • the plasmid expressing FamG domain deleted human Nell-1 (Nell-l [LamG] ), constructed from the pcDNATM3. 1 //nyr-His B Mammalian Expression Vector (Thermo Fisher Scientific, Canoga Park, CA, USA) 68 was transfected into CHO-K1 cells (ATCC ® CCF-61TM) by
  • FIPOFECTAMINE ® 3000 Thermo Fisher Scientific.
  • the His-tagged Nell-l [LamG] was then purified from the supernatant of the transient transfection product by PROBONDTM Purification System (Thermo Fisher Scientific).
  • Cntnap4 human Cntnap4
  • GenScript GenScript (Piscataway, NJ, USA).
  • the extracellular portion of Cntnap4 (Cntnap4 extra ) was subcloned into pSecTag2A Mammalian Expression Vector (Thermo Fisher Scientific) with the primers 5’-CAC GGT ACC TGG GAA TTC CTA T-3’ and 5’-TTA CTC GAG CTG CAG AGT CAC TT-3 ⁇ and then transfected into CHO-K1 cells.
  • the His-tagged Cntnap4 extra was purified from the supernatant of the transient transfection product by PROBONDTM Purification System (Thermo Fisher Scientific), and identified by Western Blot ( Figures 28A-28B).
  • HIS-SEFECT ® Nickel Magnetic Agarose Beads (Sigma- Aldrich Corp., St. Fouis, MO, USA) were selected to immobilize His-tagged Nell-1.
  • the phage display cDNA library was constructed from the Human Brain cDNA Fibrary (EMD Millipore, Billerica, MA, USA) using the novagen T7SEFECT ® System (EMD Millipore). An aliquot of the amplified phage display cDNA library was incubated with His-tagged Nell-1 coated beads for four rounds of biopanning screens.
  • the phages bound to the His-tagged Nell-1 coated beads were eluted, and 100 plaques were selected to amplify the phage DNA by PCR with the provided primers and buffers in the Novagen T7SELECT ® System. Sequences spanning over 500 bp were selected and sequenced by Laragen Inc. (Culver City, CA, USA).
  • Dissociation constant ELISA was performed per manufacturer instructions from Novagen T7 Tail-Fiber Monoclonal Antibody (EMD Millipore). In short, 10 pg/ml of His-tagged full- length Nell-1 or Nell-l [LamG] was used as bait to coat the ELISA plate (Corning Inc., New York, NY, USA). After removing the unbounded bait, the plate was blocked with 3% non-fat milk (Bio- Rad Laboratories Inc., Hercules, CA, USA) overnight at 4°C. 100 m ⁇ /well diluted phage lysate (1X10 4 - 1X10 10 phages/ml) was added to the coated plate, and incubated at 37°C for 1 hour.
  • mouse Gapdh Mm99999915_gl
  • mouse Cntnap4 Mm00519175_ml
  • mouse Cntnap3 Mm01297195_ml
  • mouse Cntnap2 Mm00470553_ml.
  • Serum-starved MC3T3-E1 pre-osteoblasts (Subclone 4, ATCC ® CRL-2593TM) and primary newborn mouse calvarial cells (NMCC) were treated with 500 ng/ml Nell-1 for 30 min, and fixed with ice-cold acetone. The fixed cells were blocked with 3% bovine serum albumin (BSA; Sigma- Aldrich Corp.) and incubated with goat anti-Cntnap4 (1 :200; Santa Cruz
  • C57BL/6 mice were bred and maintained under an institutionally approved protocol provided by the Chancellor’s Animal Research Committee at UCLA (protocol number: 2012- 041), as previously described 50 .
  • Calvarial tissues dissected from 60-day-old mice were fixed in 4% ice-cold paraformaldehyde (PFA; Sigma-Aldrich Corp.) for 24 hours, and decalcified with 19% EDTA (Sigma-Aldrich Corp.) for 14 days prior to paraffin embedding.
  • Hematoxylin and eosin (H&E) staining was performed on 5-pm sections. Images were documented by a Keyence BZ-X710 system (Itasca, IL, USA).
  • DUOLINK ® PLA assay All reagents used for DUOLINK ® PLA assay were purchased from Sigma- Aldrich Corp. Deparaffinized 60-day-old mouse calvarial sections were blocked with DUOLINK ® blocking solution at room temperature for one hour before incubation with the same primary antibodies used in IHC staining, but diluted in DUOLINK ® antibody diluent at 4°C overnight. The following day, the sections were washed, and then incubated with DUOLINK ® In Situ PLA ® Probe Anti-Rabbit MINUS and Probe Anti-Goat PLUS (for Cntnap4 and Cntnap3) or Probe Anti- Mouse PLUS (for Cntnap2) at 37°C for 60 min, following manufacturer instructions.
  • Amplification solution was mixed with polymerase and applied to the slides for 100 min at 37°C, accordingly.
  • the slides were washed, mounted with DUOLINK ® In Situ Mounting Medium with DAPI, and examined with Leica TCS SP8 Confocal Laser Scanning Platform.
  • Co-IP Co-Immunoprecipitation
  • MC3T3-E1 cells or NMCC were suspended in PBS, and incubated with Nell-1.
  • BS 3 (Thermo Fisher Scientific) was added to the cells as a cross-linker.
  • Total protein was isolated by adding cold RIPA lysis buffer (Thermo Fisher Scientific) to the collected cells, and incubating with anti-Nell- 1 antibody coated agarose beads.
  • the Nell- 1/candidate receptor(s) complex was eluted for SDS-PAGE.
  • Binding studies were performed on a Biacore 3000 instrument (Biacore AB, Uppsala, Sweden) by the UCFA Surface Plasma Resonance Core. Cntnap4 extra was immobilized on CM5 sensor chips (GE Healthcare Life Sciences, Marlborough, MA, USA) by amine coupling. The solution phase of Nell-1 was dissolved in HBS-EP buffer, which contained 0.15 M NaCl, 10 mM HEPES, pH 7.4, 3 mM EDTA, and 0.005% polysorbate 20 (GE Healthcare Life Sciences). The solution traversed through the sensors at a flow rate of 50 m ⁇ /minute. Low-retention polypropylene tubes (Corning Inc.) were used throughout.
  • MC3T3-E1 and ADTC5 cells were transfected with Cntnap4 shRNA Lentiviral particles (Santa Cruz Biotechnology) or non-target control shRNA by LIPOFECT AMINE ® 3000.
  • the positive transfected colonies were selected by Puromycin (Sigma-Aldrich Corp.) and validated by Cntnap4 mRNA expression levels in order to establish the Cntnap4 knockdown cell line.
  • Cntnap4- KD Control and stable Cntnap4 knockdown (Cntnap4- KD) MC3T3-E1 cells were seeded on 24-well plates for alkaline phosphatase (ALP), Alizarin Red, and ICC staining. Additionally, cells were seeded on 6-well plates for osteogenic genes expression assay.
  • ALP alkaline phosphatase
  • Alizarin Red Alizarin Red
  • Both cell types were cultured in osteogenic differentiation medium [a-MEM (Thermo Fisher Scientific), 10% fetal bovine serum (FBS; Thermo Fisher Scientific), 50 pg/rnl ascorbic acid (Sigma-Aldrich Corp.), and 10 mM b-glycerophosphate (Sigma-Aldrich Corp.)] with or without 500 ng/ml Nell-1 or 100 ng/ml BMP2 (Medtronic, Minneapolis, MN, USA). ALP staining and Alizarin Red staining were performed as previously described 49 . Anti-Ocn (1 :200; Santa Cruz Biotechnology) and anti-Opn (1 :200; Santa Cruz Biotechnology) antibodies were used for ICC staining.
  • a-MEM Thermo Fisher Scientific
  • FBS fetal bovine serum
  • 50 pg/rnl ascorbic acid Sigma-Aldrich Corp.
  • 10 mM b-glycerophosphate Sigma-Aldrich
  • mice Alp ⁇ 5’-ACT GAT GTG GAA TAC GAA CTG GAT GAG AAG G-3’ (SEQ ID NO: l) and 5’-CAG TCA GGT TGT TCC GAT TCA ATT CAT ACT GC-3’ (SEQ ID NO:2); mouse Collagen Ial : 5’ -CTG GCG GTT CAG GTC CAA T-3’ (SEQ ID NOG) and 5’-TTC CAG GCA ATC CAC GAG C-3’ (SEQ ID NO:4); mouse Collagen la2 5’-AAG GGT GCT ACT GGA CTC CC-3’ (SEQ ID NOG) and 5’-TTG TTA CCG GAT TCT CCT TTG G-3’ (SEQ ID NOG); mouse Ocn: 5’-CTG CCC TAA AGC CCA AAC TCT-3’ (SEQ ID NO:7) and 5’-GAG AGG ACA GGG A
  • Calvarial vaults of newborn wildtype mice were harvested and cultured as previously described 50 , and randomly assigned to each experiment group.
  • Lentiviral particles of Cntnap4 shRNA (Santa Cruz Biotechnology) and CMV-Nell-1 (GenTarget Inc., San Diego, CA, USA) were added to the culture medium at day 0 and day 1, respectively.
  • Alizarin Complexone (2 pg/ml, Sigma-Aldrich Corp.) was added to the culture medium at day 4, and the medium, containing Alizarin Complexone, was changed every 3 days thereafter.
  • the explants were fixed in 4% Paraformaldehyde at day 10.
  • the Alizarin Complexone deposition on explants was observed with an Olympus SZX12 fluorescent microscope. Overlap width of frontal and parietal bones, as well as the area of unclosed fontanel, were quantified using Image-Pro Plus in a blinded fashion (Media Cybernetics, Warrendale, PA, USA).
  • Sub-confluent control and Cntnap4- KD MC3T3-E1 cells were subjected to serum starvation for 18 hours before treatment with PBS control or 500 ng/ml Nell-1 for 10, 30, or 60 min at 37°C. Protein isolation and Western blot were performed as previously described 50 .
  • ERK1 + ERK2 (1 :1,000; Abeam), ERKl(phospho Y204) + ERK2 (phospho Y187) (1 : 1,000; Abeam), JNK1 (1 : 1,000; Abeam), JNK1 + JNK2 + JNK3 (phospho Y185 + Y185 + Y223) (1 :1,000; Abeam), p38 (1 :1,000; Abeam), p38 (phospho T180 + Y182) (1 :1,000; Abeam), activc-(i-Catcnin (1 :5,000; EMD Millipore), b-Catenin (1 : 1,000; Abeam), Axin2 (1 :1,000; Abeam), GAPDH (1 :500; Santa Cruz Biotechnology), and Histone H3 (1 : 1,000; EMD Millipore).
  • Control and stable Cntnap4- KD ADTC5 cells were cultured in 1 :1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (Thermo Fisher Scientific) supplied with 5% FBS, 10 mg/ml of human transferrin (Sigma- Aldrich Corp.) and 30 nM sodium selenite (Sigma- Aldrich Corp.) until sub-confluence. After 18-hours of serum starvation (0.1% FBS), cells were treated with 0, 400, 800, 1600, or 3200 ng/ml Nell-1 for 3 hours 44 .
  • NFact2 GenBank Accession Number AK161174.1
  • primers forward primer 5’-CTT TCA GAT GGG AAT AAA CGT C-3’ (SEQ ID NO: 13), and reverse primer 5’-TCC TAC TCA CAT AGC AAC AGA A-3’ (SEQ ID NO: 14); and Gapdh (GenBank Accession Number AK002273.1) primers: forward primer 5’-ATT CAA CGG CAC ATG CAA GG-3’ (SEQ ID NO: 15), reverse primer 5’-GAT GTT AGT GGG GTC TCG CTC-3’ (SEQ ID NO: 16), respectively 44 .
  • Hasebe A et al., Molecular biotechnology 2012; 51 :58-66.
  • a physician or orthopedic surgeon of skill in the art can treat a patient with back pain.
  • the patient experiences a traumatic accident by slipping on the ice and is experiencing continuous lower back pain, numbness in some of his extremities, and neurological impairment.
  • the doctor can perform a physical examination to test the patient’s range of motion, muscle strength, and tenderness of the injured area.
  • the doctor can then diagnosed the patient with spinal stenosis and perform a spinal tap, followed by an MRI and CT scan to confirm the diagnosis.
  • the doctor may recommend a spinal fusion surgery to alleviate the pain and neurological symptoms.
  • the doctor can perform a posterolateral fusion (PLF) to heal the injured area.
  • PPF posterolateral fusion
  • a bone graft comprising a composition described herein (e.g., a composition containing an antibody that specifically binds to Cntnap4) can be prepared and administered according the methods described herein.
  • the graft can be administered, for example, around the damaged vertebrae to encourage bone formation. Over the next 6 to 18 months, the patient can be examined to determine if the materials in the bone graft promote osteogenesis and fuse with the vertebrae to heal the painful area.
  • a physician of skill in the art can treat a patient with Alzheimer’s disease.
  • the patient experiences cognitive degeneration and loss of memory.
  • the doctor can perform a physical examination to test the patient’s memory and recall.
  • the doctor diagnoses the patient with Alzheimer’ s disease and can administer a composition described herein (e.g., a composition containing an antibody that specifically binds to Cntnap4).
  • the antibody can be administered intrathecally once a week for 26 weeks in order to trigger neurogenesis. Over the next 6 months, the patient can be examined to determine if the symptoms of the disease diminish and to determine if some cognitive ability is restored.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Veterinary Medicine (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Zoology (AREA)
  • Public Health (AREA)
  • Toxicology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Environmental Sciences (AREA)
  • Hospice & Palliative Care (AREA)
  • Epidemiology (AREA)
  • Psychiatry (AREA)
  • Biotechnology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Rheumatology (AREA)
  • Cell Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention provides a composition for treating a disorder, comprising an effective amount of an agent effective for potentiating an effective binding of blood NELL-1 to blood Cntnap4 in a mammalian subject in need thereof to achieve a normal level of blood NELL-1 binding to blood Cntnap4 in the mammalian subject, the mammalian subject has an abnormal level of blood NELL-1 binding to blood Cntnap4. Methods of making and using the composition are also provided. Also provided is a method of diagnosing a neurological disorder or a skeletal disorder. Also featured are methods and compositions for treating or reducing the symptoms of a disorder, such as a neurological disorder or a bone disorder, in particular autism and osteoporosis, respectively.

