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WO1993009798A1 - Therapeutic and diagnostic methods based on tissue specific nt-3 expression and receptor binding - Google Patents

Therapeutic and diagnostic methods based on tissue specific nt-3 expression and receptor binding Download PDF

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Publication number
WO1993009798A1
WO1993009798A1 PCT/US1992/009652 US9209652W WO9309798A1 WO 1993009798 A1 WO1993009798 A1 WO 1993009798A1 US 9209652 W US9209652 W US 9209652W WO 9309798 A1 WO9309798 A1 WO 9309798A1
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Prior art keywords
disease
nervous system
system cells
binding
disorder
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French (fr)
Inventor
C. Anthony Altar
Peter Distefano
Stanley Wiegand
Nancy Ip
Carolyn Hyman
Roseann Ventimiglia
Ronald Lindsay
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Regeneron Pharmaceuticals Inc
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Regeneron Pharmaceuticals Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • 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
    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators

Definitions

  • the present invention relates to
  • neurotrophin-3 (NT-3), a member of the NGF/BDNF/NT-3/NT-4 gene family and to therapeutic and diagnostic methods utilizing neurotrophin-3 in the treatment of neurological disorders.
  • a family of neurotrophic factors has been identified that includes ⁇ -nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF),
  • Neurotrophin-3 neurotrophin-3
  • HDNF hippocampus-derived neurotrophic factor
  • NT-4 neurotrophin-4
  • Nerve growth factor (NGF) is by far the best characterized neurotrophic factor (Levi-Montalcini and Angeletti, 1968, Physiol. Rev. 48:534-569; Thoenen and Barde, 1980, Physiol. Rev. 60:1284-13335).
  • mice submaxillary gland is a rich source of NGF allowed for the purification and amino acid sequence analysis of mouse NGF (Angelitti, et al., 1973, Biochemistry 12:100-115) and DNA sequence analysis of mouse and human NGF (Scott, et al., 1983, Nature 302: 538-540; Ullrich, et al., 1983, Nature 303:821-825).
  • Comparison of mouse and human NGF showed that the protein is conserved within animals and in support of this, NGF-like activities have been isolated from several species (Harper and Thoenen, 1981, Ann. Rev. Pharmacol. Toxicol. 21:205- 229).
  • BDNF Brain-derived neurotrophic factor
  • NT-3 which bears structural similarity to both NGF and BDNF, has been isolated from mouse (Hohn, et al., 1990, Nature, 344:339-341), rat (Maisonpierre, et al., 1990, Science 247: 1446-1451; Ernfors, et al., 1990, Proc. Natl. Acad. Sci. USA 87: 5454-5458), and human (Rosenthal, et al., 1990, Neuron 4:767-773; PCT Application No.
  • hippocampus (Ayer-LeLievre, et al., 1988, Science 240: 1339-1341; Ernfors, et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 5454-5458; Ernfors, et al., 1990, Neuron 5: 511-526; Whetmore, et al., 1991, Neurol. 109: 141-152; Hofer, et al., 1990 EMBO J., 9:2459-2464,
  • NGF, BDNF and NT-3 support the growth of both overlapping and unique sets of neuronal growth factor
  • NGF supports the development and maintenance of peripheral sympathetic and neural crest-derived sensory neurons (reviewed in Thoenen and Barde, 1980, Physiol. Rev., 60:1284-1325; Levi-Montalcini, 1987,
  • BDNF has been observed to support the survival of both placode and neural crest derived sensory neurons (Hofer and Barde, 1988,
  • NGF Basal forebrain cholinergic neurons
  • the effects of the three proteins are mediated by their interaction with specific receptors present on sensitive cells.
  • the trkA, trkB and trkC members of tyrosine protein kinases have been
  • the present invention relates to methods for the diagnosis and treatment of neurological disorders involving target cells for NT-3. Patterns of NT-3 expression in vivo and the distribution of tissue specific displaceable receptor binding sites for NT-3 in mammals are described.
  • the invention is based, in part, on the discovery that high NT-3 receptor binding densities exist in mammalian brain sections in the anterior nucleus of the olfactory bulb, layer 1 of neocortex, the nucleus of the lateral olfactory tract, the dentate gyrus, CA1, CA3 and CA4 of the hippocampus, and the caudate-putamen.
  • high NT-3 receptor binding densities exist in mammalian brain sections in the anterior nucleus of the olfactory bulb, layer 1 of neocortex, the nucleus of the lateral olfactory tract, the dentate gyrus, CA1, CA3 and CA4 of the hippocampus, and the caudate-putamen.
  • the invention is further based on the discovery that mRNA encoding the NT-3 receptor, trkC, is expressed in the striatum. Pathological conditions involving such regions which display high levels of NT-3 expression and/or moderate to high density NT-3 receptor may be diagnosed or treated in accordance with the invention.
  • the striatum is an area of the brain involved in a number of neurological disorders, including Huntington's chorea, in particular
  • the present invention provides for methods of diagnosing and treating disorders of the striatum that utilize NT-3, fragments of NT-3, NT-3 derivatives, or antibodies that bind to NT-3 or NT-3 receptors.
  • the invention is still further based on the discovery that NT-3 is retrogradely transported by certain sensory neurons as well as neurons of the hippocampus and neostriatum. Therefore, the present invention, in particular embodiments, provides for methods of diagnosing and treating disorders of sensory neurons, the hippocampus, and the neostriatum that utilize NT-3, fragments of NT-3, NT-3
  • NT- 3, fragments of NT-3, NT-3 derivatives, or antibodies that bind specifically to NT-3 receptors may be labeled and utilized in an imaging protocol to locate cells and tissues in the body that express the NT-3 receptor in vivo.
  • Anti-NT-3 antibodies may likewise be utilized to image native NT-3 bound to the surface of such target cells. Abberrant expression patterns imaged using such techniques may be analyzed to diagnose pathological conditions.
  • NT-3 compositions or anti-NT-3 antibodies may be used to potentiate or block the biological effect of NT3 on target cells and tissues in vivo.
  • Specific diseases that may be diagnosed and/or treated in accordance with the invention are described herein.
  • FIGURE 1 Amounts of [ 125 I]NT-3 bound to horizontal rat brain sections during association and dissociation binding assays. Each value is the mean ⁇ s.e.m. fmol of NT-3 bound per mg protein calculated from the dpm per section from each 4 animals.
  • n 4
  • FIGURE 4 Coronal and horizontal rat brain sections were incubated in 300 pM [ 125 I]NT-3 alone or with 300 nM NT-3 to define non-displaceable binding in adjacent sections. Note the high levels of displaceable binding in the dentate gyrus, CA pyramidal layers of hippocampus, caudate-putamen, and superficial neocortex.
  • FIGURE 5 Coronal adult cat brain sections were incubated in 300 pM [ 125 I]NT-3 alone or with 300 nM
  • NT-3 to define non-displaceable binding in adjacent sections. Note the similarity of binding as observed in the rat shown in Figure 4.
  • FIGURE 6 Horizontal sections from two adult humans were collected through the basal ganglia and incubated in [ 125 I]NT-3 alone or with 300 nM NT-3 as in Figures 4 and 5. Intense and highly displaceable labeling was present in the caudate nucleus, putamen, neocortex, and claustrum.
  • FIGURE 7 Northern blot analysis of
  • trkA 2.6 Kb Xho1 Fragment
  • trk B 1.1 Kb Hpa/Sca
  • FIGURE 8 Northern blot analysis depicting the time-course effect of NGF, BDNF and NT-3 on the induction of C-FOS.
  • C-FOS 1.1 Kb Pst1 Fragment;
  • GAPD4 1.25 Kb Pst1 Fragment.
  • FIGURE 9 Northern blot analysis of trkB in rat brain.
  • the predominant band (arrow, approximately 7.0 to 9.0 Kb) was measured by densitometric scanning, as shown in Figure 11.
  • FIGURE 10 Northern blot analysis of trkC mRNA expression in rat brain. The predominant band (arrow, approximately 15 Kb) was measured by
  • FIGURE 11 Quantitation of the predominant trkB transcript in developing rat striatum.
  • FIGURE 12 Quantitation of the predominant trkC transcript in developing rat striatum.
  • FIGURE 15 Dose-dependent induction of C-FOS expression in dissociated cerebellum cultures in vitro by neurotrophin-3.
  • FIGURE 16 Dose-dependent induction of C-FOS expression in dissociated cerebellum cultures in vitro by brain-derived neurotrophic factor.
  • FIGURE 17 Dose-dependent induction of
  • FIGURE 18 Effect of NT-3 on dopaminergic neuron survival in vitro. Cultures of E14 ventral mesencephalon were prepared as described previously (Hyman, et al., 1991, Nature 350:230-233). Following a four hour attachment period, the culture medium was switched to a serum-free formulation at which time purified human recombinant NT-3 was added to the indicated concentrations. The cultures were maintained for 7 days in vitro, then they were
  • FIGURE 19 Effect of NT-3 on dopamine uptake in vitro. Cultures were prepared as in
  • FIG. 18 Purified human recombinant NT-3 was added at the time of the culture media switch as described in Figure 18. Each concentration of NT-3 was tested in sets of five replicate dishes. Cultures were maintained for 7 days in vitro, at which time they were processed for the measurement of high affinity dopamine uptake.
  • FIGURE 20 Effect of NT-3 on GABA uptake in vitro. Cultures were prepared as in Figure 18.
  • NT-3 Purified human recombinant NT-3 was added to the cultures at the time of the media switch as described. Each concentration of NT-3 was tested in sets of five replicate dishes. Cultures were maintained in vitro for 7 days, then they were processed for the
  • FIGURE 21 Effect of NT-3 on GAD activity in vitro. Cultures were prepared as in Figure 18.
  • NT-3 Purified human, recombinant NT-3 was added at the time of the media switch as described. Each concentration of NT-3 was tested in sets of five replicate dishes. Cultures were maintained for 7 days in vitro, and were then processed for measurement of GAD activity.
  • FIGURE 22 Effect of neurotrophin concentration on choline acetyl transferase (CAT) activity in E17 cultures of rat septal cholinergic neurons.
  • CAT choline acetyl transferase
  • FIGURE 23 Retrograde transport of [ 125 I]NT-3 from the right sciatic nerve of rats. NT-3 was
  • FIGURE 24 Retrograde transport of NT-3, BDNF and NGF from the right sciatic nerve of control and pyridoxine treated rats.
  • FIGURE 25 Transport of NT-3 and BDNF in the L4-L5 spinal cord segment of control and
  • the present invention provides for the use of biologically active NT-3 molecules in diagnostic and therapeutic regimens for detecting and treating neurological disorders involving target cells for NT-3.
  • the methods of the invention also provide for inhibition or neutralization of NT-3 activity using anti-NT-3 antibodies, antisense RNA or ribozymes.
  • NT-3 derivatives of NT-3, including biologically active fragments thereof, or antibodies that bind to NT-3 receptors, including anti-idiotypic antibodies that mimic NT-3, may be used to image neuronal cells that express the NT-3 receptor. Abberant patterns of receptor expression may be analyzed to diagnose pathological-conditions.
  • the NT-3 compositions or antibodies that bind to NT-3 receptors may be conjugated to a radiolabel, radiopaque label, fluor, enzyme, etc. Such conjugates may be used
  • Imaging protocols including but not limited to CAT scans, x-rays, fluorograms, MRI or PET scans, etc. to analyze the tissue distribution of NT-3 receptor in vivo.
  • Anti-NT-3 antibodies may similarly be used in such imaging protocols to image native NT-3 bound to the surface of such target cells.
  • conjugates may also be used to identify NT-3 receptors in biopsy or autopsy specimens.
  • NT-3, or NT-3 derivatives, including biologically active fragments thereof may be administered to potentiate its biological effect on a target tissue.
  • NT-3 gene products derived from the same species for therapeutic or diagnostic purposes, although cross-species NT-3 may be useful in certain specific embodiments of the invention.
  • Neurological disorders in which stimulation of NT-3 responsive target cells corrects the disorder may be treated in this manner.
  • neutralizing anti-NT-3 antibodies may be utilized to block the effects of native NT-3 on target cells.
  • antisense RNA or ribozymes may be used to inhibit or block the production of native NT-3 or peptide antagonists of NT-3 may be utilized.
  • Neurological disorders involving the over- proliferation of cells that express the NT-3 receptor and respond to NT-3 may be treated in this manner.
  • such protocols may be used to treat neurological tumors that express the NT-3 receptor and proliferate in response to NT-3.
  • a number of disorders of neurological tissue which express NT-3 and/or the NT-3 receptor may be treated in accordance with the invention. Where peripheral nerves are involved, a clinical test may be performed to determine whether the condition improves in response to the application of NT-3 or the
  • the efficacy of the treatment may be first assessed in an appropriate animal model system.
  • disorders involving neurological tissues that express NT-3 and/or the NT-3 receptor which may be treated in accordance with the invention include but are not limited to:
  • disorders of olfaction and taste such as anosmia and dysgeusia
  • disease affecting the neocortex including but not limited to Alzheimer's disease and clinical defects produced by stroke as well as predominantly cortical dementing illnesses, including but not limited to Pick's disease, cortical-basal ganglionic degeneration, diffuse cortical Lewy body disease and Jacob-Creuzfeldt disease and including disorders involving the superficial layer of the neocortex caused by lesions or processes which compress or invade the brain from its external surface, including but not limited to consequences of brain trauma as exemplified by a laceration or contusion, subdural hematoma, epidural hematoma and tumor metastases;
  • the NT-3 utilized in accordance with the invention may be prepared by recombinant DNA
  • NT-3 may be prepared from eukaryotic cells that express recombinant NT-3 as follows.
  • Conditioned medium may be collected from a bioreactor (Charles River) seeded with eukaryotic cells expressing NT-3 and diluted with distilled water at about a 1.25:1 ratio and then may be continuously loaded at 4°C onto a 15 ml (1.6 cm i.d.) S-Sepharose
  • 10.6L (diluted) may be applied in a period of about five days.
  • the column may be washed with 50 ml of 20 mM MES buffer (a solution of 4-morpholinethane-sulfonic acid, the pH of which has been adjusted to about 5.8 with NaOH) followed by 50 ml of the same buffer containing 250 mM sodium chloride followed by the same MES buffer without sodium chloride until absorbance at 280 nm reaches baseline level.
  • the column may then be washed with about 25 ml of 20 mM bicine buffer at pH 8.5 followed by 15 ml of the same buffer containing 100 mM sodium chloride.
  • the column may subsequently be eluted with 150 ml, 100 mM to 750 mM sodium chloride gradient in 20 mM bicine buffer (pH 8.5).
  • the flow rate may be desireably about 2.5 ml/min.
  • Absorbance of the eluate may be monitored at 280 nm at a sensitivity of 0.2 absorbance unit full scale.
  • Fractions containing recombinant NT-3 may be identified by dorsal root ganglia (DRG) explant bioassay.
  • DRG dorsal root ganglia
  • the concentrated sample may then be applied onto a 120 ml (1.6 cm i.d.) Sephacryl S-100HR gel filtration column (Pharmacia) packed and equilibrated with 20 mM HEPES Buffer pH 7.6 containing 400 mM NaCl.
  • the column may be eluted at about 0.25 ml/min. 2 ml size fractions may be collected and assayed for NT-3 activity in the DRG explant bioassay.
  • the present invention also provides for fragments of NT-3 which are either antigenic or functionally active.
  • Functionally active fragments include fragments of NT-3 that are NT-3 agonists as well as fragments that are NT-3 antagonists. If it is desireable to supplement NT-3 activity, an agonist fragment may be used. If it is desireable to inhibit NT-3 activity, an antagonist fragment may be used.
  • Agonist fragments preferably comprise at least two of the three disulfide bridges found in the native NT-3 molecule and/or are capable of achieving at least about thirty percent, and preferably at least about fifty percent, of the activity of intact NT-3 measured under the same conditions, e.g. in a DRG assay.
  • NT-3 activity refers to an antibody that decreases the activity of native NT-3 in the DRG assay, described infra, by at least about 30 percent.
  • Any of the antibodies used in accordance with the invention include but are not limited to polyclonal, monoclonal, chimeric, single chain, and Fab fragments.
  • an animal host may be immunized using the appropriate immunogen formulated in an adjuvant to increase the immune response.
  • adjuvants include but are not limited to Freund's (complete and incomplete), mineral gels, such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhold limpit hemocyanin, dinitrophenol, BCG (bacille Calmette-Guerin) and
  • Monoclonal antibodies may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in
  • Antibody fragments may also be generated from whole antibody molecules using known techniques such as pepsin digestion and reduction of disulfide bonds.
  • the active ingredient which may comprise NT-3, a biologically active derivative or fragment thereof, or an antibody or antibody fragment prepared as described above should be formulated in a suitable pharmaceutical carrier for administration in vivo by any appropriate route including but not limited to injection (e.g., intravenous, intraperitoneal,
  • epithelial or mucocutaneous linings e .g. , oral mucosa, rectal and intestinal mucosa, etc.
  • sustained release implant including a cellular or tissue implant.
  • the active ingredient may be formulated in a liquid carrier such as saline, incorporated into liposomes, microcapsules, polymer or wax-based and controlled release preparations, or formulated into tablet, pill or capsule forms.
  • a liquid carrier such as saline
  • the concentration of the active ingredient used in the formulation will depend upon the effective dose required and the mode of administration used.
  • the dose used should be sufficient to achieve
  • circulating plasma concentrations of active ingredient that are efficacious.
  • a circulating serum concentration level ranging from about 1 ng/ml to 10 ⁇ g/ml may be used; preferably ranging from about 1 ng/ml to
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • NT-3 receptors are distributed in a tissue- specific fashion. Neurological disorders involving pathologies of such tissues are candidates for diagnosis and/or treatment in accordance with the invention. Diseases or disorders involving nervous system cells (including neural cells as well as supporting cells, etc.) that are responsive to NT-3 are the targets of the methods of the invention. For example, high densities of NT-3 receptor occur in the anterior nucleus of the olfactory bulb, layer 1 of the neocortex, the nucleus of the lateral olfactory tract, the dentate gyrus, CA1, CA3, and CA4 of the
  • NT-3 receptor Moderate to low densities of the NT-3 receptor occur in the neocortex nucleus accumbens, spinal gray, basolateral amygdala, interpedunucular nucleus, superior colliculus, medial septum and cerebellum. Disorders involving such regions may be diagnosed or treated using the methods of the invention.
  • NT-3 an NT-3 derivative
  • NT-3 including a biologically active agonist NT-3 fragment, or an anti-idiotypic antibody which mimics the binding and biological effects of NT-3.
  • Such treatment will stimulate the growth or differentiation or survival of the target cells .
  • Neurological disorders involving the over-proliferation of cells that express the NT-3 receptor and which proliferate in response to NT-3, or which achieve an undesireable phenotype as a result of exposure to NT-3 may be treated by inhibiting the effect or production of native NT-3. This may be accomplished by administering an effective dose of neutralizing anti-NT-3 antibody to inhibit the
  • NT-3 biological effect of NT-3; or by delivery of anti-sense RNA or ribozymes to the cellular source of NT-3 to inhibit the production of native NT-3; or by administration of an NT-3 antagonist.
  • NT-3 responsive tissues have been damaged or are in the process of degeneration
  • treatment which augments NT-3 activity may be
  • NT-3 responsive tissue has been damaged may be made by any method known in the art. For example, if neuroimaging techniques, such as CAT scan, MRI, PET scan, etc.
