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WO2019210080A1 - Méthodes et compositions pour troubles du squelette et neurologiques - Google Patents

Méthodes et compositions pour troubles du squelette et neurologiques Download PDF

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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
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WIPO (PCT)
Prior art keywords
nell
cntnap4
antibody
disorder
antigen
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Ceased
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English (en)
Inventor
B. Chia Soo
Kang Ting
Zhong Zheng
Chenshuang LI
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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    • 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
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    • 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.

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Abstract

La présente invention concerne une composition pour traiter un trouble, comprenant une quantité efficace d'un agent efficace pour potentialiser une liaison efficace du sang NELL-1 au sang Cntnap4 chez un sujet mammifère qui en a besoin pour obtenir un niveau normal de liaison du sang NELL-1 au sang Cntnap4 chez le sujet mammifère, le sujet mammifère présentant un niveau anormal de liaison du sang NELL-1 au sang Cntnap4. La présente invention concerne également des procédés de fabrication et d'utilisation de la composition. L'invention concerne également un procédé de diagnostic d'un trouble neurologique ou d'un trouble du squelette. L'invention concerne également des procédés et des compositions pour traiter ou réduire les symptômes d'un trouble, tel qu'un trouble neurologique ou un trouble osseux, en particulier l'autisme et l'ostéoporose, respectivement.
PCT/US2019/029161 2018-04-25 2019-04-25 Méthodes et compositions pour troubles du squelette et neurologiques Ceased WO2019210080A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111920800A (zh) * 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 一种千层纸素a在治疗骨质疏松和促进骨折愈合中的应用
CN111920800B (zh) * 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 一种千层纸素a在治疗骨质疏松和促进骨折愈合中的应用
CN112870361A (zh) * 2021-01-28 2021-06-01 重庆医科大学 铁死亡抑制剂在制备预防或治疗铁过载导致的骨质疏松或骨丢失的药物中的应用
CN112915207A (zh) * 2021-03-25 2021-06-08 上海市第六人民医院 Lnx2基因的缺失在治疗骨质疏松中的应用

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