Description

METHODS AND COMPOSITIONS FOR SKELETAL AND NEUROLOGICAL
DISORDERS
STATEMENT OF GOVERNMENTAL SUPPORT
[0001] This invention was made with Government support under AR 061399 and AR 066782, awarded by the National Institutes of Health. The Government has certain rights in the invention.
FIELD OF THE INVENTION
[0002] The present invention generally relates a methods and compositions for a disorder such as a neurological disorder or a bone disorder such as osteoporosis.
BACKGROUND OF THE INVENTION
[0003] Neural disorders and skeletal disorders continue to be main health challenges of human being as the aging and aged population of the world continues to grow since people are living a longer life due to improved health care and advances of various technologies. For example, autism spectrum disorder (ASD), a neural disorder, now affects about 1 % of the general population. However, after decades of investigation, the definite causes of ASD have not been fully revealed, and thus, efficient treatment is lacking to date. Similarly, while various methods of treatment of skeletal disorders have been developed, there are much room for improvement of such treatment with the aid of new insight into the various biological/biochemical processes leading to skeletal disorders.
[0004] Therefore, there is a continuing need for strategies and agents for treating or ameliorating neural and skeletal disorders.
[0005] The embodiments below address the above described problems and needs.
SUMMARY OF THE INVENTION
[0006] In one aspect of the present invention, provided is a composition for treating a disorder, which composition comprises an effective amount of an agent effective for potentiating an effective binding of blood NELL-1 to blood Cntnap4 in a mammalian subject in need thereof to achieve a normal level of blood NELL-1 binding to blood Cntnap4 in the mammalian subject, wherein the mammalian subject has an abnormal level of blood NELL-1 binding to blood Cntnap4.
[0007] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the agent comprises a Wnt/b -eaten in signaling activator.
[0008] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the agent comprises NELL-1, Cntnap4, and a combination of NELL- 1 and Cntnap4.
[0009] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the agent comprises a GSK3[> inhibitor.
[0010] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the agent comprises an agonist of GABAergic activity and/or an inhibitor of Dopaminergic activity in the subject.
[0011] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the agent further comprises integrin-bΐ.
[0012] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the agonist of GABAergic activity is Indiplon.
[0013] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the inhibitor of Dopaminergic activity is
Risperidone.
[0014] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the disorder is a neurological disorder or a skeletal disorder.
[0015] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the disorder is osteoporosis.
[0016] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the disorder is autism.
[0017] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the agent is an enhancer or inhibitor of NELL- 1 or Cntnap4.
[0018] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the agent is an exogenous gene construct expressing NELL-1 or Cntnap4.
[0019] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the composition further comprises a
pharmaceutically acceptable carrier for local or systemic delivery.
[0020] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the subject is a human being.
[0021] In another aspect of present invention, provided is a method of diagnosing a neurological disorder or skeletal disorder in a mammalian subject, which method comprises:
providing a normal level of blood NELL-1 binding to blood Cntnap4 in a normal mammalian subject in reference to a disorder selected from a neurological disorder or a bone disorder,
generating a diagnostic level of blood NELL-1 binding to blood Cntnap4 by measuring blood NELL-1 and blood Cntnap4 in the mammalian subject,
determining that the mammalian subject suffers from the neurological disorder or bone disorder if the diagnostic level of blood NELL-1 binding to blood Cntnap4 significantly deviates from the normal level of blood NELL-1 binding to blood Cntnap4.
[0022] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the disorder is a neurological disorder.
[0023] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the disorder is autism or osteoporosis.
[0024] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the subject is a human being.
[0025] In a further aspect of the present invention, provided is a method of treating or ameliorating a disorder in a mammalian subject, which method comprises administering to the mammalian subject in need thereof a composition comprising an effective amount of an agent effective for potentiating an effective binding of blood NELL- 1 to blood Cntnap4 in the mammalian subject to achieve a normal level of blood NELL-1 binding to blood Cntnap4 in the mammalian subject,
wherein the mammalian subject has an abnormal level of blood NELL-1 binding to blood Cntnap4 (e.g., compared to a control subject or a subject without a disorder, such as a bone disorder or a neurological disorder), and
wherein the disorder is a neurological disorder or a bone disorder.
[0026] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent comprises a Wnt/|l-catenin signaling activator.
[0027] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent comprises NELL-1, Cntnap4, and a combination of NELL- 1 and Cntnap4.
[0028] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent comprises a GSK3 inhibitor.
[0029] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent comprises an agonist of GABAergic activity and/or an inhibitor of Dopaminergic activity in the subject.
[0030] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent further comprises integrin-bΐ.
[0031] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agonist of GABAergic activity is Indiplon.
[0032] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the inhibitor of Dopaminergic activity is Risperidone.
[0033] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the disorder is a neurological disorder or a skeletal disorder.
[0034] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the disorder is osteoporosis.
[0035] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the disorder is autism.
[0036] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent is an enhancer or inhibitor of NELL- 1 or Cntnap4.
[0037] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent is a gene construct expressing NELL-1 or Cntnap4.
[0038] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the composition further comprises a pharmaceutically acceptable carrier for local or systemic delivery.
[0039] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the subject is a human being.
[0040] In a further aspect of the present invention, provided is a method of fabricating a composition, which method comprises:
providing an effective amount of an agent, which is capable of potentiating an effective binding of blood NELL-1 to blood Cntnap4 in a mammalian subject in need thereof to achieve a normal level of blood NELL-1 binding to blood Cntnap4 in the mammalian subject (e.g., compared to a control subject or a subject without a disorder, such as a bone disorder or a neurological disorder), and
forming the composition,
wherein the disorder is a neurological disorder or a bone disorder.
[0041] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent comprises a Wnt/|i-catenin signaling activator.
[0042] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent comprises NELL-1, Cntnap4, and a combination of NELL- 1 and Cntnap4. [0043] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent comprises a GSK3 inhibitor.
[0044] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent comprises an agonist of GABAergic activity and/or an inhibitor of Dopaminergic activity in the subject.
[0045] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent further comprises integrin-bΐ.
[0046] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agonist of GABAergic activity is Indiplon.
[0047] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the inhibitor of Dopaminergic activity is Risperidone.
[0048] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the disorder is a neurological disorder or a skeletal disorder.
[0049] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the disorder is osteoporosis.
[0050] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the disorder is autism.
[0051] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent is an enhancer or inhibitor of NELL- 1 or Cntnap4.
[0052] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent is a gene construct expressing NELL-1 or Cntnap4.
[0053] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the composition further comprises a pharmaceutically acceptable carrier for local or systemic delivery.
[0054] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the subject is a human being.
[0055] In another aspect, featured is an isolated antibody or antigen-binding fragment thereof that specifically binds an epitope present on Cntnap4. The antibody or antigen-binding fragment thereof may be an agonistic antibody. The epitope may be within a Laminin G domain of Cntnap4. The antibody or antigen-binding fragment thereof may be a humanized antibody, a human antibody, or a monoclonal antibody. The antibody or antigen-binding fragment thereof may include human constant regions. The antigen-binding fragment may be an antibody that lacks the Fc portion or is a F(ab’)2, a Fab, an Fv, or an scFv structure.
[0056] Also featured is a fusion protein containing the antibody or antigen-binding fragment thereof of any of the above embodiments fused to Nell-1 or a fragment thereof.
[0057] Also featured is a pharmaceutical composition including the antibody or antigen binding fragment thereof of any of the above embodiments in combination with, e.g., a pharmaceutically acceptable carrier, excipient, and/or diluent.
[0058] Also featured is a method of promoting bone formation (e.g., osteogenesis) in a subject in need thereof by administering to the subject the antibody or antigen-binding fragment thereof or a pharmaceutical composition containing the antibody or antigen-binding fragment thereof, such that the antibody or antigen-binding fragment thereof specifically binds to Cntnap4, thereby promoting the bone formation (e.g., osteogenesis).
[0059] In some embodiments, the subject has a bone disorder, such as osteoporosis.
[0060] Also featured is a method of promoting neurogenesis in a subject in need thereof by administering to the subject the antibody or antigen-binding fragment thereof a pharmaceutical composition including the antibody or antigen-binding fragment thereof, such that the antibody or antigen-binding fragment thereof specifically binds to Cntnap4, thereby promoting neurogenesis.
[0061] In some embodiments, the subject has a neurological disorder, such as Autism spectrum disorder.
[0062] The antibody or antigen-binding fragment thereof may be administered at a dosage of about 0.001 mg/kg/day to about 10 mg/kg/day (e.g., 0.001 to about 0.01 mg/kg/day, about 0.01 to about 0.1 mg/kg/day, about 0.1 to about 1 mg/kg/day, or about 1 to about 10 mg/kg/day). In the compositions described herein, the antibody or antigen-binding fragment thereof may be formulated at a concentration of 0.5-300 mg/mL, e.g., in a volume of 0.1-2 mL (such as a volume of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0 mg/mL).
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] Figures 1A-1C show Nell-1 structure and function. Figure 1A shows distinct Nell-1 domains. Figure IB shows known functions of Nell-1. Figure 1C shows a nonsense mutation in the mouse Nell-1 locus induced by N-ethyl-N-nitrosourea (ENU) resulted in neonatal lethality with skeletal abnormalities.
[0064] Figures 2A-2E show generation of floxed Nell-1 and Cntnap4 mice. Figure 2 A shows exon 1 was targeted for floxed Nell-1; Figure 2B shows X-gal stained and microCT scanned newborn mice of floxed Nell-1 bred with Wntl-Cre showing defect in frontal bones (*); Figure 1C shows quantitative measurements of defect area in frontal bones; Figure 2D shows exon3 was targeted for floxed Cntnap4; Figure 2E shows similar fontal bone defect was seen in Cntnap4;Wntl-Cre knockout mice (25). Scale bar in B: 2mm (L), 2.5mm (R) and in E: lmrn. **p<0.01.
[0065] Figures 3A-3F show co-localization of Nell- 1 and Cntnap4 in mouse brain. Figure 3 A shows hippocampal and thalamic areas of 2-month-old WT mouse brain. Figures 3B-3E show immuno fluorescent staining of Nell- 1 (red) and Cntnap4(green) in different areas of the mouse brain in A showing co-localization in pyramidal cells and interneurons. Figure 3F shows complete overlapping of Nell- 1 and Cntnap4 in Purkinje cells in the cerebellum. CA1, CA3: areas of hippocampal proper; DG: dentate gyrus; HTH: hypothalamus; CBF: cerebellum. Scale bar: lOOpm. Insets: enlarged images of single or double positively stained neurons.
[0066] Figures 4 A and 4B show immunohistochemistry of Nell- 1 and Cntnap4 in the human hippocampus. Figure 4A shows double positive neurons in CA1. Figure 4B shows co -localization of Nell- 1 and Cntnap4 in pyramidal cells and interneurons of CA4.
[0067] Figures 5A-5C show risperidone decreases hyperactivity and repetitive behavior in Nell- 1 1 mice. Figure 5A shows Nell-1+/- mice exhibit overgrooming behavior. Figure 5B shows risperidone, an antagonist to Dopamine, improves the marble burying score of Nell-1+/- mice. Figure 5C shows risperidone improves Nell-1+/- mice’s behavior in the three-chamber social interaction test. n=12 mice/genotype and treatment condition. *p<0.05 by Mann-Whitney U test.
[0068] Figure 6 is a summary of studies in Example 3. Notes: Nl : Nell-1; C4:Cntnap4; WT: wild type; KO: knockout; CB: craniofacial bones; ASD: autism spectrum disorders; NPC: neural progenitor cells; TEM: transmission electronic microscopy.
[0069] Figures 7A and 7B demonstrate immunohistochemistry of the mouse trigeminal ganglion (TG). Figure 7 A shows Cntnap4 positive ganglion neurons (green) and Nell-1 positive satellite cells (red). Figure 7B shows that there are more parvalbumin (PV) positive (red) than tyrosine hydroxylase (TH) positive (green) ganglion neurons in TG.
[0070] Figures 8A-8F show postnatal changes of CB by microCT in Nell-1 and Cntnap4 mutant mice. Figure 8A shows abnormal CB with hydrocephalus in KO mice at P35; Figure 8B shows similar CB changes with severely shortened frontonasal axis (red brackets) in KO mice at P20 and P50; Figures 8C-8F show premature fusion (red arrows) of ISS in KO mice at P20. Nl : Nell-1;
C4: Cntnap4; ISS: intersphenoid synchondrosis; SOS: spheno -occipital synchondrosis. Scale bar in B: lmrn.
[0071] Figures 9A-9C show double calvarial defect model in juvenile mouse. Figure 9A shows an illustration of calvarial defects and their relation to bones and sutures; Figure 9B shows two 2mm defects were created on the right frontal and left parietal bones without affecting sutures; Figure 9C shows two different tissue origins of the calvarial vault. Dotted circles in red represent bone defects; Dotted line in black represents Jugum limitants by which separates anterior and posterior frontal sutures. Bones- F:frontal; N:nasal; Oioccipital; P:parietal. Sutures- Cs:coronal; Ls:lambdoid; PFs: posterior frontal; Ss:saggital; * bone defects; CNC: cranial neural crest; PM: paraxial mesoderm. Scale bar = 1mm.
[0072] Figures 10A and 10B show Cntnap4 is indispensable for Nell-l’s activation of Wnt/b- catenin signaling pathways in osteogenesis. Figure 10A shows a diagram of Wnt/ -catenin signaling pathway, and its antagonists DKK1 and PNU74654 and agonist CHIR99021; Figure 10B shows expression of Wnt/ -catenin signaling molecules in the whole cell lysate of Control (WT) and Cntnap4-KD MC3T3-E1 cells treated with rhNell-1.
[0073] Figures l lA and 11B show Wnt inhibitors block Nell- 1 stimulation of Wnt/[i-catenin signaling and osteoblastic differentiation in CNCCs. Figures 11 A and 1 IB show gene expression of Wnt downstream and osteogenic markers at day 7 post-treatment with CNCCs. * p<0.05;
**p<0.01
[0074] Figures 12A-12C show distribution of Parvalbumin (PV) and Tyrosine Hydroxylase (TH) positive neurons in the mouse brain. Figure 12A shows PV positive pyramidal cells in hippocampal CA1; Figure 12B shows most interneurons were positively stained with PV in the hypothalamus (HTH); Figure 12C shows PV positive Purkinje cells in the cerebellum (CBL); Arrows pointing to PV+ cells.
[0075] Figure 13A and 13B show expression of active b-catenin (ABC) in neurons of the mouse brain. Figures 13A and 13B show differential intensities of ABC positive neurons (red staining pointed by arrowheads) were readily detectable in the neocortex. Some ABC positive neurons also stained strongly positive for Nell-1 (yellow staining pointed by arrows).
[0076] Figures 14A-14C demonstrate the establishment of primary mouse neuronal cell culture. Figure 14A shows primary mouse hippocampal neurons were cultured on poly-D-lysine coated coverslip at Day 5; Figure 14B shows identification of neuronal cells by MAP2 (red) and astrocytes by GFAP (green) using immunocytochemistry at Day 7 primary culture; Figure 14C shows co-localization/expression of Nell- 1 and Cntnap4 in mouse primary neuronal cells at Day 7 culture.
[0077] Figure 15 show phage biopanning diagram using His-tagged Nell- 1 -coated magnetic beads. A cDNA library was constructed from human brain mRNA and packaged with T7 phages. The library was probed with His-tagged Nell- 1 -coated magnetic beads. After four rounds of biopanning, most of the non-specific binding phages were washed off, and the remaining phages were considered binding candidates. Among the phage candidate cDNA inserts, PCR products over 500 bp in length were sequenced and analyzed.
[0078] Figures 16A-16D show confirmation of binding affinity between Cntnap4 phages and Nell-1 using a binding dissociation constant ELISA assay. Figure 16A shows the Basic Local Alignment Search Tool (BLAST) result of the amino acid sequence displayed by the T7 phage constructed with human brain cDNA matches the partial protein sequence of human Cntnap4. Query: the amino acid sequence enclosed by the T7 phage DNA; Sbjct: the matched amino acid sequence Cntnap4. The LamG domains are highlighted in pink. Figure 16B shows increasing the number of phages incubated with Nell-1 pre-coated ELISA plates, the Cntnap4 phage demonstrated significantly higher binding affinity than the control phage. Figure 16C shows Cntnap4 phage revealed high binding affinity only to full length Nell-1, and not to LamG domain- deleted Nell-1. Figure 16D shows structures of Nell- 1 and Cntnap4 and their potential interaction domains. Nell-1 is a secreted protein comprised of 810 amino acids with a molecular weight of ~90 kDa before N-glycosylation and oligomerization. It contains several structural motifs including a laminin G domain, a coiled-coil (CC) domain, five cysteine-rich (CR) domains, and six epidermal growth factor (EGF)-like domains. Cntnap4 (contactin associated protein-like 4, also known as Caspr4) is a transmembrane protein of 1310 amino acids consisting of a large extracellular domain, a single membrane-spanning domain, and a short cytoplasmic region at the carboxy-terminus. The extracellular region is composed of a discoidin-like domain (DISC), a fibrinogen-related domain (FreD), two EGF repeats, and four Faminin G domains. The cytoplasmic region contains a binding site for PDZ domains. The potential binding domain of Nell-1 and Cntnap4 is highlighted by the blue dashed line. TM: transmembrane. Mean ± S.E. of six independent experiments performed in triplicate are shown. *: P < 0.05 when compared to control phage.
[0079] Figures 17A and 17B show mRNA expression levels of Cntnap4 in 12 types of non neuron and glial cells used in various Nell-1 studies. Figure 17A shows of the 8 types of tested cell lines, the MC3T3-E1 cell line expressed the highest levels of Cntnap4. Figure 17B shows of the 4 types of tested primary cells, NMCC exhibited the highest expression levels of Cntnap4. NMCC: newborn mouse calvarial cells. mRC: mouse rib chondrocytes. hBMSC: human bone marrow stem cells. hARC: human articular chondrocytes. Mean + S.E. of six independent experiments performed in triplicate are shown.
[0080] Figures 18A-18C show Co-localization of Cntnap4 and Nell-1 in pre-osteoblastic cells and calvarial bones. Figure 18A shows confocal laser scanning microscopy (CFSM) revealed the co-localization of Nell-1 and Cntnap4 in MC3T3-E1 pre-osteoblasts after 30 minutes of incubation with exogenous recombinant human Nell-1. Co-localization can be found
predominantly on the plasma membrane. Direct Nell-1 and Cntnap4 interaction was validated by DUOFINK® proximity ligation assay (PEA). Figure 18B shows similar co-localization and protein interaction of Nell- 1 and Cntnap4 were also observed in the plasma membrane of NMCC with 30 minutes of Nell- 1 treatment. Figure 18C shows calvarial bone of P60 mice showed high- intensity double staining of Nell- 1 and Cntnap4 in the bone marrow cavity. White arrows: marrow cavity cells with both Nell-1/Cntnap4 co-localization staining and PLA signaling; yellow arrow: bone lining cells with Nell-1/Cntnap4 co-localization staining. Scale bar = 50 mhi (yellow), 500 mhi (black), mhi 100 (blue), and 20 mhi (white).
[0081] Figures 19A-19E show physical interaction between Nell-1 and Cntnap4. Figures 19A and 19B show pull-down assays were performed with (Figure 19A) MC3T3-E1 pre-osteoblasts and (Figure 19B) neonatal mouse calvarial cells (NMCC). Increased Cntnap4 was detected when beads were coated with His-tagged Nell-1. Figure 19C and 19D show co-Immunoprecipitation assay with (Figure 19C) MC3T3-E1 pre-osteoblasts and (Figure 19D) NMCC demonstrated an increase in Cntnap4 when cells were incubated with Nell-1. To confirm specificity, no Cntnap4 was detected when the agarose beads were not coated with anti-Nell- 1 antibody. E. Surface Plasmon Resonance (SPR) assays were performed to assess the binding affinity and dynamic relationship between pentameric Nell-1 [Nell- 1(5)] and the immobilized extracellular portion of Cntnap4 (Cntnap4extra), demonstrating classical ligand-receptor binding. Red dashed lines present the kinetic projection performed by Scrubber 2.0.
[0082] Figures 20A-20C show that Cntnap4 is indispensable for Nell-1 osteogenic bioactivity in vitro. Figure 20A shows alkaline phosphatase (AFP) staining on day 9 and Alizarin Red staining on day 14 revealed increased staining in the Nell-1 and BMP2 groups of control shRNA transfected MC3T3-E1 cells. In Cntnap4- KD MC3T3-E1 cells, high staining intensities of AFP and Alizarin Red were only present in the BMP2 group. Figure 20B shows a time-dependent, steady increase in Ocn and Opn staining was observed in both PBS and recombinant human Nell- 1 -treated control MC3T3-E1 cells. At each individual time point, the Nell- 1 -treated group demonstrated increased staining intensity when compared to the PBS-treated group. In Cntnap4- KD MC3T3 cells, neither PBS nor Nell-1 treatment resulted in detectable positive staining of Ocn or Opn. Figure 20C shows in control MC3T3-E1 cells, Alp, Collagen Ial, and Collagen Ia2 reached peak expression levels 9 days after stimulation, while Ocn, Opn, and Bsp displayed time- dependent patterns of increased expression. Notably, higher expression levels were detected for each gene in the Nell-1 treatment group when compared to the PBS group. However, Cntnap4- KD MC3T3-E1 cells did not exhibit significant changes in any osteogenic markers nor any differences between the PBS group and the Nell-1 group. Mean ± S.E. of six independent experiments performed in triplicate are shown. *: P <0.05 when compared to Control + PBS group. Scale bar = 100 mhi.
[0083] Figures 21A-21C show Cntnap4- knockdown blocks the osteogenic effects of Nell- 1 ex vivo. Figure 21 A shows the mineral deposition in the mouse calvarial explants during the culture period was revealed by Alizarin Complexone. Fentiviral overexpression of Nell-1 increased the density of Alizarin Complexone, however when Nell-1 was overexpressed in Cntnap4-KD samples, the Alizarin Complexone staining was comparable to the control (without Cntnap4- KD or Nell-1 overexpression). Figure 2 IB shows quantification of the maximal width of the frontal and parietal bones overlapping area. Nell-1 overexpression alone increased the overlapping area, while Cntnap4- KD alone slightly reduced the overlapping area. When the Cntnap4- KD samples were treated with Nell-1 lentiviral overexpression, the maximal width of the overlapping area remained unchanged (similar to that of the control group). Figure 21C shows quantification of the unclosed anterior fontanel area. The calvarial explants in the Nell-1 overexpression group demonstrated a completely closed fontanel, while the anterior fontanel in the control group remained open. Cntnap4- KD alone slightly inhibited the closure of the anterior fontanel. The Cntnap4-KO + Nell-1 overexpression group showed a largely open fontanel area. The edge of each calvarial bone is outlined by a white dotted line; yellow arrows represent the maximal width of the frontal and parietal bones overlapping area (in the coronal suture). P: Parietal, F: Frontal. 8 calvaria explants were used for each group. For B and C, the means were used as center values. One-way ANOVA was used for statistical analysis. *: P < 0.05, **: P < 0.01. Scale bar = 100 mhi.
[0084] Figures 22A-22C show Cntnap4 is indispensable for Nell- l’s bioactivity on the activation of MAPK and WNT signaling pathways in vitro. Figure 22 A shows the activation of MAPK signaling in Control and Cntnap4- KD MC3T3-E1 pre-osteoblasts stimulated with Nell-1. In control MC3T3-E1 cells, significantly higher levels of pERK and pJNK were detected 10 min and 30 min after Nell-1 stimulation, respectively. There was no change detected in the
phosphorylation level of P38. In Cntnap4- KD MC3T3-E1 cells, Nell-1 stimulation did not alter the expression levels of pERK and pJNK. Figures 22B and 22C show expression of Wnt signaling molecules in the (Figure 22B) whole cell lysate and (Figure 22C) cell nuclear lysate of Control and Cntnap4-KD MC3T3-E1 cells treated with Nell-1. In control MC3T3-E1 cells, Nell-1 significantly increased the expression levels of Axin2 and active b-catenin, while no effect was observed on these markers in Cntnap4- KD cells treated with Nell-1. Charts demonstrate mean relative band intensity (normalized to control MC3T3-E1 at 0 min) + S.E. for three individual experiments. *, P < 0.05 when compared with Control at 0 min; #, P < 0.05 when compared with Cntnap4- KD at 0 min.
[0085] Figure 23 is a schematic diagram of Nell- 1 signaling pathways in osteogenesis. As a secreted molecule, Nell-1 initiates cellular signaling through binding to its specific receptor, Cntnap4, on the cell surface. The MAPK and Wnt signaling pathways play critical roles in Nell-1- mediated osteogenesis. Nell-1 preferentially activates ERK and JNK in MAPK signaling, and also promotes the phosphorylation/activation of Runx2, which in turn stimulates the expression of Nell-1 and Ocn by directly binding to the OSE2 region of their promoters. In addition, Nell-1 promotes the expression of Axin2 and acti vc-()-catcnin, and increases the nuclear translocation of acti ve-[i-catenin.
[0086] Figures 24A-24F show expression of Cntnap4, Cntnap3, and Cntnap2 in vitro and in vivo. Figures 24A and 24B show Nell-1 significantly increased the levels of Cntnap4 in both (Figure 24A) MC3T3-E1 pre -osteoblasts and (Figure 24B) NMCC. On the contrary, expression of Cntnap2, which was markedly lower than that of Cntnap4, was not responsive to Nell-1 simulation. More importantly, transcription of Cntnap3 was not detectable in both MC3T3-E1 pre-osteoblasts and NMCC, regardless of Nell- 1 treatment. Figure 24C shows in accordance with the gene expression analysis, Cntnap3 protein was not detected in MC3T3-E1 pre-osteoblasts and NMCC after 30 minutes of Nell- 1 treatment. Thus, neither co-localization nor direct binding of Cntnap3 and Nell-1 was detected in vitro. Figure 24D shows neither co-localization nor direct binding of Cntnap3 and Nell-1 was detected in P60 mouse calvarial bone. Figure 24E shows minimal Cntnap2 staining was detected in vitro. Figure 24F shows only a limited number of bone marrow cells demonstrated Cntnap2/Nell-1 double staining (yellow arrows) or direct binding, as evidenced by PEA (red arrows). Mean + S.E. of three independent experiments performed in duplicate are shown. Scale bar = 50 mhi (yellow), and 20 mhi (white).
[0087] Figure 25 shows mRNA expression levels of Cntnap4 in control and Cntnap4 shRNA transfected MC3T3-E1 cells. Expression of Cntnap4 in the stable Cntnap4 knockdown MC3T3- E1 cell line was about 85% lower than the expression in control cells. Mean + S.E. of six independent experiments performed in triplicate are shown. *: P < 0.05 when compared to Control group.
[0088] Figures 26 A and 26B show Cntnap4 knockdown does not alter Nell- 1 -induced Nfatc2 expression in ATDC5 cells. Figure 26A shows Cntnap4 knockdown reduced the expression level of Nfatc2 in ATDC5 cells. Figure 26B shows Nell-1 stimulated similar levels of Nfatc2 increase in both control and Cntnap4- KD ATDC5 cells in the same dose-dependent manner. Mean + S.E. of three independent experiments performed in duplicate are shown.
[0089] Figure 27 shows Co-localization of Cntnap4 and Nell-1 in normal human hippocampus cells. Scale bar = 25 mhi.
[0090] Figures 28A and 28B show expression and purification of the extracellular portion of human Cntnap4 (Cntnap4extra). The purified extracellular portion of recombinant human Cntnap4 protein analyzed by Western blot with Rabbit-anti-Cntnap4 polyclonal antibodies: Figure 28 A shows HPA031859, Sigma-Aldrich, and Figure 28B shows HPA053742, Sigma-Aldrich.
[0091] Figure 29 demonstrates the effectiveness of the invention composition for autism.
DETAIFED DESCRIPTION OF THE INVENTION
Definitions
[0092] The term“effective amount”, as used herein, is an amount of an agent that is sufficient to produce a statistically significant, measurable change of a condition in repaired tissue using the agent disclosed herein as compared with the condition in the repaired tissue without using the agent. Such effective amounts can be gauged in clinical trials as well as animal studies. Such a statistically significant, measurable, and positive change of a condition in repaired tissue using the agent disclosed herein as compared with the condition in the repaired tissue without using the agent is referred to as being an“improved condition”.
[0093] As used herein, the term“significantly” or“significant” shall mean statistically significant.
[0094] As used herein, the term“normal level of blood NELL-1 binding to blood Cntnap4” refers to the state of binding of blood NELL-1 to blood Cntnap4 in a normal mammalian subject. This term, in the context of“in reference to a disorder selected from a neurological disorder or a bone disorder” shall mean a mammalian subject who does not suffer from or who is not disposed to develop a disorder selected from a neurological disorder or a bone disorder.
[0095] As used herein, the term“agent” refers to a substance(s) effective for potentiating an effective binding of blood NELL-1 to blood Cntnap4 in a mammalian subject. An agent can be a single substance or a combination of substances.
[0096] As used herein, the term“enhancer” refers to a substance which enhances the activity of a particular reactant, catalyst, or a biologic substance (e.g., a protein or enzyme).
[0097] As used herein, the term“agonist” refers to a chemical that binds to a receptor and activates the receptor to produce a biological response. Whereas an agonist causes an action, an antagonist blocks the action of the agonist, and an inverse agonist causes an action opposite to that of the agonist.
[0098] As used herein, the term“activator” refers to a chemical that triggers or causes to occur a chemical or biological process.
[0099] As used herein, the term“inhibitor” refers a substance which slows down or prevents a particular chemical reaction or other process or which reduces the activity of a particular reactant, catalyst, or a biologic substance (e.g., a protein or enzyme)
[0100] Lor example, an inhibitor of glycogen synthase kinase 3 beta (GSK3 ) is one that slows down or prevents a particular chemical reaction or other process or which reduces the activity of GSK3 ; an inhibitor of dopamine is one that slows down or prevents a particular chemical reaction or other process or which reduces the activity of dopamine, and an activator of Wnt/b- catenin signaling pathway is one that that triggers the Wnt/|l-catcnin signaling pathway in a subject (e.g., a human being).
[0101] Examples of GSK3 inhibitor include, but are not limited to: 1) GSK2a/ inhibitors: SB 216763, SB 415286, CHIR 98014, CHIR 99021, Bios-7- indolylmaleimidem (www.nature.com/articles/nrdl415), BIO (6-bromioindi rub in-3 -oxime ), AZD2858, and AZD1080