  • damage to a structure which exhibits binding to NT-3 for example, as set forth in Table 2, infra, treatment to augment NT-3 activity may be initiated. But damage to such a structure may also be ascertained by other techniques, including, but not limited to, biopsy or on the basis of clinical examination or history, EEG, EMG, etc.
  • a diminuition in NT-3 activity may be desireable in conditions that involve either
  • Such conditions may include tumors, for example, in which NT-3 may be employed in an autocrine loop, or in the epilepsies or certain disorders of the striatum that are associated with production of excessive involuntary movement.
  • tumors it may be possible to identify an increase in NT-3 receptors, endogenous NT-3, or responsiveness to NT-3 using histologic and/or cell culture techniques; if such a condition is determined, diminuition of NT-3 activity may be desireable.
  • NT-3 molecules or possibly anti-NT-3 antibodies may assist in the treatment of such disorders as anosmia (the absence of smell) or dysgeusia (the distortion of normal taste).
  • anosmia the absence of smell
  • dysgeusia the distortion of normal taste
  • anosmia that may be treated by therapeutic applications of .NT-3 include, but are not limited to, head trauma (which may be caused by severing of the neurons crossing through the cribriform plate as well as complications associated with the normal aging process), multiple sclerosis, Parkinson's disease or a frontal lobe tumor.
  • dysgeusia that may be treated by therapeutic applications of NT-3 include, but are not limited to, Bell's palsy, familial dysautonomia and multiple sclerosis as well as distortions of taste that arise on the basis of olfactory dysfunction.
  • NT-3 expression and receptor binding within the lateral, intermediate and medial portions of the superficial entorhinal cortex, dentate gyrus, the granule cell layer of the dentate gyrus hilus as well as the CA1, CA3 and CA4 pyramidal layers of the hippocampus.
  • the expression and binding of NT-3 within these distinct tissues of the brain indicate therapeutic applications which include but are not limited to, memory deficits resulting from global cerebral ischemia which occurs in survivors of cardiac arrest.
  • therapeutic applications of biologically active NT-3 molecules may be utilized in the treatment of
  • Alzheimer's disease This region of the brain is involved early in the pathology of the disease and there is speculation (Saper and German 1987,
  • Alzheimer's disease may initiate in this region of the brain, spread through the cortex and hippocampus, and retrogradely affect the basal forebrain and other brainstem nuclei.
  • hippocampal tissue (a) expresses both trkB and trkC, (b) NT-3 induces FOS expression of both trkB and trkC, (c) NT-3 induces FOS expression and
  • neorofilament protein produces and increase in AChE - positive and calbindin - immunopositive cells. Since patients diagnosed with Parkinson's disease, Huntington's disease and Alzheimer's disease show a marked decrease in both mRNA transcripts and protein for calbindin (lacopino and Christakos, 1990, Proc. Natl. Acad. Sci. USA 87: 4078-4082) treatment with NT-3 may be used to increase expression of calbindin in such patients. 5.4.3. THE NEOCORTEX AND SUPERFICIAL
  • the examples described infra also demonstrate NT-3 expression and binding within the neocortex and superficial layers of the neocortex.
  • Disorders involving the neocortex which occur in a number of diseases may be treated including but not limited to Alzheimer's disease stroke victims and predominantly cortical dementing illnesses, including but not limited to, Pick's disease, cortical-basal ganglionic degeneration, diffuse cortical lewy body disease and Jacob-Creutzfeld disease.
  • the pathology of human victims, in contrast to other animal models, is that predominately all layers of the cortex are affected, with the regional distribution being determined by the territory of the blocked cerebral vessel. In the area surrounding the infarction there is a zone sometimes referred to as the "ischemic penumbra" in which the blood-brain barrier is
  • NT-3 or derivative or NT-3 peptide fragment may work to minimize the clinical deficits produced by a stroke.
  • superficial layers of neocortex might be lesions or processes which compress or invade the brain from its external surface, including, but not limited to:
  • brain trauma such as laceration or contusion, subdural hematoma, epidural hematoma and tumor metastases.
  • NT-3 expression and receptor binding within the caudate-putamen.
  • Therapeutic applications of NT-3 to the caudate-putamen may be utilized to treat, by way of example and not by limitation,
  • choreoathetic dystonia syndromes due to perinatal hypoxia and striatonigral degeneration and progressive supranuclear palsy.
  • Huntington's disease is an untreatable, progressive, dominantly inherited disease with 100% penetrance. Huntington's disease characteristically presents itself in early midlife, after the
  • the pathology of Huntington's disease consists principally of degeneration of the so-called "medium spiny" neurons of the caudate nucleus and putamen.
  • Degeneration is noted in other areas of the nervous system as well, including the frontal cortex, thalamus and the deep cerebellar nuclei. These cell losses result in decreases in the amounts of the
  • Symptoms include behavioral deterioration
  • striatum ⁇ an area demonstrated herein to express high levels of NT-3 transcripts and high density NT-3 receptors ⁇ treatment with NT-3 or a biologically active derivative or fragment thereof may be used in accordance with the invention to treat Huntington's disease.
  • Chorea-acanthocytosis is a hereditary choreatic syndrome of young adults who lack the severe intellectual deterioration or behavioral disturbances of Huntington's disease. Some patients have epileptic seizures. There is neuronal cell loss in the caudate nucleus and the putamen. The disease is distinguished by its autosomal recessive inheritance, muscle
  • the present invention also involves therapeutic applications for the treatment of
  • the basal ganglia is a principal target of perinatal "hypoxic-ischemic injury resulting in the neurological impairments of "cerebral palsy".
  • cerebral palsy There are three principal clinical variants of cerebral palsy: spastic hemiplegia, spastic diplegia and
  • Therapeutic applications of the invention also include treatment of striatonigral degeneration and progressive supranuclear palsy (Steele-Richardson- Olszewski Syndrome), which are so-called “Parkinsonplus syndromes” with clinical features of Parkinson's disease which do not respond to conventional
  • NT-3 promotes neurite survival and outgrowth in the sensory neurons of dorsal root ganglion cultures.
  • In vitro cultures enriched in lumbar or cervical ganglia are more responsive to NT-3 in relation to in vitro
  • JNT-3 may act preferentially on large diameter, large fiber proprioceptive neurons such as those found in the lumbar and cervical ganglia. Therefore, NT-3 may be useful in treating peripheral neuropathies affecting these larger sensory neurons which occur in patients with diabetes or patients undergoing chemotherapy for cancer or AIDS treatment (e.g., taxol, vincristine, cisplatin, di-deoxyinosine).
  • DIAGNOSTIC APPLICATIONS e.g., taxol, vincristine, cisplatin, di-deoxyinosine.
  • a number of in vitro or in vivo diagnostic tests which involve assaying NT-3 or the NT-3 receptor may be utilized to assist in diagnosis and the
  • NT-3 may be assayed in various biopsy or autopsy tissues by methods known in the art.
  • immunoassays may be used to detect expression of the NT-3 protein.
  • assays include but are not limited to ELISA,
  • nucleotide probes that are complementary to the NT-3 sequence may be used in RNA hybridization methods in situ to detect expression of the NT-3 mRNA
  • transcripts in the biopsy or autopsy sample may include the use of PCR (polymerase chain reaction). Tissue concentrations of NT-3 protein or mRNA transcripts may be correlated with disease.
  • the NT-3 receptor may be assayed in biopsy or autopsy samples or in vivo.
  • NT-3 or NT-3 derivatives may be modified, for detection, e.g., by labeling with a radioisotope, a radioopaque compound, a fluor, an enzyme etc.
  • conjugates may be used for imaging the NT-3 receptor in biopsy or autopsy samples in vitro or for imaging in vivo.
  • antibodies that mimic NT-3 and/or bind to the NT-3 receptor may similarly be used to image the NT-3 receptor distribution in biopsy or autopsy samples in vitro or in vivo. Imaging techniques well known in the art can be used to such ends, e.g., CAT SCAN, X-ray, etc. Aberrations in the distribution of receptor may be correlated with disease.
  • NT-3 preferentially accumulates in the lumbar and cervical ganglia thus providing a method for diagnosis of sensory neuron disorders.
  • a biopsy sample of such neurons of an afflicted patient may be utilized to determine the level of NT-3 protein and to compare to the level of NT-3 from an analogous sample from a normal individual.
  • An aberrancy in the level of NT-3 may correlate with the presence of a sensory neuron disorder.
  • the actual determination of the level of NT-3 protein may be carried out by a method comprising contacting the sample with an anti-NT-3 antibody such that immunospecific binding can occur.
  • the present invention provides for a method of diagnosing NT-3 related peripheral nervous system disorders comprising
  • NT-3 responsiveness to NT-3 and indicates the presence of a peripheral nervous system disorder that is NT-3 related.
  • the present invention also provides for a method to diagnose a central nervous system disorder.
  • Evaluation of retrograde transport may be performed by any method known in the art, including but not limited to MRI, CAT, or scintillation scanning as discussed infra. Such methods may be used to identify the location of a nervous system lesion, as retrograde transport should substantially diminish upon reaching the lesion.
  • kits for such retrograde evaluation comprising in a container a detectably labeled NT-3 protein, derivative or
  • Such a label can be a radioactive isotope, or other label known in the art.
  • the present invention also provides for a method of treating sensory neuron disorders comprising administering, to a patient in need of such treatment, an effective amount of an NT-3 protein, derivative or peptide fragment capable of supporting the survival, growth and/or differentiation of motor neurons as demonstrated in an in vitro culture system.
  • effective amounts of neurotrophic factor may desirably be determined on a case by case basis, as sensory neurons from different tissue sources or from different species may exhibit different sensitivities to neurotrophic factor.
  • CAT choline acetyltransferase
  • NT-3 was produced by the preferred method described supra in Section 5.1.
  • the NT-3 aliquots were monitored for protein content by amino acid analysis and for biological activity using the dorsal root ganglia (DRG) explant culture system using muNGF as a standard.
  • DRG dorsal root ganglia
  • the NT-3 was iodinated by the
  • the [ 125 I]NT-3 was labeled to a specific activity of 2800-4400 cpm/fmol (1872-2876 Ci/mmole of NT-3) and stored at a concentration of 80-100 nM at 2-8°C. [ 125 I]NT-3 was used within 1-5 days in all studies to avoid the observed increase in non-displaceable [ 125 I]NT-3 binding that occurred after this time.
  • the brain of an adult male cat was sectioned coronally from the frontal cortex to the medulla and sections from 22 levels were collected as described for the rat. Twenty ⁇ m thick sections of a human brain were collected through the basal ganglia and included the caudate, putamen, neocortex, and adjacent fiber bundles.
  • Binding assays were conducted according to the procedure of Richardson, et al. (1986, Neurosci. 20:23-36) and with modifications for dry film
  • each section was preincubated for 1 hour at 22oC in 100 mM phosphate-buffered saline, pH 7.4, containing 0.5 mM MgCl 2 and 0.5 mM PMSF. Equivalent binding was observed for sections that were preincubated for 1, 3, or 24 hours.
  • Sections were then incubated in DMEM tissue culture medium containing high glucose, 10% heat-inactivated fetal calf serum (70° C for 0.5 hours), 25 mM HEPES buffer, 4 ⁇ g/ml leupeptin, 0.5 mM PMSF, (BRL,
  • adjacent brain sections were incubated in the same solutions with the addition of 300 nM NT-3 to define displaceable binding.
  • a range of concentrations of muNGF, hCNTF, and BDNF were each used to compete with [ 125 I]NT-3 binding in several experiments.
  • the sections were washed for 0.5 hr in the phosphate buffer. In the absence of excess unlabelled NT-3, equivalent amounts of total and non-displaceable binding were obtained with 3 minutes, 10 minutes and 2 hour washes with unlabeled buffer.
  • association experiment association experiment
  • dissociation experiment dissociation experiment
  • K d and B max values were calculated by equilibrium saturation analysis with each of four brains according to the best fit to a parabola by iterative, nonlinear regression analysis (Rodbard and Lewald, 1970, Acta Endocrinol. 147:79-103).
  • IC 50 values were calculated by the method of Bliss and
  • binding sites in horizontal and coronal sections of rat brain This binding was displaced by 70-90% by a 1000-fold greater concentration of NT-3 whereas 10-15% higher levels of non-specific binding were obtained at higher ligand concentrations. Because binding was particularly robust and displaceable in the caudate-putamen, neocortex, and hippocampus, horizontal sections that contained these areas were used for subsequent quantitative studies with
  • neocortex (anterior nucleus of the olfactory bulb, nucleus of the lateral olfactory tract), hippocampus (dentate gyrus, CA1, 3, and 4), caudate-putamen, and the neocortex (Table 2).
  • the first layer of the neocortex was prominently labeled in the frontal, parietal, and cingulate regions. Layer 1 was also heavily labeled throughout the entorhinal cortex.
  • nucleus accumbens Intermediate levels of binding were present in nucleus accumbens, basolateral amygdala, interpeduncular nucleus, ventral and dorsal horns of spinal cord, and superior colliculus. Lower amounts of specific binding were present in the lateral geniculate, medial septum, and cerebell ⁇ m. No displaceable binding was detected in white fiber buncles including the corpus callosum, anterior commissure, internal capsule, interbulbar internal capsule, or in the globus pallidus, most thalamic nuclei, hypothalamus, or in other regions of the pons, medulla, or other amygdaloid nuclei. No
  • [ 125 I]NT-3 binding sites were found in circumventricular organs such as the choroid plexus or ependymal cell layers. Specific binding was also absent in the liver, muscle, kidney, pancreas, and heart. TABLE 2
  • NT-3 binding were evaluated from visual evaluation of autoradiographs generated with 300 pM [125I]NT-3.
  • the non-specific binding defined with 300 nm NT-3 appeared uniform throughout the brain sections and accounted for only 10-20% of the total amount of bound ligand. This allowed the relative amounts of specific binding to be estimated from the total binding images. 6.3. DISCUSSION
  • heterogeneous brain binding site is nonetheless phylogenetically conserved.
  • These maps illustrate the brain regions in which endogenous NT-3 may function as a neurotrophic factor and also provide the basis for both in vitro and in vivo studies to assess the neuronal specificities and therapeutic targets of NT-3.
  • the displaceable binding of [ 125 I]NT-3 ranged from 60-90% of the total binding.
  • topography of the 10-40% non-displaceable binding probably represents NT-3 receptor-independent
  • the capacity of high and low affinity NT-3 binding sites were 26 and 170 fmol/mg protein
  • the high affinity site is at least twice as dense as that for rhNGF (Altar, et al., Proc. Natl. Acad. Sci. USA 88:281-285; Altar, et al., 1991 J. Neurosci. 11:828-836).
  • the high affinity binding site for [ 125 I]BDNF appears to be even more numerous and more ubiquitously localized than those for NGF or NT-3.
  • cholinergic areas include cranial nerve nuclei III and IV, the pedunculopontine or parabrachial cholinergic projections to thalamus, hypothalamus, and inferior colliculus, and the projection of preoptic
  • piriform cortex may contribute to the relatively intense NT-3 binding observed in these areas.
  • neurotransmitter systems in piriform and entorhinal cortex that project to hippocampus may contain these NT-3 binding sites.
  • NGF receptors are relatively low in the, adult rat cerebellum and other areas in which NGF binding sites and receptor immunostaining are quite dense.
  • the granule cell layer of cerebellum is also the most densely labeled brain area following in situ
  • trkC antisense probe hybridization with trkC antisense probe.
  • the strong, residual trkC signal generated with trkC sense cRNA probe (Lamballe, et al., 1991, Cell 66:967-979) or a relatively diminished translation of trkC message into functional receptor are two possible explanations for the presence of trkC antisense message but not NT-3 binding in the cerebellar granule cell layer.
  • trkC expression is also very pronounced in hippocampus, neocortex, and neostriatum (Lamballe et al., 1991, Cell 66: 967-979) and these areas were most intensely labeled with [ 125 I]NT-3.
  • trkC mRNA and [ 125 I]NT-3 binding will ascertain the resemblance of these two markers.
  • Hippocampi were dissected from E16-E18, or
  • E20 rat embryos of Sprague-Dawley rats and collected in F10 medium.
  • the tissues were minced, rinsed twice with F10 medium (Gibco) and trypsinized with 0.25% trypsin (Gibco) for 20 minutes at 37°C. Trypsin was inactivated by the addition of a serum-containing medium composed of minimum essential medium (MEM) supplemented with fetal calf serum (FCS, 10%), glutamine (2 mM), penicillin (25 U/ml) and
  • MEM minimum essential medium
  • FCS fetal calf serum
  • FCS fetal calf serum
  • penicillin 25 U/ml
  • polyornithine-laminin (10 ⁇ g/ml) in DME plus 10% fetal calf serum. After 4 hours of culture, the medium was changed to DME plus 1 mg/ml BSA and N2 media
  • BDNF and NT-3 Approximately 40% of the hippocampal cells showed a fos response to BDNF and NT-3. The effects of BDNF and NT-3 on fos induction were not additive. All of the cells that showed the fos response to BDNF and NT-3 were neurons. Double staining with fos and calbindin showed that calbindin-immunopositive cells were among the cell population that responded with fos induction.
  • BDNF and NT-3 were found to induce an increase (50%) in the amount of neurofilament protein. They also produced an increase in the number of AChE-positive and calbindin-immunopositive cells. In contrast, NGF had no apparent effect. Dose response studies of BDNF and NT-3 on the number of calbindin-immunopositive cells showed that the response
  • NT-3 produced a 20-fold increase in the number of calbindin-positive cells which was accompanied by an increase in calbindin-mRNA levels. Delaying the addition of NT-3 to the cultures for 4 days did not appear to affect the increase in the number of calbindin-positive cells, suggesting that NT-3 acts to induce the calbindin-phenotype instead of acting as a survival factor. Developmental profiles of the increase in calbindin-positive cells produced by BDNF and NT-3 were compared. The effect of NT-3 was much more striking for hippocampal neurons earlier in development (E16-E18) and declined later in development (E20), while the reciprocal effect was observed for BDNF.
  • the only cell populations that appeared to respond to both BDNF and NT-3 thus include AChE positive and calbindin positive cells.
  • Other cell populations examined which did not appear to respond to BDNF and NT-3 in terms of survival include GABAergic cells, glutamatergic cells and somatostatin-positive cells. Decreases of up to 60-80% in the levels of both the message and protein for calbindin were observed in the hippocampus of patients who were diagnosed as having Parkinson's, Huntington's or Alzheimer's disease (lacopino and Christakos, 1990, Proc. Natl. Acad. Sci. USA 87:4078-4082).
  • GABAergic cells GABAergic cells
  • glutamatergic cells glutamatergic cells
  • somatostatin-positive cells Decreases of up to 60-80% in the levels of both the message and protein for calbindin were observed in the hippocampus of patients who were diagnosed as having Parkinson's, Huntington's or Alzheimer's disease (lacopino and Christa
  • calbindin protein in the hippocampal neurons may have significant physiological implication in that it may be important in preventing cell death in select populations of hippocampal neurons.