Figure imgf000015_0001
2) GSK3 inhibitors: AR A014418, 1 -Azakenpaulone, TWS119, Tideglusib, IM-12, Indiruhlin,
Figure imgf000015_0002
and CP21R7 (“CP21”)
[0102] Examples of dopamine inhibitor include, but are not limited to:
1) dopamine receptor inhibitors: Benztropine mesylate, Alizapride HC1, and Amfebutamone HC1;
2) dopamine receptor antagonists: Quetiapine fumarate, Chlorpromazine HC1, Domperidone, Metoclopramide HC1, Olanzapine, Paliperidone, Amisulpride, Rotundine, Chlorprothixene, Lurasidone HC1, and Lcvosulpiridc;
3) dopamine reuptake inhibitor: 4-Hydroxy- l-methyl-4-(4-methylphenyl)-3-piperidyl 4- methylphenyl ketone, Altropane, Amfonelic acid, Amineptine, BTCP, 3C-PEP, DBL-583, Difluoropine, GBR-12783, GBR-12935, GBR-13069, GBR-13098, GYKI-52895, Iometopane, Methylphenidate, Ethylphenidate, Modafinil, Armodafmil, RTI-229, Vanoxerine, Adrafinil, Benztropine, Bupropion, Fluorenol, Medifoxamine, Metaphit, Rimcazole, Venlafaxine, Oroxylin A, Dexmethylphenidate, Difemetorex, Fencamfamine, Lefetamine, Levophacetoperane,
Mesocarb, Nomifensine, Piplintane, Pyrovalerone, Adrafmil, Armodafmil, Bupropion, Mazindol, Modafmil, Nefazondone, Sertraline, Sibutramine, Dephenylpyraline, Ethybenzatropine,
Ketamine, Nefopam, Pethidine, Tripelennamine, Cocaine, MDPV, Naphyrone, and
Phencyclidine.
[0103] Examples of Wnt/b -catenin signaling pathway activator include, but are not limited to: IM-12, AZD2858, Methyl vanillate, Sotrastaurin, Cpdl, Cpd2, Norrin, R-spondins, 2-amino-4- [3,4-(methlyenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine, SKL2001, Wnt agonist 1, and CP21.
[0104] As used herein, the term“optional” shall mean having the choice to add or not to add a technical element or feature to an embodiment of invention. As such, the term“optional” can also be construed to mean“with” or“without” a technical element or feature in an embodiment of invention.
[0105] As used herein the term“comprising” or“comprises” is used in reference to
compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.
[0106] As used herein the term“consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
[0107] As used herein, the term“desirable property” refers to any attributes of a biologies that is significant with respect to the biologies’ action as a therapeutics or biologically active agent.
Such desirable properties include, for example, blood circulation life, shelf-life, hydrophobicity or hydrophilicity, biological activity, bioavailability, cytotoxicity, non-immunogenicity, or conformational properties, etc.
[0108] Whenever used, the word“will” shall be construed as denoting a present tense or future tense action.
[0109] As used in this specification and the appended claims, the singular forms“a,”“an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to“the method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
Composition
[0110] In one aspect of the present invention, provided is a composition for treating a disorder, which composition comprises an effective amount of an agent effective for potentiating an effective binding of blood NELL-1 to blood Cntnap4 in a mammalian subject in need thereof to achieve a normal level of blood NELL-1 binding to blood Cntnap4 in the mammalian subject, wherein the mammalian subject has an abnormal level of blood NELL-1 binding to blood Cntnap4 (e.g., relative to a control subject).
[0111] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the agent comprises a Wnt/b -eaten in signaling activator.
[0112] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the agent comprises NELL-1, Cntnap4, and a combination of NELL- 1 and Cntnap4.
[0113] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the agent comprises a GSK3 inhibitor.
[0114] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the agent comprises an agonist of GABAergic activity and/or an inhibitor of Dopaminergic activity in the subject.
[0115] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the agent further comprises integrin-bΐ.
[0116] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the agonist of GABAergic activity is Indiplon.
[0117] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the inhibitor of Dopaminergic activity is
Risperidone.
[0118] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the disorder is a neurological disorder or a skeletal disorder.
[0119] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the disorder is osteoporosis.
[0120] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the disorder is autism.
[0121] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the agent is an enhancer or inhibitor of NELL- 1 or Cntnap4.
[0122] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the agent is an exogenous gene construct expressing NELL-1 or Cntnap4. [0123] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the composition further comprises a
pharmaceutically acceptable carrier for local or systemic delivery.
[0124] In some embodiments of the invention composition, optionally in combination with any of the various embodiments disclosed herein, the subject is a human being.
[0125] In some embodiments of the invention method, optionally in combination with any or all of the various embodiments disclosed herein, the composition is a formulation for systemic or local delivery.
[0126] In some embodiments of the invention method, optionally in combination with any or all of the various embodiments of the present invention, the at least one desirable property is selected from the group consisting of blood circulation life, shelf-life, hydrophobicity or hydrophilicity, biological activity, bioavailability, cytotoxicity, non-immunogenicity, or conformational properties, etc.
[0127] In some embodiments, the invention composition can include a carrier. Further, the composition can be formulated into various formulations for a desired mode of delivery.
Neurogenesis-Associated Protein Cntnap4 as a
Specific Cell Surface Receptor of Osteogenic Ligand Nell-1
[0128] Neurological disorders, such as Autism spectrum disorder (ASD), are lifelong neurodevelopmental disability conditions that affects a great portion of general population.
However, after decades of investigation, the definite causes of these disorders have not been fully revealed. In the present invention, by revealing the essential function of Nell- 1 in neurogenic development and neurological disorders such as ASD, this invention provides the basis for more definitive clinical diagnosis in post-natal individuals as well as fetal screening for
neurodevelopmental abnormalities.
[0129] Meanwhile, since Nell- 1 is a soluble extracellular matrix protein, Nell-based therapies exhibit higher potential and feasibility than other membrane protein-based therapies to treat neurological disorders.
[0130] Furthermore, Nell-1 also exhibits significant pro-osteogenic bioactivities. Therefore, Nell- 1 -based treatment can replace the complex combinatory treatment strategy of neural related and osteoporosis drugs for the patients suffering from neurodevelopmental disability accompanied by bone related diseases.
[0131] Antibodies
[0132] The agent that binds to Cntnap4 to activate osteogenesis and/or neurogenesis may be an antibody or an antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof may be agonistic. For example, the antibody may bind may specifically bind to Cntnap4 and activate the receptor, e.g., to promote osteogenesis and/or neurogenesis. The antibody or antigen-binding fragment thereof may bind to any suitable epitope on Cntnap4 in order initiate downstream signaling, e.g., via Wnt and MAPK signaling pathways. The antibody may bind in the same location as Nell-1 and mimic Nell-1 binding. The antibody may bind allosterically, e.g., outside of the Nell-1 binding pocket, but cause Cntnap4 to be more likely to bind Nell-1.
[0133] Cntnap4 is a transmembrane protein of 1310 amino acids consisting of a large extracellular domain, a single membrane-spanning domain, and a short cytoplasmic region at the carboxy-terminus. The extracellular region is composed of a discoidin-like domain (DISC), a fibrinogen-related domain (FreD), two EGF repeats, and four Laminin G domains. Thus, the epitope bound by the antibody or antigen-binding fragment thereof may be located within any of the foregoing domains or a portion thereof. The antibody or antigen-binding fragment thereof may bind to an epitope within at least one of the Laminin G domains (See FIGS. 2A-2D).
[0134] The epitope bound by the antibody or antigen-binding fragment thereof may include a region within Cntnap4, such as within residues 1-100, 100-200, 200-300, 300-400, 400-500, 500- 600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, or 1300-1310. The epitope may include a portion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more residues of Cntnap4.
[0135] Also featured are fusion proteins including an antibody or antigen-binding fragment thereof fused to Nell-1 or a fragment thereof. The fusion proteins may be used to promote osteogenesis and/or neurogenesis. Nell-1 may be fused at the N-terminus or the C-terminus of the antibody. The antibody may target Nell-1 to a Cntnap4 receptor, e.g., to direct Nell-1 to a specific tissue type or the enhance binding by Nell-1 to the Cntnap4 receptor.
[0136] Evitove mapping
[0137] Antibodies can be optimized to target a preferred epitope on Cntnap4 in order to exhibit desirable functional properties. For example, methods for identifying the particular epitope to which an antibody binds are known to those skilled in the art. Standard techniques include peptide scanning, in which overlapping, short peptides (for example, 10-30 amino acids, e.g., 20, in length) derived from the full length protein to which the antibody binds are individually tested for their ability to bind the antibody. From such experiments, the region of the protein to which the antibody binds can then be determined.
[0138] Site-directed mutagenesis can also be used to identify the antigenic region(s) of Cntnap4. In this approach, point mutations are systematically introduced into the target polypeptide and the ability of the antibody to bind the peptide with mutations at various positions is used to determine whether a particular region of that protein contains the epitope to which the antibody binds.
[0139] Antibody epitopes can also be identified using high-throughput mutagenesis techniques, such as Shotgun Mutagenesis (Integral Molecular, Inc., Philadelphia, Pa.), which can be used to generate large numbers of mutations within the target protein. Such methodologies permit efficient identification of epitopes within the protein.
[0140] To determine if various antibodies to Cntnap4 bind similar epitopes, an in vitro competitive blockade assay can be performed.
[0141] Generation of additional antibodies
[0142] Additional antibodies (e.g., monoclonal, polyclonal, poly-specific, or mono-specific antibodies) against Cntnap4 can be made, e.g., using any of the numerous methods for making antibodies known in the art. In one example, a coding sequence for a fragment of Cntnap4 is expressed as a C-terminal fusion with glutathione S-transferase (GST) (Smith et al., Gene 67:31- 40, 1988). The fusion protein is purified on glutathione-Sepharose beads, eluted with glutathione, cleaved with thrombin (at an engineered cleavage site), and purified for immunization of rabbits. Primary immunizations are carried out with Freund’s complete adjuvant and subsequent immunizations with Freund’s incomplete adjuvant. Antibody titers are monitored by Western blot and immunoprecipitation analyses using the thrombin-cleaved protein fragment of the GST fusion protein. Immune sera are affinity purified using CNBr-Sepharose-coupled protein.
Antiserum specificity can be determined using a panel of unrelated GST proteins.
[0143] As an alternate or adjunct immunogen to GST fusion proteins, peptides corresponding to relatively unique immunogenic regions of a polypeptide described herein can be generated and coupled to keyhole limpet hemocyanin (KLH) through an introduced C-terminal lysine.
Antiserum to each of these peptides is similarly affinity purified on peptides conjugated to BSA, and specificity is tested by ELISA or Western blot analysis using peptide conjugates, or by Western blot or immunoprecipitation using the polypeptide expressed as a GST fusion protein.
[0144] Alternatively, monoclonal antibodies that specifically bind Cntnap4 can be prepared using standard hybridoma technology (see, e.g., Kohler et al., Nature 256:495-7, 1975; Kohler et al., Eur. J. Immunol. 6:511-9, 1976; Kohler et al., Eur. J. Immunol. 6:292-5, 1976; Hammerling et al., Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981). Once produced, monoclonal antibodies can also be tested for specific recognition by Western blot or
immunoprecipitation analysis. Alternatively, monoclonal antibodies can be prepared using a polypeptide described hereinc and a phage display library (Vaughan et al., Nat. Biotechnol. 14:309-14, 1996).
[0145] Cntnap4 epitopic fragments can be generated by standard techniques, e.g., using PCR and cloning the fragment into a pGEX expression vector. These fragments can be used to produce anti-Cntnap4 antibodies that target a particular epitope. Fusion proteins are expressed in E. coli and purified using a glutathione agarose affinity matrix. To minimize potential problems of low affinity or specificity of antisera, two or three such fusions can be generated for each protein, and each fusion can be injected into at least two rabbits. Antisera are raised by injections in a series, and can include, for example, at least three booster injections.
[0146] Useful antibodies for binding Cntnap4 in order to promote osteogenesis and/or neurogenesis can be identified in several different screening assays. First, antibodies can be assayed by ELISA to determine whether they are specific for the immunizing antigen (i.e., a Cntnap4 epitope described herein). Using standard techniques, ELISA plates can be coated with immunogen, the antibody can be added to the plate, washed, and the presence of bound antibody can be detected by using a second antibody specific for the Ig of the species in which the antibody was generated.
[0147] A functional in vitro assay can also be used to screen the antibodies, e.g., using an osteogenesis assay as described herein.
[0148] Humanized antibodies
[0149] The invention also features humanized antibodies that bind to Cntnap4 in order to activate osteogenesis and/or neurogenesis. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as“import” residues, which are typically taken from an“import” variable domain. Humanization can be essentially performed following the method of Winter and co workers (Jones et ah, Nature 321 :522-5, 1986; Riechmann et al., Nature 332:323-7, 1988;
Verhoeyen et al., Science 239: 1534-6, 1988), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies can be chimeric antibodies (U.S. Patent No. 4,816,567), in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which at least some hypervariable region residues as well as other variable region residues are substituted by residues from analogous sites in, e.g., rodent antibodies.
[0150] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies can be important to reduce antigenicity. According to the so-called“best- fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework for the humanized antibody. See, e.g., Sims et al., J. Immunol. 151 :2296-308, 1993; Chothia et al., J. Mol. Biol. 196:901-17, 1987. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies. See, e.g., Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-9, 1992; Presta et al ., J. Immunol. 151 :2623-32, 1993.
[0151] Humanized antibodies can also be produced that retain high affinity for the antigen (e.g., Cntnap4) and other favorable biological properties, such as potentiating osteogenesis and/or neurogenesis. To achieve this goal, according to one method, humanized antibodies can be prepared by analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.
[0152] Human antibodies
[0153] Human antibodies that bind Cntnap4 can be constructed by combining Fv clone variable domain sequence(s) selected from human-derived phage display libraries with known human constant domain sequences(s) (Hoogenboom et al., J. Mol. Biol. 227:381-8, 1992; Marks et al., J. Mol. Biol. 222:581-97, 1991). These antibodies can be used to bind Cntnap4 to activate osteogenesis and/or neurogenesis. Alternatively, human monoclonal antibodies can be made by the hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor, J. Immunol. 133:3001-5, 1984; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol. 147: 86-95, 1991.
[0154] Antibody fragments
[0155] Also featured are antibody fragments that comprise a portion of an intact antibody that binds Cntnap4, in particular, the antigen binding region thereof. Examples of antibody fragments include Fab, Fab’, F(ab’)2 , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. These fragments may be used to bind Cntnap4 in order to active osteogenesis and/or neurogenesis.
[0156] Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual“Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab’)2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
[0157] Fv is the minimum antibody fragment which contains a complete antigen-binding site. In one embodiment, a two -chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a“dimeric” structure analogous to that in a two-chain Fv species. It is in this configuration that the three hypervariable regions (HVRs) of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs (i.e. complementarity determine regions (CDRs) confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
[0158] The Fab fragment contains the heavy- and light-chain variable domains and also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab’-SH is the designation herein for Fab’ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab’) 2 antibody fragments originally were produced as pairs of Fab’ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
[0159] Single-chain Fv or scFv antibody fragments comprise the VH and VL domains of antibody, where these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see, e.g., Pluckthiin, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer- Verlag, New York, 1994), pp. 269-315.
[0160] Various techniques have been developed for the production of antibody fragments.
Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., J. Biochem. Biophys. Methods 24: 107-17, 1992; and Brennan et al., Science 229:81-3, 1985). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and ScFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries. Alternatively, Fab’-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab’)2 fragments (Carter et al.,
Bio/Technology 10: 163-7, 1992). In another approach, F(ab’)2 fragments are isolated directly from recombinant host cell culture. Fab and F(ab’)2 fragment with increased in vivo half-life comprising salvage receptor binding epitope residues are described in U.S. Patent No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.
Carriers
[0161] The present invention involves compositions useful for practicing the therapeutic methods described herein. Therapeutic compositions contain a physiologically tolerable carrier together with an active agent as described herein, dissolved or dispersed therein as an active ingredient. In a preferred embodiment, the therapeutic composition is not immunogenic when administered to a mammal or human patient for therapeutic purposes. As used herein, the terms“pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like. A pharmaceutically acceptable carrier will not promote the raising of an immune response to an agent with which it is admixed, unless so desired. The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectable either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified or presented as a liposome composition. The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient. The therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of an active agent used in the methods described herein that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
[0162] Pharmaceutically acceptable carrier is well known in the art. Examples of such carrier includes, e.g., salient, for liquid or suspension formulations, natural or synthetic polymeric materials for burst or sustained release formulations or targeted delivery formulations. Some examples of the carriers are further described in detail below.
Polymeric Materials
[0163] In some embodiments, the carrier disclosed herein can be a polymeric material Exemplary polymeric material that can be used here include but are not limited to a biocompatible or bioabsorbable polymer that is one or more of poly(DL-lactide), poly(L-lactide), poly(L-lactide), poly(L-lactide-co-DL-lactide), polymandelide, polyglycolide, poly(lactide-co-glycolide), poly(D,L-lactide-co-glycolide), poly(L-lactide-co-glycolide), poly(ester amide), poly(ortho esters), poly(glycolic acid-co-trimethylene carbonate), poly(D,L-lactide-co-trimethylene carbonate), poly(trimethylene carbonate), poly(lactide-co-caprolactone), poly(glycolide-co- caprolactone), poly(tyrosine ester), polyanhydride, derivatives thereof. In some embodiments, the polymeric material comprises a combination of these polymers.
[0164] In some embodiments, the polymeric material comprises poly(D,L-lactide- co-glycolide). In some embodiments, the polymeric material comprises poly(D,L-lactide). In some
embodiments, the polymeric material comprises poly(L-lactide). [0065] Additional exemplary polymers include but are not limited to poly(D-lactide) (PDLA), polymandelide (PM), polyglycolide (PGA), poly(L-lactide-co-D,L-lactide) (PLDLA), poly(D,L-lactide) (PDLLA), poly(D,L-lactide-co-glycolide) (PLGA) and poly(L-lactide-co-glycolide) (PLLGA). With respect to PLLGA, the stent scaffolding can be made from PLLGA with a 25 mole% of GA between 5-15 mol%. The PLLGA can have a mole% of (LA:GA) of 85: 15 (or a range of 82:18 to 88:12), 95:5 (or a range of 93:7 to 97:3), or commercially available PLLGA products identified as being 85: 15 or 95 :5 PLLGA. The examples provided above are not the only polymers that may be used. Many other examples can be provided, such as those found in Polymeric Biomaterials, second edition, edited by Severian Dumitriu; chapter 4.
[0165] In some embodiments, polymers that are more flexible or that have a lower modulus that those mentioned above may also be used. Exemplary lower modulus bioabsorbable polymers include, polycaprolactone (PCL), poly(trimethylene carbonate) (PTMC), polydioxanone (PDO), poly(3-hydrobutyrate) (PHB), poly(4-hydroxybutyrate) (P4HB), poly(hydroxyalkanoate) (PHA), and poly(butylene succinate), and blends and copolymers thereof.
[0166] In exemplary embodiments, higher modulus polymers such as PLLA or PLLGA may be blended with lower modulus polymers or copolymers with PLLA or PLGA. The blended lower modulus polymers result in a blend that has a higher fracture toughness than the high modulus polymer. Exemplary low modulus copolymers include poly(L-lactide)-b-polycaprolactone (PLLA-b-PCL) or poly(L-lactide)-co-polycaprolactone (PLLA-co-PCL). The composition of a blend can include 1-5 wt% of low modulus polymer.
[0167] More exemplary polymers include but are not limited to at least partially alkylated polyethyleneimine (PEI); at least partially alkylated poly(lysine); at least partially alkylated polyornithine; at least partially alkylated poly(amido amine), at least partially alkylated homo- and co-polymers of vinylamine; at least partially alkylated acrylate containing aminogroups, copolymers of vinylamine containing aminogroups with hydrophobic monomers, copolymers of acrylate containing aminogroups with hydrophobic monomers, and amino containing natural and modified polysaccharides, polyacrylates, polymethacryates, polyureas, polyurethanes, polyolefins, polyvinylhalides, polyvinylidenehalides, polyvinylethers, polyvinylaromatics, polyvinylesters, polyacrylonitriles, alkyd resins, polysiloxanes and epoxy resins, and mixtures thereof.
[0168] Additional examples of biocompatible biodegradable polymers include, without limitation, polycaprolactone, poly(L-lactide), poly(D,L-lactide), poly(D,L-lactide-co- PEG) block copolymers, poly(D,L-lactide-co-trimethylene carbonate), poly(lactide-co- glycolide), polydioxanone (PDS), polyorthoester, polyanhydride, poly(glycolic acid-co-trimethylene carbonate), polyphosphoester, polyphosphoester urethane, poly(amino acids), polycyanoacrylates, poly (trimethylene carbonate), poly(iminocarbonate), polycarbonates, polyurethanes, polyalkylene oxalates, polyphosphazenes, PHA-PEG, and combinations thereof. The PHA may include poly(a- hydroxy acids), poly(P-hydroxyacid) such as poly(3-hydroxybutyrate) (PHB), poly(3- hydroxybutyrate-co-valerate) (PHBV), poly(3-hydroxyproprionate) (PHP), poly(3- hydroxyhexanoate) (PHH), or poly (4 -hydroxy acid) such as poly poly(4-hydroxybutyrate), poly(4- hydroxy valerate), poly(4-hydroxyhexanoate), poly(hydroxyvalerate), poly(tyrosine carbonates), poly(tyrosine arylates), poly(ester amide), polyhydroxyalkanoates (PHA), poly(3- hydroxyalkanoates) such as poly(3-hydroxypropanoate), poly(3-hydroxybutyrate), poly(3- hydroxy valerate), poly(3-hydroxyhexanoate), poly(3-hydroxyheptanoate) and poly(3- hydroxyoctanoate), poly(4-hydroxyalkanaote) such as poly(4-hydroxybutyrate), poly(4- hydroxy valerate), poly(4-hydroxyhexanote), poly(4-hydroxyheptanoate), poly(4- hydroxyoctanoate) and copolymers including any of the 3-hydroxyalkanoate or 4- hydroxyalkanoate monomers described herein or blends thereof, poly(D,L-lactide), poly(L- lactide), polyglycolide, poly(D,L-lactide-co-glycolide), poly(L-lactide-co-glycolide), polycaprolactone, poly(lactide-co-caprolactone), poly(glycolide-co-caprolactone),
poly(dioxanone), poly(ortho esters), poly(anhydrides), poly(tyrosine carbonates) and derivatives thereof, poly(tyrosine ester) and derivatives thereof, poly(imino carbonates), poly(glycolic acid- co-trimethylene carbonate), polyphosphoester, polyphosphoester urethane, poly(amino acids), polycyanoacrylates, poly(trimethylene carbonate), poly(iminocarbonate), polyphosphazenes, silicones, polyesters, polyolefms, polyisobutylene and ethylene-alphaolefin copolymers, acrylic polymers and copolymers, vinyl halide polymers and copolymers, such as polyvinyl chloride, polyvinyl ethers, such as polyvinyl methyl ether, polyvinylidene halides, such as polyvinylidene chloride, polyacrylonitrile, polyvinyl ketones, polyvinyl aromatics, such as polystyrene, polyvinyl esters, such as polyvinyl acetate, copolymers of vinyl monomers with each other and olefins, such as ethylene-methyl methacrylate copolymers, acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetate copolymers, polyamides, such as Nylon 66 and polycaprolactam, alkyd resins, polycarbonates, polyoxymethylenes, polyimides, polyethers, poly(glyceryl sebacate), polypropylene fumarate), poly(n-butyl methacrylate), poly(sec-butyl methacrylate), poly(isobutyl methacrylate), poly(tert-butyl methacrylate), poly(n-propyl methacrylate), poly(isopropyl methacrylate), poly(ethyl methacrylate), poly(methyl methacrylate), epoxy resins, polyurethanes, rayon, rayon-triacetate, cellulose acetate, cellulose butyrate, cellulose acetate butyrate, cellophane, cellulose nitrate, cellulose propionate, cellulose ethers, carboxymethyl cellulose, polyethers such as poly(ethylene glycol) (PEG), copoly(ether-esters) (e.g. poly(ethylene oxide-co-lactic acid) (PEO/PLA)), polyalkylene oxides such as poly(ethylene oxide), polypropylene oxide), polypther ester), polyalkylene oxalates, phosphoryl choline containing polymer, choline, poly(aspirin), polymers and co-polymers of hydroxyl bearing monomers such as 2-hydroxyethyl methacrylate (HEMA), hydroxypropyl methacrylate (HPMA), hydroxypropylmethacrylamide, PEG acrylate (PEGA), PEG methacrylate, methacrylate polymers containing 2-methacryloyloxyethyl- phosphorylcholine (MPC) and n-vinyl pyrrolidone (VP), carboxylic acid bearing monomers such as methacrylic acid (MA), acrylic acid (AA), alkoxymethacrylate, alkoxyacrylate, and 3- trimethylsilylpropyl methacrylate (TMSPMA), poly(styrene-isoprene-styrene)-PEG (SIS-PEG), polystyrene-PEG, polyisobutylene-PEG, polycaprolactone-PEG (PCL-PEG), PLA-PEG, poly(methyl methacrylate), MED610, poly(methyl methacrylate)-PEG (PMMA-PEG), polydimethylsiloxane-co-PEG (PDMS-PEG), poly(vinylidene fiuoride)-PEG (PVDF-PEG), PLURONIC™ surfactants (polypropylene oxide-co-polyethylene glycol), poly(tetramethylene glycol), hydroxy functional poly( vinyl pyrrolidone), biomolecules such as collagen, chitosan, alginate, fibrin, fibrinogen, cellulose, starch, dextran, dextrin, hyaluronic acid, fragments and derivatives of hyaluronic acid, heparin, fragments and derivatives of heparin, glycosamino glycan (GAG), GAG derivatives, polysaccharide, elastin, elastin protein mimetics, or combinations thereof.
[0169] In some embodiments, polyethylene is used to construct at least a portion of the device. For example, polyethylene can be used in an orthopedic implant on a surface that is designed to contact another implant, as such in a joint or hip replacement. Polyethylene is very durable when it comes into contact with other materials. When a metal implant moves on a polyethylene surface, as it does in most joint replacements, the contact is very smooth and the amount of wear is minimal. Patients who are younger or more active may benefit from polyethylene with even more resistance to wear. This can be accomplished through a process called crosslinking, which creates stronger bonds between the elements that make up the polyethylene. The appropriate amount of crosslinking depends on the type of implant. For example, the surface of a hip implant may require a different degree of crosslinking than the surface of a knee implant.
[0170] Additional examples of polymeric materials can be found, for example, in US Pat. No. 6,127,448 to Domb, US Pat. Pub. No. 2004/0148016 by Klein and Brazil, US Pat. Pub. No. 2009/0169714 by Burghard et ah, US Pat. No. 6,406,792 to Briquet et al, US Pat. Pub. No. 2008/0003256 by Martens et al, each of which is hereby incorporated by reference herein in its entirety.
Methods of Diagnosis
[0171] In another aspect of present invention, provided is a method of diagnosing a neurological disorder or skeletal disorder in a mammalian subject, which method comprises:
providing a normal level of blood NELL-1 binding to blood Cntnap4 in a normal mammalian subject in reference to a disorder selected from a neurological disorder or a bone disorder,
generating a diagnostic level of blood NELL-1 binding to blood Cntnap4 by measuring blood NELL-1 and blood Cntnap4 in the mammalian subject,
determining that the mammalian subject suffers from the neurological disorder or bone disorder if the diagnostic level of blood NELL-1 binding to blood Cntnap4 significantly deviates from the normal level of blood NELL-1 binding to blood Cntnap4.
[0172] In some embodiments of the method, optionally in combination with any of the various embodiments disclosed herein, the disorder is a neurological disorder.
[0173] In some embodiments of the method, optionally in combination with any of the various embodiments disclosed herein, the disorder is autism or osteoporosis.
[0174] In some embodiments of the method, optionally in combination with any of the various embodiments disclosed herein, the subject is a human being.
Method of Use
[0175] In a further aspect of the present invention, provided is a method of treating or ameliorating a disorder in a mammalian subject, which method comprises administering to the mammalian subject in need thereof a composition comprising an effective amount of an agent effective for potentiating an effective binding of blood NELL- 1 to blood Cntnap4 in the mammalian subject to achieve a normal level of blood NELL-1 binding to blood Cntnap4 in the mammalian subject,
wherein the mammalian subject has an abnormal level of blood NELL-1 binding to blood Cntnap4, and
wherein the disorder is a neurological disorder or a bone disorder.
[0176] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent comprises a Wnt/(i-catcnin signaling activator.
[0177] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent comprises NELL-1, Cntnap4, and a combination of NELL- 1 and Cntnap4.
[0178] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent comprises a GSK3 inhibitor.
[0179] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent comprises an agonist of GABAergic activity and/or an inhibitor of Dopaminergic activity in the subject.
[0180] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent further comprises integrin-bΐ.
[0181] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agonist of GABAergic activity is Indiplon.
[0182] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the inhibitor of Dopaminergic activity is Risperidone.
[0183] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent is an antibody or antigen-binding fragment thereof that binds to Cntnap4, as described herein.
[0184] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the disorder is a neurological disorder or a skeletal disorder. [0185] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the disorder is osteoporosis.