  • Striatal neuronal cultures were prepared from E17 rat brains as follows: striatal tissue was minced in calcium- and magnesium-free Hank's balanced salt solution and dissociated by enzymatic treatment with 0.25% trypsin and DNAase (0.2 mg/ml) followed by mechanical trituration. Dissociated cells were seeded at a density of 8 ⁇ 10 6 cells on 100 mm dishes which had been previously coated with polylysine and
  • Striatal and hippocampal astrocyte cultures were prepared from P1 rat brains as follows: tissues were minced and enzymatically dissociated by treatment 0.25% trypsin and DNAase 0.2 mg/ml). Following a 5 minute centrifugation at low speed, and an
  • the cell suspension was dissociated mechanically by trituration.
  • Cells were then passed through a Nitex filter cartridge and seeded at a density of 71,000 cells/cm 2 in T75 tissue culture flasks in DME-FCS.
  • Immunocytochemical staining was used to determine the purity of the astrocyte and neuronal cultures.
  • Antibodies to glial fibrillary acidic protein (specific to astrocytes) and neurofilament protein (specific to neurons) were used in combination with other cell-type-specific markers to delineate the cellular composition of the cultures.
  • Total RNA was prepared from striatal RNA
  • neuronal cultures at 4 DIV
  • striatal astrocyte cultures at 28 DIV
  • RNA samples were prepared from striatal or whole brain tissue samples by extraction in 0.3M LiCl/6M urea followed by phenol/chloroform extraction (Auffray and Rougeon, 1980, Eur. J. Biochem 107:303-314).
  • RNAs (10 ug/lane) were fractionated by electrophoresis through 1% agarose-formaldehyde gels (Bothwell et al., 1990, in: Methods for Cloning and Analysis of Eukaryotic Genes, pp. 42-43, ed., Jones and Bartlett, Boston) followed by capillary transfer to nylon membranes. Probes to trkB (1.1 kb, spanning the intracytoplasmic tyrosine kinase domain) and trkC (800 bp, spanning the
  • intracytoplasmic tyrosine kinase domain were labeled by random hexamer labeling with 32 P dCTP (Stratagene Prime It).
  • Membranes were hybridized overnight in 0.5 sodium phosphate buffer, pH 7.9 , containing 1 percent bovine serum albumin, 7 percent SDS and 100 ⁇ g/ml sonicated salmon sperm at 65°C. Filers were rinsed briefly in 2 ⁇ SSC and 0.1 percent SDS and then washed twice in 1 ⁇ SSC and 0.1 percent SDS and exposed to
  • TrkC mRNA expression was detected in rat striatum as early as E17 ( Figure 10). Expression of trkC mRNA reaches a peak between P7 and P20, where a 1.5-1.9 fold increase in transcript level is detected relative to whole adult rat brain ( Figure 12).
  • trkC mRNA is expressed in cultured striatal neurons (4 DIV) but not in striatal or hippocampal astrocytes (28 DIV).
  • BDNF and NT-3 may act as trophic factors in the striatum.
  • Chick dorsal root ganglia When cultured either as explants or dissociated neuron-enriched cultures, sensory neurons of the chick dorsal root ganglia (DRG) are responsive to a greater or lesser degree to NGF, BDNF, NT-3, NT-4 and CNTF. Chick embryo DRG neurons die within 24 hours of being placed in culture in the absence of any neurotrophic factor. Each of the above factors can support the survival and outgrowth of neurites of some of these neurons, ranging from 10-60%, depending on the factor, stage of neuronal development etc. It appears that each of the above neurotrophic factors has both distinct and overlapping specificities towards sup-populations of DRG neurons, although there is no clear evidence as to which sub-types of sensory neurons are supported by which neurotrophic factor. This example defines the comparative specificity of NT-3, NGF and BDNF.
  • DRG were collected from chick embryos at developmental stages ranging from E6 - E10. Starting in the sacral region ganglia from right and left sides were collected in pairs along the entire neural axis. 5-6 ganglia at, each level or pools of sacral, lumbar, thoracic or cervical ganglia were explanted in
  • Ganglia were cultured in F14 medium + 5% horse serum in the presence or absence of 5 ng/ml, NGF, BDNF or NT-3. After 24 hours the. extent of fiber outgrowth was measured on an arbitrary scale of 0 to +5, 0 being virtually no fibers,. +5 being a profuse halo of fibers (the saturating level seen with NGF, the most potent of the factors in this assay). 9.1.2. DISSOCIATED NEURON-ENRICHED CULTURES
  • Lumbar and thoracic ganglia from E8 embryos were collected separately, dissociated with 0.25% trypsin and freed of non-neuronal cells (Lindsay et al, 1985, Develop. Biol. 112, 319-328).
  • Purified neurons were seeded on a substrate of polyornithine- laminin at 8,000 neurons per 35 mm dish. Cells were cultured in the presence of F14 medium containing 5% horse serum. A dose response ranging from 1 pg to 10 ng of either NGF, BDNF or NT-3 was carried out. After 48 hours the number of process bearing neurons was determined in triplicate cultures (Lindsay et al., 1985, Develop. Biol, 12: 319-328). 9.2. RESULTS AND DISCUSSION
  • NGF supported the survival of a similar percentage of either lumbar or thoracic DRG neurons (40-50%). Although BDNF was less effective than NGF, the effects of BNDF were essentially the same towards lumbar or thoracic neurons - supporting survival of around 30% of the neurons. In contrast, NT-3
  • the DRG of the lumbar and cervical enlargements contain more large-diameter, large fiber proprioceptive sensory neurons than either sacral or thoracic ganglia.
  • NT-3 has selective activity on large-diameter, large fiber DRG neurons.
  • FOS IMMUNOHISTOCHEMISTRY Cells were treated for 30-300 minutes with BDNF, NT-3, or NGF at a concentration of 0.1-1 ng/ml at the end of the culture period. Cells were then rinsed once in BME and prefixed for 10 min in 2% paraformaldehyde in BME before fixation at room temperature in 4% paraformaldehyde in PBS for 30 min. After permeabilization with 0.1% triton for 10 min and blocking in 10% normal goat serum (NGS) and 1% BSA for 90 min, the cells were incubated for 2 days at 4°C with anti-FOS antibody (Oncogene) diluted 1/2000 in PBS with 5% NGS.
  • NGS normal goat serum
  • DAB hydrochloride
  • This example discloses the efficacy of neurotrophin 3 in supporting the survival or
  • E14 rat brain The preparation of cultures from E14 rat brain was carried out as described by Hyman, et al., 1991, Nature 350: 230-233. Briefly, all cultures were prepared from the ventral mesencephalon dissected from 14-day-old embryonic rats (E14). Typically, pooled tissue from two or three litters of rat embryos from timed-mated Sprague Dawley rats was trypsinized
  • the single-cell suspension was seeded onto 35-mm dishes (precoated with polyornithine and laminin) containing growth medium to give a density of 5 ⁇ 10 4 cells cm -2 .
  • MEM medium supplemented with glutamine (2 mM), glucose (6 mg/ml -1 ), penicillin G (0.5 U/ml -1 ), streptomycin (5 ⁇ g/ml -1 ) and FCS (7.5%) to allow cell attachment
  • cells were cultured in the presence or absence of BDNF in a serum-free, defined medium (Hyman et al., 1991, Nature 350:230-232) except that insulin was included at 20 ng/ml -1 .
  • To visualize dopaminergic cells cultures were fixed with 4% paraformaldehyde, washed extensively, permeabilized with 0.02% saponin in Sorensen's buffer with 1.5% horse serum and stained with a mouse monoclonal
  • GAD enzyme activity was determined according to the method of Kimura and Kuriyama (1975, Jpn J. Pharm. 25:189-195) by measuring the release of 14 CO 2 from L-[1- 14 C] glutamic acid. Cells on 35 mm dishes were lysed with 30 ⁇ l of a solution containing 50 mM KH 2 PO 4 (pH 7.2) and 0.25% Triton X-100, scraped and collected. Five microliters of the cell lysate was assayed for GAD enzyme activity.
  • the reaction mixture contained 0.57 mM of L-[1- 14 C] glutamic acid (NEN, NEC-715, 52.6 mCi/mmol), glutamic acid (3 mM), pyridoxal phosphate (0.2 mM) and AET (1 mM), in a KH 2 PO 4 buffer (50 mM, pH 7.2). Under these reaction conditions, the enzyme reaction was found to be linear for up to 2.5 hours.
  • Each treatment group had five 35 mm dishes, 3 of which were marked with a (-) and two with a (+). All culture dishes were washed once with incubation buffer and once with warm buffer containing +/- BZT and DABA as appropriate. To each culture dish was added 0.8 ml of +/- BZT and DABA buffer prior to incubation at 37°C for 5 minutes; followed by the addition of 0.2 ml of 3 H-DA (final concentration of 50 nM) and 14 C-GABA (final concentration of 500nM). Uptake of 3 H-DA + 14 C-GABA was allowed to proceed at 37°C for 15 minutes. The culture dishes were then placed on ice and the incubation solution was
  • the cultures were washed three times with an incubation buffer having the following composition: 100 ⁇ M pargyline, 1 mM ascorbate, 10 ⁇ M aminooxyacetic acid and 2 mM-beta-alanine. Cultures were then preincubated for 5 minutes at 37oC in incubation buffer; replicate cultures were preincubated in buffer additionally containing 5 ⁇ M benztropine (BZT) and 1 mM 2,4 diamino-n-butyric acid (DABA). 50 nM 3 H-DA and 500 nM 14 C-GABA were then added for 15 minutes at 37°C. The uptake of label was stopped by placing the dishes on a bed of ice and rinsing with ice coId buffer. The samples were solubilized with 0.5 N NaOH and 3 H and 14 C was measured by liquid scintillation counting. Specific uptake was defined as the following composition: 100 ⁇ M pargyline, 1 mM ascorbate, 10 ⁇ M aminooxyace
  • Dopaminerigic markers were also analyzed via immunocytochemical staining for tyrosine hydroxylase (Hyman, et al., 1991, Nature 350: 230-233).
  • a dose response test of NT-3 was carried out to determine if cells cultured in the presence of this factor would show an increased survival of
  • NT-3 induced a maximal effect of a 2.8 fold increase in 3 H-GABA uptake activity when included in the culture medium at a concentration of 20 ng/ml for a period of 7 days in vitro (Figure 20).
  • the results show similar fold effects in assays conducted to examine independent phenotypic marker activities, indicating that the NT-3 acts as a survival-promoting activity for these neurons.
  • the results show similar fold effects in assays conducted to examine independent phenotypic marker activities, indicating that the NT-3 acts as a survival-promoting activity for these neurons.
  • NT-3 may stimulate the GABA uptake activity of the cells (a measure of their metabolic activity) independently of exerting its effect on the GAD activity.
  • the septal region from Sprague-Dawley rats after 17 days of gestation was dissected free from the surrounding tissue. Tissue fragments were pooled, washed three times with Hams F-10, and then
  • tissue culture dish transferred to a 35mm tissue culture dish and minced.
  • a single cell suspension was made by incubating the tissue with 0.25% trypsin for 20 minutes at 37°C.
  • the cells were dissociated by passing the fragments repeatedly through the constricted tip of a Pasteur pipet. The dissociated cells were then centrifuged at 500 ⁇ g for 45 seconds.
  • the cells were plated into 6 mm wells which had been coated with polyornithine (10 ⁇ g/ml) and laminin (10 ⁇ g/ml). The cell viability was checked, after 24 hours in culture, by the ability of the cells to exclude trypan blue.
  • the normal growth medium, 5HS/N3, for cultures composed of neurons and glia contained:
  • the growth medium was removed from the cultures by rinsing the cells twice with 100 ⁇ l of PBS.
  • the cells were lysed via one freeze-thaw cycle and a 15 minute incubation at 37°C in 50 mM KH 2 PO 4 pH 6.7, containing 200 mM NaCl and 0.25% (v/v) Triton X-100.
  • Two microliters of the cell lysate was removed and assayed for CAT activity according to the microFonnum procedure (Fonnum, 1975, J. Neurochem. 24:407- 409).
  • the final substrate composition consisted of 0.2 mM [ 14 C] Acetyl-CoA (NEN, 54.4, mCi/mmol), 300 mM NaCl, 8 mM choline bromide, 20 mM
  • acetyltransferase was tested by the addition of a specific inhibitor of CAT activity, N-hydroxyethyl-4- (1-napthylvinyl)pyridium (HNP), during the assay
  • Neurotrophin-3 slightly increased the level of CAT activity after a 7-day treatment period at a concentration of 100 ng/ml (Figure 22).
  • NGF nerve growth factor
  • Quantitative data are expressed as cpm in L4 plus L5 DRG and compared to L4, L5 cpm in the non-injected side.
  • [ 125 I]NT-3 was shown to be retrogradely transported from the crushed adult rat sciatic nerve to the L4, L5 DRG ( Figure 23).
  • the transport was specific in that a 100-fold excess of unlabeled NT-3 was able to block 80-90% of the transport.
  • rats were anesthetized with chloral hydrate (170 mg/kg) mixed with pentobarbital (35.2 mg/kg).
  • the right sciatic nerve was exposed and 2 ⁇ l of 125 I-labeled NT-3 or BDNF or NGF was injected into the nerve at the level of the obturator internus tendon. Wounds were sutured and the rats allowed to recover for 18 hr. At this time rats were killed by decapitation and the lumbar 4th (L4) and 5th dorsal root ganglia (DRG) removed and placed in 4%
  • L4-L5 spinal cord segment was also dissected and counted in fixative. Samples were counted in a gamma counter for 1 minute and the counts per minute for L4 plus L5 DRGs were assessed.
  • Figure 24 shows that all three labeled neurotrophins were transported to the ipsilateral (right) but not contralateral (left) DRG when injected into the right sciatic nerves of control rats.
  • Figure 24 also shows that [ 125 -I]NT-3 and [ 125 I]BDNF transport in the DRG were reduced 71% and 60%, respectively, in pyridoxine treated rats (p ⁇ 0.01). No significant change was observed for 125 I-NGF in pyridoxine treated rats. Interestingly, the transport of NT-3 and BDNF was not significantly altered in the spinal cords of pyridoxine treated rats compared to controls
  • Neurotrophin-3 was radioiodinated as described supra in Example Section 14.1.2.
  • NT-3 is retrogradely transported from the hippocampus to the neurons of the medial septum/diagonal band, although proportionately many fewer counts were transported for NT-3 than NGF.
  • Film and emulsion autoradiographic experiments (Table 5) showed that labeling associated with both neurotrophic factors was well localized to magnocellular neurons of the medial septum and diagonal band, cells which are known to provide the cholinergic input to hippocampus.
  • Magnocellular neurons were very densely labeled and quite numerous in NGF-injected animals compared to animals injected with NT-3. However, NT-3 was found also to be transported by a population of smaller neurons within the medial septum/diagonal band.
  • NT-3 was also transported by large
  • NT-3 were immunoreactive for tyrosine hydroxylase, a marker for the dopaminergic neurons of the pars compacta.
  • NT-3 was transported to several additional sites in the rat forebrain. As is the case for NGF, we have demonstrated transport of radiolabelled NT-3 to the supramammillary nucleus, but here the pattern of labeling is more intense than is apparent for NGF. Additionally, NT-3 was transported to a number of brain regions which do not appear to transport NGF at all. After injection into the dentate gyrus of the hippocampus, a few labeled cells were present bilaterally within the CA4/hilus
  • HPC dorsal hippocampus
  • MS/OB medial septum/diagonal band of Broca
  • V Mes
  • ventral mesencephalon r
  • right side l
  • left side All injections (striatum and hippocampus) were made on the right side of the brain.
  • Numbers with T prefix in the trophic factor column are animal code numbers. All other numbers represent cpm in the brain area indicated.

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Abstract

The present invention provides for therapeutic and diagnostic method based on human NT-3 expression, specifically the potential to treat Alzheimer's disease, Huntington's disease as well as disorders or diseases of the peripheral nervous system. The present invention also provides for the survival-promoting activity of NT-3 on dopaminergic neuronal populations. The present invention additionally provides for therapeutic and diagnostic applications dependent on the ability of NT-3 to be retrogradely transported in both the central and peripheral nervous system.

Description

THERAPEUTIC AND DIAGNOSTIC METHODS BASED ON
TISSUE SPECIFIC NT-3 EXPRESSION AND RECEPTOR BINDING
1. INTRODUCTION
The present invention relates to
neurotrophin-3 (NT-3), a member of the NGF/BDNF/NT-3/NT-4 gene family and to therapeutic and diagnostic methods utilizing neurotrophin-3 in the treatment of neurological disorders.
2. BACKGROUND OF THE INVENTION Neurotrophic factors play an important role in both the development and maintenance of the
vertebrate nervous system, where they support neuronal survival and differentiation. Widespread neuronal cell death accompanies normal development of the central and peripheral nervous systems, and apparently plays a crucial role in regulating the number of neurons which project to a given target field (Berg, D.K., 1982, Neuronal Development 297-331; Cowan et al., 1984, Science 225:1258-1265). Ablation and transplantation studies of peripheral target tissue during development have shown that neuronal cell death results from competition among neurons for limiting amounts of neurotrophic factors produced in their projection fields.
A family of neurotrophic factors has been identified that includes β-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF),
neurotrophin-3 (NT-3; also known as hippocampus-derived neurotrophic factor, HDNF) and neurotrophin-4 (NT-4). Nerve growth factor (NGF) is by far the best characterized neurotrophic factor (Levi-Montalcini and Angeletti, 1968, Physiol. Rev. 48:534-569; Thoenen and Barde, 1980, Physiol. Rev. 60:1284-13335). The discovery that the male mouse submaxillary gland is a rich source of NGF allowed for the purification and amino acid sequence analysis of mouse NGF (Angelitti, et al., 1973, Biochemistry 12:100-115) and DNA sequence analysis of mouse and human NGF (Scott, et al., 1983, Nature 302: 538-540; Ullrich, et al., 1983, Nature 303:821-825). Comparison of mouse and human NGF showed that the protein is conserved within animals and in support of this, NGF-like activities have been isolated from several species (Harper and Thoenen, 1981, Ann. Rev. Pharmacol. Toxicol. 21:205- 229). Subsequently, DNA sequences from bull (Meier, et al., 1986, EMBO J. 5: 1489-1493); chick (Meir, et al., 1986, EMBO J. 5: 1489-1493; Ebendal, et al., 1986, EMBO J. 5: 1483-1487; Wion, et al., 1986, FEBS Letters 203:83-86); cobra (Selby, et al., 1987, J. Neurosci. Res. 18:293-298); rat (Whittemore, et al., 1988, J. Neurosci. Res. 20:403-410); and guinea pig (Schwartz, et al., 1989, Neurochem. 52:1203-1209) NGFs were determined.
Brain-derived neurotrophic factor (BDNF) was first isolated from pig brain (Barde, et al., 1982, EMBO J. 1:549-553) and subsequently cloned as a cDNA from this tissue (Leibrock, et al., 1989 Nature
341:149-152; PCT application number PCT/US90/04915, filed August 29, 1990).
The gene for NT-3, which bears structural similarity to both NGF and BDNF, has been isolated from mouse (Hohn, et al., 1990, Nature, 344:339-341), rat (Maisonpierre, et al., 1990, Science 247: 1446-1451; Ernfors, et al., 1990, Proc. Natl. Acad. Sci. USA 87: 5454-5458), and human (Rosenthal, et al., 1990, Neuron 4:767-773; PCT Application No.
PCT/US90/04916, filed August 29, 1990, Publication No. W091/03569; Maisonpierre et al., 1991, Genomics
10:558-568). The three factors show approximately 55% amino acid similarity to each other, and most sequence differences are present in five regions that contain amino acid motifs characteristic of each protein.
All three factors are expressed in specific sets of neurons in the brain; the highest levels of mRNA for all three factors was observed in the
hippocampus (Ayer-LeLievre, et al., 1988, Science 240: 1339-1341; Ernfors, et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 5454-5458; Ernfors, et al., 1990, Neuron 5: 511-526; Whetmore, et al., 1991, Neurol. 109: 141-152; Hofer, et al., 1990 EMBO J., 9:2459-2464,
Phillips et al., 1990, Science 250:290-294). The localization of NT-3 mRNA by Northern blot analysis or in situ hybridization reveals a heterogenous
distribution, with high levels in heart, spleen, skin, gut, brain, lung, liver, and muscle (Hohn, et al, 1990, Nature 344: 339-341, Ernfors, et al, 1990, Neuron 5:511-526; Maisonpierre, et al., 1990, Neuron 5 : 501-509; Maisonpierre, et al., 1990, Science 247:1446-1451).
NGF, BDNF and NT-3 support the growth of both overlapping and unique sets of neuronal
populations as measured in vitro in explanted chick ganglia or dissociated neuronal cultures. (Hohn, et al., 1990, Nature, 344: 339-341; Maisonpeirre, et al., 1990, Science, 247: 1446-1451; Ernfors, et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 5454-5458; Rosenthal, et al., 1990, Neuron 4: 767-773).
NGF supports the development and maintenance of peripheral sympathetic and neural crest-derived sensory neurons (reviewed in Thoenen and Barde, 1980, Physiol. Rev., 60:1284-1325; Levi-Montalcini, 1987,
Science, 237:1154-1162). BDNF has been observed to support the survival of both placode and neural crest derived sensory neurons (Hofer and Barde, 1988,
Nature, 331:261-262). In the brain, NGF has been shown to support basal forebrain cholinergic neurons (reviewed in Whittemore and Seiger, 1987, Brain Res., 434:439-464; Thoenen, et al., 1987, Rev. Physiol.
Biochem. Pharmacol., 105:145-178: Ebendal, 1989, Prog. Growth Factor Res. 1: 143-159) and BDNF has been shown to stimulate the survival of these neurons in vitro (Alderson, et al., 1990, Neuron 5: 297-306). NT-3 has been observed to support the growth and survival of certain subsets of central and peripheral neurons (Maisonpierre, et al., 1990, Neuron 5: 501-509;
Maisonpierre, et al., 1990, Science 247:1446-1451;
Rosenthal, et al., 1990, Neuron 4:767-773; Hohn, et al., 1990, Nature 344:339-341).
The effects of the three proteins are mediated by their interaction with specific receptors present on sensitive cells. The trkA, trkB and trkC members of tyrosine protein kinases have been
identified as receptors for NGF, BDNF and NT-3
respectively (trkA: Kaplan, et al., 1991, Nature
350:158-160; Klein, et al., 1991, Cell 65:189-197); (trkB: Klein, et al., 1989, EMBO J. 8:3701-3709;
Squinto, et al.. Cell 65:885-893); and (trkC:
Lamballe, et al., 1991, Cell 66: 967-979). The existence of trk protein kinases that serve
preferentially as high affinity receptors for NGF, BDNF, and NT-3, respectively, indicate unique but as yet unknown brain distributions of high affinity binding sites for these three neurotrophic factors. Although [125I]NT-3 binding to trkc-transfected 3T3 fibroblasts is highly specific and associates to these cells with high and low affinities (Lamballe, et al., 1991, Cell 66: 967-979) no information has been
available, prior to the present invention, concerning [125I]NT-3 binding sites in brain or other mammalian tissues.
3. SUMMARY OF THE INVENTION
The present invention relates to methods for the diagnosis and treatment of neurological disorders involving target cells for NT-3. Patterns of NT-3 expression in vivo and the distribution of tissue specific displaceable receptor binding sites for NT-3 in mammals are described.
The invention is based, in part, on the discovery that high NT-3 receptor binding densities exist in mammalian brain sections in the anterior nucleus of the olfactory bulb, layer 1 of neocortex, the nucleus of the lateral olfactory tract, the dentate gyrus, CA1, CA3 and CA4 of the hippocampus, and the caudate-putamen. Moderate to low NT-3
receptor binding densities exist in the neocortex nucleus accumbens, spinal gray, basolateral amygdala, interpeduncular nucleus, superior colliculus, medial septum and cerebellum. The invention is further based on the discovery that mRNA encoding the NT-3 receptor, trkC, is expressed in the striatum. Pathological conditions involving such regions which display high levels of NT-3 expression and/or moderate to high density NT-3 receptor may be diagnosed or treated in accordance with the invention.
Because the striatum is an area of the brain involved in a number of neurological disorders, including Huntington's chorea, in particular
embodiments, the present invention provides for methods of diagnosing and treating disorders of the striatum that utilize NT-3, fragments of NT-3, NT-3 derivatives, or antibodies that bind to NT-3 or NT-3 receptors. The invention is still further based on the discovery that NT-3 is retrogradely transported by certain sensory neurons as well as neurons of the hippocampus and neostriatum. Therefore, the present invention, in particular embodiments, provides for methods of diagnosing and treating disorders of sensory neurons, the hippocampus, and the neostriatum that utilize NT-3, fragments of NT-3, NT-3
derivatives, or antibodies that bind to NT-3 or NT-3 receptors.
In diagnostic applications, for example, NT- 3, fragments of NT-3, NT-3 derivatives, or antibodies that bind specifically to NT-3 receptors, may be labeled and utilized in an imaging protocol to locate cells and tissues in the body that express the NT-3 receptor in vivo. Anti-NT-3 antibodies may likewise be utilized to image native NT-3 bound to the surface of such target cells. Abberrant expression patterns imaged using such techniques may be analyzed to diagnose pathological conditions.
For therapeutic applications, NT-3 compositions or anti-NT-3 antibodies may be used to potentiate or block the biological effect of NT3 on target cells and tissues in vivo. Specific diseases that may be diagnosed and/or treated in accordance with the invention are described herein.
4. DESCRIPTION OF THE FIGURES
FIGURE 1. Amounts of [125I]NT-3 bound to horizontal rat brain sections during association and dissociation binding assays. Each value is the mean ± s.e.m. fmol of NT-3 bound per mg protein calculated from the dpm per section from each 4 animals.
(Top) Association of 200 pM [125I]NT-3 at seven
incubation times. Three hundred nM NT-3 defined non- displaceable binding and sections were washed for one hr following incubation. NSB = non-specific binding; SB = specific binding. (Bottom) After a 3.5 hr equilibrium association with 200 pM [125I]NT-3, the labeled sections were rinsed for 5 seconds and placed in 70 ml PBS that contained 200 nM NT-3.
FIGURE 2. (Left) Equilibrium association of total, non-specific, and specific [125I]NT-3 binding to horizontal brain sections (n = 4 sections/point).
(Right) Bound versus B/F plot of the specific binding data in the adjacent figure. The two populations of specific [125I]NT-3 binding sites are represented by the two linear functions. Affinity and capacity values for these separate curves and the complete curve are given in Table 1.
FIGURE 3. Inhibition of [125I]NT-3 binding to horizontal brain sections by NT-3 (IC50 = 420+ 60 pM; nH = 1.2 ± 0.26) and BDNF (IC50 = 230 ± 100 pM for 10-1000 pM range of BDNF; nH = 0.76 ± 0.18; IC50 = 37 ± 2.9 nM for 0.2 - 100 nM range of BDNF) but only by murine NGF or CNTF (IC50 > 100 nM). n = 4
brains/point; sem = ± 5-20% at each concentration.
FIGURE 4. Coronal and horizontal rat brain sections were incubated in 300 pM [125I]NT-3 alone or with 300 nM NT-3 to define non-displaceable binding in adjacent sections. Note the high levels of displaceable binding in the dentate gyrus, CA pyramidal layers of hippocampus, caudate-putamen, and superficial neocortex.
FIGURE 5. Coronal adult cat brain sections were incubated in 300 pM [125I]NT-3 alone or with 300 nM
NT-3 to define non-displaceable binding in adjacent sections. Note the similarity of binding as observed in the rat shown in Figure 4. FIGURE 6. Horizontal sections from two adult humans were collected through the basal ganglia and incubated in [125I]NT-3 alone or with 300 nM NT-3 as in Figures 4 and 5. Intense and highly displaceable labeling was present in the caudate nucleus, putamen, neocortex, and claustrum.
FIGURE 7. Northern blot analysis of
hippocampus neurons (lane A), hippocampus astrocytes (lane B) and adult brain (Lane C) utilizing 32P-labeled probes specific for (A) trkA; (B) trkB; (C) trkC. trk A = 2.6 Kb Xho1 Fragment; trk B = 1.1 Kb Hpa/Sca
Fragment; trk C = 800 bp Eco RI Fragment.
FIGURE 8. Northern blot analysis depicting the time-course effect of NGF, BDNF and NT-3 on the induction of C-FOS. C-FOS = 1.1 Kb Pst1 Fragment;
GAPD4 = 1.25 Kb Pst1 Fragment.
FIGURE 9. Northern blot analysis of trkB in rat brain. The predominant band (arrow, approximately 7.0 to 9.0 Kb) was measured by densitometric scanning, as shown in Figure 11.
FIGURE 10. Northern blot analysis of trkC mRNA expression in rat brain. The predominant band (arrow, approximately 15 Kb) was measured by
densitometric scanning, as shown in Figure 11.
FIGURE 11. Quantitation of the predominant trkB transcript in developing rat striatum.
Densitometric scanning of a one week exposure of a Northern blot was used to obtain data. Levels are standardized relative to whole adult rat brain.
FIGURE 12. Quantitation of the predominant trkC transcript in developing rat striatum.
Densitometric scanning of a one week exposure of a Northern blot was used to obtain data. Levels are standardized relative to whole adult rat brain. FIGURE 13. Bar graphs showing the maximal fiber outgrowth response of E6, E8 and E10 chick embryo to NGF, BDNF and NT-3, as determined from full dose response curves (not shown). Open bars = NGF; Cross-hatched bars = BDNF; Solid Bars = NT-3. Each bar is the mean +/- the s.e.m of 5 or more ganglia at each level scored at 24h. S, L, T, and C are sacral, lumbar, thoracic and cervical ganglia respectively. NGF was purified mouse salivary gland NGF; BDNF and NT-3 were both recombinant human proteins produced and purified to homogeneity by Amgen Inc., (obtained from Dr. James Miller).
FIGURE 14. Dose-response curves indicating the survival of lumbar or thoracic DRG neurons from E8 chick embryos cultured for 48 hours with NGF, BDNF or NT-3. Horizontal axis = concentration of neurotrophic factor (ng/ml); vertical axis = % neurite bearing cells.
FIGURE 15. Dose-dependent induction of C-FOS expression in dissociated cerebellum cultures in vitro by neurotrophin-3.
FIGURE 16. Dose-dependent induction of C-FOS expression in dissociated cerebellum cultures in vitro by brain-derived neurotrophic factor.
FIGURE 17. Dose-dependent induction of
C-FOS expression in dissociated cerebellum cultures in vitro by nerve growth factor.
FIGURE 18. Effect of NT-3 on dopaminergic neuron survival in vitro. Cultures of E14 ventral mesencephalon were prepared as described previously (Hyman, et al., 1991, Nature 350:230-233). Following a four hour attachment period, the culture medium was switched to a serum-free formulation at which time purified human recombinant NT-3 was added to the indicated concentrations. The cultures were maintained for 7 days in vitro, then they were
processed for TH immunocytochemistry. Each
concentration of NT-3 was tested in triplicate dishes.
FIGURE 19. Effect of NT-3 on dopamine uptake in vitro. Cultures were prepared as in
Figure 18. Purified human recombinant NT-3 was added at the time of the culture media switch as described in Figure 18. Each concentration of NT-3 was tested in sets of five replicate dishes. Cultures were maintained for 7 days in vitro, at which time they were processed for the measurement of high affinity dopamine uptake.
FIGURE 20. Effect of NT-3 on GABA uptake in vitro. Cultures were prepared as in Figure 18.
Purified human recombinant NT-3 was added to the cultures at the time of the media switch as described. Each concentration of NT-3 was tested in sets of five replicate dishes. Cultures were maintained in vitro for 7 days, then they were processed for the
measurement of high affinity GABA uptake.
FIGURE 21. Effect of NT-3 on GAD activity in vitro. Cultures were prepared as in Figure 18.
Purified human, recombinant NT-3 was added at the time of the media switch as described. Each concentration of NT-3 was tested in sets of five replicate dishes. Cultures were maintained for 7 days in vitro, and were then processed for measurement of GAD activity.
FIGURE 22. Effect of neurotrophin concentration on choline acetyl transferase (CAT) activity in E17 cultures of rat septal cholinergic neurons.
FIGURE 23. Retrograde transport of [125I]NT-3 from the right sciatic nerve of rats. NT-3 was
injected alone or was co-injected with either NGF or BDNF. FIGURE 24. Retrograde transport of NT-3, BDNF and NGF from the right sciatic nerve of control and pyridoxine treated rats.
FIGURE 25. Transport of NT-3 and BDNF in the L4-L5 spinal cord segment of control and
pyridoxine treated rats.
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention provides for the use of biologically active NT-3 molecules in diagnostic and therapeutic regimens for detecting and treating neurological disorders involving target cells for NT-3. The methods of the invention also provide for inhibition or neutralization of NT-3 activity using anti-NT-3 antibodies, antisense RNA or ribozymes.
For diagnostic applications, NT-3, derivatives of NT-3, including biologically active fragments thereof, or antibodies that bind to NT-3 receptors, including anti-idiotypic antibodies that mimic NT-3, may be used to image neuronal cells that express the NT-3 receptor. Abberant patterns of receptor expression may be analyzed to diagnose pathological-conditions. To this end, the NT-3 compositions or antibodies that bind to NT-3 receptors may be conjugated to a radiolabel, radiopaque label, fluor, enzyme, etc. Such conjugates may be used
in vivo in appropriate imaging protocols, including but not limited to CAT scans, x-rays, fluorograms, MRI or PET scans, etc. to analyze the tissue distribution of NT-3 receptor in vivo. Anti-NT-3 antibodies may similarly be used in such imaging protocols to image native NT-3 bound to the surface of such target cells.
These conjugates may also be used to identify NT-3 receptors in biopsy or autopsy specimens. For therapeutic applications, NT-3, or NT-3 derivatives, including biologically active fragments thereof, may be administered to potentiate its biological effect on a target tissue. Generally, it is preferable to use NT-3 gene products derived from the same species for therapeutic or diagnostic purposes, although cross-species NT-3 may be useful in certain specific embodiments of the invention.
Neurological disorders in which stimulation of NT-3 responsive target cells corrects the disorder may be treated in this manner. Where inhibition of NT-3 is desired, neutralizing anti-NT-3 antibodies may be utilized to block the effects of native NT-3 on target cells. Alternatively, antisense RNA or ribozymes may be used to inhibit or block the production of native NT-3 or peptide antagonists of NT-3 may be utilized. Neurological disorders involving the over- proliferation of cells that express the NT-3 receptor and respond to NT-3 may be treated in this manner.
For example, such protocols may be used to treat neurological tumors that express the NT-3 receptor and proliferate in response to NT-3.
A number of disorders of neurological tissue which express NT-3 and/or the NT-3 receptor may be treated in accordance with the invention. Where peripheral nerves are involved, a clinical test may be performed to determine whether the condition improves in response to the application of NT-3 or the
inhibition of NT-3. Where the central nervous system or brain is involved, the efficacy of the treatment may be first assessed in an appropriate animal model system.
Disorders involving neurological tissues that express NT-3 and/or the NT-3 receptor which may be treated in accordance with the invention include but are not limited to:
(i) disorders of olfaction and taste, such as anosmia and dysgeusia;
(ii) diseases affecting the superficial entorhinal cortex, dentate gyrus and the CA1, CA3 and CA4 layer of hippocampus, including but not limited to Alzheimer's disease and memory defects resulting from global ischemia as occurs in survivors of cardiac arrest;
(iii) disease affecting the neocortex, including but not limited to Alzheimer's disease and clinical defects produced by stroke as well as predominantly cortical dementing illnesses, including but not limited to Pick's disease, cortical-basal ganglionic degeneration, diffuse cortical Lewy body disease and Jacob-Creuzfeldt disease and including disorders involving the superficial layer of the neocortex caused by lesions or processes which compress or invade the brain from its external surface, including but not limited to consequences of brain trauma as exemplified by a laceration or contusion, subdural hematoma, epidural hematoma and tumor metastases;
(iv) diseases affecting the caudateputamen, including but not limited to
Huntington's disease, chorea-acanthocytosis, choreoathetotic or dystonic syndromes due to perinatal hypoxia as well as striatonigral degeneration and progressive supranuclear palsy, so called "Parkinson-plus syndromes"; (v) disorders of dopaminergic and GABAergic neuronal populations in the substantia nigra; and
(vi) disorders of sensory neurons, particularly proprioceptive neurons in the cervical or lumbar regions of the spinal cord.
The invention is described in greater detail in the subsections below.
5.1. PRODUCTION OF NT-3
The NT-3 utilized in accordance with the invention may be prepared by recombinant DNA
techniques and/or chemical synthetic methods which are well known to those skilled in the art. For example, such methods are described in PCT application number
PCT/US90/04916, publication number W091/03569, filed
August 29, 1990, which is incorporated by reference herein in its entirety.
in a preferred, nonlimiting embodiment of the invention, NT-3 may be prepared from eukaryotic cells that express recombinant NT-3 as follows.
Conditioned medium may be collected from a bioreactor (Charles River) seeded with eukaryotic cells expressing NT-3 and diluted with distilled water at about a 1.25:1 ratio and then may be continuously loaded at 4°C onto a 15 ml (1.6 cm i.d.) S-Sepharose
Fast Flow (Pharmacia) column. A total volume of about
10.6L (diluted) may be applied in a period of about five days. The column may be washed with 50 ml of 20 mM MES buffer (a solution of 4-morpholinethane-sulfonic acid, the pH of which has been adjusted to about 5.8 with NaOH) followed by 50 ml of the same buffer containing 250 mM sodium chloride followed by the same MES buffer without sodium chloride until absorbance at 280 nm reaches baseline level. The column may then be washed with about 25 ml of 20 mM bicine buffer at pH 8.5 followed by 15 ml of the same buffer containing 100 mM sodium chloride. The column may subsequently be eluted with 150 ml, 100 mM to 750 mM sodium chloride gradient in 20 mM bicine buffer (pH 8.5). The flow rate may be desireably about 2.5 ml/min. Absorbance of the eluate may be monitored at 280 nm at a sensitivity of 0.2 absorbance unit full scale. Fractions containing recombinant NT-3 may be identified by dorsal root ganglia (DRG) explant bioassay. The fractions with the highest specific activity may be combined and concentrated to about 2 ml by high pressure membrane ultrafiltration using 3 KDa Omega type membrane. The concentrated sample may then be applied onto a 120 ml (1.6 cm i.d.) Sephacryl S-100HR gel filtration column (Pharmacia) packed and equilibrated with 20 mM HEPES Buffer pH 7.6 containing 400 mM NaCl. The column may be eluted at about 0.25 ml/min. 2 ml size fractions may be collected and assayed for NT-3 activity in the DRG explant bioassay.