[0186] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the disorder is autism.
[0187] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent is an enhancer or inhibitor of NELL- 1 or Cntnap4.
[0188] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent is a gene construct expressing NELL-1 or Cntnap4.
[0189] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the composition further comprises a pharmaceutically acceptable carrier for local or systemic delivery.
[0190] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the subject is a human being.
[0191] Neurological disorders, such as Autism spectrum disorder (ASD), are lifelong
neurodevelopmental disability conditions that affects a great portion of general population. However, after decades of investigation, the definite causes of these disorders have not been fully revealed.
[0192] By revealing the essential function of Nell-1 in neurogenic development and neurological disorders such as ASD, this invention provides the basis for more definitive clinical diagnosis in post natal individuals as well as fetal screening for neurodevelopmental abnormalities.
[0193] Meanwhile, since Nell-1 is a soluble extracellular matrix protein, Nell-based therapies exhibit higher potential and feasibility than other membrane protein-based therapies to treat neurological disorders.
[0194] Lurthermore, Nell-1 also exhibits significant pro-osteogenic bioactivities. Therefore, Nell-1- based treatment can replace the complex combinatory treatment strategy of neural related and osteoporosis dmgs for the patients suffering from neurodevelopmental disability accompanied by bone related diseases
Method of Fabrication
[0195] In a further aspect of the present invention, provided is a method of fabricating a composition, which method comprises:
providing an effective amount of an agent, which is capable of potentiating an effective binding of blood NELL- 1 to blood Cntnap4 in a mammalian subject in need thereof to achieve a normal level of blood NELL-1 binding to blood Cntnap4 in the mammalian subject, and
forming the composition, wherein the disorder is a neurological disorder or a bone disorder.
[0196] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent comprises a Wnt/(i-catcnin signaling activator.
[0197] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent comprises NELL-1, Cntnap4, and a combination of NELL- 1 and Cntnap4.
[0198] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent comprises a GSK3 inhibitor.
[0199] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent comprises an agonist of GABAergic activity and/or an inhibitor of Dopaminergic activity in the subject.
[0200] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent further comprises integrin-bΐ.
[0201] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agonist of GABAergic activity is Indiplon.
[0202] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the inhibitor of Dopaminergic activity is Risperidone.
[0203] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the disorder is a neurological disorder or a skeletal disorder.
[0204] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the disorder is osteoporosis.
[0205] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the disorder is autism.
[0206] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent is an enhancer or inhibitor of NELL- 1 or Cntnap4.
[0207] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent is a gene construct expressing NELL-1 or Cntnap4.
[0208] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the agent is an antibody or antigen-binding fragment thereof that binds to Cntnap4, as described herein.
[0209] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the composition further comprises a pharmaceutically acceptable carrier for local or systemic delivery.
[0210] In some embodiments of the invention method, optionally in combination with any of the various embodiments disclosed herein, the subject is a human being.
Dosage and Administration
[0211] Dosages of an agent for treating a disorder as described herein (e.g., a bone disorder or a neurological disorder) varies according to different disorders, gender types, and age groups. In some embodiments, the dosage of an agent as described herein ranges from 0.0005 mg/kg body weight to 1 g/kg body weight. In some embodiments, the dosage range is from 0.001 mg/kg body weight to 0.5 g/kg body weight, from 0.0005 mg/kg body weight to 0.1 g/kg body weight, from 0.001 mg/kg body weight to 0.05 g/kg body weight.
[0212] In some embodiments, the agent is an antibody or antigen-binding fragment thereof, which may be administered at a dosage of about 0.001 mg/kg/day to about 10 mg/kg/day (e.g., 0.001 to about 0.01 mg/kg/day, about 0.01 to about 0.1 mg/kg/day, about 0.1 to about 1 mg/kg/day, or about 1 to about 10 mg/kg/day).
[0213] As another alternative, a dosage form of an agent described herein is selected for localized delivery and is not necessarily selected with regard to body weight or to achieve a certain serum level, but to achieve a localized effect, e.g., as for a localized injection, implantation or other localized administration to the eye.
[0214] Administration of the doses recited above can be repeated for a limited period of time. In some embodiments, the doses are given once a day, multiple times a day, for example but not limited to three times a day, once every other day, once a week, once a month, once every 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months, or once a year. In a preferred embodiment, the doses recited above are administered daily for several days, weeks, or months. The duration of treatment depends upon the subject’s clinical progress and responsiveness to therapy. Continuous, relatively low maintenance doses are contemplated after an initial higher therapeutic dose.
[0215] Agents useful in the methods and compositions described herein can be administered topically, intravenously (by bolus or continuous infusion), orally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, intracavity, intrathecally, or parenterally, or can be delivered by peristaltic means, if desired, or by other means known by those skilled in the art. It is preferred that the agents for the methods described herein are administered topically to the eye. For the treatment of tumors, the agent can be administered systemically, or alternatively, can be administered directly to the tumor e.g., by intratumor injection or by injection into the tumor’s primary blood supply.
[0216] Therapeutic compositions containing at least one agent disclosed herein can be conventionally administered in a unit dose. The term“unit dose” when used in reference to a therapeutic composition refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e., carrier, or vehicle.
[0217] The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered and timing depends on the subject to be treated, capacity of the subject’s system to utilize the active ingredient, and degree of therapeutic effect desired. An agent can be targeted by means of a targeting moiety, such as e.g., an antibody or targeted liposome technology. Antibody-based or non-antibody-based targeting moieties can be employed to deliver a ligand or the inhibitor to a target site. Preferably, a natural binding agent for an unregulated or disease associated antigen is used for this purpose.
[0218] Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are particular to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for administration are also variable, but are typified by an initial administration followed by repeated doses at one or more intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.
[0219] An agent may be adapted for catheter-based delivery systems including coated balloons, slow-release drug-eluting stents or other drug-eluting formats, microencapsulated PEG liposomes, or nanobeads for delivery using direct mechanical intervention with or without adjunctive techniques such as ultrasound.
[0220] It is understood that the foregoing detailed description and the following examples are illustrative only and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments, which will be apparent to those of skill in the art, may be made without departing from the spirit and scope of the present invention. Further, all patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents. [0221] The following examples illustrate rather than limit the embodiments of the present invention.
[0222] Example 1. General description of studies of Nell-l/Cntnap4 interactions on osteogenesis and neural system functions
[0223] Summary
[0224] We first identified high Nell-1 expression in the prematurely fused sutures of craniosynostosis patients, and later verified Nell-l’s osteogenic property in transgenic Nell-1 overexpression mice. Recently, we identified Cntnap4, a member of the neurexin superfamily of transmembrane molecules, to exert a novel ligand/receptor interaction with Nell-1 in osteoblasts. Specifically, Cntnap4 binding is specific and necessary for Nell-1 mediated Wnt signaling during osteogenesis in calvarial cells. Moreover, inactivation of either Cntnap4 or Nell-1 in cranial neural crest cells (CNCCs) using Wntl-Cre resulted in remarkably similar calvarial bone defects. These data support the existence of a Nell-1/Cntnap4 functional axis in intramembranous osteogenesis.
[0225] On the other hand, Cntnap4 is a known interneuron presynaptic membrane protein.
Global loss of Cntnap4 differentially inhibits GABAergic output and augments dopaminergic transmission, and induces autism spectrum disorder (ASD)-like behaviors in mice. Notably, Nell- 1 is highly expressed in the brain, and co-localizes with Cntnap4 in both mouse and human brains. Moreover, we observed Nell-1 haploinsufficient mice to also exhibit ASD-like behaviors.
Therefore, it leads to the following— that Nell-1 has dual roles in the brain and craniofacial bones (CB) via a novel interaction between Nell-1 and Cntnap4, and that disruption of the Nell- 1/Cntnap4 functional axis will not only induce deficits in CB, but also interfere with neural transmission in the brain. Studies on such are summarized as follows.
Functional interaction of Nell- 1/Cntnap4 in craniofacial skeletal and brain tissues.
[0226] We previously demonstrated the major roles of Nell- 1 and Cntnap4 in normal CB development. Here, we first detect the spatiotemporal tissue distribution/co-localization of Nell- 1 and Cntnap4 in both CB and brain during normal growth and development in WT mice at embryonic, neonatal, and postnatal stages. Here the functional contributions of Nell-1/Cntnap4 in the postnatal CB and brain are specified using tissue specific knockout Nell-1 and Cntnap4 mice. Wntl-Cre mice targeting CNNCs-derived CB and trigeminal ganglions (TG), and PV-2A-Cre mice targeting parvalbumin positive (PV+) GABAergic neurons in the brain and TG are bred with the existing Nell-lfl/fl and Cntnap4fl/fl mice to obtain Nell-lWntlKO, Cntnap4WntlKO, Nell- 1PV-2AKO, and Cntnap4PV-2AKO mice. Longitudinal measurements of CB growth by live microCT and observation of ASD-like manifestations by behavioral assessments are correlated with morphological changes of CB, brain, and the craniofacial neuroskeletal interface where TG sensory fibers innervate CB. Mechanism of the Nell-1/Cntnap4 axis in CB repair and regeneration
[0227] To further delineate the mechanism of Nell-1’s osteogenic effects in craniofacial skeletal tissues within the context of its interaction with Cntnap4, double calvarial defects in the frontal bones (CNCC -derived) and parietal bones (non-CNCC-derived) of Cntnap4WntlKO mice are created. Here, the healing process of these defects are evaluated using Nell-1 protein by live microCT and histomorphometry analyses. The major molecular events of the Nell-1/Cntnap4 interaction during intramembranous osteogenesis are dissected with CNCCs. Specifically, how integrin bΐ, a cell surface Nell-1 binding partner, and glycogen synthase kinasc-3(i (GSK3[i ), a crucial regulator of b-catenin phosphorylation, modulate Nell-1/Cntnap4 interaction-mediated Wnt^-catenin signaling activation during CNCC osteogenesis are determined. The study of ΰ8K3b inhibitor as an agonist of Wnt^-catenin signaling forms a comparative baseline for the neuronal studies as described below.
Specific effects of Nell- 1/Cntnap4 interaction on brain GABAergic transmission
[0228] In contrast to Cntnap4, Nell-l’s function in the brain, despite its high expression, is completely unknown. With the preliminary finding that adult ENU-induced Nell-1
haploinsufficient mice exhibit ASD-like behaviors that are similar to those seen in global Cntnap4 mutant mice, we propose that abnormal Nell-1/Cntnap4 interactions in the relevant neurons may be responsible for these ASD-like behaviors. As decreased GABAergic output by PV+ neurons in the brain has been linked to the pathogenesis of ASD-like behaviors in global Cntnap4 mutant mice, in studies according to the description below, the modulatory effects of GABAergic augmentation by Indiplon, an agonist of GABA, on ASD-like manifestations in Nell-lPV-2AKO and Cntnap4PV-2AKO mice are tested. This defines precisely the functional involvement of Nell- 1/Cntnap4 on GABAergic transmission and its role in ASD pathogenesis. Further, we assess Wnt^-catenin signaling in Nell-1/Cntnap4 interaction-mediated GABA release, and the possible additive or synergistic effects of GSK3[i inhibitors with Nell-1 protein in activating Wnt^-catenin signaling and functions in mouse neuronal cells. It is expected to identify Wnt^-catenin signaling via Nell-1/Cntnap4 interactions as a major molecular mechanism for Nell-l’s novel roles in neural tissues (please also see Examples 1 and 2, supra )
[0229] In sum, the studies described above specifically investigate the dual roles and mechanisms of Nell- 1 in both craniofacial skeletal and neural tissues through a newly identified Nell-1/Cntnap4 interaction, which shows that Nell-1 is not only effective for patients with skeletal conditions, but also for patients with cognitive and/or degenerative neural conditions.
Detailed description
1. Significance of NELL-1 and Cntnap4 on osteogenesis and neural function
1.1 Nell-1 is a unique osteogenic factor with multifaceted advantageous features. [0230] Neural EGFL like 1 (Nell-1; also known as Nel-like molecule, type 1) is a unique multimeric secretory protein (1) (Figure 1A). Watanabe et al. first sequenced Nell-1 from a human fetal brain cDNA library (2) while our group first identified Nell-l’s osteogenic function from its high expression in active bone formation sites in human craniosynostosis (CS) patients (3). Through gain- and loss-of-function models, we have demonstrated the essential role of Nell-1 in craniofacial and appendicular skeletogenesis (4-6). For example, Nell-1 loss-of-function newborn mice demonstrated reduced calvarial bone growth, enlarged sagittal sutures, and short body lengths (1, 4, 7) (Figure 1C). Reaffirming the importance of Nell-1 in human development, a similar phenotype of delayed cranial fontanelle and suture closure with short body stature was recently reported in a 3-year-old Japanese girl that had a de novo hemizygous interstitial deletion of chromosome I lpl4.1-pl5.3, the gene locus for Nell-1 (11). Excitingly, the osteogenic potential and therapeutic capacity of Nell- 1 has been validated in various small and large animal models (1, 12-19) without the appreciable off-target effects seen with bone morphogenetic protein 2 (BMP2; INFUSE®, Medtronic) (12, 20-23). It has been shown that Nell-1 has excellent safety profile with documented tumor-suppressive properties and provides significant benefits in preventing osteoporotic bone loss, and its anti-inflammatory and adipogenic inhibition properties (5, 18, 20, 21, 24) (Figure IB).
1.2 Cntnav4, a novel susceptibility factor of Autism Spectrum Disorders (ASPs), is a specific cell surface recevtor of Nell-1 in yromotine osteogenesis.
[0231] In our search to find the specific cell surface receptor for Nell- 1 -mediated signaling in osteogenesis, we identified Nell-1 as a novel ligand for Cntnap4 (25). Contactin-associated protein-like 4 (Cntnap4, also known as Caspr4) is a transmembrane neurexin superfamily member with vital functions in neurodevelopment, neurocognition, and the pathogenesis of
neuropsychiatric disorders (26-29). Cntnap4 critically regulates brain interneuron synaptic transmission by increasing GABAergic, while decreasing dopaminergic activity (27). Global Cntnap4 knockout mice exhibit relatively decreased GABAergic, and increased dopaminergic activity that associates with repetitive, ASD-like behaviors that is rescued by pharmacological dampening of dopaminergic signaling or augmentation of GABAergic signaling (27).
Importantly, GABA is the major inhibitory neurotransmitter in the brain and ASD has been increasingly linked to brain inhibitory circuit dysfunction (30). Concomitantly, we demonstrated that Cntnap4 is functionally required for Nell-1 stimulation of Wnt/b -eaten in signaling pathway in vitro, and for Nell-1 mediated-osteogenesis in vivo (25). Specific inactivation of either Cntnap4 or Nell-1 in cranial neural crest cells (CNCCs) using Wntl-Cre resulted in remarkably similar calvarial bone defects (Figures 2A-2E) (25).
_ Table 1. Growth assessment of craniofacial bones at postnatal stages _
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000037_0004
Figure imgf000037_0002
Figure imgf000037_0003
[0232] Taken together, these data reveal not only a novel and critical role of Cntnap4 in neural tissues— but also the necessity of ligand/receptor interactions between Nell- 1 and Cntnap4 during craniofacial skeletogenesis. However, the sufficiency and requirement of Cntnap4 in the repair and regeneration of Nell- 1 -mediated craniofacial bone defects and its underlying mechanisms remain to be systematically investigated. To this end, the broader goal of deciphering the potential functions of Nell-1/Cntnap4 in other tissues must be achieved, which is addressed in the studies described below.
1.3 Potential impacts of the Nell-1/Cntnav4 functional axis in the neural system and craniofacial neuroskeletal interface.
[0233] Nell- 1 protein has long been known to be highly expressed in the brain with unknown neural functions. The identification of a neuronally expressed molecule, Cntnap4, as Nell-l’s receptor in mediating osteogenesis led us to further postulate that there may be potential Nell-1 functions or Nell-1/Cntnap4 interactions in the neural system and at the craniofacial neuroskeletal interface where the innervation of nerve fibers meet the craniofacial bones (25, 31). While past studies showed either Nell- 1 or Cntnap4 expression in the hippocampus, inferior olivary nucleus, and spinal cord (3, 26, 27, 32, 33), we are the first to perform co-localization studies of Nell- 1 and Cntnap4 in the central nervous system (Figure 3). Our detailed in situ Nell-1 and Cntnap4 co localization studies in mouse brain revealed for the first time significant overlap of Nell- 1 and Cntnap4 protein in human hippocampal pyramidal cells and interneurons that might be critical for regulating excitatory (e.g., Dopamine) and inhibitory (e.g., GABA) neurotransmitters in the brain (Figures 4A and 4B). Concordantly, transcriptome analyses have implicated both Nell-1 and Cntnap4 involvement in several neurodegenerative and neuropsychiatric disorders such as ASD (34, 35) and Alzheimer’s disease (36). Excitingly, our preliminary study also revealed that, like global Cntnap4 knockout mice, which exhibit diminished GABAergic and increased
Dopaminergic activity (27), ENU-induced adult Nell-1 haploinsufficient mice also exhibit ASD- like behaviors that are rescued by repressing Dopamine activity (Figures 5A-5C). Moreover, recent work shows that neurotransmitters at the neuroskeletal interface can have profound effects on skeletal growth (37, 38), while other studies suggest that the skeleton also orchestrates the development and cognitive functions of the nervous system (38-40). Indeed, analysis of ASD has revealed a high association with craniofacial defects (41). Consequently, although the emerging field of neuroskeletal biology has focused primarily on the trunk skeleton (42-44) with particular emphasis on the pathogenesis of adolescent idiopathic scoliosis (45), our preliminary data presented here highlight the need for more fundamental neuroskeletal biology studies of the craniofacial system. The discovery of the Nell-1/Cntnap4 functional axis may provide a novel signaling framework for both neural tissue and craniofacial neuroskeletal interface studies during development and growth, as well as in health and disease (39, 46).
2. Cntnp4 Interactions with Nell-1 (NELL-1/Cntnap4 Interaction ) on osteogenesis and neural system development and functions:
[0234] Our preliminary data showing how a neuronal expressing molecule, Cntnap4, is Nell-l’s receptor in mediating osteogenesis and the striking Nell-1 and Cntnap4 co-localization patterns in brain provide a strong scientific premise for innovative study described herein. 2.1. Novel Cntnap4 interaction with Nell-1 during craniofacial skeleton development and srowth.
[0235] The identification and functional validation of a known presynaptic membrane molecule, Cntnap4, as a cell surface specific receptor of Nell- 1 in osteoblast cells (25), was a significant finding that prompted us to propose more comprehensive investigation of Nell-1 in the context of Nell-1/Cntnap4 interactions during craniofacial skeletal development and growth. The molecular mechanisms leading to activation of the Wnt/|l-catenin pathway by Nell-1/Cntnap4 signaling during craniofacial skeleton development and growth are explored through in vitro experiments with CNCCs and in vivo studies as described herein, which lead to findings of novel Cntnap4 interactions during craniofacial skeletogenesis and at the craniofacial neuroskeletal interface that extend beyond its originally described presynaptic membrane functions (27).
2.2. Studies of novel Nell-1/Cntnav4 interactions in bone and brain on Nell-1 and Cntnap4 conditional knockout mice
[0236] Nell- 1 is recognized to be critical for mouse development, particularly in the craniofacial skeletal system (4, 5, 7, 47). However, it remains unclear how Nell-1 affects postnatal growth of craniofacial bones and the brain due to lack of appropriate mouse models (4). Significantly, we have successfully generated and characterized two new mouse lines of floxed Nell-1 and floxed Cntnap4 (Figures 2A-2E). These mice when bred with Wntl-Cre allow us to target the CNCC derived craniofacial bones (CB) (e.g., frontal bone) and trigeminal ganglions (TG) for studies described below (48, 49). In addition, as parvalbumin positive (PV+) GABAergic inhibitory neurons are critical for ASD pathogenesis (27) and comprise a significant portion of the TG (see Figures 7A and 7B), we use PV-2A-Cre to determine the effect of Nell- 1 or Cntnap4 deficiency on PV+ neurons in the brain as well as on TG innervation of the CB at the neuroskeletal interface. Specifically, the craniofacial neuroskeletal interface is where sensory fibers, a large majority of which are from the TG, innervate the CB (50). Due to the developmental contribution of CNCC to the TG and the preponderance of PV+ neurons in the TG (see Figures 7A and 7B), both Wntl and PV driver mouse lines when crossed with Floxed Nell-1 or Cntnap4 will result in defects in CB innervation at the neuroskeletal interface. We also take advantage of floxed Cntnap4-EGFP mice (Figure 2D) or using R26R tdTomato reporter mouse line (JAX Stock# 007909) to perform in vivo lineage tracing during CB defect repair. These new models serve as the basis of and facilitate investigation of Nell-1, Cntnap4 and Nell-1/Cntnap4 interactions in CB, the brain, and at the craniofacial neuroskeletal interface.
2.3. Uncovering novel Nell-1’s functions in the nervous system.
[0237] The mammalian Nell-1 gene was first cloned in a rat brain cDNA library and identified as a nuclear protein binding partner (1-3). Since then, there have been minimal studies on Nell-l’s role in the nervous system (31). Our groundbreaking finding of presynaptic membrane protein, Cntnap4 (27), as Nell-1’s specific receptor combined with the distinct co-localization of Nell- 1 and Cntnap4 in neural tissues (Figures. 18 and 19), and the presence of ASD-like behavioral changes in Nell-1 haploinsufficient mice that is rescued by repressing Dopamine activity (Figures 5A-5C) led us to propose studies on the unrecognized roles of Nell- 1 in the nervous system and at the craniofacial neuroskeletal interface. The potential importance of Nell-1/Cntnap4 interaction in neural tissue development and the pathogenesis of ASD-like manifestations in Nell-1 and/or Cntnap4 deficient mice are further explored. The primary focus of these studies on neural effects is on GABAergic neurons as outlined below due to the fact that GABA is the major inhibitory neurotransmitter in the brain and ASD has been increasingly linked to brain inhibitory circuit dysfunction (30) (see again section A2) and due to the broader distribution of PV+ inhibitory neurons compared with tyrosine hydroxylase positive (TH+) dopaminergic neurons in brain areas where Nell-1 and Cntnap4 extensively co-express and co-localize (refer to Figures 12A-12C).
2.4. Significance of the studies.
[0238] We believe findings described herein would not only lead to a fuller understanding of Nell-1 and Cntnap4 functions in craniofacial bone formation and healing, but also lead to novel breakthroughs in our understanding of how incurable neurocognitive disorders such as ASD may arise— as well as to identify novel molecular targets for future therapeutic development.
3. Design and protocol of studies:
[0239] The studies described herein on the roles of Nell-1/Cntnap4 interactions in CB and neural tissues are all based on strong scientific premises with preliminary data support. The design and protocols of studies involve strong scientific rigor with justification for animal numbers and consideration of sex as a biological variable (SABV). Firstly, the spatiotemporal distribution/co localization of Nell- 1 and Cntnap4 in normal CB and brain tissues is defined. In addition, the functional contribution of Nell- 1 and Cntnap4 to the development and growth of these tissues are characterized and the involvement at the craniofacial neuroskeletal interface are explored using normal wild type and cell lineage specific (Wntl-Cre and PV-2A-Cre) knockout mice, respectively. Cntnap4Wntlknockout mice are used to evaluate the requirement for Cntnap4 during Nell- 1 -mediated calvarial bone defect repair as well as investigate Wnt/()-catcnin signaling activation by Nell-1/Cntnap4 interaction during craniofacial osteogenesis. Effects of Nell- 1 and Nell-1/Cntnap4 interaction in neural tissues are assessed through Cntnap4pv 2Aknockout and pharmacological GABAergic augmentation approaches. Influences and signaling pathways of the Nell-1/Cntnap4 interaction in modulating the release of the key inhibitory neurotransmitter, GABA, are defined. The studies described here unravel the roles and underlying molecular mechanisms of Nell- 1 and the Nell-1/Cntnap4 interaction in CB, the brain, and at the craniofacial neuroskeletal interface, to support development of better therapies for skeletal, brain, and neuroskeletal pathologies (Figure 6).
3.1. Functional interaction of Nell-1/Cntnap4 in craniofacial skeletal and brain tissues.
3.1(A): Spatiotemporal tissue distribution/co-localization of Nell-1 and Cntnao4 in craniofacial skeletal and neural tissues during normal development and growth in wild type mice.
[0240] Preliminary Data and Discussion. In addition to identifying Cntnap4 as the first specific cell surface receptor of Nell- 1 protein in mediating osteogenesis (25), we also demonstrated co localization of Nell- 1 and Cntnap4 in neurons of the mouse hippocampus, hypothalamus, cerebral cortex, and cerebellum (Figure 3) as well as in the TG (Figures 7 A and 7B). Our findings not only align with previously published data on CNS Nell-1 or Cntnap4 spatial gene expression patterns (3, 26, 32), but also provide novel evidence of their overlap. To better determine the functional involvement of Nell-1/Cntnap4 in craniofacial skeletal and neural tissues, we propose foundational studies to reveal the spatiotemporal tissue distribution and co-localization of Nell- 1 and Cntnap4 in the CB, brain, and at craniofacial neuroskeletal interfaces such as between the CB and the TG, whereby the peripheral nervous tissues of sensory and sympathetic fibers coming from TG innervate the CB during development and postnatal growth. Here we propose, but are not limited to, that Nell-1 and Cntnap4 exhibit similar spatiotemporal expression patterns with areas of co-localization in the CB, brain, and craniofacial neuroskeletal interfaces during development and growth.
[0241] Materials and procedures . Based on initial data in Figures. 18, 19, and 22, the spatiotemporal tissue distribution and co-localization of Nell- 1 and Cntnap4 in the mouse craniofacial bone, brain (the major structures/areas including the hippocampus, hypothalamus, cerebral cortex, and cerebellum), and craniofacial neuroskeletal interfaces (e.g., TG connections to craniofacial bones) are evaluated and determined. For neuroskeletal interface studies, we examine craniofacial bone innervation by the peripheral sensory (Calcitonin-gene-related peptide positive, CGRP) and sympathetic (Dopamine b-hydroxylase positive, DBH) nerve fibers in the calvarial bones of wild type mice at multiple developmental and postnatal stages (50-53) along with Nell-1 and Cntnap4.
[0242] Methodologies and Analyses. (1) Examination of the spatiotemporal tissue distribution and co-localization of Nell- 1 and Cntnap4 in embryonic and adult CB and brain tissues and the TG-CB using wild type (WT) C57BL/6J mice of both sexes: Transcardial perfusion with 4% paraformaldehyde for postnatal (P7 -juvenile; PI 4-youth; P60-adult) mouse brain tissue harvesting along with the CB tissues are performed. The CB is decalcified in 19%EDTA at 40C. The decalcified CB and neural tissues undergo frozen and paraffin sectioning for histology, respectively. We perform H&E as well as Nissl’s for neurons, and Goldner trichrome for skeletal tissues for all samples. Immunohistochemistry (IHC) for Nell-1 (R&D) and Cntnap4 (Sigma) is conducted on tissue sections of the postnatal tissues as well as on emersion-fixed neonatal and embryonic (E12.5, 14.5, 16.5, 18.5) tissues, respectively. Next, simultaneous double
immunostaining is followed using Donkey anti-sheep IgG conjugated with Alexa Fluoro-594 and Goat anti-rabbit IgG conjugated with Alexa Fluoro-488. Confocal laser scanning microscopy (CLSM) is used for co-localization imaging in addition to epi-fluorescent microscopy (Keyence, Japan). DUOLINK® (Sigma-Aldrich) proximity ligation assay (PLA) is conducted for validation of direct interaction between Nell-1 and Cntnap4 in situ as we did previously (25). To reveal the types of Nell- 1 and Cntnap4 positive neural cells, the antibodies against parvalbumin (R&D), tyrosine hydroxylase (Abeam), GABA (Sigma), Dopamine, MAP2 and GFAP (ThermoFisher) are also used for neural tissues IHC. The antibodies against active b-catenin (clone 8E7 of anti-ABC from Millipore), osteocalcin and cathepsin K (Santa Cruz) are also included for neural tissue and/or CB IHC. (2) Detection of the innervation of the craniofacial bones: At the neuroskeletal interface of TG-CB, particularly at the coronal and sagittal sutures which are the major sites of innervation from the TG (50), we use decalcified whole-head preparations of male and female wild type mice at both the juvenile stage (P7) and the mature adult stage (P60) for
immunostaining of both the sensory nerve fiber (CGRP+) (51, 52) and the sympathetic nerve fiber (DBH+), which modulates bone mass accrual in long bones (53). Nell-1, Cntnap4, and the major neurotransmitters GABA, Dopamine, and Glutamate are detected using double immunostaining (37). (3) Ultrastructural characterization of synapses in hippocampal neurons and neuroskeletal interfaces in frontal bones relevant to Nell-1/Cntnap4 localization: The hippocampal area and calvarial frontal bone is selected for transmission electronic microscopy (TEM). Selection of these tissues/areas will allow comparative studies after introducing Wnt-Cre (targeting CNCCs for frontal bones) and PV-2A-Cre (targeting PV+ neurons in the hippocampus) for cell-specific knockouts. To locate Nell-1 and/or Cntnap4 at synapses or neuroskeletal interfaces, we perform immunoelectron microscopy using both 6 nm and 12 nm colloidal gold labeled detecting antibodies (Jackson ImmunoResearch Faboratories, Inc.). The ultrastructural relationship of the nerve fibers and CB tissues are determined using our previous protocols (54) with some modifications. We use 8 WT mice of each sex at the indicated postnatal stages and 5 litters of mice at the indicated embryonic and neonatal stages for staining and analyses (see Vertebrate Animals).