The present invention also provides for fragments of NT-3 which are either antigenic or functionally active. Functionally active fragments include fragments of NT-3 that are NT-3 agonists as well as fragments that are NT-3 antagonists. If it is desireable to supplement NT-3 activity, an agonist fragment may be used. If it is desireable to inhibit NT-3 activity, an antagonist fragment may be used.
Agonist fragments preferably comprise at least two of the three disulfide bridges found in the native NT-3 molecule and/or are capable of achieving at least about thirty percent, and preferably at least about fifty percent, of the activity of intact NT-3 measured under the same conditions, e.g. in a DRG assay. 5.2. PRODUCTION OF NT-3 ANTIBODIES
Various procedures known in the art may be used for the production of antibodies that bind to and neutralize NT-3 activity, that bind to the NT-3 receptor, or anti-idiotypes that mimic NT-3 or its receptor. As used herein, an antibody that
neutralizes NT-3 activity refers to an antibody that decreases the activity of native NT-3 in the DRG assay, described infra, by at least about 30 percent. Any of the antibodies used in accordance with the invention include but are not limited to polyclonal, monoclonal, chimeric, single chain, and Fab fragments.
For the production of antibodies, an animal host may be immunized using the appropriate immunogen formulated in an adjuvant to increase the immune response. Such adjuvants include but are not limited to Freund's (complete and incomplete), mineral gels, such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhold limpit hemocyanin, dinitrophenol, BCG (bacille Calmette-Guerin) and
Corynebacterium parvum.
Monoclonal antibodies may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in
culture. These include but are not limited to the hybridoma technique originally described by Kohler &
Milstein (1975, Nature, 256:495-497); the more recent human B-cell hybridoma technique (Cote et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030); the EBV-hybridoma technique (Cole et al., 1985, in Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, pp. 77- 96); the production of chimeric antibodies- (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-6855;
Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985, Nature 314:452-454). Alternatively, single chain antibodies (U.S. Patent No. 4,946,778) or the construction of Fab expression libraries (Huse et al., 1989, Science 246:1275-1281) can be used to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. Antibody fragments may also be generated from whole antibody molecules using known techniques such as pepsin digestion and reduction of disulfide bonds.
5.3. FORMULATION OF THE ACTIVE INGREDIENT The active ingredient, which may comprise NT-3, a biologically active derivative or fragment thereof, or an antibody or antibody fragment prepared as described above should be formulated in a suitable pharmaceutical carrier for administration in vivo by any appropriate route including but not limited to injection (e.g., intravenous, intraperitoneal,
intramuscular, subcutaneous, endoneural, perineural, intraspinal, intraventricular, intrathecal etc.), by absorption through epithelial or mucocutaneous linings (e .g. , oral mucosa, rectal and intestinal mucosa, etc.); or by a sustained release implant, including a cellular or tissue implant.
Depending upon the mode of administration, the active ingredient may be formulated in a liquid carrier such as saline, incorporated into liposomes, microcapsules, polymer or wax-based and controlled release preparations, or formulated into tablet, pill or capsule forms.
The concentration of the active ingredient used in the formulation will depend upon the effective dose required and the mode of administration used.
The dose used should be sufficient to achieve
circulating plasma concentrations of active ingredient that are efficacious. For example, when NT-3 is the active ingredient, a circulating serum concentration level ranging from about 1 ng/ml to 10 μg/ml may be used; preferably ranging from about 1 ng/ml to
100 ng/ml. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
5.4. THE TREATMENT OF NEUROLOGICAL DISORDERS As demonstrated by the working examples described herein, the expression of NT-3 and
distribution of NT-3 receptors occurs in a tissue- specific fashion. Neurological disorders involving pathologies of such tissues are candidates for diagnosis and/or treatment in accordance with the invention. Diseases or disorders involving nervous system cells (including neural cells as well as supporting cells, etc.) that are responsive to NT-3 are the targets of the methods of the invention. For example, high densities of NT-3 receptor occur in the anterior nucleus of the olfactory bulb, layer 1 of the neocortex, the nucleus of the lateral olfactory tract, the dentate gyrus, CA1, CA3, and CA4 of the
hippocampus, and the caudate-putamen. Moderate to low densities of the NT-3 receptor occur in the neocortex nucleus accumbens, spinal gray, basolateral amygdala, interpedunucular nucleus, superior colliculus, medial septum and cerebellum. Disorders involving such regions may be diagnosed or treated using the methods of the invention.
Neurological disorders of the foregoing tissues in which the NT-3 target cell is
underrepresented, or in which cells bearing the NT-3 receptor are damaged or are in the process of
degeneration, may be treated by the administration of an effective dose of NT-3, an NT-3 derivative
including a biologically active agonist NT-3 fragment, or an anti-idiotypic antibody which mimics the binding and biological effects of NT-3. Such treatment will stimulate the growth or differentiation or survival of the target cells . Neurological disorders involving the over-proliferation of cells that express the NT-3 receptor and which proliferate in response to NT-3, or which achieve an undesireable phenotype as a result of exposure to NT-3 may be treated by inhibiting the effect or production of native NT-3. This may be accomplished by administering an effective dose of neutralizing anti-NT-3 antibody to inhibit the
biological effect of NT-3; or by delivery of anti-sense RNA or ribozymes to the cellular source of NT-3 to inhibit the production of native NT-3; or by administration of an NT-3 antagonist.
For example, if NT-3 responsive tissues have been damaged or are in the process of degeneration, treatment which augments NT-3 activity may be
desireable. The determination that NT-3 responsive tissue has been damaged may be made by any method known in the art. For example, if neuroimaging techniques, such as CAT scan, MRI, PET scan, etc.
localizes damage to a structure which exhibits binding to NT-3, for example, as set forth in Table 2, infra, treatment to augment NT-3 activity may be initiated. But damage to such a structure may also be ascertained by other techniques, including, but not limited to, biopsy or on the basis of clinical examination or history, EEG, EMG, etc.
A diminuition in NT-3 activity may be desireable in conditions that involve either
proliferation or overactivity of cells or tissues that are responsive to NT-3. Such conditions may include tumors, for example, in which NT-3 may be employed in an autocrine loop, or in the epilepsies or certain disorders of the striatum that are associated with production of excessive involuntary movement. In the case of tumors, it may be possible to identify an increase in NT-3 receptors, endogenous NT-3, or responsiveness to NT-3 using histologic and/or cell culture techniques; if such a condition is determined, diminuition of NT-3 activity may be desireable.
Various diseases or conditions that occur in the target tissues which may be treated in accordance with the invention are described in the subsections below. 5.4.1. DISORDERS OF THE CENTRAL
OLFACTORY PATHWAY
A high NT-3 binding density exists in the anterior nucleus of the olfactory bulb, the nucleus of the lateral olfactory tract and the olfactory
tubercula. The expression and binding of NT-3 within the olfactory pathway indicate therapeutic
applications not only in disorders of olfaction, but also in phylogenetically similar taste receptors of the tongue. By way of example, and not by limitation, application of therapeutic quantities of biologically active NT-3 molecules or possibly anti-NT-3 antibodies may assist in the treatment of such disorders as anosmia (the absence of smell) or dysgeusia (the distortion of normal taste). Examples of anosmia that may be treated by therapeutic applications of .NT-3 include, but are not limited to, head trauma (which may be caused by severing of the neurons crossing through the cribriform plate as well as complications associated with the normal aging process), multiple sclerosis, Parkinson's disease or a frontal lobe tumor. Examples of dysgeusia that may be treated by therapeutic applications of NT-3 include, but are not limited to, Bell's palsy, familial dysautonomia and multiple sclerosis as well as distortions of taste that arise on the basis of olfactory dysfunction.
5.4.2. DISORDERS OF THE HYPOTHALAMUS
AND LIMBIC SYSTEM
The examples described infra demonstrate NT-3 expression and receptor binding within the lateral, intermediate and medial portions of the superficial entorhinal cortex, dentate gyrus, the granule cell layer of the dentate gyrus hilus as well as the CA1, CA3 and CA4 pyramidal layers of the hippocampus. The expression and binding of NT-3 within these distinct tissues of the brain indicate therapeutic applications which include but are not limited to, memory deficits resulting from global cerebral ischemia which occurs in survivors of cardiac arrest. Additionally,
therapeutic applications of biologically active NT-3 molecules may be utilized in the treatment of
Alzheimer's disease. This region of the brain is involved early in the pathology of the disease and there is speculation (Saper and German 1987,
Neuroscience 23:389-398) that Alzheimer's disease may initiate in this region of the brain, spread through the cortex and hippocampus, and retrogradely affect the basal forebrain and other brainstem nuclei.
The examples described herein demonstrate the effect of NT-3 on hippocampal tissue. An in vitro rat hippocampal culture system was utilized to show that hippocampal tissue (a) expresses both trkB and trkC, (b) NT-3 induces FOS expression of both trkB and trkC, (c) NT-3 induces FOS expression and
neorofilament protein, and (d) produces and increase in AChE - positive and calbindin - immunopositive cells. Since patients diagnosed with Parkinson's disease, Huntington's disease and Alzheimer's disease show a marked decrease in both mRNA transcripts and protein for calbindin (lacopino and Christakos, 1990, Proc. Natl. Acad. Sci. USA 87: 4078-4082) treatment with NT-3 may be used to increase expression of calbindin in such patients. 5.4.3. THE NEOCORTEX AND SUPERFICIAL
LAYERS OF NEOCORTEX
The examples described infra also demonstrate NT-3 expression and binding within the neocortex and superficial layers of the neocortex. Disorders involving the neocortex which occur in a number of diseases may be treated including but not limited to Alzheimer's disease stroke victims and predominantly cortical dementing illnesses, including but not limited to, Pick's disease, cortical-basal ganglionic degeneration, diffuse cortical lewy body disease and Jacob-Creutzfeld disease. The pathology of human victims, in contrast to other animal models, is that predominately all layers of the cortex are affected, with the regional distribution being determined by the territory of the blocked cerebral vessel. In the area surrounding the infarction there is a zone sometimes referred to as the "ischemic penumbra" in which the blood-brain barrier is
relatively leaky, and neurons are threatened with, but not suffering from, fatal ischemic damage. It is within this area that a biologically active NT-3 or derivative or NT-3 peptide fragment may work to minimize the clinical deficits produced by a stroke.
A specific therapeutic target of the
superficial layers of neocortex might be lesions or processes which compress or invade the brain from its external surface, including, but not limited to:
consequences of brain trauma, such as laceration or contusion, subdural hematoma, epidural hematoma and tumor metastases.
5.4.4. THE CAUDATE-PUTAMEN
The examples described infra further
demonstrate NT-3 expression and receptor binding within the caudate-putamen. Therapeutic applications of NT-3 to the caudate-putamen may be utilized to treat, by way of example and not by limitation,
Huntington's disease, chorea-acanthocytis,
choreoathetic dystonia syndromes due to perinatal hypoxia and striatonigral degeneration and progressive supranuclear palsy.
Huntington's disease is an untreatable, progressive, dominantly inherited disease with 100% penetrance. Huntington's disease characteristically presents itself in early midlife, after the
reproductive portion of the life cycle is over. The pathology of Huntington's disease consists principally of degeneration of the so-called "medium spiny" neurons of the caudate nucleus and putamen.
Degeneration is noted in other areas of the nervous system as well, including the frontal cortex, thalamus and the deep cerebellar nuclei. These cell losses result in decreases in the amounts of the
neurotransmitter amino acid GABA, as well as several neuropeptides (reviewed in Martin, 1984, Neurology 34:
1059-1072). Symptoms include behavioral deterioration
(psychiatric disturbances including depression, schizophreniform psychoses as well as dementia;
Mayeux, et al., 1986, Ann. Neurol. 20: 727-731) involuntary motor movements and progressive loss of motor co-ordination including eventual loss of the ability to speak and swallow. The disease is
uniformly fatal. Currently it is estimated that there are over 20,000 victims of Huntington's disease in the United States alone. This figure does not include individuals who are not under medical care for lack of available treatment options or those who carry the gene but are not yet manifesting symptoms. The gene for Huntington's disease has been localized to the distal portion of human chromosome 4p (Gusella, et al., 1983, Nature 306:234-238), but the exact nature of the product of the disease gene is as yet
undetermined. The availability of a genetic marker would enable treatment of the disease before patients become symptomatic, thus optimizing the opportunity for neurotrophic factor "rescue" of degenerating, or yet-to-degenerate, neurons. Recent observations suggest that indeed there is a progressive and
geographical pattern to the loss of striatal neurons in Huntington's disease; that is, that the disease process does not strike cells randomly throughout affected areas of the brain at all times in the illness. Thus, treatment of at-risk or early
symptomatic intervals could stop the course of the disease in its track. The observations of Carrasco and Mukherji (1986, Lancet 1: 1388-1389) indicate that at-risk individuals without overt symptoms of the disease may already have up to 80% cell loss in the caudate nucleus and 40% loss in the putamen. Since neurodegeneration as a result of Huntington's disease is most prominent in the basal ganglia, most
particularly the striatum ╌ an area demonstrated herein to express high levels of NT-3 transcripts and high density NT-3 receptors ╌ treatment with NT-3 or a biologically active derivative or fragment thereof may be used in accordance with the invention to treat Huntington's disease.
Chorea-acanthocytosis is a hereditary choreatic syndrome of young adults who lack the severe intellectual deterioration or behavioral disturbances of Huntington's disease. Some patients have epileptic seizures. There is neuronal cell loss in the caudate nucleus and the putamen. The disease is distinguished by its autosomal recessive inheritance, muscle
wasting, high arches to the feet and acanthocytic erythrocytes on peripheral blood smears. There is currently no treatment for this disorder. However, therapeutic treatment of biologically active NT-3 or a derivative or an NT-3 peptide fragment may be
effective in combating neuronal cell loss in the caudate nucleus and putamen.
The present invention also involves therapeutic applications for the treatment of
choreoathetotic dystonia syndromes caused by perinatal hypoxia. The basal ganglia is a principal target of perinatal "hypoxic-ischemic injury resulting in the neurological impairments of "cerebral palsy". There are three principal clinical variants of cerebral palsy: spastic hemiplegia, spastic diplegia and
"double athetosis": In the former two syndromes, disability is due to a stroke-like lesion of one or both descending corticospinal tracts, respectively. In "Double Athetosis" the symptoms are in reality those of bilateral dystonic choreoathetosis due to ischemic necrosis of intrinsic neurons of the caudate nucleus and putamen. The resultant scarring leaves this region with a mottled appearance on pathological specimens. Since most births in the US occur in a hospital, and risk factors for cerebral hypoxic injury are recognized, acute intervention should be feasible. Early administration of NT-3, at a time when neonatal injury is in progress or has just occurred, could result in the rescue of potentially damaged neurons in this disorder.
Therapeutic applications of the invention also include treatment of striatonigral degeneration and progressive supranuclear palsy (Steele-Richardson- Olszewski Syndrome), which are so-called "Parkinsonplus syndromes" with clinical features of Parkinson's disease which do not respond to conventional
antiparkinson medication. The reason for this drug- resistance is that the neurons of the caudate-putamen, upon which these drugs must act in order to exert their therapeutic effects, are dead or degenerated. These disorders account for up to 10% of patients in major Parkinson's disease clinics (Stacy and Jankovic, Neurol. Clin. North America 2:473-487). Therefore, early administration of biologically active NT-3 or a derivative or an NT-3 peptide fragment is indicated for treatment.
5.4.5. THE EFFECT OF NT-3 ON SURVIVAL AND
NEURITE OUTGROWTH IN SENSORY NEURONS
As shown in the examples, infra. NT-3 promotes neurite survival and outgrowth in the sensory neurons of dorsal root ganglion cultures. In vitro cultures enriched in lumbar or cervical ganglia are more responsive to NT-3 in relation to in vitro
cultures enriched in either sacral or thoracic ganglia of similar ages. These results indicate that JNT-3 may act preferentially on large diameter, large fiber proprioceptive neurons such as those found in the lumbar and cervical ganglia. Therefore, NT-3 may be useful in treating peripheral neuropathies affecting these larger sensory neurons which occur in patients with diabetes or patients undergoing chemotherapy for cancer or AIDS treatment (e.g., taxol, vincristine, cisplatin, di-deoxyinosine). 5.5. DIAGNOSTIC APPLICATIONS
A number of in vitro or in vivo diagnostic tests which involve assaying NT-3 or the NT-3 receptor may be utilized to assist in diagnosis and the
development of a therapeutic regimen. For example, NT-3 may be assayed in various biopsy or autopsy tissues by methods known in the art. In this regard, using anti-NT-3 antibodies immunoassays may be used to detect expression of the NT-3 protein. Such assays include but are not limited to ELISA,
radioimmunoassays, competitive and noncompetitive immunoassays, displacement immunoassays,
immunohistological procedures, etc. Alternatively, nucleotide probes that are complementary to the NT-3 sequence may be used in RNA hybridization methods in situ to detect expression of the NT-3 mRNA
transcripts in the biopsy or autopsy sample. Such techniques may include the use of PCR (polymerase chain reaction). Tissue concentrations of NT-3 protein or mRNA transcripts may be correlated with disease.
Alternatively, the NT-3 receptor may be assayed in biopsy or autopsy samples or in vivo. To this end, NT-3 or NT-3 derivatives may be modified, for detection, e.g., by labeling with a radioisotope, a radioopaque compound, a fluor, an enzyme etc. Such conjugates may be used for imaging the NT-3 receptor in biopsy or autopsy samples in vitro or for imaging in vivo. In another embodiment, antibodies that mimic NT-3 and/or bind to the NT-3 receptor may similarly be used to image the NT-3 receptor distribution in biopsy or autopsy samples in vitro or in vivo. Imaging techniques well known in the art can be used to such ends, e.g., CAT SCAN, X-ray, etc. Aberrations in the distribution of receptor may be correlated with disease.
5.5.1. DIAGNOSIS OF SENSORY
NEURON DISORDERS
As demonstrated by the examples described infra. NT-3 preferentially accumulates in the lumbar and cervical ganglia thus providing a method for diagnosis of sensory neuron disorders. A biopsy sample of such neurons of an afflicted patient may be utilized to determine the level of NT-3 protein and to compare to the level of NT-3 from an analogous sample from a normal individual. An aberrancy in the level of NT-3 may correlate with the presence of a sensory neuron disorder. The actual determination of the level of NT-3 protein may be carried out by a method comprising contacting the sample with an anti-NT-3 antibody such that immunospecific binding can occur.
5.5.2. RETROGRADE AXONAL TRANSPORT OF
NT-3 AND THERAPEUTIC AND
DIAGNOSTIC APPLICATIONS
The retrograde axonal transport of NT-3 in sensory and central nervous system neurons is
described in the examples infra. This phenomenon can be assayed and used to devise effective therapies for sensory neuron disorders. To this end, an effective amount of an NT-3 protein, derivative or peptide factor to support the survival, growth, and/or
differentiation of sensory neurons is administered to a patient in need of treatment. The specific
retrograde transport of NT-3 can be used to indicate whether neurons are responsive to NT-3 in normal or diseased states. Therefore, the present invention provides for a method of diagnosing NT-3 related peripheral nervous system disorders comprising
injecting a detectably labeled NT-3 protein or peptide into a peripheral nerve and determining whether the labeled NT-3 protein or peptide is retrogradely transported, in which a failure to be retrogradely transported positively correlates with lack of
responsiveness to NT-3 and indicates the presence of a peripheral nervous system disorder that is NT-3 related.
The present invention also provides for a method to diagnose a central nervous system disorder. Evaluation of retrograde transport may be performed by any method known in the art, including but not limited to MRI, CAT, or scintillation scanning as discussed infra. Such methods may be used to identify the location of a nervous system lesion, as retrograde transport should substantially diminish upon reaching the lesion.
The invention further provides kits for such retrograde evaluation comprising in a container a detectably labeled NT-3 protein, derivative or
fragment. Such a label can be a radioactive isotope, or other label known in the art.
The present invention also provides for a method of treating sensory neuron disorders comprising administering, to a patient in need of such treatment, an effective amount of an NT-3 protein, derivative or peptide fragment capable of supporting the survival, growth and/or differentiation of motor neurons as demonstrated in an in vitro culture system.
In in vitro embodiments, including, but not limited to those described infra, effective amounts of neurotrophic factor may desirably be determined on a case by case basis, as sensory neurons from different tissue sources or from different species may exhibit different sensitivities to neurotrophic factor. For any particular culture, it may be desirable to construct a dose response curve that correlates neurotrophic factor concentration and motor neuron response. To evaluate sensory neuron survival, growth, and/or differentiation, one can compare motor neurons exposed to an NT-3 protein, derivative or peptide fragment to sensory neurons not exposed to an NT-3 protein, derivative or peptide fragments, using, for example, vital dyes to evaluate survival, phase- contrast microscopy and/or neurofilament stain to measure neurite sprouting, or techniques that measure the bioactivity of motor neuron-associated compounds, such as choline acetyltransferase (CAT), or any other methods known in the art.
6. EXAMPLE: HIGH AFFINITY BINDING SITES
IN RAT. CAT AND HUMAN BRAIN
6.1. MATERIALS AND METHODS
6.1.1 PRODUCTION AND PURIFICATION OF NT-3
Purified NT-3 was produced by the preferred method described supra in Section 5.1. The NT-3 aliquots were monitored for protein content by amino acid analysis and for biological activity using the dorsal root ganglia (DRG) explant culture system using muNGF as a standard.
6.1.2 IODINATION OF NT-3
The NT-3 was iodinated by the
lactoperoxidase method. The [125I]NT-3 was labeled to a specific activity of 2800-4400 cpm/fmol (1872-2876 Ci/mmole of NT-3) and stored at a concentration of 80-100 nM at 2-8°C. [125I]NT-3 was used within 1-5 days in all studies to avoid the observed increase in non-displaceable [125I]NT-3 binding that occurred after this time.
The biological activity of [125I]NT-3 was also established with the DRG assay which showed it to be at least as active as its unlabeled counterpart. The demonstrable retrograde transport of [125I]NT-3 from crushed sciatic nerve to dorsal root ganglion which was specifically blocked by unlabeled NT-3 (DiStefano et al., 1991, Soc. Neurosci. Ab. 17:1121) and the indistinguishable potencies of NT-3 and [125I]NT-3 in the DRG assay both indicated that the radioiodinated NT-3 used was biologically active.
6.1.3. PREPARATION OF RAT, CAT,
AND HUMAN BRAIN SECTIONS
Male Sprague-Dawley rats (200-250 grams, Zivic-Miller) maintained on a 12:12 hour light:dark cycle and ad libitum access to food and water were sacrificed by carbon dioxide asphyxiation. Within 5 minutes of death, the brain of each rat was frozen in isopentane at -15°C and these tissues were used for association, dissociation, and equilibrium saturation analyses. Serial, 12 μm-thick coronal and horizontal sections were collected at 0.30-0.50 mm intervals from the frontal cortex to the medulla at the level of the dorsal motor nucleus of the vagus (coronal sections) or through the olfactory bulbs, caudate-putamen, and cerebellum (horizontal sections). Sections were thaw-mounted onto gelatin-coated glass microscope slides and stored frozen for up to one month at -70°C.
The brain of an adult male cat was sectioned coronally from the frontal cortex to the medulla and sections from 22 levels were collected as described for the rat. Twenty μm thick sections of a human brain were collected through the basal ganglia and included the caudate, putamen, neocortex, and adjacent fiber bundles.
6.1.4. [125I]NT-3 BINDING ASSAY
Binding assays were conducted according to the procedure of Richardson, et al. (1986, Neurosci. 20:23-36) and with modifications for dry film
autoradiography of [125I]rhNGF (Altar, et al. 1991, Proc. Natl. Acad. Sci. U.S.A. 88:281-285).
After thawing, each section was preincubated for 1 hour at 22ºC in 100 mM phosphate-buffered saline, pH 7.4, containing 0.5 mM MgCl2 and 0.5 mM PMSF. Equivalent binding was observed for sections that were preincubated for 1, 3, or 24 hours.
Sections were then incubated in DMEM tissue culture medium containing high glucose, 10% heat-inactivated fetal calf serum (70° C for 0.5 hours), 25 mM HEPES buffer, 4 μg/ml leupeptin, 0.5 mM PMSF, (BRL,
Gaithersburg, MD, first dissolved to 0.1 mg/ml
isopropyl alcohol), 0.5 mM MgCl2, and 10 pM to 10 nM [125I]NT-3 (equilibrium saturation analysis) or 200-300 pM [125I]NT-3 (all other assays) based on a dimeric molecular weight of 26,000. Slides containing
adjacent brain sections were incubated in the same solutions with the addition of 300 nM NT-3 to define displaceable binding. A range of concentrations of muNGF, hCNTF, and BDNF were each used to compete with [125I]NT-3 binding in several experiments. Following the incubation, the sections were washed for 0.5 hr in the phosphate buffer. In the absence of excess unlabelled NT-3, equivalent amounts of total and non-displaceable binding were obtained with 3 minutes, 10 minutes and 2 hour washes with unlabeled buffer.
The association and dissociation of 300 pm [125I]NT-3 binding after a 1 hour wash (association experiment) or 3.5 hour incubation (dissociation experiment) was also determined. After washing, sections were fixed for 10 minutes in 4%
paraformaldehyde at 22°C, rinsed for 2 seconds in water at 22°C, and dried within 5 minutes by a stream of room temperature air. The labeled sections and 125I-containing radioactivity standards (Amersham, Inc.) were exposed at room temperature for 2-5 days
([125I]NT-3) to 125I-sensitive film (Hyperfilm, Amersham, Inc.).
6.1.5. DATA ANALYSIS
After generation of autoradiographs, the horizontal sections were scraped from the slides with a razor blade, placed into 12 × 75 nun borosilicate glass tubes, and counted for radioactive decay using a gamma counter. Kinetic determinations of Kd values were calculated for 4 brains used in association and dissociation experiments (Weiland and Molinoff, 1981, Life Sci. 29:313-330). Equilibrium dissociation and association rate constants were obtained under
pseudo-first order conditions, since much less than 10% of free ligand was bound to sections after 3 hours of incubation. Kd and Bmax values were calculated by equilibrium saturation analysis with each of four brains according to the best fit to a parabola by iterative, nonlinear regression analysis (Rodbard and Lewald, 1970, Acta Endocrinol. 147:79-103). IC50 values were calculated by the method of Bliss and
James (1966, Biometrics 22, 573-580). Statistical analyses for between-group comparisons were made with
Dunnet's t-test following a one-way analysis of variance. 6.2. RESULTS
In preliminary studies, 40-400 pM concentrations of [125I]NT-3 were found to optimally label a dense and heterogeneously distributed
population of binding sites in horizontal and coronal sections of rat brain. This binding was displaced by 70-90% by a 1000-fold greater concentration of NT-3 whereas 10-15% higher levels of non-specific binding were obtained at higher ligand concentrations. Because binding was particularly robust and displaceable in the caudate-putamen, neocortex, and hippocampus, horizontal sections that contained these areas were used for subsequent quantitative studies with
200-300 pM [125I]NT-3.
[125I]NT-3 associated to and dissociated from its displaceable binding site in horizontal sections (n = 4 brains) in a consistent manner across brain regions. The binding varied by 4-20% (s.e.m.) at association time points and by 10-25% at dissociation time points. The association of 200 pM [125I]NT-3 increased until 2 hr, at which time equilibrium was achieved and no additional binding was obtained during an additional 6 hours (Figure 1, top). The association rate constant for binding to these sections was 0.0074 × 109 min-1 mole-1. Sections incubated for 3.5 hour with 200 pM [125I]NT-3 were washed in buffer containing 200 nM NT-3 for 2 minutes to 18 hours revealed a
dissociation of specific [125I]NT-3 binding at a rate of 19.4 × 10-4 min-1 (Figure 1, bottom). The ratio of the kinetically-derived dissociation and association rate constants (k-1/k1; Weiland and Molinoff, 1981, Life Sci. 29:313-330) gave an average equilibrium dissociation constant (Kd) value 227 ± 28 pM.
Additional horizontal brain sections equilibrated with [125I]NT-3 concentrations ranging from 10 pM to 9 nM [125I]NT-3 revealed saturable,
displaceable binding that was clearly biphasic from the saturation isotherm (Figure 2, left) and the B/F reciprocal plot (Figure 2, right). The saturation in displaceable binding from 10-750 pM allowed a good fit for Kd and Bmaxvalues by non-linear regression analysis. The Kd was 269 ± 64 and the capacity of binding was 26 ± 3 fmol/mg protein (Table 1). A second range of concentrations from 0.75 - 9 nM revealed a second plateau of specific binding that revealed a lower affinity (Kd= 2.8 ± 0.34 nM) and higher capacity (Bmax = 170 ± 14 fmol/mg protein) site (Table 1). The Bmax value calculated for the entire 10 pM - 9 nM range was approximately an additive function of the separate Bmax values for the high and low affinity sites (Table 1). In contrast, [125I]rhNGF associated more quickly and dissociated more slowly than did [125I]NT-3 and bound with about 4- to 10-fold greater affinity (Table 1).
NT-3 competed with 300 pM [125I]NT-3 for a single population of binding sites (nH = 1.2 ± 0.26) with an IC50of 420 ± 60 pM (Figure 3). BDNF also competed for most of 300 pM [125I]NT-3 binding but did so in a biphasic manner (IC50 values of 230 ± 100 pM; nH = 0.76 ± 0.18; and 37 ± 2.9 nM; nH = 0.94 ± 0.05)
(Figure 3). In contrast, [125I]NT-3 binding was not prevented by coincubation with up to 100 nM of either
CNTF or muNGF.
TABLE 1
CAPACITY AND AFFINITY OF LOW AND HIGH AFFINITY [125I] NT-3 AND [125I] rhNGF-BINDING Concentration Range Kd Bmax (fmol/mg prot)
[125I] NT-3
10 - 750 pM 269 pM ± 64 26 ± 3
0.75 - 10 nM 2.8 nM ± 0.34** 170 ± 15** 10 pM - 10 nM 3.0 nM ± 0.28** 200 ± 6**
[12SI]rhNGF*
(4-294 pM) 69 pM ± 9.7 9.9 ± 1.8 Kinetic [125I]NT-3 [125I]rhNGF*
Determinations
Association k1 0.0074 × 109 M-1 0.066 × 109 M-1 min-1 min-1
Dissociation k-1 19.4 × 10-4 min-1 8.9 × 10-4 min-1 Kd (k-1/k1) 227 ± 28 pM 28 ± 11 pM
*Average values from Altar, et al., 1991, Proc. Natl. Acad. Sci. USA 88:281-285.
Horizontal sections of rat forebrain (n=4/group) as shown in figure 4 were incubated in 10 pM to 10 nM
[125I]NT-3 with or without 300 nM NT-3 to define non-specific binding. The Kd and Bmax values are calculated as mean ± sem.
**p < 0.01 versus high affinity [125I]NT-3 binding, Student's t-test. Autoradiographs of either coronal or
horizontal sections revealed a pattern of specific
[125I]NT-3 binding that was clearly different from that observed for murine or human [125I]NGF (Riopelle, et al., 1987, Neurochem. Res. 12:923-928; Richardson, et al., 1986, J. Neurosci 6:2312-2321; Ravich and
Kreutzberg, 1987, Neurosci. 20:23-36; Altar, et al.,
1991, Proc. Natl. Acad. Sci. USA 88:281-285; Altar, et al., 1991, J. Neurosci, 11:828-836). The highest levels of binding were found in olfactory pathways
(anterior nucleus of the olfactory bulb, nucleus of the lateral olfactory tract), hippocampus (dentate gyrus, CA1, 3, and 4), caudate-putamen, and the neocortex (Table 2). In particular, the first layer of the neocortex was prominently labeled in the frontal, parietal, and cingulate regions. Layer 1 was also heavily labeled throughout the entorhinal cortex.
Intermediate levels of binding were present in nucleus accumbens, basolateral amygdala, interpeduncular nucleus, ventral and dorsal horns of spinal cord, and superior colliculus. Lower amounts of specific binding were present in the lateral geniculate, medial septum, and cerebellμm. No displaceable binding was detected in white fiber buncles including the corpus callosum, anterior commissure, internal capsule, interbulbar internal capsule, or in the globus pallidus, most thalamic nuclei, hypothalamus, or in other regions of the pons, medulla, or other amygdaloid nuclei. No
[125I]NT-3 binding sites were found in circumventricular organs such as the choroid plexus or ependymal cell layers. Specific binding was also absent in the liver, muscle, kidney, pancreas, and heart. TABLE 2
RELATIVE DISTRIBUTION OF SPECIFIC
[125I]NT-3 BINDING SITES IN RAT BRAIN
High Binding
Anterior nucleus of olfactory bulb
Nucleus of lateral olfactory tract
Lateral, intermediate, and medial portions
of superficial entorhinal cortex
Dentate gyrus
CA1, CA3, CA4 pyramidal layer of hippocampus
granule cell layer of dentate gyrus hilus
Layer 1, frontal, parietal, cingulate cortices
Olfactory tubercle
Medium Binding
neocortex (layers 2-6)
nucleus accumbens
caudate-putamen
basolateral amygdala
interpeduncular nucleus
spinal gray
superior colliculus
Low Binding
superior geniculate
medial septum
cerebellum
No Detectable Binding
globus pallidus
pars compacta or reticulata of substantia nigra
medial lemniscus
thalamus
internal capsule
ependymal layer
choroid plexus
corpus callosum
interbulbar anterior commissure
ventricle walls
skeletal muscle, liver, kidney, pancreas, heart
The relative amounts of NT-3 binding were evaluated from visual evaluation of autoradiographs generated with 300 pM [125I]NT-3. The non-specific binding defined with 300 nm NT-3 appeared uniform throughout the brain sections and accounted for only 10-20% of the total amount of bound ligand. This allowed the relative amounts of specific binding to be estimated from the total binding images. 6.3. DISCUSSION
Receptor autoradiography with biologically active [125I]NT-3 has revealed that this ligand binds with high affinity in a saturable, reversible, and pharmacologically specific manner to rat brain. The similar distribution in rat, cat (see Figure 5), and human (see Figure 6) brain suggests that this
heterogeneous brain binding site is nonetheless phylogenetically conserved. These maps illustrate the brain regions in which endogenous NT-3 may function as a neurotrophic factor and also provide the basis for both in vitro and in vivo studies to assess the neuronal specificities and therapeutic targets of NT-3. Thus, considerations of the kinetics,
pharmacology, and localization of these sites will be addressed.
6.3.1 CHARACTERISTICS OF [125I]NT-3
BINDING TO ITS HIGH AFFINITY SITE
The displaceable binding of [125I]NT-3 ranged from 60-90% of the total binding. The uniform
topography of the 10-40% non-displaceable binding probably represents NT-3 receptor-independent
association of tracer to diverse tissue components, including myelinated fiber bundles and non-specific protein binding sites. Nevertheless, neither fiber bundles nor a variety of brain regions bound [125I]NT-3, demonstrating the regional selectivity of this ligand for discrete sites. A proportionately greater amount of non-displaceable binding has been observed for
[125I]muNGF binding to tissue sections (Riopelle, et al., 1987, Neurochem. Res. 2:928-928; Richardson, et al., 1987, J. Neurosci. 6:2312-2321), and cultured cells (Landreth and Shooter, 1980, Proc. Natl. Acad. Sci. USA 77:4751-4755; Meakin and Shooter, 1991, Neuron 6 :153-163). The decrease in non-specific
[125I]rhNGF binding measured in spinal cord sections (Jakeman, et al., 1990, Abstract, p. 21. Development and Plasticity of the Spinal Cord, Columbus, OH and in brain (Altar, et al., 1991, Proc. Natl. Acad. Sci. 88:281-285; Altar, et al., 1991, J. Neurosci.
11:882-836) with the additional protein carriers used presently may have contributed to the high specificity of the NT-3 signal obtained here. Nevertheless, the [125I]NT-3 binding was more readily displaceable in the presence of unlabeled homologous ligand (80-90%) than obtained for [12SI]rhNGF (60-85%; Altar, et al., 1991, Proc. Natl. Acad. Sci. USA 88:281-285: Altar, et al., 1991, J. Neurosci, 11:828-836) under identical
conditions.
The displacement of [125I]NT-3 after its association to brain sections was minimal in the absence of unlabeled NT-3 and proceeded over several hours in the presence of 1000-fold excess NT-3. This requirement for excess homologous ligand to promote the dissociation of high affinity neurotrophin ligand binding has also been reported for muNGF (Sutter et al., 1979, J. Biol. Chem. 254:5972-5982). rhNGF
(Altar, et al., 1991, Proc. Natl. Acad. Sci. USA
88.:281-285) , and BDNF (Rodriguez-Tebar, et al., 1990, Neuron 4:487-492).
[12SI]NT-3 bound to two distinct sites in rat brain sections that were each mostly displaced by 300 nM NT-3. The high affinity site was rigorously
characterized because its 227-269 pM affinity constant approximates the 200-300 pM concentrations used to define the kinetics, pharmacology, and distribution of [125I]NT-3 binding to rat, cat, and human brain. The site is preferentially and completely occupied by NT-3. Interestingly, part of [125I]NT-3 was displaced by an equal concentration of BDNF and not at all by even 100 nM NGF or CNTF. The ability of NT-3 and BDNF but not NGF to recognize the trkB receptor (Squinto, et al., 1991, Cell 65:885-893; Kaplan, et al., 1991, Nature 360:158-160; Bothwell, 1991, Cell 65:915-918) is consistent with the pharmacological profile for
[125I]NT-3 binding.
The capacity of high and low affinity NT-3 binding sites were 26 and 170 fmol/mg protein,
respectively. Thus, the high affinity site is at least twice as dense as that for rhNGF (Altar, et al., Proc. Natl. Acad. Sci. USA 88:281-285; Altar, et al., 1991 J. Neurosci. 11:828-836). The high affinity binding site for [125I]BDNF appears to be even more numerous and more ubiquitously localized than those for NGF or NT-3.