[0243] Results: We would observe: (1) similar spatiotemporal tissue distribution patterns and co localization for Nell-1 and Cntnap4 in craniofacial skeletons and neural tissues during development and growth; (2) significantly higher Nell-1 and Cntnap4 levels in embryonic and juvenile (P7) relative to adult (P60) tissues, similar to the skeletal tissue stage dependent expression of Nell-1 (18); (3) Nell-1/Cntnap4 co-localization/interactions at neuronal synapses and at the craniofacial neuroskeletal interface with potential differences in the types and levels of neurotransmitter expression; (4) most Nell-1 and Cntnap4 expressing cells to be PV+ in the P60 adult brain similar to what our preliminary data demonstrated.
3.1(B): Relative contributions of Nell-1 and Cntnav4 to postnatal erowth and functions of the craniofacial skeleton and brain usins cell Unease specific knockout mice.
[0244] Preliminary Data and discussion: Nell-1 is known to significantly impact craniofacial bone development via osteogenic cells (4, 5, 7, 47). However, it remains unknown how Nell-1 and Cntnap4 may interact to modulate postnatal growth of the craniofacial skeletal and brain tissues. For studies here we have created separate, cell lineage specific Nell-1 and Cntnap4 knockout mice (see again preceding descriptions). We use the tissue spatiotemporal distribution pattern of Nell-1/Cntnap4 from above and Cntnap4 knockout mice to specifically focus on Nell- 1/Cntnap4 interactions in the craniofacial skeleton and brain during postnatal growth and function. Notably, we found similar craniofacial bone defect phenotypes in Nell-1 and Cntnap4 conditional (Wntl-Cre for CNCCs) knockout mice at neonatal and postnatal stages (Figures 2A- 2E, 8). Meanwhile, global Cntnap4 knockout mice are known to exhibit ASD manifestations (27), while we observed our ENU-induced Nell-1 haploinsufficient adult mice to exhibit similar ASD- like behaviors (e.g., repetitive behaviors, social interaction deficits) as those described in the Cntnap4 knockout mice (Figures 5A-5C). Thus, here we propose, but are not limited to, that lineage specific knockout of Nell-1 or Cntnap4 will disrupt Nell-1/Cntnap4 interactions in osteogenic and neuronal cells to significantly impact postnatal craniofacial skeletal growth, brain function, and the craniofacial neuroskeletal interface by altering, in part, osteoblastic
differentiation, neuronal growth, function, and/or neurotransmitter release.
[0245] Materials and Procedures. We use Wntl-Cre for the CB derived from CNCCs, and PV- 2A-Cre for relevant brain areas including the neocortex, hippocampus, hypothalamus and cerebellum where PV+ neurons are abundant (refer to Figures 12A-12C), to make specific knockout mice by breeding established floxed Nell-1 and Cntnap4 mice with these Cre mouse lines (see Vertebrate animals). The R26R tdTomato reporter mouse line (JAX Stock# 007909) is also used in certain experiments for cell lineage tracing and cell specific knockout validation. Live microCT is utilized on postnatal mice to obtain whole skull scanning data for longitudinal measurements following published standard protocols by Jackson Laboratory (56) and our previous report (18). We conduct relevant behavioral tests (see list of tests in Vertebrate Animals) to examine the functional involvement of Nell- 1 and/or Cntnap4 on obsessive/repetitive and abnormal social behaviors with the precision of cell-specific inactivation. To reveal changes at the tissue and cellular level, we implement dynamic histomorphometric analysis, EdU pulse and chase experiments, and pathological evaluation of CBs and neural tissues as well as evaluation of the craniofacial neuroskeletal interface at selected stages. Both male and female mice are used. Methodologies and Analyses: (1) The Nell-lfl/fl; Wntl-Cre and Cntnap4fl/fl; Wntl-Cre mice are used to evaluate the effects of Nell- 1 and Cntnap4 on postnatal growth of the CB: We have successfully generated CNCCs specific Nell-1 and Cntnap4 knockout mouse lines (Nell- lWntlKO and Cntnap4WntlKO) by breeding existing floxed Nell-1 (6, 13, 18) and floxed Cntnap4 mice (25) with Wntl-Cre (JAX Stock# 022501), respectively (Figures 2A-2E). The CBs of CNCCs-derived calvarial vault and base in particular is the major subject of detailed analysis (57, 58); (2) Precise assessment of postnatal craniofacial bone growth by dynamic microCT: We make microCT-based precise measurements of the dynamic changes of both the calvarial vault and base using a comprehensive protocol established by Jackson Laboratory (56). Accordingly,
16 mice from each group (8 males and 8 females for each genotype) undergo live microCT scanning (Skyscan 1176, Bruker-microCT, Kontich, Belgium) of the whole skull at P7 (juvenile), P14 (youth), and P60 (adult) when the changes of the growing calvarial vault and cranial base are at the highest peak (56) (Table 1). 3D reconstructions of the skull is used for measurement of selected parameters (Table 2) (6, 18). (3) Dynamic histomorphometric analysis of craniofacial bones: The same mice at P14 and P60 in each group of Table 4 receive two injections of calcine (@20mg/kg BW) at 6 and 3 days for P14 mice, and at 12 and 5 days for P60 mice prior to sacrifice to identify the regions of active bone matrix formation. The undecalcified craniofacial bone sections are prepared after microCT scanning for dynamic bone histomorphometric analysis and histological evaluation of osteoids as described in our previous study (18). (4) Generation of Nell-lfl/fl; PV-2A-Cre and Cntnap4fl/fl; PV-2A-Cre mice to precisely define the functional involvement of Nell- 1 and/or Cntnap4 on abnormal behaviors: PV-2A-Cre knock-in mice express the Cre protein in PV+ interneurons of the neocortex, hippocampus, and cerebellum where Cntnap4 also expresses highly (59, 60). The Nell-lPV-2AKO and Cntnap4PV-2AKO mice are generated by crossing existing floxed Nell-1 and floxed Cntnap4 mouse lines with PV-2A-Cre line (JAX Stock# 008069). Conventional behavioral phenotyping assays for mouse models of autism is utilized for P60 and P180 mice as we did for our preliminary data using ENU-induced Nell-1 mice (see Vertebrate Animals) (61). The treatment groups and number of animals are summarized in Table 6. (5) Histological analyses of CB and neural tissues along with EdU pulse- chase procedure: Tissue fixation/processing, IHC along with selected osteogenic and neural markers, and confocal microscopy approaches are the same as those described above. In particular, we correlate the craniofacial skeletal deformities in Nell-1 and Cntnap4 knockout mutants with possible pathological alterations of the innervations and/or abnormal expression of major neurotransmitter at the craniofacial neuroskeletal interfaces. EdU is pulsed at 72 hours prior to sacrifice, and then chased for cell proliferation until sacrifice. We performed a power analysis for the sample size estimation of CB measurements with significant differences being detected using the Students' t-test (two-tailed, equal variance) between corresponding measurements at the compared ages with 80% power (alpha = 0.05). Based on our preliminary data using 12 mice per group for behavioral testing (Figures 5A-5C), we propose 16 mice of each gender per group to optimally power the new behavioral studies (Table 3).
[0246] Results: We would observe: (1) greater changes in the transverse and vertical measurements at P7 and P14 in both Nell-lWntlKO and Cntnap4WntlKO mutant offspring; (2) ASD-like behaviors in Nell-lPV-2AKO and, in agreement with published data (27), Cntnap4PV- 2AKO mice when compared to WT control mice. This would verify that precise inactivation of Nell-1 or Cntnap4 in PV+ GABAergic neurons can induce ASD-like behaviors; (3) potentially altered innervation patterns in sensory nerve fibers and/or of neurotransmitter levels at the craniofacial neuroskeletal interface in Nell-lWntlKO and Cntnap4WntlKO relative to WT mice. 3.2. Mechanisms of the Neill /Cntnav4 axis in CB repair and reeeneration
3.2(A): Healins process of calvarial bone defects in Cntnao4 deficient mice
[0247] Preliminary Data and Discussion : With the knowledge and experience gained from the experiments described above and relevant previous studies (14, 62), we use a 2mm mouse double calvarial bone defect model that we developed (14, 63) and modified according to Quarto et al. (62, 64) involving both the frontal and parietal bones. To mimic the early ages of children in clinical need for improved graft materials and osteogenic factors during cranioplasty (65), we make double calvarial defects in juvenile Cntnap4Wnt-lKO mice generated according to the description above. This will enable maximal differentiation of Nell- 1 vs. Cntnap4 effects on postnatal CB defect repair. Figure 9A and 9B shows our successful optimization of the model; we noted that it requires 6 full weeks to completely regenerate the 2mm bone defects without therapeutic intervention. The different embryological origins of the frontal and parietal bones (Figure 9C) will allow selective Wntl -mediated Cntnap4 knockout of CNCCs in the frontal bone, but not the parietal bone, in each Cntnapt4Wnt-lKO mouse as an intra-animal control. Here we propose, but are not limited to, that the interaction and signaling of Nell-1/Cntnap4 is required for postnatal CB defect repair and regeneration.
[0248] Materials and Procedures : Double calvarial defects in juvenile Cntnap4 knockout mice are created and then recombinant human NELL-1 protein (rhNELL-1) delivered thereto (Aragen Bioscience Inc., see Authentication of key biological or chemical resources) using an established protein delivery protocol. This enables us to compare the crucial role of Nell-1/Cntnap4 interactions in frontal bone defects that have CNCC sources of Cntnap4 knocked out and in parietal bone defects with normal Cntnap4 expression. MicroCT with morphological analyses are used to monitor and evaluate the healing progress with strong scientific rigor.
[0249] Methodologies and Analyses: (1) Creation of double calvarial bone defects in the frontal and parietal bones of Cntnap4WntlKO juvenile mice: We create 2mm calvarial defects in Cntnap4WntlKO mice at P7 as well as in relevant floxed Cntnap4 control mice without Cre expression (see detailed procedures in Vertebrate Animals). [0250] The treatment groups are summarized in Table 4. Animal numbers based on a published study similar to the proposed experiment (56). With an alpha = 0.05 and power = 0.80, the projected sample size needed with this effect size (GPower 3.1) (66) is approximately N = 3. As such, our sample size of N = 5 per group for each gender at each timepoint will be adequate. (2) Delivery of rhNELL protein to the calvarial defect: We use a Poly(lactic-co-glycolic) Acid (PLGA) scaffold for loading and delivery of rhNELL- 1 at 200ng/defect and recombinant BMP2 control (Infuse by Medtronic) at 200ng/defect as we previously reported (14). (3) Imaging analyses of the calvarial bone defect healing process: We assess relative defect healing at 2 and 6 weeks postoperatively by serial live microCT scans (see Vertebrate Animals) and static microCT imaging after sacrifice. We perform quantitative measurements of bone volume (BV) and bone density (BD) of the healing defects along with other bone parameters using a previously established protocol (6, 18). (4) Histology evaluation: Upon completion of microCT scanning, the 4% paraformaldehyde-fixed samples are decalcified in 19% EDTA and embedded in paraffin. H&E, Goldener trichrome, ALP, and TRAP enzymatic histochemistry staining are performed as we previously described (18). Immunohistochemistry is performed using antibodies against bone markers including osteopontin (OPN) and osteocalcin (OCN), Wnt signaling markers including active b-catenin (ABC) and Axin2, as well as Nell-1 (R&D), Cntnap4 (Sigma) and VEGL (Santa Cruz Biotechnology) using the ABC colorimetric method (Vector Laboratories, Burlingame, CA). (5) Tracking Cntnap4 knockout CNCCs during calvarial bone regeneration by fluorescent imaging: To trace the involvement of mutant CNCCs to the Nell- 1 -stimulated defect regeneration process, Cntnap4WntlKO and/or Cntnap4Wntl-R26RtdTomatoKO mice are used for tracking CNCCs in the healing frontal bone defect similar to what we performed in a previous study(6). We also assess cell proliferation at 2 weeks post-operation by EdU pulse labeling 4 hours prior to sacrifice and correlate with labeled Cntnap4 KO CNCCs.
Table 4. Therapeutic evaluation of calvarial defect in mice with various genotypes
Figure imgf000046_0001
Figure imgf000046_0002
Figure imgf000046_0003
_ [0251] Results We would see (1) significant bone regeneration of a similar degree by rhNELL-1 protein in the frontal and parietal bones of wild type mice in accordance with our past studies; (2) minimal to no bone regeneration by Nell-1 protein in the frontal bone of Cntnap4WntlKO mice with significant bone regeneration in the parietal bone, if Cntnap4 is indeed required for Nell-l’s osteogenic effect in craniofacial bone repair. We also expect (3) similar bone regeneration in all BMP2 treated defects, irrespective of frontal vs. parietal bone defects or of genotype, since BMP2 uses a different signaling pathway than Nell-1/Cntnap4. In addition, we expect (4) less involvement and proliferation of fluorescent labeled Cntnap4 KO CNCCs in the healing frontal bone defect of Cntnap4WntlKO mice.
3.2(B): Molecular events of the N ell-1 /Cntnap4 interaction durine osteosenesis with CNCCs
[0252] Preliminary Data and Discussion Mechanistically, Nell-1 activates Wnt/b -eaten in during osteogenesis (18, 47, 67, 68) with Integrin bΐ as a key Nell-1 binding partner for Wnt/b- catenin signaling (18, 69-71). In addition, we show that Cntnap4 is functionally required for Nell- 1 stimulation of Wnt^-catenin signaling in osteogenic-committed cells (Figures 10A and 10B). We also demonstrated in CNCCs that both intracellular (i.e., PNU74654) and surface (i.e.,
DKK1) Wnt inhibitors can block Nell-1 mediated osteogenic effects, with PNU74654 providing more complete blockage of Nell-l’s effects (Figures 11A and 1 IB) (also see Figure 10A).
Meanwhile, glycogen synthase kinase^ (ΌdK3b ) inhibitors are recognized as potent activators of the Wnt^-catenin signaling pathway as CSK3[l mediated b-catenin phosphorylation causes its destabilization (72, 73) (see Figure 10A). Besides its importance in augmenting Wnt signaling, GSK-3 inhibitors are thought to regulate many cognitive-related processes such as neurogenesis, synaptic plasticity and neural cell survival and are increasingly studied as therapeutic agents for degenerative, cognitive, and psychiatric neural conditions (74, 75). Collectively, these data provide the scientific premise for our proposal that Cntnap4 and Integrin bΐ are necessary co factors for Nell- 1 -mediated Wnt^-catenin signaling in CNCCs during osteogenesis and that Wnt agonist, ΰdK3b inhibitor, may work additively or synergistically with Nell-1/Cntnap4 to increase Wnt^-catenin signaling.
[0253] Materials and Procedures. First, we determine if pre-treatment with neutralizing antibodies targeting cell surface Integrin bΐ *p<0.05; **p<0.01 affect rhNEFF-1 activated Wnt/b- catenin signaling in WT and Cntnap4WntlKO CNCCs. Next, we study the transcriptional, translational, and post-translational levels of ΰdK3b upon rhNEFF-1 stimulation with or without ΰdK3b inhibitors and correlate with Wnt^-catenin signaling activity and osteogenesis using responsive CNCCs and MC3T3-E1 cells.
[0254] Methodologies and Analyses. (1) Preparation of primary CNCCs and MC3T3 cells with genetic modifications: Primary CNCCs are isolated from the frontonasal bones of WT and Cntnap4WntlKO mice generated for studies described above. The MC3T3-E1 cells and established Cntnap4 knockout clones (25) are also tested in addition to the primary Cntnap4 KO CNCCs. (2) TOP-FLASH in vitro screening: The b-Catenin/Wnt TCF/LEF Response Element Luciferase Reporter Lentivims (G&P Biosciences, Santa Clara, CA) is used to transduce the CNCCs and MC3T3 cells for screening rhNELL-1 activation (at 800ng/ml and 1600ng/ml) of Wnt/ b-catenin signaling. To assess the necessity of integrin bΐ in Nell- 1 -mediated Wnt signaling activation in the context of Cntnap4, pre-treat the cells are pre-treated with Anti- Integrin bΐ Antibody, clone P5D2 (MilliporeSigma, Burlington, MA) or isotype IgG control using luciferase protocol and Wnt3a as a positive control similar to what we described (18). Next, CNCCs and MC3T3 cells that respond positively to TOP-FLASH screening are selected and expanded for subsequent analyses. (3) Levels and the phosphorylation status of GSK3[i and b-catenin (cytoplasm vs nuclear): TOP-FLASH responsive CNCCs are stimulated for 10, 30, 60, and 120 minutes by rhNELL-1 at 800 and 1600 ng/ml (Aragen Bioscience Inc). We then measure GSK3[i and b-catenin (cytoplasmic vs nuclear) levels by qPCR, Western blot, and fluorescent immunocytochemistry as we have described (18, 21, 47). We also use Western blots with specific antibodies against serine, threonine, and tyrosine following immunoprecipitation of GSK3[i and b-catenin using respective antibodies to investigate the phosphorylation status of GSK3[i and b- catenin similar to what we did in a previous Runx2 study(47). (4) Assessing if rhNELL-1 and Inhibitors of GSK3[i synergistically impact osteogenesis: While Nell-1 has been identified as a novel Wnt agonist (21), GSK3[i inhibitors have been intensely studied as mechanistically distinct activators of the Wnt^-catenin signaling pathway (76). Here we use WT and Cntnap4WntlKO CNCCs to assess how GSK3[i inhibitors affect Wnt/[i-catenin signaling in the presence of rhNELL-1 during osteogenesis. We select two small molecule inhibitors of GSK3[i, CHIR98014 and CHIR99021 (@0.2-2mM) (Sigma-Aldrich) (72), to investigate their capacity in promoting osteogenesis either alone or in combination with rhNELL-1 through Wnt^-catenin signaling activation (Figure 10A). We detect the expression levels of Dishevelled protein (DVL), Axin2, and b-catenin by qPCR and/or Western blot. The Wnt canonical pathway downstream molecules, c-myc and cyclin Dl, along with key osteogenic marker genes, Ocn and Runx2, are also analyzed to assess Wnt signaling activation. We use ALP and Alizarin Red staining for in vitro osteogenesis of CNCCs and MC3T3 cells as described (18, 47) and correlate with Wnt b-catenin signaling status. Wnt3a (at 50-100ng/ml) is used as a positive control for the in vitro experiments described herein. Thirty newborns of each genotype of mutant mice, inclusive of both sexes, are used for primary CNCCs isolation and the in vitro experiments (see Vertebrate Animals).
[0255] Results. It is expected that effective Wnt^-catenin signaling activation requires intact Nell-1/Cntnap4/Integrin bΐ interaction. The Wnt agonist, GSK3[i inhibitors, may work additively or synergistically with Nell-1/Cntnap4 to increase Wnt^-catenin signaling. 3.3. Explore the specific effects ofNell-l/Cntnap4 interaction on brain GABAereic transmission
3.3(A): Modulatory effects of GABAereic augmentation with Indiylon on ASD-like manifestations in PV+ neurons in specific Nell-1 and Cntnap4 knockout mice
[0256] Preliminary Data and Discussion. Cntnap4 critically regulates brain interneuron synaptic transmission by increasing GABAergic activity while decreasing dopaminergic activity (27). Global Cntnap4 knockout mice exhibit relatively decreased GABAergic activity and increased dopaminergic activity that is associated with repetitive, ASD-like behaviors that are rescued by pharmacological dampening of dopaminergic signaling or augmentation of GABAergic signaling (27). Excitingly, we have identified Nell-1 as the first ligand capable of binding to Cntnap4 protein in the hippocampal pyramidal cells and intemeurons of mouse and human brains (Figures 3 and, 4) (25). The broad areas of co-localized Nell-1 and Cntnap4 in the principal neurons and intemeurons of the olfactory bulb, neocortex, hypothalamus, and cerebellum in the mouse brain are readily detectable (Figure 3). Even more exciting, using conventional behavioral phenotyping assays for mouse models of autism (77, 78), we showed that Nell-1 haploinsufficient adult mice exhibit increased repetitive behaviors and social interaction deficits similar to those seen in Cntnap4 global knockout mice that are repressed by Risperidone (Figures 5A-5C). Thus, the Nell- 1/Cntnap4 interaction might serve as a new therapeutic target of drugs known for augmenting GABA (e.g., Indiplon) and/or repressing Dopamine (e.g., Risperidone) to reverse the ASD-like phenotypes seen in both global Cntnap4 (27) and Nell-1 knockout mice (Figures 5A-5C). By virtue of the broad distribution of PV+ neurons in the brain (Figures 12A-12C) and their role in the pathogenesis of ASD in Cntnap4 mutant mice (27), we primarily focus on the specific role of Nell-1/Cntnap4 interaction in PV+ GABAergic neurons during ASD development. Here we propose, but are not limited to, that enhancing the action of the inhibitory neurotransmitter, GABA, with Indiplon specifically improve ASD-like behaviors in both Nell-1 and Cntnap4 PV+ neuron specific knockout mice.
[0257] Materials and Procedures. To determine the functional involvement of Nell- 1 and Cntnap4 or Nell-1/Cntnap4 interaction on obsessive/repetitive and abnormal social behaviors in mice and to determine the role of GABAergic signaling in this process, we administer Indiplon to the PV+ neuron specific Nell-1 and Cntnap4 knockout mice. We correlate and assess in vivo imaging of the brain and postmortem brain histology with abnormal behavioral manifestations in the mice.
[0258] Methodologies and Analyses. (1) Responsiveness of ASD-like behaviors in Nell-lPV- 2AKO, Cntnap4PV-2AKO, and compound Nell-lPV-2ACntnap4PV-2AKO mice to Indiplon: We administer Indiplon (6mg/kg) to 2 month (P60) and 6 month (PI 80) old mice created according to the above description by oral gavage (79) in a 45% 2 -hydro xypropyl-cyclodextrin suspension (HBC; Sigma/ RBI) as previously reported (27, 80). Responsiveness to Indiplon are assessed by behavioral tests [e.g., heightened startle or pre-pulse inhibition (PPI) defect] described in the Cntnap4 model (27). Table 5 shows the treatment groups after power analysis for animal numbers. (2) In vivo imaging of brain activity by microPET: To determine changes in brain metabolism in mutant vs. WT mice ± Indiplon intervention, we use [18F]-fluorodeoxy glucose ([18FJ-FDG) to assess cerebral glucose metabolism, as abnormal [18F]-FDG uptake correlates highly with many neuropsychiatric pathologies (81-83). We perform concomitant microCT and microPET for hard and soft tissue correlation with [18FJ-FDG imaging as previously described (18). (3) Postmortem neuroanatomical assessment: We assess perfusion-fixed brain tissue sections from the neocortex, hippocampus, hypothalamus, and cerebellum as these are major areas of focus in many mouse autism models (84-86). We perform regular HE, special Nissl, and Kluver-Barrera (KB) staining as well as double IHC of the neural markers as described above. Qualitative and quantitative analyses of neuronal morphological changes and immunostaining patterns are conducted as previously described (18, 31). (4) Ultrastructural resolution of synaptic changes by TEM with immunogold labeling: To differentiate pathological changes in the synapses of involved neurons, we use the same TEM and immunogold labeling protocols as described above to conduct comparative studies among treated WT and KO animals.
Figure imgf000050_0001
[0259] Results. It is expected that: (1) Either Nell-1 or Cntnap4 knockout in PV+ neurons will decrease GABAergic signaling to increase ASD-like behavioral abnormalities; (2) Indiplon will increase GABAergic signaling in Nell-lPV-2AKO or Cntnap4PV-2AKO to reverse some ASD- like behavioral abnormalities; (3) There will be some degree of correlation between abnormal ASD-like behaviors with alterations in microPET, neuroanatomical, and ultrastructural assessments of specific brain areas. 3.3(B): Wnt/B-catenin sisnalins in Nell- 1/Cntnav4 interaction-mediated GABA release
[0260] Preliminary Data and Discussion. The Wnt/b -eaten in pathway is actively involved in neural maturation and synaptogenesis (87), as well as in regulation of neurotransmitter release (87, 88). Meanwhile, Wnt/b -eaten in signaling activators such as ΰ8K3b inhibitors (see the Rationale above) are thought to regulate many cognitive-related processes such as neurogenesis, synaptic plasticity, and neural cell survival (75). Interestingly, a Wnt^-catenin signaling enhancer (89), ligand of Numb protein X2 (Lnx2), also binds to the intracellular domain of Cntnap4 in neuronal cells (28). With our confirmation that Wnt^-catenin signaling in osteoblastic cells requires intact Nell-1/Cntnap4 interaction (Figures 10A and 10B), and that active b-catenin (ABC) is differentially distributed in various neurons of the mouse brain (Figures 13 A and 13B), here we propose, but are not limited to, that rhNELL- 1 stimulation of neurons will result in Nell- 1/Cntnap4 interactions that activate Wnt^-catenin signaling to increase GABA release and/or GABAergic activity— and that GSK3[i inhibitors will further enhance rhNELL- 1 effects.
[0261] Materials and Procedurs. Primary hippocampal neuronal cells, neocortex neuronal cells, and neural progenitor cells (NPCs) from WT and specific mutant mice of Nell-lPV-2AKO and Cntnap4PV-2AKO are used along with neurogenic cell line SH-SY5Y cells (neuroblastoma cell lines). GABA release, calcium imaging, and the relevant gene expression of Cntnap4 and Wnt/b- catenin signaling molecules, particularly b-catenin and GSK3[i, are assayed after rhNELL- 1 stimulation.
[0262] Ligures 14A-14C demonstrates establishment of primary mouse neuronal cell culture. Ligure 14A shows the primary mouse hippocampal neurons were cultured on poly-D-lysine coated coverslip at Day 5; Ligure 14B shows identification of neuronal cells by MAP2 (red) and astrocytes by GLAP (green) using immunocytochemistry at Day 7 primary culture; Ligure 14C shows co-localization/expression of Nell- 1 and Cntnap4 in mouse primary neuronal cells at Day 7 culture.
[0263] Methodologies and Analyses. (1) Isolation of primary neuronal cells and neural progenitor cells: Hippocampal and neocortical neurons are isolated from the brain tissue of WT, Nell-lPV-2AKO and Cntnap4pv 2AKO mice (see Vertebrate Animals) per established protocols (90, 91). The neuronal induction of neuroblastoma cell line, SH-SY5Y, is also be performed as published (92) and used in parallel with primary neuronal cells. We assess the co-localization of Nell-1 and Cntnap4 on isolated primary neurons using fluorescent immunocytochemistry and confocal microscopy as we previously described in MC3T3 cells (25); (2) Detection of GABA neuro transmitter release upon rhNELL- 1 stimulation: Primary neuronal cells of various genotypes are seeded on poly-D-lysine pre-coated vessels in serum-free, complete neural basal medium (ThermoPisher). We apply rhNELL- 1 (at 400-2000ng/ml) to the cell culture and collect the conditioned medium at 30min post stimulation for mouse GABA ELISA (G-Biosciences, St. Louis, MO, U.S.A). (3) Lunctional identification of Nell-1/Cntnap4 interaction in neuronal cells and neural progenitor cells (NPCs): To better understand the role of Nell- 1 in neural cells, the effects of rhNELL-1 on the growth and maturation of neuronal cell neurites, on cell proliferation of NPCs, and on neuronal differentiation of NPCs and the neuroblastoma cell line, SH-SY5Y, are studied using standard protocols (92, 93). Cntnap4 KO cells and the Wnt inhibitors (Dkk-1 and PNU74564) are utilized to validate their necessity in Nell-1 mediated neural effects as we previously described for osteogenesis studies (18, 21) (also see Ligures 10A and 10B); (4) Assessing how GSK3 inhibitors affect Nell-1/Cntnap4 activation of Wnt/|l-catcnin signaling in primary neuronal cells: We extract the cytoplasmic and nuclear proteins of neuronal cells and NPCs stimulated by rhNELL-1 with and without GSK3 inhibitor, CHIR99021, to detect the level and phosphorylation status of b-catenin and GSK3|1 by Western Blot as described above.
We assay the gene expression of Wnt/|l-catcnin downstream targets using qPCR. Lunctional calcium imaging using Fluo-4 (ThermoLisher) with primary neuronal cells upon rhNELL-1 stimulation is explored and correlated with activation of Wnt/ -catenin signaling (94-96). We use 30 mutant mice of each genotype with both sexes for primary neuronal cell isolation and for the in vitro experiments (see Vertebrate Animals).
[0264] Results. It is expected to observe increased GABA release from neuronal cells after rhNELL-1 stimulation when the Nell-1/Cntnap4 axis is intact. Nell-1 may have significant positive impacts on neurite growth in neuronal cells, on NPC proliferation and on neuronal differentiation of neuroblastoma cell line, SH-SY5Y, by activating Wnt/|l-catcnin signaling (28). GSK3 inhibitors may significantly enhance the neurogenic effects of Nell- 1.
3.4. Statistical Analysis.
[0265] We consult with/use the UCLA Statistical Biomathematical Consulting Clinic for all statistical analyses. We use Origin Pro for standard statistical analyses for qRT-PCR and Western- blotting data, or in ArrayStudio for log transformed metabolomics profiling data. The One-way ANOVAis used to test whether at least two unknown means are all equal or whether at least one pair of means is different. Welch’s two-sample t-testis used to test whether two unknown means are different from two independent groups. The Two-way ANOVAis used to interpret the interaction of two factors, such as genotypes and Nell- 1 treatment. R program is used to achieve Hierarchical clustering with the Euclidean distance and Principal Components Analysis (PC A).
4. Conclusion
[0266] The studies provide (1) novel insight into Nell-l’s role in the nervous system and in the pathogenesis of ASD-like conditions, and (2) mechanistic insight of the Nell-1/Cntnap4 functional axis in neuroskeletal development, growth, and disease that will significantly impact growth and regeneration of postnatal craniofacial bones in cases of disease or injury. References
1. Zhang X, et al., Journal of dental research. 2010;89(9):865-78. Epub 2010/07/22. doi: 10.1177/0022034510376401. PubMed PMID: 20647499; PMCID: 2959101.
2. Watanabe TK, et al., Genomics. 1996;38(3):273-6. PubMed PMID: 8975702.
3. Ting K, et al., Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research. 1999; 14(l):80-9. doi: 10.1359/jbmr.l999.14.1.80.
PubMed PMID: 9893069.
4. Desai J, et al., Human molecular genetics. 2006;15(8): 1329-41. doi: 10.1093/hmg/ddl053. PubMed PMID: 16537572.
5. Zhang X, et al., The Journal of clinical investigation. 2002; 110(6): 861-70. doi:
10.1172/JCI15375. PubMed PMID: 12235118; PMCID: 151127.
6. Qi H, et al., Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research. 2018. Epub 2018/10/24. doi: 10.1002/jbmr.3615.
PubMed PMID: 30352124.
7. Zhang X, et al., J Craniofac Surg. 2012;23(l):61-6. doi:
10.1097/SCS.0b013e318240c8c4. PubMed PMID: 22337375; PMCID: PMC3282020.
8. Andres RH, et al., Brain Res Bull. 2008;76(4):329-43. doi:
10.1016/j .brainresbull.2008.02.035. PubMed PMID: 18502307.
9. Chilibeck PD, et al., Medicine and science in sports and exercise. 2015;47(8): 1587-95. doi: 10.1249/MSS.0000000000000571. PubMed PMID: 25386713.
10. Moon A, et al., Journal of Obesity & Weight Loss Therapy. 2016;6:298. doi:
doi: 10.4172/2165-7904.1000298.
11. Dateki S, et al., Am J Med Genet A. 2017;173(l):217-20. doi: 10.1002/ajmg.a.37978. PubMed PMID: WOS:000393134900032.
12. Siu RK, et al., Tissue engineering Part A. 2011;17(7-8): 1123-35. doi:
10.1089/ten.TEA.2010.0486. PubMed PMID: 21128865; PMCID: 3063712.
13. James AW, et al., JCI Insight. 2017;2(12). Epub 2017/06/15. doi:
10.1172/jci.insight.92573. PubMed PMID: 28614787; PMCID: PMC5470886.
14. Aghaloo T, et al., Am J Pathol. 2006;169(3):903-15. doi:
10.2353/ajpath.2006.051210. PubMed PMID: 16936265; PMCID: PMC1698834.
15. Li W, et al., Tissue engineering Part A. 2010;16(9):2861-70. doi:
10.1089/ten. tea.2009.0550. PubMed PMID: 20528102; PMCID: 2928135.
16. Li W, et al., Plastic and reconstructive surgery. 2011;127(2):580-7. doi:
10.1097/PRS .0b013e3181 fed5 ae . PubMed PMID: 21285762; PMCID: 3089952.
17. Kwak J, et al., Tissue engineering Part A. 2013;19(3-4):426-36. doi:
10.1089/ten.TEA.2012.0042. PubMed PMID: 23083222; PMCID: 3542871. 18. James AW, et al., Nat Commun. 2015;6:7362. Epub 2015/06/18. doi:
10.1038/ncomms8362. PubMed PMID: 26082355; PMCID: PMC4557288.
19. Shen J, et al., Biochemical and biophysical research communications. 2015;460(2):368- 74. doi: 10.1016/j.bbrc.2015.03.040. PubMed PMID: 25791475.
20. Shen J, et al., Tissue Eng Pt A. 2013;19(21-22):2390-401. doi:
10.1089/ten. tea.2012.0519. PubMed PMID: WOS:000326038000010.
21. Shen J, et al., Am J Pathol. 2016;186(2):419-34. doi: 10.1016/j.ajpath.2015.10.011. PubMed PMID: 26772960; PMCID: 4729267.
22. James AW, et al., Biochemical and biophysical research communications.
2011 ;411 ( 1 ) : 126-31. doi: 10.1016/j hbrc.2011.06.111. PubMed PMID : WOS :000293368000022.
23. Xue J, et al., Bone. 2011;48(3):485-95. doi: 10.1016/j.bone.2010.10.166. PubMed PMID: 20959151.
24. Jin Z, et al., Oncogene. 2007;26(43):6332-40. doi: 10.1038/sj.onc.l210461. PubMed PMID: 17452981.
25. Li C, et al., Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.
2018;33(10): 1813-25. Epub 2018/06/16. doi: 10.1002/jbmr.3524. PubMed PMID: 29905970.
26. Spiegel I, et al., Molecular and cellular neurosciences. 2002;20(2):283-97. PubMed PMID: 12093160.
27. Karayannis T, et al., Nature. 2014;511(7508):236-40. Epub 2014/05/30. doi:
10.1038/nature 13248. PubMed PMID: 24870235; PMCID: PMC4281262.
28. Yin FT, et al., Stem Cells Dev. 2015;24(5):640-52. doi: 10.1089/scd.2014.0261. PubMed PMID: WOS :000349824400009.
29. Baig DN, et al., Brain Research Bulletin. 2017;129:82-90.
30. Pizzarelli R, et al., Neural Plast. 2011;2011 :297153. Epub 2011/07/19. doi:
10.1155/2011/297153. PubMed PMID: 21766041; PMCID: PMC3134996.
31. Zhang X, et al., Laboratory investigation; a journal of technical methods and pathology. 2006;86(7):633-44. doi: 10.1038/labinvest.3700430. PubMed PMID: 16652108.
32. Nakamura R, et al., The Journal of biological chemistry. 2012;287(5):3282-91. doi:
10.1074/jbc.Ml l 1.281485. PubMed PMID: 22157752; PMCID: 3270983.
33. Kuroda S, et al., Biochemical and biophysical research communications. 1999;265(1):79- 86. doi: 10.1006/bbrc.1999.1638. PubMed PMID: 10548494.
34. Connolly JJ, et al., Child development. 2013;84(1): 17-33. doi: 10.1111/j.1467- 8624.2012.01838.x. PubMed PMID: 22935194.
35. Butler MG, et al., International journal of molecular sciences. 2015;16(3):6464-95. doi: 10.3390/ijms 16036464. PubMed PMID: 25803107; PMCID: 4394543. 36. Zhang L, et al., Genetics and molecular research : GMR. 2015; 14(2):7218-32. doi:
10.4238/2015 June.29.15. PubMed PMID: 26125932.
37. Spencer GJ, et al., FEBS Lett. 2004;559(l-3):6-12. Epub 2004/02/13. doi:
10.1016/S0014-5793(04)00053-5. PubMed PMID: 14960299.
38. Chamouni A, et al., Rev Endocr Metab Dis. 2015;16(2):99-113. doi: 10.1007/sl 1154- 015-9312-5. PubMed PMID: WOS:000360393400004.
39. Masi L., et al., the official journal of the Italian Society of Osteoporosis, Mineral Metabolism, and Skeletal Diseases. 2012;9(l):13-6. PubMed PMID: 22783328; PMCID:
3392669.
40. Rousseaud A, et al., Horm Mol Biol Clin I. 2016;28(2):69-83. doi: 10.1515/hmbci-2016- 0030. PubMed PMID:
WOS:000389638600001.
41. Registry TNBD. Autistic Spectrum Disorders Report. 2009.
42. Ducy P, A, et al., Cell. 2000; 100(2): 197-207. Epub 2000/02/05. PubMed PMID:
10660043.
43. Elefteriou F, et al., Nature. 2005;434(7032):514-20. Epub 2005/02/23. doi:
10.1038/nature03398. PubMed PMID: 15724149.
44. Patel MS, et al., Calcified tissue international 2007;80(5):337-47. Epub 2007/04/19. doi: 10.1007/s00223 -007 -9015-3. PubMed PMID: 17440766.
45. Burwell RG, et al., Scoliosis. 2009;4:24. Epub 2009/11/03. doi: 10.1186/1748-7161-4-24. PubMed PMID: 19878575; PMCID: PMC2781798.
46. Karsenty G, et al., Nature. 2012;481(7381):314-20. doi: 10.1038/naturel0763. PubMed PMID: WOS:000299210600033.
47. Zhang X, et al., Journal of bone and mineral research : the official journal of the
American Society for Bone and Mineral Research. 2011 ;26(4):777-91. doi: 10.1002/jbmr.267. PubMed PMID: 20939017; PMCID: PMC3179324.
48. D'Amico-Martel A, et al., Am J Anat. 1983;166(4):445-68. Epub 1983/04/01. doi:
10.1002/aja.1001660406. PubMed PMID: 6858941.
49. O'Rahilly R, et al., J Anat. 2007;211(3):335-51. Epub 2007/09/13. doi: 10.1111/j.1469- 7580.2007.00773.x. PubMed PMID: 17848161; PMCID: PMC2375817.
50. Kosaras B, et al., J Comp Neurol. 2009;515(3):331-48. doi: 10.1002/cne.22049. PubMed PMID: 19425099; PMCID: PMC2710390.
51. Alvarez FJ, et al., J Neurocytol. 1991 ;20(9):716-31. PubMed PMID: 1960536.
52. Ichikawa H, et al., Neuroscience. 2002;113(3):537-46. PubMed PMID: 12150774.
53. Takeda S, et al., J Neuroendocrinol. 2008;20(6):802-7. doi: 10.1111/j.1365- 2826.2008.01732.x. PubMed PMID: 18601702. 54. Khorasani H, et al., Am J Pathol. 2011 ;178(2):621-8. Epub 2011/02/02. doi:
10.1016/j.ajpath.2010.10.019. PubMed PMID: 21281794; PMCID: PMC3070584.
55. Zhang Y, et al., Biomaterials. 2014;35(24) :6614-21. Epub 2014/05/14. doi:
10.1016/j .biomaterials .2014.04.061. PubMed PMID: 24818884.
56. Vora SR, et al., Front Physiol. 2015;6:417. doi: 10.3389/fphys.2015.00417. PubMed PMID: 26793119; PMCID: PMC4709510.
57. Hsu W, et al., Dev Dyn. 2010;239(l):338-45. Epub 2009/08/05. doi:
10.1002/dvdy.22044. PubMed PMID: 19653308; PMCID: PMC2797833.
58. Chai Y, et al., Development. 2000;127(8): 1671-9. Epub 2000/03/22. PubMed PMID: 10725243.
59. He M, et al., Neuron. 2016;92(2):555. Epub 2016/10/21. doi:
10.1016/j.neuron.2016.10.009. PubMed PMID: 27764676.
60. Taniguchi H, et al., Neuron. 2011;71(6):995-1013. doi: 10.1016/j.neuron.2011.07.026. PubMed PMID: 21943598; PMCID: PMC3779648.
61. Silverman JL, et al., Nat Rev Neurosci. 2010;l l(7):490-502. doi: 10.1038/nrn2851. PubMed PMID: 20559336; PMCID: PMC3087436.
62. Quarto N, et al., Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 2010;25(7):1680-94. doi:
10.1359/jbmr.091116. PubMed PMID: 19929441; PMCID: PMC3154006.
63. James AW, et al., Stem Cells Transl Med. 2012;l(9):673-84. doi: 10.5966/sctm.2012- 0053. PubMed PMID: 23197874; PMCID: PMC3659737.
64. Li S, et al., PloS one. 2015;10(10):e0138059. doi: 10.1371/journal.pone.0138059.
PubMed PMID: 26431534; PMCID: PMC4592195.
65. Lam S, et al., Craniomaxillofac Trauma Reconstr. 2015;8(2): 159-70. Epub 2015/05/23. doi: 10.1055/S-0034-1395880. PubMed PMID: 26000090; PMCID: PMC4428737.
66. Faul F, et al., Behav Res Methods. 2007;39(2): 175-91. PubMed PMID: 17695343.
67. Bokui N, et al., FEBS Lett. 2008;582(2):365-71. doi: 10.1016/j.febslet.2007.12.006. PubMed PMID: 18082140; PMCID: 2959102.
68. Chen F, et al., International orthopaedics. 2012;36(10):2181 -7. doi: 10.1007/s00264-012- 1590-x. PubMed PMID: 22797704; PMCID: 3460081.
69. Shen J, et al., J Cell Biochem. 2012;113(12):3620-8. Epub 2012/07/19. doi:
10.1002/j cb .24253. PubMed PMID: 22807400.
70. Hasebe A, et al., FEBS Lett. 2012;586(16):2500-6. Epub 2012/06/26. doi:
10.1016/j.febslet.2012.06.014. PubMed PMID: 22728432.
71. Nakamura Y, et al., The Journal of biological chemistry. 2014;289(14):9781-94. Epub 2014/02/25. doi: 10.1074/jbc.M 113.507020. PubMed PMID: 24563467; PMCID: PMC3975024. 72. Huang J, et al., Sci Rep. 2017;7:40716. Epub 2017/01/18. doi: 10.1038/srep40716. PubMed PMID: 28094799; PMCID: PMC5240561.
73. Wu D, et al., Trends Biochem Sci. 2010;35(3):161-8. Epub 2009/11/04. doi:
10.1016/j.tibs.2009.10.002. PubMed PMID: 19884009; PMCID: PMC2834833.
74. Cohen P, et al., Nat Rev Drug Discov. 2004;3(6):479-87. Epub 2004/06/03. doi:
10.1038/nrdl415. PubMed PMID: 15173837.
75. O'Leary O, et al., CNS Drugs. 2015;29(1):1-15. Epub 2014/11/09. doi: 10.1007/s40263- 014-0213-z. PubMed PMID: 25380674.
76. Metcalfe C, et al., J Cell Sci. 2011;124(Pt 21):3537-44. Epub 2011/11/16. doi:
10.1242/jcs.091991. PubMed PMID: 22083140.
77. Crawley JN, et al., Brain Pathol. 2007;17(4):448-59. doi: 10.111 l/j.1750- 3639.2007.00096.x. PubMed PMID: 17919130.
78. Angoa-Perez M, et al., J Vis Exp. 2013(82):50978. doi: 10.3791/50978. PubMed PMID: 24429507; PMCID: PMC4108161.
79. Foster AC, et al., J Pharmacol Exp Ther. 2004;311(2):547-59. Epub 2004/07/17. doi: 10.1124/jpet.l03.063487. PubMed PMID: 15256538.
80. Petroski RE, et al., J Pharmacol Exp Ther. 2006;317(l):369-77. Epub 2006/01/10. doi:
10.1124/jpet.105.096701. PubMed PMID: 16399882.
81. Silverman DH, et al., JAMA. 2001;286(17):2120-7. Epub 2001/11/22. PubMed PMID: 11694153.
82. von Horsten S, et al., Human molecular genetics. 2003;12(6):617-24. Epub 2003/03/07. PubMed PMID: 12620967.
83. Garcia-Alloza M, , et al., Neuromolecular Med. 2004;6(l):65-78. Epub 2005/03/23. doi: 10.1385/NMM:6: 1 :065. PubMed PMID: 15781977.
84. Ellegood J, et al., Neurotherapeutics. 2015;12(3):521-33. Epub 2015/06/04. doi:
10.1007/sl3311-015-0360-z. PubMed PMID: 26036957; PMCID: PMC4489953.
85. Wang SS, et al., Neuron. 2014;83(3):518-32. Epub 2014/08/08. doi:
10.1016/j.neuron.2014.07.016. PubMed PMID: 25102558; PMCID: PMC4135479.
86. Tsai PT, et al., Nature. 2012;488(7413):647-51. Epub 2012/07/06. doi:
10.1038/nature 11310. PubMed PMID: 22763451 ; PMCID: PMC3615424.
87. Rosso SB, et al., Frontiers in cellular neuroscience. 2013;7:103. doi:
10.3389/fncel.2013.00103. PubMed PMID: 23847469; PMCID: 3701138.
88. Ciani L, et al., Nature Communications. 2015;6. doi: Artn 830210.1038/Ncomms9302. PubMed PMID: WC>S:000363020100006.
89. Camps J, et al., Cancer research. 2013;73(6):2003-13. doi: 10.1158/0008-5472.CAN-12- 3159. PubMed PMID: 23319804; PMCID: 4729305. 90. Brewer GJ, et al., Nature protocols. 2007 ;2(6): 1490-8. doi: 10.1038/nprot.2007.207. PubMed PMID: 17545985.
91. Rietze RL, et al., Nature. 2001;412(6848):736-9. Epub 2001/08/17.
doi: 10.1038/35089085. PubMed PMID: 11507641.
92. Rogers MV, et al., Neuroscience. 1994;60(2):479-94. Epub 1994/05/01. PubMed PMID: 7521024.
93. Chen G, et al., FASEB J. 2004;18(10): 1162-4. Epub 2004/05/11. doi: 10.1096/fj.04- 1551fje. PubMed PMID: 15132987.
94. Martin VV, et al., Cell Calcium. 2004;36(6):509-14. Epub 2004/10/19. doi:
10.1016/j .ceca.2004.05.002. PubMed PMID: 15488600.
95. Cameron M, et al., PloS one. 2016;l l(5):e0155468. Epub 2016/05/18. doi:
10.1371/journal.pone.0155468. PubMed PMID: 27183102; PMCID: PMC4868260.
96. Chen Y, et al., Molecular pain. 2017;13: 1744806917748051. Epub 2017/12/08. doi: 10.1177/1744806917748051. PubMed PMID: 29212403; PMCID: PMC5731619.
97. De Calisto J, et al., Development. 2005;132(l l):2587-97. Epub 2005/04/29. doi:
10.1242/dev .01857. PubMed PMID: 15857909.
98. Sugimura R, et al., Birth Defects Res C Embryo Today. 2010;90(4):243-56. Epub 2010/12/25. doi: 10.1002/bdrc.20195. PubMed PMID: 21181886.
99. Jager C, et al., Curr Protoc Mouse Biol. 2016;6(3):333-42. Epub 2016/09/02. doi:
10.1002/cpmo.l5. PubMed PMID: 27584556.
100. Lewis AE, et al., Dev Biol. 2013;379(2):229-34. Epub 2013/05/08. doi:
10.1016/j.ydbio.2013.04.026. PubMed PMID: 23648512; PMCID: PMC3804302.
101. Madisen L, et al., Nat Neurosci. 2010;13(l):133-40. Epub 2009/12/22. doi:
10.1038/nn.2467. PubMed PMID: 20023653; PMCID: PMC2840225.
102. Savitt JM, et al., The Journal of neuroscience : the official journal of the Society for Neuroscience. 2005;25(29):6721-8. Epub 2005/07/22. doi: 10.1523/JNEUROSCI.0760-05.2005. PubMed PMID: 16033881.
103. Hippenmeyer S, et al., PLoS Biol. 2005;3(5):el59. Epub 2005/04/20. doi:
10.1371/journal.pbio.0030159. PubMed PMID: 15836427; PMCID: PMC 1084331.
104. Kalueff AV, et al., Nat Protoc. 2007;2(10):2538-44. doi: 10.1038/nprot.2007.367. PubMed PMID: 17947996.
105. Deacon RM, et al., Nat Protoc. 2006;l(l):122-4. doi: 10.1038/nprot.2006.20. PubMed PMID: 17406223.
106. Terzian AL, et al., Front Behav Neurosci. 2011;5:49. doi: 10.3389/fnbeh.2011.00049. PubMed PMID: 21887137; PMCID: PMC3156975. 107. Valsamis B, et al., J Vis Exp. 2011(55) :e3446. doi: 10.3791/3446. PubMed PMID:
21912367; PMCID: PMC3217252.
108. Han S, et al., Nature. 2012;489(7416):385-90. doi: 10.1038/naturel l356. PubMed PMID: 22914087; PMCID: 3448848.
109. Waif AA, et al., Nat Protoc. 2007;2(2):322-8. doi: 10.1038/nprot.2007.44. PubMed PMID: 17406592; PMCID: PMC3623971.
110. Poliak DD, et al., Nat Protoc. 2010;5(5):954-62. doi: 10.1038/nprot.2010.64. PubMed PMID: 20431541.
111. Shiotsuki H, et al., J Neurosci Methods. 2010; 189(2): 180-5. doi:
10.1016/j.jneumeth.2010.03.026. PubMed PMID: 20359499.
112. Hagihara H, et al., Journal of visualized experiments : JoVE. 2009(33). doi:
10.3791/1543. PubMed PMID: 19920804; PMCID: 3142893.
113. Fu H, et al., Mol Ther. 2003;8(6):911-7. Epub 2003/12/11. PubMed PMID: 14664793.
114. Pieper AA, et al., Proc Natl Acad Sci U S A. 2005;102(39): 14052-7. Epub 2005/09/21. doi: 10.1073/pnas .0506713102. PubMed PMID: 16172381; PMCID: PMC1216832.
115. Ono N, et al., Dev Cell. 2014;29(3):330-9. Epub 2014/05/16. doi:
10.1016/j.devcel.2014.03.014. PubMed PMID: 24823376; PMCID: PMC4083679.
116. Ono N, et al., Nat Cell Biol. 2014; 16(12): 1157-67. Epub 2014/11/25. doi:
10.1038/ncb3067. PubMed PMID: 25419849; PMCID: PMC4250334.
117. Wilk K, et al., Stem Cell Reports. 2017;8(4):933-46. Epub 2017/04/04. doi:
10.1016/j.stemcr.2017.03.002. PubMed PMID: 28366454; PMCID: PMC5390237.
118. Chan CK, et al., Cell. 2015;160(l-2):285-98. Epub 2015/01/17. doi:
10.1016/j. cell.2014.12.002. PubMed PMID: 25594184; PMCID: PMC4297645.
119. Karra D, et al., J Neurosci. 2010;30(18):6171-7. Epub 2010/05/07. doi:
10.1523/JNEUROSCI.0183-10.2010. PubMed PMID: 20445041.
120. Sariyer IK. Methods Mol Biol. 2013;1078: 133-9. Epub 2013/08/27. doi: 10.1007/978-1- 62703-640-5_l l. PubMed PMID: 23975826; PMCID: PMC5130101.
121. Watson DJ, et al., Mol Ther. 2002;5(5 Pt l):528-37. Epub 2002/05/07. doi:
10.1006/mthe.2002.0584. PubMed PMID: 11991743.
[0267] Example 2. Studies on Neuro genesis- Associated Protein Cntnap4 as a Specific Cell
Surface Receptor of Osteogenic Ligand Nell-1 - Abstract
[0268] Secretory protein Neural EGFL Like 1 (Nell-1) was originally identified using a human fetal brain cDNA library. Accumulating bioinformatics analyses predict Nell-1 as a suspicious target in multiple neurodegenerative and neurocognitive disorders; however, the first insight into Nell-1’s biological function was derived from its prominent overexpression in actively fusing sutures of human craniosynostosis patients. Gain- and loss-of-function studies confirmed Nell-1’s indispensable role in bone development, while Nell-1 administration confirmed its potent osteoinductivity in small and large animal models. Although Nell-1 is known to regulate osteogenesis via Wnt and MAPK signaling, the lack of Nell- 1 specific cell surface receptor(s) identification has impeded mechanistic studies. Herein, we identify and validate a novel ligand/receptor interaction between Nell-1 and contactin associated protein-like 4 (Cntnap4) for Nell- 1 -mediated osteogenesis. Cntnap4 is a transmembrane neurexin superfamily member located on the presynaptic membrane of interneurons, has vital neurogenesis and central nervous system functions, and is associated with multiple neurodegenerative and neuropsychiatric disorders; nevertheless, it has no known osteogenic function. Our present data shows Nell-1 binding with Cntnap4 on the surface of osteogenic-committed cells in vitro and in vivo, and exhibiting high- affinity interactions with Cntnap4. Moreover, Cntnap4 knockdown blocks Nell- 1 -responsive Wnt and MAPK signaling, and effectively abolishes Nell-l’s osteogenic effects. The identification of this new interaction between a neurogenesis-associated receptor Cntnap4 and osteogenic ligand Nell-1 facilitates basic and translational studies on osteogenic roles of Nell-1/Cntnap4 signaling, and also has broader impacts on the growing research field of neuroskeletal interplay in homeostasis and diseases of the nervous and skeletal systems.
Introduction
[0269] Neural EGFL Like 1 (Nell-1; also known as Nel-like molecule, type 1) is a unique multimeric secretory protein1. Watanabe et al. first identified Nell-1 from a human fetal brain cDNA library2, while our group recognized its high expression in actively fusing human calvarial sutures from craniosynostosis (CS) patients3. Over the next 18 years, using various gain- and loss- of-function models, we demonstrated Nell-l’s essential role in craniofacial and appendicular skeletogenesis4 15, as well as its interplay with runt-related transcription factor 2 (Runx2, also known as Cbfal)1, 7, Wnt16 18, and MAPK19, 20 signaling during osteogenesis. We also developed the translational applications of recombinant human Nell-1 as a potentially safer alternative to the main FDA approved growth factor for bone regeneration, bone morphogenetic protein 2
(BMP2)17, 21, 22. Intriguingly, the highest Nell-1 expression occurs in developing and adult neural systems1, and now, accumulating transcriptome analyses reveal novel and potentially critical functions for Nell-1 in neurodevelopment23 25, neurodegenerative disorders26 28, and even neurocognitive dysfunctions such as autism spectrum disorders (ASD)29 32.
[0270] Nell-1 is highly conserved across species with 92.6% homology between human and rat1. Devoid of a transmembrane domain, Nell-1 is a secretory extracellular protein1, 33. Nell-1 contains multiple defined structural motifs, including a secretory signal peptide, an N-terminal thrombospondin- 1 -like (TSPN) module [which overlaps with a laminin G (LamG) domain], a coiled-coil region, several von Willebrand factor-like (vWF) repeats with five cysteine residues [also known as chordin-like, cysteine-rich (CR) domains], and six epidermal growth factor (EGF)-like repeats1 3, 33, 34. The combination of the TSPN/LamG module, CR domains, and EGF- like repeats in Nell-1 suggests specific ligand-receptor interactions3, 34, 35, but Nell-1 does not appear to have interactions with known EGF-like receptors3, 4. While we demonstrated that Nell-1 requires Wnt and MAPK signaling during osteogenesis16 20, multiple attempts to search for a Nell- 1 specific cell surface receptor, including the use of yeast two-hybrid systems4, were unsuccessful until now.
[0271] Herein, we describe contactin associated protein-like 4 (Cntnap4, also known as Caspr4), a transmembrane neurexin superfamily member with vital functions in neurodevelopment and neurocognition36 38, as the first specific cell surface receptor for Nell-1 mediated signaling. First, to identify potential receptors for Nell-1, we constructed a phage display using a human brain cDNA library, where Nell-1 is known to be highly expressed1, 2, 33. This revealed the binding between Cntnap4 and Nell-1. Next, we verified co-localization of Nell- 1 and Cntnap4 in osteogenic-committed cells through confocal laser scanning microscopy (CLSM) and in situ proximity ligation assay (PLA). Physical binding between Nell-1 and Cntnap4 was further confirmed by pull-down and co-immunoprecipitation (Co-IP) assays, as well as by surface plasmon resonance (SPR) analysis that revealed a classical ligand/receptor, high-binding affinity between Nell-1 and Cntnap4. Using Cntnap4 knock-down, we demonstrated that Cntnap4 is functionally required for Nell-1 stimulation of Wnt and MAPK signaling in vitro, and Nell-1 mediated osteogenesis of cranial suture explants ex vivo. Taken together, our data reveal the critical neurogenesis-associated protein36 38, Cntnap4, as a specific cell surface receptor for Nell-1 during osteogenesis. More importantly, we have identified Nell-1, which itself is also highly expressed in neural tissues, as the first extracellular ligand to Cntnap4. Cntnap4 belongs to a larger category of synaptic cell adhesion molecules (SCAMs)39 that have been implicated in a wide variety of neurodevelopmental and neuropsychiatric disorders. This seminal study on Nell- 1/Cntnap4 may provide a foundational framework for future studies on neurogenesis, osteogenesis, and excitingly, the field of neuroskeletal biology. In particular, these interactions permitting significant crosstalk between the brain and bone may determine health and disease during development and homeostasis.
Results - T7 Phage Particles with 1st and 2nd LamG Extracellular Domains of Cntnap4 Exhibit High Binding Affinity to Nell-1 Protein
[0272] The mammalian neural EGFL Like 1 ( Nell-1 ; also known as Nel-like molecule, type 1) gene and its related gene Nell-2 were originally cloned from a human brain cDNA library based on their similarity to the chicken neural epidermal growth factor (EGF)-like molecule ( Nel ) gene2, with Nell-2 being the mammalian ortholog of chicken Nel 40. In order to identify native binding protein(s) of Nell- 1, we utilized a human brain cDNA library to construct a T7 phage display cDNA library for biopanning. After four rounds of biopanning screens with His-tagged Nell- 1 -coated magnetic beads, the phages bound to the His-tagged Nell-1 coated beads were eluted. 100 plaques were selected for phage DNA amplification by PCR, and the resultant DNA fragments over 500 bp were sequenced (Figure 15). Twenty-two Nell- 1 -binding candidates were isolated, eight of which were transmembrane proteins (Table 6). It is worth noting that we repeatedly detected phage particles harboring a sequence that aligned with the 1st and 2nd LamG extracellular domains of Cntnap4 (Figure 16A). This phage containing the 1st and 2nd LamG extracellular domains of Cntnap4 exhibited a high binding affinity to the full-length Nell-1 protein (Figures 16B-16D), which also has an N-terminal LamG domain1, 2.
Table 6. List of Nell- 1 binding candidate protein screened by phage biopanning
Transmembrane protein
Protein Name Gene Name
Amyloid beta A4 protein App
Annexin A13 Anxal3
Contactin-associated protein-like 4 Cntnap4
FXYD domain-containing ion transport regulator 6 Fxyd6
Myelin basic protein Mbp
Reticulon-3 Rtn3
Suppressor of tumorigenicity 7 protein-like St7I
Vesicular glutamate transporter 1 Slcl7a7
Others
Protein Name Gene Name
ATP-dependent RNA helicase DDX42 (EC 3.6.4.13)
Ddx42
(DEAD box protein 42)
Cholecystokinin Cck
DUSP 16 protein Duspl6
E3 ubiquitin-protein ligase NEDD4-like Nedd4l
Epidermal growth factor-like protein 7 Efgl7
Eukaryotic translation initiation factor 2 subunit 3 Eif2s3
Formin-binding protein 4 Fnbp4
Hypermethylated in cancer 2 protein Hic2
Nicotinamide mononucleotide adenylyltransferase 2 Nmnat2 Pre-B-cell leukemia transcription factor interacting protein
Pbxipl
Proactivator polypeptide Psap
Protein phosphatase 1 regulatory subunit 14A PpplrMa
S-phase kinase-associate protein 1 Skpl
Tropomodulin 3 Tmod3
[0273] The Nel protein family (Nel-described in chicken and fish; Nell-1 and Nell- 2 -described in human and mammals, with Nell-2 being the ortholog to chicken and fish Nel40) exhibits similar domain organization34. Nell-1 and Nel can form homo- or hetero-complexes with each other through their N-terminal LamG/TSPN domains prior to secretion40, but do not form hetero complexes40 with thrombopondin-1 (TSP-1), despite the Nel family and TSP-1 sharing similar structure and heparin-binding capability in their respective LamG/TSPN regions33, 40, 41. Of interest, the N-terminus LamG/TSPN domain of Nell- 1 is also required for the Nell-1/Cntnap4 interaction, as the deletion of the LamG/TSPN domain from Nell-1 eliminated nearly all binding of the Cntnap4 phage to Nell-1 (Figure 16C). Overall, the LamG/TSPN domains of Nel proteins appear to be important for homo/hetero-complex formation with other Nel family members, as well as for binding to Cntnap4.
Nell-1 and Cntnap4 Bind on the Plasma Membrane of Osteogenic-Committed Cells
[0274] As Nell-1 is known to regulate osteogenic and chondrogenic differentiation42 46, we first examined Cntnap4 gene expression in twelve Nell- 1 -responsive cell lines capable of
osteochondral differentiation7, 17· 20· 44· 47-50 (Table 7). Overall, Cntnap4 expression was highest in the highly osteogenic-committed MC3T3-E1 cell line51, 52 (Figure 17A), relative to less differentiated cell lines (such as C3H10T1/253, 54, ST-255, 56, 143B57, 58, MG6357, 59, and SaoS257, 58), predominantly adipo genic-committed cell lines (such as M2-10B460) or chondrogenic-committed cell lines (such as ATDC561). The greater Cntnap4 expression pattern found in cells with greater osteogenic capability was also observed in primary cells. Primary newborn mouse calvarial cells (NMCC7) exhibited significantly higher levels of Cntnap4 expression than primary mouse rib chondrocytes, human articular chondrocytes, or human bone marrow stem cells (Figure 17B).
Table 7. Cell types used for Cntnap4 expression screening
Cell Type Species Source
C3H10T1/2 (Clone 8: ATCC®CCL-226™) mouse embryo/sarcoma M2-10B4 (ATCC®CRL- 1972™) mouse bone marrow/stroma cell line
MC3T3-E1 (Subclone 4: ATCC®CRL-2593™) mouse calvaria
ST-2 (DSMZ ACC 333) mouse bone marrow/stroma ATDC5 (HPA 99072806) mouse teratocarcinoma 143B (ATCC®CRF-8303™) human osteosarcoma MG63 (ATCC®CRF-1427™) human osteosarcoma SaoS2 (ATCC®HTB-85™) human osteosarcoma newborn mouse calvaria cells (NMCC) mouse calvaria primary mouse rib chondrocytes (mRC) mouse rib cage cell human bone marrow stromal cells (hBMSC) human bone marrow human articular chondrocytes (hARC) human articular cartilage
[0275] Moreover, Nell-1 significantly upregulated Cntnap4 expression in both MC3T3-E1 pre osteoblasts and primary NMCC (Figures 24A and 24B). To determine if Nell- 1 and Cntnap4 co localize, we incubated MC3T3-E1 pre -osteoblasts and primary NMCC with exogenous recombinant human Nell-1. CLSM confirmed the co-localization of Nell- 1 and Cntnap4 on the plasma membrane of MC3T3-E1 and primary NMCC, while PLA confirmed a direct Nell- 1/Cntnap4 interaction (Figures 18A andl8B). In vivo, we also detected Nell-1/Cntnap4 binding, most prominently in the marrow cavity of mouse calvaria (Figure 18C).
[0276] To further validate the physical interaction between Nell-1 and Cntnap4, we used His- tagged Nell-1 to successfully pull down Cntnap4 from the lysate of MC3T3-E1 pre-osteoblasts and primary NMCC without the use of cross-linking reagents (Figures 19A andl9B). This demonstrates a strong interaction between Cntnap4 and Nell-1. Additionally, Co-IP assays using an antibody against Nell-1 confirmed that Cntnap4 efficiently binds Nell-1 in both MC3T3-E1 pre-osteoblasts and primary NMCC (Figures 19C and 19D). To quantify the binding affinity, we performed SPR analysis. This revealed a classical ligand/receptor interaction62 between Nell-1 and the immobilized extracellular portion of Cntnap4 (Cntnap4extra) with a binding affinity of KD = 32.8 ± 0.9 nM, ka = (2.39 ± 0.05) x 105 1/Ms, and kd = 0.0078 ± 0.0002 1/s (Figure 19E). These data further corroborate that Cntnap4 is a novel cell surface receptor of Nell- 1 in osteogenic- committed cells.
[0277] Although extracellular ligands for protein members of Cntnap family have not been previously described, Cntnap proteins are known to share intracellular ligands. For instance, amyloid beta precursor protein binding family A member 1 (APBA1, also known as Mintl) is an intracellular ligand for both Cntnap3 and Cntnap4, while calcium/calmodulin dependent serine protein kinase (CASK) intracellularly binds both Cntnap2 and Cntnap436. Thus, it is possible that Nell-1, as the first extracellular ligand of Cntnap4 to be identified, may also bind Cntnap2 and Cntnap3 in osteogenic-committed cells. However, we did not detect any Cntnap3 expression in cultured MC3T3-E1 pre-osteoblasts or primary NMCC, or in mouse calvarial bone cells in vivo (Figures 24A-24D). We did, however, detect Cntnap2 expression in both MC3T3-E1 pre osteoblasts and NMCC, but its expression was significantly lower than that of Cntnap4 and was not induced by Nell-1 stimulation, unlike Cntnap4 (Figures 24A and 24B). Moreover, unlike Cntnap4, a very limited number of bone marrow cells exhibited binding between Nell-1 and Cntnap2 (Figures 24E and 24F). Collectively, these results suggest that Cntnap4, but not Cntnap2 or Cntnap3, interacts with Nell-1 in osteogenic-committed cells.
Binding to Cntnap4 is Required for Nell- 1 -Responsive Osteogenesis
[0278] To determine the necessity of Cntnap4 for Nell- 1 -mediated osteogenesis, we established stable Crefreap4-knockdown (Cntnap4-KD) MC3T3-E1 cells using Cntnap4 shRNA (Figure 25). Nell-1 protein treatment increased alkaline phosphatase (ALP) and Alizarin Red staining in control scramble-shRNA transfected MC3T3-E1 cells, while only negligible staining was detected in Nell-1 treated Cntnap4- KD MC3T3-E1 cells (Figure 20A). In contrast, control MC3T3-E1 cells and Cntnap4- KD MC3T3-E1 cells exhibited similar robust osteogenic responses to bone morphogenetic protein 2 (BMP2), an osteogenic protein with a signaling pathway distinct from Nell- 11 (Figure 20A). These data indicate that Cntnap4- knockdown specifically targeted Nell-1, but not BMP2-mediated MC3T3-E1 cell osteogenic differentiation. In addition, Nell-1 protein significantly enhanced osteocalcin (Ocn) and osteopontin (Opn) expression in control MC3T3-E1 pre-osteoblasts, while no positive staining for either of these two osteogenic markers was observed in Nell-1 treated Cntnap4- KD MC3T3-E1 cells (Figure 20B). Concomitant gene expression profiling of osteogenic markers Alp, Collagen led, Collagen led, Ocn, Opn, and bone sialoprotein (Bsp) (Figure 20C) revealed inhibited osteogenic differentiation in Cntnap4-KD MC3T3-E1 cells, and consequent lack of response to Nell-1. These data show that Cntnap4 is indispensable for the osteogenic bioactivity of Nell- 1.
[0279] To further confirm the role of Cntnap4 in Nell- 1 -mediated osteogenesis, we transduced neonatal mouse calvarial explants with control (empty), CMV-Nell-1, Cntnap4 shRNA, or CMV- N ell-1 + Cntnap4 shRNA lentiviral particles, and cultured them ex vivo. After 10 days of culture, the explants transfected with CMV-Nell-1 exhibited increased bone formation (delineated by increased mineral apposition in Alizarin Complexone) (Figure 21 A), increased bony overlaps between the parietal and frontal bones (in the coronal sutures) (Figure 2 IB), and narrowed anterior fontanels (Figure 21C), relative to the explants transfected with control lentiviral particles. Moreover, Cntnap4-KD completely ablated the osteogenic effects of Nell-1 overexpression in the calvarial explants transfected with CMV-Nell-1 (Figures 21A-21C). These data further demonstrate the essential role of Cntnap4 in Nell- 1 -mediated osteogenesis and calvarial bone development.
[0280] Although Nell-1 is also a major regulator of chondrogenic differentiation and maturation43, 46, 63 , chondrogenic committed ATDC5 cells exhibited notably lower Cntnap4 expression (Figures 20A-20B). Meanwhile, expression of nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 2 (Nfatc2), a known Nell-1 primary response gene in ATDC5 cells44, was decreased by Cntnap4- KD alone (without Nell-1) in ATDC5 cells (Figure 26A). However, Cntnap4-KD did not affect dose-dependent Nell-1 stimulated increases of Nfatc2 expression in Cntnap4-KD ATDC5 cells (Figure 26B). It appears that, although both Nell-1 and Cntnap4 can regulate Nfatc2 expression, Nell-1 can induce Nfatc2 in chondrogenic-committed cells independently of Cntnap4. Given the high expression of Cntnap4 in Nell- 1 -responsive osteogenic-committed cells (e.g., MC3T3-E1 cells and NMCC), and the low expression in less osteogenic-committed, but highly Nell- 1 -responsive cells, such as ADTC5 cells44, Cntnap4 is not likely to be the only cell surface receptor for Nell-1. Nell-1 may interact with different receptors in a cell-type and differentiation stage specific manner.
Cntnap4 Mediates Nell- 1 -Stimulated MAPK and Wnt Signaling in Osteogenic-Committed Cells
[0281] Cntnap4- KD specifically perturbed the robust osteogenic response of MC3T3-E1 cells to Nell-1, but not BMP2 (Figure 20 A), suggesting that Cntnap4 is specifically involved in osteogenesis regulated by Nell-1. Since Nell-1 is known to activate Wnt and MAPK transduction during osteogenesis18, 50, we examined the effect of Cntnap4-KD on Wnt and MAPK signaling pathways during osteogenic differentiation. In agreement with our previous studies, Nell-1 induced high ERK and JNK phosphorylation/activation levels in control MC3T3-E1 cells.
Conversely, Nell- 1 -responsive ERK or JNK activation was markedly diminished in Cntnap4-KD MC3T3-E1 cells (Figure 22A). Similarly, whereby Nell-1 treatment significantly elevated intracellular and nuclear levels of Axin2 and active b-catenin in control MC3T3-E1 pre osteoblasts, Cntnap4- KD completely abolished Nell- 1 -responsive Wnt signaling (Figures 22B and 22C). These findings demonstrate that Cntnap4 is critical and specific for Nell-1 activation of MAPK and Wnt signaling in osteogenic-committed cells (Figure 23).
Discussion
[0282] In the last two decades, the osteogenic potential of Nell- 1 has been validated in rat calvarial10 and femoral segmental13 defect models, rat12, 14, 22, sheep9, and non-human primate64 spinal fusion models, and mouse16, 18, rat15, and sheep18 osteoporotic models. Importantly, Nell-1 signaling pathways (Figure 23) are distinct from BMP216, 19 21, 65. As a consequence, well- documented off-target BMP2 effects such as post-operative inflammation, osteoclastogenesis, adipogenesis, and ectopic bone formation have not been observed after Nell-1 administration9, 21’ 66, 67. Thus, Nell-1 may hold great promise for future clinical development as a safer therapeutic for bone repair and regeneration1, 18.
[0283] In our past investigation of Nell- 1 binding proteins, we described intracellular complex formation68 between all-trans retinoic acid induced differentiation factor [ATRAID; also known as apoptosis related protein 3 (APR3)] and Nell-1 in osteosarcoma- and kidney-derived cell lines68. Since ATRAID was a protein mainly present on the lysosomal membrane69, it was not considered a suitable candidate for extracellular Nell- 11 binding and signal transduction. Instead, ATRAID, due to its similarities with lysosomal-associated membrane protein 1/269, may play a role in endocytosis and/or transportation of Nell-168. This may also explain our previous observation that ATRAID can bind with multiple truncated Nell-1 constructs with or without the N-terminal LamG domain68. Previously, we also reported the binding of Nell- 1 C-terminal sequence with integrin-bΐ49, 70, and that integrin-bΐ knock-down inhibited Nell- 1 -responsive Wnt- signal transduction18. Integrin-bΐ, however, demonstrates relatively promiscuous binding with a broad range of molecules in various cell types (including osteoblasts and chondrocytes)70 72, and is less likely to be a Nell- 1 -specific receptor. Nevertheless, since either integrin-filis or Cntnap4 knock-down can inhibit Nell- 1 -responsive Wnt-signal transduction, cross-talk may occur between integrin-bΐ and Cntnap4-mediated Nell- 1 -responsive signal transduction. Structurally, integrin-bΐ binds to the C-terminus of Nell-170, while Cntnap4 binds to the Nell-l’s N-terminus. As such, the possibility that Nell-l-integrin-bΐ binding facilitates Nell-1 ligand-receptor complex formation with Cntnap4 to stimulate downstream signal transduction remains for future investigation.