6.3.2 DISTRIBUTION OF NT-3 BINDING SITES
The topography of [125I]NT-3 binding to rat brain sections did not resemble the distribution of [125I]rhNGF. binding to similar sections (Richardson et al., 1986, J. Neurosci 6:2312-2321; Riopelle et al., 1987, Neurochem. Res. 12:923-928; Ravich and
Kreutzberg, 1987, Neurosci. 10:23-36; Altar et al., 1991, Proc. Natl. Acad. Sci. USA 88:281-285). For example, none of the prominent cholinergic nuclei in the diagonal band, medial septum, nucleus basalis, fifth cranial nerve nucleus, or globus pallidus (Kasa, 1986, Nature 331:261-262) were labeled by [125I]NT-3 , whereas these areas are prominently labeled with
[125I]rhNGF or by immunostaining with the 192 MAb to the low affinity subunit of the NGF receptor (eg., Woolf, et al., 1989, Neurosci. 30: 143-152; Pioro and Cuello, 1990, Neurosci 34:57-87). Some hindbrain areas known to contain cholinergic markers are devoid of both NGF (Woolf, et al., 1989, Neurosci. 30:143-152) and NT-3 binding sites. These "NT-3/NGF receptor-poor"
cholinergic areas include cranial nerve nuclei III and IV, the pedunculopontine or parabrachial cholinergic projections to thalamus, hypothalamus, and inferior colliculus, and the projection of preoptic
magnocellular cholinergic neurons to the ventral tegmental area. Cholinergic projections (Kasa, 1986, Proc. Neurobiol. 26:211-272) from the diagonal band to the NGF receptor-poor (Altar et al., 1991, Proc. Natl. Acad. Sci. USA 88:281-285) piriform cortex, habenula, and entorhinal cortex may contribute to the relatively intense NT-3 binding observed in these areas.
Alternatively, the glutamatergic or other
neurotransmitter systems in piriform and entorhinal cortex that project to hippocampus may contain these NT-3 binding sites.
The relative paucity of NT-3 binding sites in adult cerebellum is in marked contrast to the dense labeling of cerebellum by [125I]NGF (Cohen-Cory, et al., 1989, Exp. Neurol. 105:104-109; Altar, et al., 1991, Proc. Natl. Acad. Sci. USA 88:281-285) or by
immunostaining for the NGF receptor (Pioro and Cuello, 1988, Brain Res. 455:182-186; Pioro and Cuello, 1990, Neurosci. 34:57-87). While emulsion autoradiography will help resolve the presence of NT-3 binding sites in this and several other regions, it is clear that NT-3 receptors are relatively low in the, adult rat cerebellum and other areas in which NGF binding sites and receptor immunostaining are quite dense. The granule cell layer of cerebellum is also the most densely labeled brain area following in situ
hybridization with trkC antisense probe. The strong, residual trkC signal generated with trkC sense cRNA probe (Lamballe, et al., 1991, Cell 66:967-979) or a relatively diminished translation of trkC message into functional receptor are two possible explanations for the presence of trkC antisense message but not NT-3 binding in the cerebellar granule cell layer.
Nevertheless, trkC expression is also very pronounced in hippocampus, neocortex, and neostriatum (Lamballe et al., 1991, Cell 66: 967-979) and these areas were most intensely labeled with [125I]NT-3. Clearly, a direct comparison of trkC mRNA and [125I]NT-3 binding to adjacent sections of rat brain will ascertain the resemblance of these two markers.
The strength of [125I]NT-3 binding was also evident in cat and human brain sections (See Figure 5 and Figure 6, respectively). Importantly, the
topography of these sites closely resembled the pattern observed in rat brain. Several subtler aspects of [125I]NT-3 binding to rat brain, such as the dense labeling of superficial neocortex and uniform binding throughout the caudate-putamen, were conserved in cat or human brain. Unlike the rat, the heterogeneity of binding was somewhat diminished in cat, although this may be only an apparent feature due to the more gradual changes in binding observed throughout the larger cat brain.
7. EXAMPLE: THE COMPARATIVE EFFECT OF
NEUROTROPHINS IN HIPPOCAMPUS OF EMBRYONIC RAT
Hippocampi were dissected from E16-E18, or
E20 rat embryos of Sprague-Dawley rats, and collected in F10 medium. The tissues were minced, rinsed twice with F10 medium (Gibco) and trypsinized with 0.25% trypsin (Gibco) for 20 minutes at 37°C. Trypsin was inactivated by the addition of a serum-containing medium composed of minimum essential medium (MEM) supplemented with fetal calf serum (FCS, 10%), glutamine (2 mM), penicillin (25 U/ml) and
streptomycin (25 μg/ml). Dissociated cells obtained by gentle trituration were collected and centrifuged at low speed (500 rpm) for 30 seconds. The
centrifugation was repeated twice, and the cell pellets were then resuspended in serum-containing medium. Hippocampal neurons were plated onto
polyornithine-laminin (10 μg/ml) in DME plus 10% fetal calf serum. After 4 hours of culture, the medium was changed to DME plus 1 mg/ml BSA and N2 media
supplement (Bottenstein, et al., Methods Enzymol.
58:94-109) and 1 mM pyruvate, at which time each respective neurotrophin was added. The media was changed every three to four days, with re-addition of the factor.
7.2. RESULTS AND DISCUSSION mRNA levels for trkA, trkB and trkC were examined in these cultures. As shown in Figure 7 (A), while there was a detectable level of trkA message in adult brain, there was no detectable trkA mRNA in either hippocampal neurons nor astrocytes. The mRNA level for trkB and trkC in the hippocampal cultures were comparable to that seen in adult brain
Figure 7(B) and 7(C), but were absent in astrocytes. The presence of trkB and C, but not trkA, on these hippocampal neurons correlates well with the finding that these cells are responsive to BDNF and NT-3, but not NGF.
The effects of neurotrophins on the induction of one of the immediate early genes (fos) were examined (Figure 8). The induction of fos mRNA by BDNF and NT-3 peaked at about 1 hour, followed by an induction of fos protein which peaked at about
2-3 hours and persisted for at least 6 hours.
Approximately 40% of the hippocampal cells showed a fos response to BDNF and NT-3. The effects of BDNF and NT-3 on fos induction were not additive. All of the cells that showed the fos response to BDNF and NT-3 were neurons. Double staining with fos and calbindin showed that calbindin-immunopositive cells were among the cell population that responded with fos induction.
BDNF and NT-3 were found to induce an increase (50%) in the amount of neurofilament protein. They also produced an increase in the number of AChE-positive and calbindin-immunopositive cells. In contrast, NGF had no apparent effect. Dose response studies of BDNF and NT-3 on the number of calbindin-immunopositive cells showed that the response
saturated at about 3 ng/ml. NT-3 produced a 20-fold increase in the number of calbindin-positive cells which was accompanied by an increase in calbindin-mRNA levels. Delaying the addition of NT-3 to the cultures for 4 days did not appear to affect the increase in the number of calbindin-positive cells, suggesting that NT-3 acts to induce the calbindin-phenotype instead of acting as a survival factor. Developmental profiles of the increase in calbindin-positive cells produced by BDNF and NT-3 were compared. The effect of NT-3 was much more striking for hippocampal neurons earlier in development (E16-E18) and declined later in development (E20), while the reciprocal effect was observed for BDNF. As summarized in Table 3, the only cell populations that appeared to respond to both BDNF and NT-3 thus include AChE positive and calbindin positive cells. Other cell populations examined which did not appear to respond to BDNF and NT-3 in terms of survival include GABAergic cells, glutamatergic cells and somatostatin-positive cells. Decreases of up to 60-80% in the levels of both the message and protein for calbindin were observed in the hippocampus of patients who were diagnosed as having Parkinson's, Huntington's or Alzheimer's disease (lacopino and Christakos, 1990, Proc. Natl. Acad. Sci. USA 87:4078-4082). In
addition, it has recently been shown that the presence of calbindin in specific regions of the hippocampal formation may be positively correlated with the relative resistance of such neurons to seizure-induced neuronal damage (Sloviter, J. Comp. Neurol.
290:183-196). This example discloses that BDNF, and, to a larger extent, NT-3, were able to induce
calbindin protein in the hippocampal neurons. Such an increase in Ca-binding capacity may have significant physiological implication in that it may be important in preventing cell death in select populations of hippocampal neurons.
TABLE 3
HIPPOCAMPAL NEURONS
TRKA -
TRKB +
TRKC +
NGF BDNF NT3
FOS INDUCTION - + +
NEUROFILAMENT - + +
GABA UPTAKE - - -
GABA POSITIVE - - -
CELLS
GLUTAMATE UPTAKE - - -
ACHE POSITIVE - ++ +
CELLS
CALBINDIN POSITIVE
CELLS - + +++
SST POSITIVE CELLS - - - VIP-IR - + -
8. EXAMPLE: EFFECT OF NEUROTROPHINS
ON STRIATAL NEURONS
The effect of neurotrophins on cultured striatal neurons is of interest in order to develop a model as to therapeutic application for addressing
Huntington's disease (discussed supra in Section
5.1.4.). Huntington's is an autosomal dominant condition that results in progressive
neurodegeneration reflected in defects in cognition, motor control and affect. Deterioration is most prominent in the basal ganglia, particularly the striatum, which appears to be the site of onset of the disease. This example discloses levels of mRNA expression by Northern blot analysis for the
neurotrophins and their receptors in vivo and in vitro. 8.1. MATERIALS AND METHODS
8.1.1. TISSUE DISSOCIATION AND CULTURE
Brain tissues were dissected from Sprague- Dawley rats at the following stages of development: embryonic day 17 (E17), postnatal days 1(P1), 7, 14, 20, and adult. Tissues for in vivo analysis were frozen immediately on dry ice.
Striatal neuronal cultures were prepared from E17 rat brains as follows: striatal tissue was minced in calcium- and magnesium-free Hank's balanced salt solution and dissociated by enzymatic treatment with 0.25% trypsin and DNAase (0.2 mg/ml) followed by mechanical trituration. Dissociated cells were seeded at a density of 8 × 106 cells on 100 mm dishes which had been previously coated with polylysine and
laminin. After an initial 4 hour incubation in medium composed of Dulbecco's Modified Eagle's Medium and 10% fetal calf serum (DME-FCS), medium was replaced with serum-free N2 medium (Bottenstein, et al., 1979, Meth. Enzymol. 58:94-109).
Striatal and hippocampal astrocyte cultures were prepared from P1 rat brains as follows: tissues were minced and enzymatically dissociated by treatment 0.25% trypsin and DNAase 0.2 mg/ml). Following a 5 minute centrifugation at low speed, and an
additional 5 minute centrifugation at 300 rpm, the cell suspension was dissociated mechanically by trituration. Cells were then passed through a Nitex filter cartridge and seeded at a density of 71,000 cells/cm2 in T75 tissue culture flasks in DME-FCS.
Medium was replaced on days 1, 3, 5, 7, and 9 after seeding. On days 7 and 9, flasks were shaken to remove neurons, macrophages and 02-A progenitor cells. Astrocytes were passaged at 10 days in vitro (DIV). 8.1.2. IMMUNOCYTOLOGICAL STAINING
Immunocytochemical staining was used to determine the purity of the astrocyte and neuronal cultures. Antibodies to glial fibrillary acidic protein (specific to astrocytes) and neurofilament protein (specific to neurons) were used in combination with other cell-type-specific markers to delineate the cellular composition of the cultures.
8.1.3. RNA ISOLATION
Total RNA was prepared from striatal
neuronal cultures (at 4 DIV), striatal astrocyte cultures (at 28 DIV) and hippocampal astrocyte
cultures (at 28 DIV) by extraction (Chomczynski and Rougeon, 1980, Anal. Biochem 162: 156-159). Total RNA was prepared from striatal or whole brain tissue samples by extraction in 0.3M LiCl/6M urea followed by phenol/chloroform extraction (Auffray and Rougeon, 1980, Eur. J. Biochem 107:303-314). RNAs (10 ug/lane) were fractionated by electrophoresis through 1% agarose-formaldehyde gels (Bothwell et al., 1990, in: Methods for Cloning and Analysis of Eukaryotic Genes, pp. 42-43, ed., Jones and Bartlett, Boston) followed by capillary transfer to nylon membranes. Probes to trkB (1.1 kb, spanning the intracytoplasmic tyrosine kinase domain) and trkC (800 bp, spanning the
intracytoplasmic tyrosine kinase domain) were labeled by random hexamer labeling with 32P dCTP (Stratagene Prime It). Membranes were hybridized overnight in 0.5 sodium phosphate buffer, pH 7.9 , containing 1 percent bovine serum albumin, 7 percent SDS and 100 μg/ml sonicated salmon sperm at 65°C. Filers were rinsed briefly in 2 × SSC and 0.1 percent SDS and then washed twice in 1 × SSC and 0.1 percent SDS and exposed to
X-ray film for one week. Quantitation of transcript levels was accomplished by densitometric scanning on an 1-KB laser densitometer.
8.2. RESULTS AND DISCUSSION
Examination of trkB expression in rat striatum revealed that expression can be detected in embryonic day 17 brain, with a dramatic increase in expression occurring between E17 and postnatal day 1 (Figure 9). A peak in expression was observed at postnatal day 14. Expression of trkB mRNA was detected in cultured E17 striatal neurons (4 DIV), but not in astrocytes (28 DIV) prepared from either P1 striatum or P1 hippocampus. Densitometric
quantitation of transcript levels (Figure 11) showed that expression of trkB mRNA at P14 is elevated 1.15 fold relative to levels in whole adult brain.
TrkC mRNA expression was detected in rat striatum as early as E17 (Figure 10). Expression of trkC mRNA reaches a peak between P7 and P20, where a 1.5-1.9 fold increase in transcript level is detected relative to whole adult rat brain (Figure 12).
Expression drops off sharply in adulthood. Similar to trkB expression, trkC mRNA is expressed in cultured striatal neurons (4 DIV) but not in striatal or hippocampal astrocytes (28 DIV).
The expression in developing rat striatum of mRNA for trkB and trkC suggests that BDNF and NT-3 may act as trophic factors in the striatum.
9. EXAMPLE: COMPARATIVE EFFECT OF NEUROTROPHIN-3 ON SURVIVAL AND
NEURITE OUTGROWTH ON SPINAL SENSORY NEURONS LOCATED IN LUMBAR,
CERVICAL. SACRAL AND THORACIC GANGLIA
When cultured either as explants or dissociated neuron-enriched cultures, sensory neurons of the chick dorsal root ganglia (DRG) are responsive to a greater or lesser degree to NGF, BDNF, NT-3, NT-4 and CNTF. Chick embryo DRG neurons die within 24 hours of being placed in culture in the absence of any neurotrophic factor. Each of the above factors can support the survival and outgrowth of neurites of some of these neurons, ranging from 10-60%, depending on the factor, stage of neuronal development etc. It appears that each of the above neurotrophic factors has both distinct and overlapping specificities towards sup-populations of DRG neurons, although there is no clear evidence as to which sub-types of sensory neurons are supported by which neurotrophic factor. This example defines the comparative specificity of NT-3, NGF and BDNF.
9.1. MATERIALS AND METHODS
9.1.1. EXPLANT CULTURES
DRG were collected from chick embryos at developmental stages ranging from E6 - E10. Starting in the sacral region ganglia from right and left sides were collected in pairs along the entire neural axis. 5-6 ganglia at, each level or pools of sacral, lumbar, thoracic or cervical ganglia were explanted in
collagen gel matrix (Lindsay and Peter, 1984,
Neuroscience, 12, 45-51). Ganglia were cultured in F14 medium + 5% horse serum in the presence or absence of 5 ng/ml, NGF, BDNF or NT-3. After 24 hours the. extent of fiber outgrowth was measured on an arbitrary scale of 0 to +5, 0 being virtually no fibers,. +5 being a profuse halo of fibers (the saturating level seen with NGF, the most potent of the factors in this assay). 9.1.2. DISSOCIATED NEURON-ENRICHED CULTURES
Lumbar and thoracic ganglia from E8 embryos were collected separately, dissociated with 0.25% trypsin and freed of non-neuronal cells (Lindsay et al, 1985, Develop. Biol. 112, 319-328). Purified neurons were seeded on a substrate of polyornithine- laminin at 8,000 neurons per 35 mm dish. Cells were cultured in the presence of F14 medium containing 5% horse serum. A dose response ranging from 1 pg to 10 ng of either NGF, BDNF or NT-3 was carried out. After 48 hours the number of process bearing neurons was determined in triplicate cultures (Lindsay et al., 1985, Develop. Biol, 12: 319-328). 9.2. RESULTS AND DISCUSSION
As shown in Figure 13, the effects of NGF on DRG explants from E6, E8 or E10 DRG was very uniform regardless of the segmental level - sacral, lumbar, thoracic and cervical ganglia showed an equally high response, with scores of 4-5 at saturating levels of NGF (open bars of Figure 13). BDNF was less potent than NGF with scores of 2-3, but the effects of BDNF were uniform along the neural axis with sacral, lumbar, thoracic and cervical ganglia showing
essentially the same response. In contrast to NGF and BDNF, the effects of NT-3 varied depending on the segmental level of the DRG. Sacral and thoracic ganglia showed only weak fiber outgrowth/in response to NT-3 at all ages between E6 and E10. Lumbar and cervical ganglia were much more responsive to NT-3, with lumbar ganglia being the most responsive with scores of 3-4. This differential effect of NT-3 on lumbar and cervical ganglia compared to sacral and thoracic ganglia was similar at all ages studied. Under the more exacting conditions of dissociated neuron-enriched cultures, similar results were obtained to those seen with explants as shown in Figure 14. NGF supported the survival of a similar percentage of either lumbar or thoracic DRG neurons (40-50%). Although BDNF was less effective than NGF, the effects of BNDF were essentially the same towards lumbar or thoracic neurons - supporting survival of around 30% of the neurons. In contrast, NT-3
supported survival of 2.5-fold more lumbar DRG neurons (30%) than thoracic neurons.
The DRG of the lumbar and cervical enlargements contain more large-diameter, large fiber proprioceptive sensory neurons than either sacral or thoracic ganglia. Thus the data indicate that NT-3 has selective activity on large-diameter, large fiber DRG neurons.
10. EXAMPLE: INDUCTION OF FOS PROTEIN
AFTER TREATMENT WITH THE NEUROTROPHINS IN CEREBELLUM IN VITRO
10.1. MATERIALS AND METHODS
10.1.1. TISSUE DISSOCIATION AND CULTURE
Cerebella from timed-pregnant adult Sprague-Dawley rats (Zivic-Miller, PA) were removed at
gestation day 16 (E 16) and dissected, on ice, free from the surrounding tissues. Cerebella from 3-4 litters of the same age, determined by the crown-to-rump length, were pooled and washed three times in cold Ham's F 10, supplemented with 25 mM Hepes and L-glutamine, before being minced in a 35 mm tissue culture dish. A single cell suspension was obtained by incubating the tissue with 0.25% trypsin (GIBCO) in Ham's F 10 for 15 min at 37°C. The cell suspension was then transferred to a trypsin inhibitor solution, containing growth medium (see below) and 50 μg/ml deoxyribonuclease type I (Sigma). Following 5 min inactivation at room temperature the cells were dissociated in growth medium; BME (GIBCO) supplemented with 6 g/1 glucose (Sigma), 5% horse serum (GIBCO), 1% (v/v) N2 additives (Bottenstein, 1983 in "Advances in Cell. Neurobiol. Vol. 4, Federoff and Hertz, eds., Academic Press, New York pp. 333-379), 0.5% (v/v) glutamine (2 mM, GIBCO) and 0.25% (v/v) penicillin- streptomycin (10 U/ml and 10 μg/ml respectively,
GIBCO) , by passing the fragments repeatedly through a constricted tip of a Pasteur pipette. Single cell suspensions were collected after three titurations and subsequently centrifuged twice at 55 × g for 45 seconds. The loose pellets of cells were combined and resuspended in growth medium. The density and
viability of cells were assessed using a hemocytometer and trypan blue staining, respectively. Cells were diluted to approximately 106 cells/ml and plated in growth medium (see above) on 24-well (Falcon) culture dishes, precoated with polyornithine (10 μg/ml, Sigma) and laminin (10 μg/ml). The medium was changed to serumfree growth medium (defined medium) 5-6 hours after plating and then changed after four days. The cells were kept in 5% CO2 humidified environment at 37°C for 8 days. The plating density used for the experiments described in the present study was
1500-1600 cells/mm2.