[0284] Although previous Nell-1 studies have focused primarily on bone, Nell-1 was first identified in fetal and adult human brain2, and abundant Nell-Fs expression is detected in developing and adult nervous systems33. Transmembrane receptor Cntnap4, first identified from a fetal mouse brain cDNA library, is abundantly expressed in the central nervous system and during neurogenesis36. Considering that the spatial expression patterns of Nell- 1 and Cntnap4 are largely overlapped in the central nervous system hippocampus, inferior olive nucleus, and spinal cord33, 36, 40, we successfully detected the binding between Nell-1 and Cntnap4 by T7 phage display from a human brain cDNA library. Since Cntnap4 is a member of the neurexin superfamily of SCAMs39, which play important roles in connecting pre- and post-synapses that are critical to synapse development and cortical interneuron function36, 37, 39, recognizing Cntnap4 as a specific, functional receptor for Nell-1 formation in osteogenic-committed cells is unexpected, but highly significant, to elucidate the mechanisms mediating the neural-bone interactions required for the regeneration of skeletal system. This is particularly important in the dental and craniofacial skeletal system for which development and restoration is largely orchestrated by the nervous system73.
[0285] Intriguingly, Cntnap4 and other neurexin superfamily members have been identified as susceptible molecules associated with neuropsychiatric disorders such as ASD74 76 and neurodegenerative disorders including age-related disease, cognitive impairment, late onset Alzheimer’s disease, Alzheimer’s disease, and Parkinson’s disease77. Murine Cntnap4- null mice demonstrate significant interneuron dysfunction with abnormally diminished GABAergic and increased dopaminergic synaptic transmission, and display perseverative behaviors that mimic human ASD patients37. Thus, human genetic linkage studies and murine models strongly implicate Cntnap4 in neurobiological diseases. By revealing the attendant expression and function of Cntnap4 in Nell-1 responsive osteogenic-commitment cells, our study indicates that Cntnap4 may represent a novel set of receptors that play essential roles in both nervous and skeletal tissues. While previous studies revealed CASK and/or APBA136 interactions with the cytoplasmic domain of Cntnap members, no extracellular binding proteins or ligands to Cntnap4 have been identified so far. We are the first to describe Nell-1, which itself is also highly expressed in neural tissues1, 2, 25, 33, as the first extracellular ligand to Cntnap4.
[0286] Previously, we found that Nell-1 overexpression in mice results in craniofacial anomalies and neural tube defects during the late embryonic development stage, which represent the phenomena observed in acrania78, a rare human congenital disease that is often associated with exencephaly79. Additionally, our current ex vivo results showed that interfering with the Nell- 1/Cntnap4 signaling pathway significantly delayed suture closure (Figures 21A-21C), which is in accordance to our previous finding that Nell- 1 -deficiency leads to postponed suture closure resembling cleidocranial dysplasia (CCD)8. These phenomena strongly indicate that the ligand/receptor binding between Nell-1 and Cntnap4 is critical for osteogenesis, and impairments are associated with craniofacial disorders.
[0287] While the influence of the central nervous system on development and morphogenesis is widely recognized80, recent studies suggest that the skeleton also orchestrates the development and cognitive functions of nervous system80 82. Indeed, analysis of ASD has revealed a high association with craniofacial defects83, and a mouse model of human multiple hereditary exostoses (MHE)84, a genetic condition with abnormal bone growth, was able to replicate some of the autistic behaviors observed in human MHE patients85. In addition, some CCD patients also develop cognitive disorders due to currently unknown mechanisms82. Because CCD is mainly caused by mutation or deletion of Runx2S6, a transcription factor regulating expression of Ocn by binding the osteoblast-specific element 2 (OSE2) in the Ocn promoter87, this observation may relate to the recent discovery of Ocn’s roles in brain development and function88. As a hormone carrying out the ability to cross the blood-brain barrier and placenta, Ocn regulates development and function of nervous system through the endocrine system88. However, although G protein- coupled receptor family C group 6 member A (Gprc6a) has been recognized as the receptor of Ocn in the reproductive system89, 90 and glucose metabolism91, because Gprc6a deficiency in mice does not replicate the neurotransmitter and behavior abnormalities seen in the Ocn-deficient animals82, 88, the receptor transducing Ocn signals in the brain is still unknown. Therefore, in order to develop effective and specific therapeutics for the unique skeletal-neural associated conditions, such as CCD and ASD, the nature of interactions between brain and skeletal tissues requires further investigation.
[0288] Excitingly, the newly identified ligand/receptor interaction of Nell- 1 and Cntnap4 provides novel insight into the skeletal-neural functional axis, and may enrich the understanding of the relationships between neural dysfunctions and skeletal diseases. Our previous studies have shown that Runx2 directly regulates Nell-1 expression by binding the OSE2 segment in the Nell-1 promoter7, and Nell-1, in turn, enhances Runx2 phosphorylation and activity50 by stimulating the MAPK signaling pathway19 (Figure 23). In this study, we show that Cntnap4 mediated the Nell-1- responsive Ocn expression in osteogenic-committed cells (Figures 20B and 20C), which suggests that there is a potential Nell-1/Cntnap4 pathway in regulating Ocn-mediated endocrinal modulation among different tissues and organs, including bone and the brain80. Moreover, our ongoing studies have found co-localized Nell-1 and Cntnap4 in the human hippocampus (Figure 27) as well as in osteogenic-committed cells. It is highly possible that, in addition to the well- known BMP-mediated signal transduction92, Nell-1/Cntnap4 represents an alternative pathway that orchestrates the skeletal-neural functional axis. Furthermore, a growing body of transcriptome analysis indicates both Nell-1 and Cntnap4 are potential loci of several neurodegenerative and neuropsychiatric disorders, such as ASD31, 32, 74 76 and Alzheimer’s disease28, 11. Our preliminary studies revealed that, like Cntnap4-mutant mice37, Nell-1 -mutant mice also have aberrant behaviors which can be rescued by specific neuropsychiatric pharmaceutical intervention (unpublished data). Further investigations are currently ongoing to demonstrate the functional involvement of Nell-1/Cntnap4 axis in the brain, as we have already shown in bone tissue.
[0289] Taken together, our current studies support the growing consensus that there is significant crosstalk between the brain and bone during development and homeostasis81, 93, and potentially, in neurodevelopmental disorders. Discovering the Nell-1/Cntnap4 functional axis may provide a novel foundational mechanistic framework for future studies in neuroskeletal biology.
Materials and Methods - Nell-1 Protein Purification
[0290] Recombinant full-length human Nell-1 with polyhistidine-tag (His-tagged Nell-1) was provided by Katayama Inc. (Amagasaki-city, Hyogo, Japan) with a purity of 98%, and used as bait in the phage display study. Recombinant full-length human Nell- 1 without tags was provided by Aragen Bioscience Inc. (Morgan Hill, CA, USA) with a purity of 92% 17 for all functional studies. The plasmid expressing FamG domain deleted human Nell-1 (Nell-l[LamG]), constructed from the pcDNA™3. 1 //nyr-His B Mammalian Expression Vector (Thermo Fisher Scientific, Canoga Park, CA, USA)68 was transfected into CHO-K1 cells (ATCC® CCF-61™) by
FIPOFECTAMINE® 3000 (Thermo Fisher Scientific). The His-tagged Nell-l[LamG] was then purified from the supernatant of the transient transfection product by PROBOND™ Purification System (Thermo Fisher Scientific).
Cntnap4 Plasmid Construction and Recombinant Protein Purification
[0291] The open reading frame sequence of human Cntnap4 (NM_033401) was obtained from GenScript (Piscataway, NJ, USA). The extracellular portion of Cntnap4 (Cntnap4extra) was subcloned into pSecTag2A Mammalian Expression Vector (Thermo Fisher Scientific) with the primers 5’-CAC GGT ACC TGG GAA TTC CTA T-3’ and 5’-TTA CTC GAG CTG CAG AGT CAC TT-3\ and then transfected into CHO-K1 cells. Next, the His-tagged Cntnap4extra was purified from the supernatant of the transient transfection product by PROBOND™ Purification System (Thermo Fisher Scientific), and identified by Western Blot (Figures 28A-28B).
Phage Display Biopanning
[0292] HIS-SEFECT® Nickel Magnetic Agarose Beads (Sigma- Aldrich Corp., St. Fouis, MO, USA) were selected to immobilize His-tagged Nell-1. The phage display cDNA library was constructed from the Human Brain cDNA Fibrary (EMD Millipore, Billerica, MA, USA) using the novagen T7SEFECT® System (EMD Millipore). An aliquot of the amplified phage display cDNA library was incubated with His-tagged Nell-1 coated beads for four rounds of biopanning screens. Subsequently, the phages bound to the His-tagged Nell-1 coated beads were eluted, and 100 plaques were selected to amplify the phage DNA by PCR with the provided primers and buffers in the Novagen T7SELECT® System. Sequences spanning over 500 bp were selected and sequenced by Laragen Inc. (Culver City, CA, USA).
Dissociation Constant Enzyme-Linked Immunosorbent Assay (ELISA)
[0293] Dissociation constant ELISA was performed per manufacturer instructions from Novagen T7 Tail-Fiber Monoclonal Antibody (EMD Millipore). In short, 10 pg/ml of His-tagged full- length Nell-1 or Nell-l[LamG] was used as bait to coat the ELISA plate (Corning Inc., New York, NY, USA). After removing the unbounded bait, the plate was blocked with 3% non-fat milk (Bio- Rad Laboratories Inc., Hercules, CA, USA) overnight at 4°C. 100 mΐ/well diluted phage lysate (1X104 - 1X1010 phages/ml) was added to the coated plate, and incubated at 37°C for 1 hour. Unbound phages were thoroughly washed out with IX Tris-buffered saline with 0.1% Tween 20 (TBST) buffer. T7 Tail-Fiber Monoclonal Antibody (1 :2000) was added for 1-hour incubation prior to the addition of horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (1 : 1000; Abeam, Cambridge, MA, USA). 1-STEP™ Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was utilized for developing. After acidification by 2M sulfuric acid (Sigma-Aldrich Corp.), absorbance of the reaction product was measured by an Epoch Microplate spectrophotometer (BioTek, Winooski, VT, USA) at 450 nm. Each concentration of phage was tested in triplicate, while empty T7 phages (without a cDNA insert) were used as the negative controls using the same process.
Gene Expression Profiling [0294] Based on previously published functional investigation of Nell- 11, 17, 49, 5°, 12 different cell lines and primary cells isolated from bone or relevant tissues were selected for use in the current study (Table 7). Total RNA was isolated from subconfluent cells by TRIZOL® Reagent, followed by DNase treatment. 1 pg RNA was injected for reverse transcription (RT) with the ISCRIPT™ Reverse Transcription Supermix for RT-qPCR (Bio-Rad Laboratories Inc). 1 mΐ RT product was used for real-time PCR with SSO ADVANCED™ Universals Probes Supermix (Bio-Rad Laboratories Inc.) and TAQMAN® primers/probe sets (Thermo Fisher Scientific) on a
QuantStudio3 system (Thermo Fisher Scientific) in triplicate. The following were used as specific TAQMAN® primer and probe sets: human Gapdh: Hs02786624_gl, human Cntnap4:
Hs00369159_ml, mouse Gapdh : Mm99999915_gl, mouse Cntnap4 : Mm00519175_ml, mouse Cntnap3: Mm01297195_ml, and mouse Cntnap2: Mm00470553_ml.
Immunocvtochemistrv (ICC) Staining
[0295] Serum-starved MC3T3-E1 pre-osteoblasts (Subclone 4, ATCC® CRL-2593™) and primary newborn mouse calvarial cells (NMCC) were treated with 500 ng/ml Nell-1 for 30 min, and fixed with ice-cold acetone. The fixed cells were blocked with 3% bovine serum albumin (BSA; Sigma- Aldrich Corp.) and incubated with goat anti-Cntnap4 (1 :200; Santa Cruz
Biotechnology, Inc., Santa Cruz, CA, USA), goat anti-Cntnap3 (1 :50; Santa Cruz Biotechnology), mouse anti-Cntnap2 (1 :50; Abeam), and rabbit anti-Nell-1 (1 :75; Allele Biotech, San Diego, CA) antibodies in IHC-TEK™ Antibody Diluent (pH 7.4; IHC World, LLC, Woodstock, MD) overnight at 4°C. Following three washes with lx PBST, cells were incubated with FITC- conjugated anti-rabbit IgG and Texas Red-conjugated anti-Goat IgG secondary antibodies (Abeam), and counterstained with DAPI Fluorescence Stain (Cell Biolabs, Inc., San Diego, CA, USA). Co-localization of Nell- 1 and Cntnap4 was documented by confocal laser scanning microscopy (CLSM) on a Leica TCS SP8 Confocal Laser Scanning Platform coupled with Leica Application Suite X software (Buffalo Grove, IL, USA).
Histological Analysis
[0296] C57BL/6 mice were bred and maintained under an institutionally approved protocol provided by the Chancellor’s Animal Research Committee at UCLA (protocol number: 2012- 041), as previously described50. Calvarial tissues dissected from 60-day-old mice were fixed in 4% ice-cold paraformaldehyde (PFA; Sigma-Aldrich Corp.) for 24 hours, and decalcified with 19% EDTA (Sigma-Aldrich Corp.) for 14 days prior to paraffin embedding. Hematoxylin and eosin (H&E) staining was performed on 5-pm sections. Images were documented by a Keyence BZ-X710 system (Itasca, IL, USA).
Immunohistochemical (IHC) Staining
[0297] IHC staining was performed on 5-pm 60-day-old mouse calvarial tissue sections and normal human hippocampus tissue slides (Abeam) with the same antibodies used for ICC staining. Images were documented with Leica TCS SP8 Confocal Laser Scanning Platform. DUOLINK® Proximity Ligation Assay (PLA1
[0298] All reagents used for DUOLINK ® PLA assay were purchased from Sigma- Aldrich Corp. Deparaffinized 60-day-old mouse calvarial sections were blocked with DUOLINK ® blocking solution at room temperature for one hour before incubation with the same primary antibodies used in IHC staining, but diluted in DUOLINK® antibody diluent at 4°C overnight. The following day, the sections were washed, and then incubated with DUOLINK® In Situ PLA® Probe Anti-Rabbit MINUS and Probe Anti-Goat PLUS (for Cntnap4 and Cntnap3) or Probe Anti- Mouse PLUS (for Cntnap2) at 37°C for 60 min, following manufacturer instructions.
Amplification solution was mixed with polymerase and applied to the slides for 100 min at 37°C, accordingly. The slides were washed, mounted with DUOLINK® In Situ Mounting Medium with DAPI, and examined with Leica TCS SP8 Confocal Laser Scanning Platform.
Pull-Down Assay
[0299] Commercially available Pull-Down PolyHis Protein: Protein Interaction Kit (Thermo Fisher Scientific) was used in this study. In brief, prey protein was isolated from MC3T3-E1 cells or NMCC, and applied to the spin column that was pre-incubated with His-tagged Nell-1, as the bait protein. The bait-prey complex was eluted for SDS-PAGE.
Co-Immunoprecipitation (Co-IP) Assay
[0300] For the Co-IP assay, MC3T3-E1 cells or NMCC were suspended in PBS, and incubated with Nell-1. BS3 (Thermo Fisher Scientific) was added to the cells as a cross-linker. Total protein was isolated by adding cold RIPA lysis buffer (Thermo Fisher Scientific) to the collected cells, and incubating with anti-Nell- 1 antibody coated agarose beads. The Nell- 1/candidate receptor(s) complex was eluted for SDS-PAGE.
Surface Plasmon Resonance (SPR) Analysis
[0301] Binding studies were performed on a Biacore 3000 instrument (Biacore AB, Uppsala, Sweden) by the UCFA Surface Plasma Resonance Core. Cntnap4extra was immobilized on CM5 sensor chips (GE Healthcare Life Sciences, Marlborough, MA, USA) by amine coupling. The solution phase of Nell-1 was dissolved in HBS-EP buffer, which contained 0.15 M NaCl, 10 mM HEPES, pH 7.4, 3 mM EDTA, and 0.005% polysorbate 20 (GE Healthcare Life Sciences). The solution traversed through the sensors at a flow rate of 50 mΐ/minute. Low-retention polypropylene tubes (Corning Inc.) were used throughout. Each experiment was repeated in triplicate. Raw data were processed by subtracting the responses in the reference flow cell and the buffer blanks (double referencing). In previous studies, recombinant human Nell-1 had a molecular weight of 140 kD under reducing conditions and over 700 kD under non-reduced conditions, which suggests that Nell-1 may be a pentameric protein and that oligomerization is important for Nell’s bioactivity1, 65. Therefore, kinetic studies between pentameric Nell-1 and the extracellular portion of Cntnap4 (Cntnap4extra) were analyzed with Scrubber 2.0 (BioLogic Software Pty Ltd., Campbell, Australia). Affinity characters were calculated by plotting the analysis concentration versus the biosensor response, and expressed in resonance unit (RU).
Small Hairpin RNA (shRNA) Transfection
[0302] MC3T3-E1 and ADTC5 cells were transfected with Cntnap4 shRNA Lentiviral particles (Santa Cruz Biotechnology) or non-target control shRNA by LIPOFECT AMINE® 3000. The positive transfected colonies were selected by Puromycin (Sigma-Aldrich Corp.) and validated by Cntnap4 mRNA expression levels in order to establish the Cntnap4 knockdown cell line.
In Vitro Osteogenic Differentiation Assays
[0303] Control and stable Cntnap4 knockdown (Cntnap4- KD) MC3T3-E1 cells were seeded on 24-well plates for alkaline phosphatase (ALP), Alizarin Red, and ICC staining. Additionally, cells were seeded on 6-well plates for osteogenic genes expression assay. Both cell types were cultured in osteogenic differentiation medium [a-MEM (Thermo Fisher Scientific), 10% fetal bovine serum (FBS; Thermo Fisher Scientific), 50 pg/rnl ascorbic acid (Sigma-Aldrich Corp.), and 10 mM b-glycerophosphate (Sigma-Aldrich Corp.)] with or without 500 ng/ml Nell-1 or 100 ng/ml BMP2 (Medtronic, Minneapolis, MN, USA). ALP staining and Alizarin Red staining were performed as previously described49. Anti-Ocn (1 :200; Santa Cruz Biotechnology) and anti-Opn (1 :200; Santa Cruz Biotechnology) antibodies were used for ICC staining. The following specific PCR primer sets were used for real-time PCR analysis: mouse Alp·. 5’-ACT GAT GTG GAA TAC GAA CTG GAT GAG AAG G-3’ (SEQ ID NO: l) and 5’-CAG TCA GGT TGT TCC GAT TCA ATT CAT ACT GC-3’ (SEQ ID NO:2); mouse Collagen Ial : 5’ -CTG GCG GTT CAG GTC CAA T-3’ (SEQ ID NOG) and 5’-TTC CAG GCA ATC CAC GAG C-3’ (SEQ ID NO:4); mouse Collagen la2 5’-AAG GGT GCT ACT GGA CTC CC-3’ (SEQ ID NOG) and 5’-TTG TTA CCG GAT TCT CCT TTG G-3’ (SEQ ID NOG); mouse Ocn: 5’-CTG CCC TAA AGC CCA AAC TCT-3’ (SEQ ID NO:7) and 5’-GAG AGG ACA GGG AGG ATC AA-3’ (SEQ ID NOG); mouse Opn: 5’-AGC AAG AAA CTC TTC CAA GCA A-3’ (SEQ ID NO:9) and 5’- GTG AGA TTC GTC AGA TTC ATC CG-3’ (SEQ ID NO:10); mouse Bsp: 5’-ATG GAG ACG GCG ATA GTT CC-3’ (SEQ ID NO:l 1) and 5’-CTA GCT GTT ACA CCC GAG AGT-3’
(SEQ ID NO: 12).
Mouse Calvarial Bone Explant Cultivation
[0304] Calvarial vaults of newborn wildtype mice were harvested and cultured as previously described50, and randomly assigned to each experiment group. Lentiviral particles of Cntnap4 shRNA (Santa Cruz Biotechnology) and CMV-Nell-1 (GenTarget Inc., San Diego, CA, USA) were added to the culture medium at day 0 and day 1, respectively. Alizarin Complexone (2 pg/ml, Sigma-Aldrich Corp.) was added to the culture medium at day 4, and the medium, containing Alizarin Complexone, was changed every 3 days thereafter. The explants were fixed in 4% Paraformaldehyde at day 10. The Alizarin Complexone deposition on explants was observed with an Olympus SZX12 fluorescent microscope. Overlap width of frontal and parietal bones, as well as the area of unclosed fontanel, were quantified using Image-Pro Plus in a blinded fashion (Media Cybernetics, Warrendale, PA, USA).
Western Blot
[0305] Sub-confluent control and Cntnap4- KD MC3T3-E1 cells were subjected to serum starvation for 18 hours before treatment with PBS control or 500 ng/ml Nell-1 for 10, 30, or 60 min at 37°C. Protein isolation and Western blot were performed as previously described50. The following primary antibodies were employed: ERK1 + ERK2 (1 :1,000; Abeam), ERKl(phospho Y204) + ERK2 (phospho Y187) (1 : 1,000; Abeam), JNK1 (1 : 1,000; Abeam), JNK1 + JNK2 + JNK3 (phospho Y185 + Y185 + Y223) (1 :1,000; Abeam), p38 (1 :1,000; Abeam), p38 (phospho T180 + Y182) (1 :1,000; Abeam), activc-(i-Catcnin (1 :5,000; EMD Millipore), b-Catenin (1 : 1,000; Abeam), Axin2 (1 :1,000; Abeam), GAPDH (1 :500; Santa Cruz Biotechnology), and Histone H3 (1 : 1,000; EMD Millipore).
In Vitro Nfatc2 Expression Analysis
[0306] Control and stable Cntnap4- KD ADTC5 cells were cultured in 1 :1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (Thermo Fisher Scientific) supplied with 5% FBS, 10 mg/ml of human transferrin (Sigma- Aldrich Corp.) and 30 nM sodium selenite (Sigma- Aldrich Corp.) until sub-confluence. After 18-hours of serum starvation (0.1% FBS), cells were treated with 0, 400, 800, 1600, or 3200 ng/ml Nell-1 for 3 hours44. For real-time PCR, Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) was used with NFact2 (GenBank Accession Number AK161174.1) primers: forward primer 5’-CTT TCA GAT GGG AAT AAA CGT C-3’ (SEQ ID NO: 13), and reverse primer 5’-TCC TAC TCA CAT AGC AAC AGA A-3’ (SEQ ID NO: 14); and Gapdh (GenBank Accession Number AK002273.1) primers: forward primer 5’-ATT CAA CGG CAC ATG CAA GG-3’ (SEQ ID NO: 15), reverse primer 5’-GAT GTT AGT GGG GTC TCG CTC-3’ (SEQ ID NO: 16), respectively44.
Statistical Analysis
[0307] All statistical analyses were conducted in consultation with the UCLA Statistical Biomathematical Consulting Clinic. Initial sample numbers were determined using power analysis to give a = 0.05 and power = 0.08. Statistical significance was performed with OriginPro 8 (Origin Lab Corp., Northampton, MA, USA) and included the one-way ANOVA and two- sample f-test. Individual comparisons between two groups were determined by the Mann-Whitney test for non-parametric data. Statistical significance was determined at the P < 0.05 level.
References 1. Zhang X, et al., Journal of dental research 2010; 89:865-878.
2. Watanabe TK, et al., Genomics 1996; 38:273-276.
3. Ting K, et al., Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 1999; 14:80-89.
4. Zhang X, et al., The Journal of clinical investigation 2002; 110:861-870.
5. Zhang X, et al., Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 2003; 18:2126-2134.
6. Desai J, et al., Human molecular genetics 2006; 15: 1329-1341.
7. Truong T, et al., Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 2007; 22:7-18.
8. Zhang X, et al., The Journal of craniofacial surgery 2012; 23:61-66.
9. Siu RK, et al., Tissue engineering Part A 2011; 17:1123-1135.
10. Aghaloo T, et al., The American journal of pathology 2006; 169:903-915.
11. Aghaloo T, et al., Molecular therapy : the journal of the American Society of Gene Therapy 2007; 15:1872-1880.
12. Lu SS, et al., The spine journal : official journal of the North American Spine Society 2007; 7:50-60.
13. Li W, et al., Plastic and reconstructive surgery 2011 ; 127:580-587.
14. Li W, et al., Tissue engineering Part A 2010; 16:2861-2870.
15. Kwak J, et al., Tissue engineering Part A 2013; 19:426-436.
16. Aaron James JS, et al., J Bone Miner Res 28 (Suppl 1). Baltimore, Maryland, USA 2013.
17. Shen J, et al., The American journal of pathology 2016; 186:419-434.
18. James AW, et al., Nature communications 2015; 6:7362.
19. Bokui N, et al., FEBS letters 2008; 582:365-371.
20. Chen F, et al., International orthopaedics 2012; 36:2181-2187.
21. Shen J, et al., Tissue Eng Pt A 2013; 19:2390-2401.
22. Yuan W, et al., Journal of orthopaedic science : official journal of the Japanese Orthopaedic Association 2013; 18:646-657.
23. McClelland S, et al., eLife 2014; 3:e01267.
24. Hackett TA, et al., BMC genomics 2015; 16:606.
25. Bakken TE, et al., Human molecular genetics 2015; 24:4327-4339.
26. Dunckley T, et al., The New England journal of medicine 2007; 357:775-788.
27. Zuccato C, et al., The Journal of neuroscience : the official journal of the Society for Neuroscience 2007; 27:6972-6983.
28. Zhang L, et al., Genetics and molecular research : GMR 2015; 14:7218-7232.
29. Logotheti M, , et al., TheScientificWorldJoumal 2013; 2013:685917. 30. Lanz TA, et al., Molecular autism 2013; 4:45.
31. Connolly JJ, et al., Child development 2013 ; 84:17-33.
32. Butler MG, et al., International journal of molecular sciences 2015; 16:6464-6495.
33. Kuroda S, et al., Biochemical and biophysical research communications 1999; 265:79-86.
34. Abreu JG, et al., Gene 2002; 287:39-47.
35. Campbell ID, et al., Philos T Roy Soc B 1991; 332:165-170.
36. Spiegel I, et al., Molecular and cellular neurosciences 2002; 20:283-297.
37. Karayannis T, et al., Nature 2014; 511 :236-240.
38. Yin FT, et al., Stem cells and development 2015; 24:640-652.
39. Baig DN, et al., Brain Research Bulletin 2017; 129:82-90.
40. Nakamura R, et al., The Journal of biological chemistry 2012; 287:3282-3291.
41. Takahashi K, et al., FEBS letters 2015; 589:4026-4032.
42. Knothe Tate ML, et al., The international journal of biochemistry & cell biology 2008; 40:2720-2738.
43. Lee M, et al., Tissue engineering Part A 2010; 16:1791-1800.
44. Chen W, et al., Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 2011; 26: 1230-1241.
45. Li C, et al., The American journal of pathology 2016.
46. Li CS, et al., Tissue engineering Part A 2016; 22:272-285.
47. Nakamura Y, et al., The Journal of biological chemistry 2014; 289:9781-9794.
48. Hasebe A, et al., Molecular biotechnology 2012; 51 :58-66.
49. Shen J, et al., Journal of cellular biochemistry 2012; 113:3620-3628.
50. Zhang X, et al., Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 2011; 26:777-791.
51. Arnsdorf EJ, et al., ProQuesst LLC 2008.
52. Yazid MD, et al., Cancer Cell Int 2010; 10.
53. Taylor SM, et al., Cell 1979; 17:771-779.
54. Ahrens M, et al., DNA Cell Biol 1993; 12:871-880.
55. Ogawa M, et al., Embo J 1988; 7:1337-1343.
56. Ding J, et al., Biosci Biotech Bioch 2003; 67:314-321.
57. Mohseny AB, et al., Laboratory Investigation 2011; 91 :1195-1205.
58. Thompson L, et al., J Orthop Res 2012; 30:831-844.
59. Tsai SW, et al., PloS one 2012; 7.
60. Rohilla M, et al., Clin Breast Cancer 2015; 15:335-342.
61. Yao Y, et al., Journal of cellular biochemistry 2013; 114:1223-1229.
62. Pattnaik P, et al., Applied biochemistry and biotechnology 2005; 126:79-92. 63. Siu RK, et al., Tissue engineering Part A 2012; 18:252-261.
64. Shen J, et al., Orthopaedic Research Society (ORS) 2015 Annual Meeting. Las Vegas, Nevada Orthopaedic Research Society 2015:1577.
65. Aghaloo T, et al., Journal of oral and maxillofacial surgery : official journal of the American Association of Oral and Maxillofacial Surgeons 2010; 68:300-308.
66. James AW, et al., Biochemical and biophysical research communications 2011; 411 :126- 131.
67. Xue J, et al., Bone 2011; 48:485-495.
68. Zou X, et al., FEBS letters 2011 ; 585:2410-2418.
69. Ding X, et al., Biochemical and biophysical research communications 2015; 460:915-922.
70. Hasebe A, et al., FEBS letters 2012; 586:2500-2506.
71. Goessler UR, et al., International journal of molecular medicine 2008; 21 :271-279.
72. Loeser RF, et al., Matrix Biol 2014; 39:11-16.
73. Adameyko I, et al., Frontiers in physiology 2016; 7:49.
74. O'Roak BJ, et al., Nature 2012; 485:246-250.
75. Wang LS, , et al., BMC medical genetics 2010; 11 : 134.
76. Ding Y, et al., Frontiers in Biology 2015; 10:239-251.
77. Iakoubov L, et al., PloS one 2013; 8:e79790.
78. Zhang X, et al., Laboratory investigation; a journal of technical methods and pathology 2006; 86:633-644.
79. Matsumoto A, et al., Anat Embryol 2002; 205:7-18.
80. Chamouni A, et al., Rev Endocr Metab Dis 2015; 16:99-113.
81. Masi L, Clinical cases in mineral and bone metabolism : the official journal of the Italian Society of Osteoporosis, Mineral Metabolism, and Skeletal Diseases 2012; 9: 13-16.
82. Rousseaud A, et al., Horn Mol Biol Clin 12016; 28:69-83.
83. Registry TNBD. Autistic Spectrum Disorders Report. 2009.
84. Matsumoto K, et al., Proceedings of the National Academy of Sciences of the United States of America 2010; 107:10932-10937.
85. Irie F, et al., Proceedings of the National Academy of Sciences of the United States of America 2012; 109:5052-5056.
86. Ott CE, et al., Human mutation 2010; 3LE1587-1593.
87. Franceschi RT, et al., Connect Tissue Res 2003; 44:109-116.
88. Oury F, et al., Cell 2013; 155:228-241.
89. Oury F, et al., Cell 2011; 144:796-809.
90. Oury F, et al., Journal Of Clinical Investigation 2013; 123:2421-2433.
91. Pi M, et al., Journal Of Bone And Mineral Research 2011; 26:1680-1683. 92. Liu A, et al., Nature reviews Neuroscience 2005; 6:945-954.
93. Karsenty G, et al., Nature 2012; 481 :314-320.
[0308] Example 3. Behavioral studies on ENU mice
[0309] Mutant mice of N-ethyl-N-nitrosourea (ENU)-induced mutations in NELL-1 were created and studied using behavioral tests. The results are summarized in Table 8 and Figure 29.
Table 8. Results of several behavioral tests on the ENU mice.
Behavioral Domain Mouse Behavioral Test Results in Nell-1
Figure imgf000078_0001
Genera! Activity Open Field Arena (OFAt Norm.?!
Motor Coc'dination Rotarod Performance Nor 3i
Learning and Memory Cotext Fear conditioning Norms!
Anxiety Sevated Pius Mate (PPM) Decreased
inrate Defer isve Behavior,. Reduced at beginning,
Activity Burst
Pain Sensitity then norma!
Sensory Reactivity Startle Response increased
Sensorimotor integration Pre-puise inhibition (P?i) increased
Repetitive Behavior Marble Burying increased
Repetitive Behavior Grooming increased
Social interaction 3 chamber social interaction Deficit
[0310] In the studies summarized by Figure 29, each of the ENU mutant mice exhibits repetitive behavior (marble burying). An amount of anti-autism drug was administered to the ENU mutant mouse by injection. Figure 29 shows that, as compared to control (blue bar), the drug reduced the marble burying behavior of ENU mice by a significant factor.
[0311] Further tests were performed on the ENU mutant mice. Injection of an anti-autism medicine into the ENU mice caused the repetitive behavior (marble burying) of the mice to be recovered (data not shown).
[0312] Those skilled in the art will know, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These and all other equivalents are intended to be encompassed by the following claims.
[0313] Example 4: Posterolateral Fusion (PLF)
[0314] According to the methods disclosed herein, a physician or orthopedic surgeon of skill in the art can treat a patient with back pain. The patient experiences a traumatic accident by slipping on the ice and is experiencing continuous lower back pain, numbness in some of his extremities, and neurological impairment. The doctor can perform a physical examination to test the patient’s range of motion, muscle strength, and tenderness of the injured area. The doctor can then diagnosed the patient with spinal stenosis and perform a spinal tap, followed by an MRI and CT scan to confirm the diagnosis. After confirming the diagnosis, the doctor may recommend a spinal fusion surgery to alleviate the pain and neurological symptoms. The doctor can perform a posterolateral fusion (PLF) to heal the injured area. A bone graft comprising a composition described herein (e.g., a composition containing an antibody that specifically binds to Cntnap4) can be prepared and administered according the methods described herein. The graft can be administered, for example, around the damaged vertebrae to encourage bone formation. Over the next 6 to 18 months, the patient can be examined to determine if the materials in the bone graft promote osteogenesis and fuse with the vertebrae to heal the painful area.
[0315] Example 5: Treatment of Alzheimer’s disease
[0316] According to the methods disclosed herein, a physician of skill in the art can treat a patient with Alzheimer’s disease. The patient experiences cognitive degeneration and loss of memory. The doctor can perform a physical examination to test the patient’s memory and recall. The doctor then diagnoses the patient with Alzheimer’ s disease and can administer a composition described herein (e.g., a composition containing an antibody that specifically binds to Cntnap4). The antibody can be administered intrathecally once a week for 26 weeks in order to trigger neurogenesis. Over the next 6 months, the patient can be examined to determine if the symptoms of the disease diminish and to determine if some cognitive ability is restored.

Claims

Claims
1. A composition for treating a disorder, comprising an effective amount of an agent effective for potentiating an effective binding of blood NELL-l to blood Cntnap4 in a mammalian subject in need thereof to achieve a normal level of blood NELL-l binding to blood Cntnap4 in the mammalian subject, wherein the mammalian subject has an abnormal level of blood NELL-l binding to blood Cntnap4.
2. The composition of claim 1, wherein the agent comprises:
a) NELL-l,
b) Cntnap4,
c) a combination of NELL-l and Cntnap4,
d) an agonist of GABAergic activity,
e) an inhibitor of Dopaminergic activity,
f) a Wnt/ -catenin signaling activator, or
g) a combination of any of a) through f).
3. The composition of claim 2, wherein the agent further comprises integrin-bT
4. The composition of claim 2, wherein the Wnt/ -catenin signaling activator is a GSK3 inhibitor.
5. The composition of claim 2 wherein the agonist of GABAergic activity is Indiplon.
6. The composition of claim 2, wherein the inhibitor of Dopaminergic activity is Risperidone.
7. The composition of claim 1, wherein the disorder is a neurological disorder or a skeletal disorder.
8. The composition of claim 1, wherein the disorder is osteoporosis.
9. The composition of claim 1, wherein the disorder is autism.
10. The composition of claim 1, wherein the agent is an enhancer or inhibitor of NELL-l or Cntnap4.
11. The composition of claim 1, wherein the agent is an exogenous gene construct expressing NELL-
1 or Cntnap4.
12. The composition of claim 1, further comprising a pharmaceutically acceptable carrier for local or systemic delivery.
13. The composition of claim 1, wherein the subject is a human being.
14. A method of diagnosing a neurological disorder or skeletal disorder in a mammalian subject, comprising:
providing a normal level of blood NELL-1 binding to blood Cntnap4 in a normal mammalian in reference to a disorder selected from a neurological disorder or a bone disorder,
generating a diagnostic level of blood NELL-1 binding to blood Cntnap4 by measuring blood NELL-1 and blood cntnap4 in the mammalian subject,
determining that the mammalian subject suffers from the neurological disorder or bone disorder if the diagnostic level of blood NELL-1 binding to blood Cntnap4 significantly deviates from the normal level of blood NELL-1 binding to blood Cntnap4.
15. The method of claim 14, wherein the disorder is a neurological disorder.
16. The method of claim 14, wherein the disorder is autism or osteoporosis.
17. The method according to claim 14, wherein the subject is a human being.
18. A method of treating or ameliorating a disorder in a mammalian subject, comprising
administering to the mammalian subject in need thereof a composition comprising an effective amount of an agent effective for potentiating an effective binding of blood NELL-1 to blood Cntnap4 in the mammalian subject to achieve a normal level of blood NELL-1 binding to blood Cntnap4 in the mammalian subject,
wherein the mammalian subject has an abnormal level of blood NELL-1 binding to blood Cntnap4, and
wherein the disorder is a neurological disorder or a bone disorder.
19. The method of claim 18, wherein the agent comprises:
a) NELL- 1,
b) Cntnap4,
c) a combination of NELL-1 and Cntnap4,
d) an agonist of GABAergic activity,
e) an inhibitor of Dopaminergic activity,
f) a Wnt/ -catenin signaling activator, or
g) a combination of any of a) through f).
20. The method of claim 19, wherein the Wnt^-catenin signaling activator is 0.8K3b.
21. The method of claim 19, wherein the agent further comprises integrin-bΐ.
22. The method of claim 19, wherein the agonist of GABAergic activity is Indiplon.
23. The method of claim 19, wherein the inhibitor of Dopaminergic activity is Risperidone.
24. The method of claim 18, wherein the disorder is a neurological disorder or a skeletal disorder.
25. The method of claim 18, wherein the disorder is osteoporosis.
26. The method of claim 18, wherein the disorder is autism.
27. The method of claim 18, wherein the agent is an enhancer or inhibitor of NELL-1 or Cntnap4.
28. The method of claim 18, wherein the agent is a gene construct expressing NELL-1 or Cntnap4.
29. The method of claim 18, wherein the composition further comprises a pharmaceutically acceptable carrier for local or systemic delivery.
30. The method of claim 18, wherein the subject is a human being.
31. A method of fabricating a composition, comprising:
providing an effective amount of an agent, which is capable of potentiating an effective binding of blood NELL-1 to blood Cntnap4 in a mammalian subject in need thereof to achieve a normal level of blood NELL-1 binding to blood Cntnap4 in the mammalian subject, and
forming the composition,
wherein the composition is according to any one of claims 1-13.
32. A fusion protein comprising Nell-1 or a fragment thereof fused to an isolated antibody or antigen binding fragment thereof that specifically binds an epitope present on Cntnap4.
33. The fusion protein of claim 32, wherein the antibody or antigen-binding fragment thereof is an agonistic antibody.
34. The fusion protein of claim 32 or 33, wherein the antibody or antigen-binding fragment thereof binds to an epitope present within a Laminin G domain of Cntnap4.
35. The fusion protein of any one of claims 32-34, wherein the antibody or antigen-binding fragment thereof comprises human constant regions.
36. The fusion protein of any one of claims 32-35, wherein the antibody or antigen-binding fragment thereof is a humanized antibody, a human antibody, or a monoclonal antibody.
37. The fusion protein of any one of claims 32-36, wherein the antigen-binding fragment is an antibody that lacks the Lc portion or is a L(ab’)2, a Lab, an Lv, or an scLv structure.
38. A pharmaceutical composition comprising the fusion protein of any one of claims 32-37 and a pharmaceutically acceptable carrier.
39. A method of promoting osteogenesis in a subject in need thereof comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds an epitope present on Cntnap4 or the fusion protein of any one of claims 32-37, or the pharmaceutical composition of claim 38, wherein the antibody or antigen-binding fragment thereof specifically binds to Cntnap4, thereby promoting osteogenesis.
40. The method of claim 39, wherein the subject has a bone disorder.
41. The method of claim 40, wherein the bone disorder is osteoporosis.
42. A method of promoting neurogenesis in a subject in need thereof comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds an epitope present on Cntnap4 or the fusion protein of any one of claims 32-37, or the pharmaceutical composition of claim 38, wherein the antibody or antigen-binding fragment thereof specifically binds to Cntnap4, thereby promoting neurogenesis.
43. The method of claim 42, wherein the subject has a neurological disorder.
44. The method of claim 43, wherein the neurological disorder is Autism spectrum disorder.
45. The method of any one of claims 39-44, wherein the antibody or antigen-binding fragment thereof or the fusion protein is administered at a dosage of about 0.001 mg/kg/day to about 10 mg/kg day.
46. The method of any one of claims 39-45, wherein the antibody or antigen-binding fragment thereof is an agonistic antibody.
47. The method of any one of claims 39-46, wherein the antibody or antigen-binding fragment thereof binds to an epitope present within a Laminin G domain of Cntnap4.
48. The method of any one of claims 39-47, wherein the antibody or antigen-binding fragment thereof comprises human constant regions.
49. The method of any one of claims 39-48, wherein the antibody or antigen-binding fragment thereof is a humanized antibody, a human antibody, or a monoclonal antibody.
50. The method of any one of claims 39-49, wherein the antigen-binding fragment is an antibody that lacks the Fc portion or is a F(ab’)2, a Fab, an Fv, or an scFv structure.
51. The fusion protein of claim 32, wherein the antibody or antigen-binding fragment thereof comprises human constant regions.
52. The fusion protein of claim 32, wherein the antibody or antigen-binding fragment thereof is a humanized antibody, a human antibody, or a monoclonal antibody.
53. The fusion protein of claim 32, wherein the antigen-binding fragment is an antibody that lacks the Fc portion or is a F(ab’)2, a Fab, an Fv, or an scFv structure.
54. A pharmaceutical composition comprising the fusion protein of claim 32 and a pharmaceutically acceptable carrier.
55. A method of promoting osteogenesis in a subject in need thereof comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds an epitope present on Cntnap4, the fusion protein claim 32, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof or the fusion protein of claim 32, wherein the antibody or antigen binding fragment thereof specifically binds to Cntnap4, thereby promoting osteogenesis.
56. The method of claim 55, wherein the subject has a bone disorder.
57. The method of claim 56, wherein the bone disorder is osteoporosis.
58. A method of promoting neurogenesis in a subject in need thereof comprising administering to an subject an antibody or antigen-binding fragment thereof that specifically binds an epitope present on Cntnap4, the fusion protein of claim 32, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof or the fusion protein of claim 32, wherein the antibody or antigen binding fragment thereof specifically binds to Cntnap4, thereby promoting neurogenesis.
59. The method of claim 58, wherein the subject has a neurological disorder.
60. The method of claim 59, wherein the neurological disorder is Autism spectrum disorder.
61. The method of any one of claim 55, wherein the antibody or antigen-binding fragment thereof or the fusion protein is administered at a dosage of about 0.001 mg/kg/day to about 10 mg/kg day.
62. The method of claim 55, wherein the antibody or antigen -binding fragment thereof is an agonistic antibody.
63. The method of claim 55, wherein the antibody or antigen-binding fragment thereof binds to an epitope present within a Laminin G domain of Cntnap4.
64. The method of claim 55, wherein the antibody or antigen -binding fragment thereof comprises human constant regions.
65. The method of claim 55, wherein the antibody or antigen-binding fragment thereof is a humanized antibody, a human antibody, or a monoclonal antibody.
66. The method of claim 55, wherein the antigen-binding fragment is an antibody that lacks the Fc portion or is a F(ab’)2, a Fab, an Fv, or an scFv structure.
PCT/US2019/029161 2018-04-25 2019-04-25 Methods and compositions for skeletal and neurological disorders Ceased WO2019210080A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201862662435P 2018-04-25 2018-04-25
US62/662,435 2018-04-25

Publications (1)

Publication Number Publication Date
WO2019210080A1 true WO2019210080A1 (en) 2019-10-31

Family

ID=68294122

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2019/029161 Ceased WO2019210080A1 (en) 2018-04-25 2019-04-25 Methods and compositions for skeletal and neurological disorders

Country Status (1)

Country Link
WO (1) WO2019210080A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111920800A (en) * 2020-08-26 2020-11-13 中国人民解放军海军军医大学第一附属医院 Application of oroxylin A in treating osteoporosis and promoting fracture healing
CN112870361A (en) * 2021-01-28 2021-06-01 重庆医科大学 Use of iron death inhibitor for preparing medicine for preventing or treating osteoporosis or bone loss caused by iron overload
CN112915207A (en) * 2021-03-25 2021-06-08 上海市第六人民医院 Application of LNX2 gene deletion in treatment of osteoporosis

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005100604A2 (en) * 2004-04-01 2005-10-27 Sequenom, Inc. Methods for identifying risk of osteoarthritis and treatments thereof
WO2009058690A2 (en) * 2007-10-30 2009-05-07 The Regents Of The University Of California Nell-1 compositions
US8012996B2 (en) * 1993-03-01 2011-09-06 Children's Medical Center Corporation Methods and composition for inhibition of angiogenesis
WO2012149472A2 (en) * 2011-04-27 2012-11-01 Ignite Institute For Individualized Health Methods, compositions, and kits for treating and preventing neurological conditions
WO2016109449A1 (en) * 2014-12-29 2016-07-07 The Board Of Trustees Of The Leland Stanford Junior University Methods of diagnosing autism spectrum disorders

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8012996B2 (en) * 1993-03-01 2011-09-06 Children's Medical Center Corporation Methods and composition for inhibition of angiogenesis
WO2005100604A2 (en) * 2004-04-01 2005-10-27 Sequenom, Inc. Methods for identifying risk of osteoarthritis and treatments thereof
WO2009058690A2 (en) * 2007-10-30 2009-05-07 The Regents Of The University Of California Nell-1 compositions
WO2012149472A2 (en) * 2011-04-27 2012-11-01 Ignite Institute For Individualized Health Methods, compositions, and kits for treating and preventing neurological conditions
WO2016109449A1 (en) * 2014-12-29 2016-07-07 The Board Of Trustees Of The Leland Stanford Junior University Methods of diagnosing autism spectrum disorders

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
HU ET AL.: "Anti-angiogenesis therapy based on the bone marrow-derived stromal cells genetically engineered to express sFlt-1 in mouse tumor model", BMC CANCER, vol. 8, no. 1, 23 October 2008 (2008-10-23), pages 1 - 10, XP021042944, DOI: 10.1186/1471-2407-8-306 *
LI ET AL.: "Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-I)-Responsive Osteogenesis", JOURNAL OF BONE AND MINERAL RESEARCH, vol. 33, no. 10, 15 June 2018 (2018-06-15), pages 1813 - 1825, XP055647922, DOI: 10.1002/jbmr.3524 *
T. KARAYANNIS ET AL.: "Cntnap4/Caspr4 Differentially Contributes to GABAergic and Dopaminergic Synaptic Transmission", NATURE, vol. 511, no. 7508, 25 May 2014 (2014-05-25), pages 236 - 240, XP055647912 *
WANG ET AL.: "NEL-Like Molecule-1 (NeIl1) Is Regulated by Bone Morphogenetic Protein 9 (BMP9) and Potentiates BMP9-Induced Osteogenic Differentiation at the Expense of Adipogenesis in Mesenchymal Stem Cells", CELLULAR PHYSIOLOGY AND BIOCHEMISTRY, vol. 41, no. 2, 30 January 2017 (2017-01-30), pages 484 - 500, XP055647915 *
YIN ET AL.: "Caspr4 Interaction with LNX2 Modulates the Proliferation and Neuronal Differentiation of Mouse Neural Progenitor Cells", STEM CELLS AND DEVELOPMENT, vol. 24, no. 5, 3 October 2014 (2014-10-03), pages 640 - 652, XP055647918, DOI: 10.1089/scd.2014.0261 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111920800A (en) * 2020-08-26 2020-11-13 中国人民解放军海军军医大学第一附属医院 Application of oroxylin A in treating osteoporosis and promoting fracture healing
CN111920800B (en) * 2020-08-26 2023-06-13 中国人民解放军海军军医大学第一附属医院 Application of oroxylin A in treating osteoporosis and promoting fracture healing
CN112870361A (en) * 2021-01-28 2021-06-01 重庆医科大学 Use of iron death inhibitor for preparing medicine for preventing or treating osteoporosis or bone loss caused by iron overload
CN112915207A (en) * 2021-03-25 2021-06-08 上海市第六人民医院 Application of LNX2 gene deletion in treatment of osteoporosis

Similar Documents

Publication Publication Date Title
US11773171B2 (en) WNT surrogate molecules and uses thereof
AU2020210134B2 (en) Use of myostatin inhibitors and combination therapies
Pirruccello-Straub et al. Blocking extracellular activation of myostatin as a strategy for treating muscle wasting
EP2732823B1 (en) Modulation of the TrpV : Vps10p-domain receptor system for the treatment of pain
RU2760875C1 (en) Antibodies specific to hyperphosphorylated tau-protein and their application methods
JP6066912B2 (en) DKK1 antibody and method of use thereof
JP2024001292A (en) Multispecific Wnt alternative molecules and their uses
CN102316888A (en) Synaptogenic regulation and control
WO2019210080A1 (en) Methods and compositions for skeletal and neurological disorders
JP2015133984A (en) Compositions and methods for inhibiting cripto/grp78 complex formation and signaling
KR20220131223A (en) How to use a DKK-1 inhibitor to treat cancer
Ishihara et al. Blocking insulin-like growth factor 1 receptor signaling pathway inhibits neuromuscular junction regeneration after botulinum toxin-A treatment
Nevo et al. Fibrosis inhibition and muscle histopathology improvement in laminin‐α2‐deficient mice
JP2024079697A (en) Transferrin receptor 2 antagonists and agonists for use in treating bone disorders
US11548942B2 (en) Methods of treating a wasting syndrome, increasing growth hormone levels, and increasing GHSR activity with a LEAP2 antibody
US20090068102A1 (en) Treating stroke
US20240352102A1 (en) Compositions for treating tauopathies and methods of use thereof
Marchbank Dual Roles for NBR1 in Bone Remodelling and Autophagosomal Protein Degradation
HK40097101A (en) Use of myostatin inhibitors and combination therapies
WO2012167032A1 (en) Compositions and methods for treating pain
Nyman Targeting Transforming Growth Factor Beta to Enhance the Fracture Resistance of Bone
US20170369578A1 (en) Regeneration of aged satellite cells
Sullivan Elucidating the disease relevance and function of the ALS/FTLD-associated protein C9orf72
EA039569B1 (en) Antibodies specific for hyperphosphorylated tau and methods of use thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19791463

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19791463

Country of ref document: EP

Kind code of ref document: A1