10.1.2. FOS IMMUNOHISTOCHEMISTRY Cells were treated for 30-300 minutes with BDNF, NT-3, or NGF at a concentration of 0.1-1 ng/ml at the end of the culture period. Cells were then rinsed once in BME and prefixed for 10 min in 2% paraformaldehyde in BME before fixation at room temperature in 4% paraformaldehyde in PBS for 30 min. After permeabilization with 0.1% triton for 10 min and blocking in 10% normal goat serum (NGS) and 1% BSA for 90 min, the cells were incubated for 2 days at 4°C with anti-FOS antibody (Oncogene) diluted 1/2000 in PBS with 5% NGS. After several washes in PBS the cultures were incubated with biotinylated goat-anti-rabbit IgG (Vector lab, ABC-Elite Kit) at 1/1000 in PBS with 1% NGS for 90 min at room temperature. The ABC complex was diluted 1/200 in PBS and incubated for 40 min. As a substrate, 3,3-diaminobenzidine
hydrochloride (DAB; Sigma) was used at a final
concentration of 0.3 mg/ml in 0.2 M acetate/ 10 mM imidazole/ 80 mM NiSO4 buffer.
10.2. RESULTS
Neurotrophin 3 and BDNF induced C-FOS protein expression in a dose-dependent manner with time in culture (Figure 15 and Figure 16),
respectively. Maximal number of cells stained for C-FOS protein were found after 150 min. However, no response was found after treatment with NGF
(Figure 17). 11. EXAMPLE: EFFECT OF NEUROTROPHIN 3 IN THE E14 VENTRAL
MESENCEPHALIC CULTURE
This example discloses the efficacy of neurotrophin 3 in supporting the survival or
phenotypic marker expression of both the dopaminergic and GABA neuronal populations present in the
developing substantia nigra. 11.1. MATERIALS AND METHODS
11.1.1. TISSUE DISSOCIATION AND CULTURE
The preparation of cultures from E14 rat brain was carried out as described by Hyman, et al., 1991, Nature 350: 230-233. Briefly, all cultures were prepared from the ventral mesencephalon dissected from 14-day-old embryonic rats (E14). Typically, pooled tissue from two or three litters of rat embryos from timed-mated Sprague Dawley rats was trypsinized
(0.125%; Worthington) in F12 medium (Gibco) for 20 minutes at 37°C. After washing in growth medium containing 7.5% FCS, the tissue was centrifuged at low speed for 5 minutes and the pellet was dissociated by trituration. After allowing 1-2 minutes for
nondispersed cell clumps to settle, the single-cell suspension was seeded onto 35-mm dishes (precoated with polyornithine and laminin) containing growth medium to give a density of 5 × 104 cells cm-2. After an overnight incubation in MEM medium supplemented with glutamine (2 mM), glucose (6 mg/ml-1), penicillin G (0.5 U/ml-1), streptomycin (5 μg/ml-1) and FCS (7.5%) to allow cell attachment, cells were cultured in the presence or absence of BDNF in a serum-free, defined medium (Hyman et al., 1991, Nature 350:230-232) except that insulin was included at 20 ng/ml-1. To visualize dopaminergic cells, cultures were fixed with 4% paraformaldehyde, washed extensively, permeabilized with 0.02% saponin in Sorensen's buffer with 1.5% horse serum and stained with a mouse monoclonal
antibody to rat TH (Boehringer-Mannheim). Primary antibody binding was visualized using a Vectastain ABC kit (Vector Labs). 11.1.2. ASSAY FOR GAD ENZYME ACTIVITY
GAD enzyme activity was determined according to the method of Kimura and Kuriyama (1975, Jpn J. Pharm. 25:189-195) by measuring the release of 14CO2 from L-[1-14C] glutamic acid. Cells on 35 mm dishes were lysed with 30 μl of a solution containing 50 mM KH2PO4 (pH 7.2) and 0.25% Triton X-100, scraped and collected. Five microliters of the cell lysate was assayed for GAD enzyme activity. In a typical assay, the reaction mixture contained 0.57 mM of L-[1-14C] glutamic acid (NEN, NEC-715, 52.6 mCi/mmol), glutamic acid (3 mM), pyridoxal phosphate (0.2 mM) and AET (1 mM), in a KH2PO4 buffer (50 mM, pH 7.2). Under these reaction conditions, the enzyme reaction was found to be linear for up to 2.5 hours. The
incubation proceeded for a period of 2 hours at 37°C, and was terminated by injecting 25 μl of 8N H2SO4 into the reaction mixture. The incubation was then
continued for another 60.minutes. 14CO2 released was trapped in Hyamine base solution, and was counted.
11.1.3. ASSAY FOR GABA/DA UPTAKE Simultaneous uptake of GABA and DA was measured in the following incubation buffer:
125 mM NaCl, 5mM KCl, 5.6 mM glucose, 1mM CaCl,
1.2 mM KH2PO4, 1.2mM Mg SO4, 25mM HEPES, adjusted to pH 7.4 and stored at room temperature. On the day of use the incubation buffer was brought to 100 nM pargyline, 1mM ascorbate, 10nM aminooxyacetic acid and 2mM beta-alanine. The buffer was divided in four aliquots, of which two were brought to 5nM benztropine mesylate (BZT) and 1nM 2,4 diamino-n-butyric acid (DABA). Two aliquots (+ and - BZT and DABA) were warmed to 37°C and the remaining 2 were stored in a refrigerator. Each treatment group had five 35 mm dishes, 3 of which were marked with a (-) and two with a (+). All culture dishes were washed once with incubation buffer and once with warm buffer containing +/- BZT and DABA as appropriate. To each culture dish was added 0.8 ml of +/- BZT and DABA buffer prior to incubation at 37°C for 5 minutes; followed by the addition of 0.2 ml of 3H-DA (final concentration of 50 nM) and 14C-GABA (final concentration of 500nM). Uptake of 3H-DA + 14C-GABA was allowed to proceed at 37°C for 15 minutes. The culture dishes were then placed on ice and the incubation solution was
aspirated off. The culture dishes were then washed 3× with cold incubation solution (+/- BZT and DABA) and once with cold standard PBS. To each sample 0.5 ml of 0.5N NaOH was added and the samples were incubated at room temperature for at least one hour, upon which time the entire sample was added to 10 ml of Ultima gold and 3H and 14C were counted on program #15 on a Packard scintillation counter.
The cultures were washed three times with an incubation buffer having the following composition: 100 μM pargyline, 1 mM ascorbate, 10 μM aminooxyacetic acid and 2 mM-beta-alanine. Cultures were then preincubated for 5 minutes at 37ºC in incubation buffer; replicate cultures were preincubated in buffer additionally containing 5 μM benztropine (BZT) and 1 mM 2,4 diamino-n-butyric acid (DABA). 50 nM 3H-DA and 500 nM 14C-GABA were then added for 15 minutes at 37°C. The uptake of label was stopped by placing the dishes on a bed of ice and rinsing with ice coId buffer. The samples were solubilized with 0.5 N NaOH and 3H and 14C was measured by liquid scintillation counting. Specific uptake was defined as the
difference between that uptake measured in the absence of BZT and DABA (total) from the uptake measured in the presence of BZT and DABA.
Dopaminerigic markers were also analyzed via immunocytochemical staining for tyrosine hydroxylase (Hyman, et al., 1991, Nature 350: 230-233).
11.2. RESULTS AND DISCUSSION
A dose response test of NT-3 was carried out to determine if cells cultured in the presence of this factor would show an increased survival of
dopaminergic neurons over the course of 7 days in vitro (Figure 18). Treatment of cultures with NT-3 caused approximately a 2.5 fold elevation in the number of dopaminergic neurons (detected by staining with antibody to TH) after 7 days in vitro. This effect was saturable at a concentration of 5 μg/ml. At concentrations of 25 and 50 ng/ml, the effect was significantly reduced.
When high affinity 3H-DA uptake was measured in a dose response test of NT-3, there was
approximately a 2 fold increase in this activity after treatment of cultures with NT-3 for 7 days in vitro (Figure 19). This effect had a similar dose response profile to the TH immunocytochemical staining
experiment described supra.
To determine if NT-3 treatment of the cultures exerts effects on other populations of neurons other than the dopaminergic cells, assays which assess the activity and/or survival of GABAergic neurons were carried out. Cultures which had been exposed to varying concentrations of NT-3 for a period of 7 days in vitro were processed for the measurements of both high affinity GABA uptake and GAD activity.
NT-3 induced a maximal effect of a 2.8 fold increase in 3H-GABA uptake activity when included in the culture medium at a concentration of 20 ng/ml for a period of 7 days in vitro (Figure 20).
When GAD enzyme activity was measured in cultures exposed to varying concentrations of NT-3 for 7 days, a maximal increase of 2.3 fold was obtained for cultures maintained in the presence of 50 ng/ml NT-3 (Figure 21).
This example discloses that NT-3 effects both the dopaminergic and GABAergic neuronal
populations in the embryonic substantia nigra of the rat. In the case of the dopaminergic system, the results show similar fold effects in assays conducted to examine independent phenotypic marker activities, indicating that the NT-3 acts as a survival-promoting activity for these neurons. In the case of the
GABAergic population of neurons, there was a more pronounced increment in the high affinity GABA uptake activity than that observed for measurement of GAD activity. This set of data suggests that NT-3 may stimulate the GABA uptake activity of the cells (a measure of their metabolic activity) independently of exerting its effect on the GAD activity.
12. EXAMPLE: EFFECT OF NEUROTROPHIN-3 ON CHOLINE ACETYLTRANSFERASE (CAT)
ACTIVITY IN E17 CULTURES OF RAT
SEPTAL CHOLINERGIC NEURONS
12.1. MATERIALS AND METHODS
12.1.1. TISSUE DISSOCIATION AND CULTURE
The septal region from Sprague-Dawley rats after 17 days of gestation was dissected free from the surrounding tissue. Tissue fragments were pooled, washed three times with Hams F-10, and then
transferred to a 35mm tissue culture dish and minced. A single cell suspension was made by incubating the tissue with 0.25% trypsin for 20 minutes at 37°C.
Following the inactivation of the trypsin by a five minute incubation at room temperature in growth medium (infra), containing 50μg/ml deoxyribonuclease type 1 (Sigma), the cells were dissociated by passing the fragments repeatedly through the constricted tip of a Pasteur pipet. The dissociated cells were then centrifuged at 500 × g for 45 seconds. The
supernatant was removed and recentrifuged. The loose cell pellets were resuspended and combined in growth medium, and the cell yield was determined by use of a hemocytometer. Finally, the cells were plated into 6 mm wells which had been coated with polyornithine (10 μg/ml) and laminin (10 μg/ml). The cell viability was checked, after 24 hours in culture, by the ability of the cells to exclude trypan blue.
The normal growth medium, 5HS/N3, for cultures composed of neurons and glia contained:
5% (v/v) horse serum (Gibco), 1% N3 additives (v/v) (Romijn, et al., 1982, Dev. Brain Res. 2:583-589), 0.5% (v/v) glutamine (200 mM, Gibco), and 0.25% (v/v) penicillin-streptomycin (10,000 units/ml, 10,000 mcg/ml respectively, Gibco) in Dulbecco's modified Eagle's medium (DMEM). Neuronal-enriched cultures were prepared by replacing the growth medium, five to six hours after plating, with DMEM containing: 1% N3 additives, 0.5% glutamine, and 0.25% penicillin and streptomycin. In both conditions, treatment with cytosine arabinoside (1 μM for 24 hours) was used to limit glial cell proliferation.
12.1.2. ASSAY OF CHOLINE ACETYLATRANSFERASE
ACTIVITY
The growth medium was removed from the cultures by rinsing the cells twice with 100 μl of PBS. The cells were lysed via one freeze-thaw cycle and a 15 minute incubation at 37°C in 50 mM KH2PO4 pH 6.7, containing 200 mM NaCl and 0.25% (v/v) Triton X-100. Two microliters of the cell lysate was removed and assayed for CAT activity according to the microFonnum procedure (Fonnum, 1975, J. Neurochem. 24:407- 409). The final substrate composition consisted of 0.2 mM [14C] Acetyl-CoA (NEN, 54.4, mCi/mmol), 300 mM NaCl, 8 mM choline bromide, 20 mM
ethylenediaminetetraacetic acid, and 0.1 mM
neostigmine in 50 mM NaH2PO4 (pH 7.4) buffer. At these enzyme and substrate concentrations, the enzymatic reaction was linear for 90-120 minutes. The
specificity of the induction for choline
acetyltransferase was tested by the addition of a specific inhibitor of CAT activity, N-hydroxyethyl-4- (1-napthylvinyl)pyridium (HNP), during the assay
(White and Cavallito, 1970, J. Neurochem.
17:1579-1589).
12.2. RESULTS
Neurotrophin-3 slightly increased the level of CAT activity after a 7-day treatment period at a concentration of 100 ng/ml (Figure 22).
13. EXAMPLE: RETROGRADE AXONAL TRANSPORT
OF [125-I]NT-3 IN SENSORY NEURONS
The rationale for determining retrograde transport of NT-3 in NT-3 responsive neurons derives from studies showing that the related neurotrophin, nerve growth factor (NGF), is a target-derived, retrogradely transported survival factor for
peripheral sympathetic and sensory neurons (Thoenen and Barde, 1980, Physiolog. Rev. 60:1284).
Quantitative retrograde transport studies of exogenous (Hendry, et al., 1974, Brain Res. 68:103) and endogenous (Korsching and Thoenen, 1983, Neurosci. Lett. 39:1) NGF have shown that the transport is specific and saturable. The elucidation of transport mechanisms for NT-3 and related neurotrophins will give insight to (1) routes of administration of NT-3, and (2) specific classes of neurons which may respond to NT-3 in the course of regeneration or pathogenic degeneration.
13.1. MATERIALS AND METHODS
13.1.1. ANIMAL TREATMENTS
Male Sprague-Dawley rats (200-250 g) were anesthetized with a mix of chloral hydrate and
pentobarbital. The right sciatic nerve was exposed and a crush was made 0.3 cm distal to the tendon of the obturator internus muscle. [125I]NT-3 was injected into the crush site with a Hamilton syringe in a volume of 2 ul over a 2 minute time period. The wounds were sutured and the animals allowed to recover for 18 hours. At this time the rats were sacrificed and the fourth and fifth lumbar (L4 and L5) dorsal root ganglia (DRG) were removed from the right
(ipsilateral) and left (contralateral) sides, immersed in 1% SDS, and counted for 1 minute in a gamma
counter. For another group of animals the ganglia were excised and immersion fixed in 4%
paraformaldehyde for processing of DRG's for
autoradiography. Quantitative data are expressed as cpm in L4 plus L5 DRG and compared to L4, L5 cpm in the non-injected side.
13.1.2. [125-I]-LABELED NEUROTROPHIN PREPARATION
Six to 10 μg of purified NT-3 were iodinated by the lactoperoxidase method (Marchalonis, 1969,
Biochem. J. 113:299-305). Incorporation of iodine was routinely 70-80% and specific activities ranged from 150-200 cpm/pg. The NT-3 was brought up in 1.0% BSA containing 0.01% protamine sulfate in phosphate buffered saline.
13.2. RESULTS
[125I]NT-3 was shown to be retrogradely transported from the crushed adult rat sciatic nerve to the L4, L5 DRG (Figure 23). The transport was specific in that a 100-fold excess of unlabeled NT-3 was able to block 80-90% of the transport.
Interestingly, the transport of NT-3 was only
partially inhibited by a 100-fold excess of BDNF or NGF, indicating a component of transport specific to NT-3 that is not sensitive to the other neurotrophins.
14. EXAMPLE: THE USE OF RETROGRADE AXONAL
TRANSPORT OF NT-3 TO MONITOR SENSORY
NEURON DEFICIT IN PYRIDOXINE TREATED RATS
14.1. MATERIALS AND METHODS
14.1.1. ANIMAL TREATMENTS
Female Sprague Dawley rats (200-250 g) were administerecT'800 mg/kg pyridoxine-HCl
intraperitoneally daily for 12 days. After the injections, rats were anesthetized with chloral hydrate (170 mg/kg) mixed with pentobarbital (35.2 mg/kg). The right sciatic nerve was exposed and 2 μl of 125I-labeled NT-3 or BDNF or NGF was injected into the nerve at the level of the obturator internus tendon. Wounds were sutured and the rats allowed to recover for 18 hr. At this time rats were killed by decapitation and the lumbar 4th (L4) and 5th dorsal root ganglia (DRG) removed and placed in 4%
paraformaldehyde. The L4-L5 spinal cord segment was also dissected and counted in fixative. Samples were counted in a gamma counter for 1 minute and the counts per minute for L4 plus L5 DRGs were assessed.
14.1.2. [125I]-LABELED NEUROTROPHIN PREPARATION NT-3, BDNF and NGF were purified and radioiodinated using the lactoperoxidase method. Free iodine was removed from each preparation by dialysis. Specific activities for 125I-labeled neurotrophins ranged from 2800-5800 cpm/fmol. All 125-I-labeled proteins were 90-99% as active as their unlabeled counterparts as assessed in the E8 chick DRG neurite outgrowth assay.
14.2. RESULTS AND DISCUSSION
Figure 24 shows that all three labeled neurotrophins were transported to the ipsilateral (right) but not contralateral (left) DRG when injected into the right sciatic nerves of control rats. Figure 24 also shows that [125-I]NT-3 and [125I]BDNF transport in the DRG were reduced 71% and 60%, respectively, in pyridoxine treated rats (p<0.01). No significant change was observed for 125I-NGF in pyridoxine treated rats. Interestingly, the transport of NT-3 and BDNF was not significantly altered in the spinal cords of pyridoxine treated rats compared to controls
(Figure 25).
The results demonstrate that examination of retrograde transport of neurotrophins may give
diagnostic clues into the underlying neural
degenerative mechanisms in peripheral neuropathies, whether they be induced chemically (as in the case here), mechanically or genetically. 15. EXAMPLE: RETROGRADE TRANSPORT OF [125I]-NT-3 IN THE CENTRAL NERVOUS SYSTEM
15.1. MATERIALS AND METHODS
15.1.1. ANIMAL TREATMENTS
Male Sprague-Dawley rats were anesthetized with chloral hydrate-pentobarbital and fixed in a stereotaxic apparatus. Small volumes of [125I]-labeled trophic factors (0.2-0.5 μl) were injected slowly into the hippocampus or neostriatum by way of a
borosilicate glass micropipette. Equivalent amounts of [125I]-NGF was injected in control animals to verify the specificity of the patterns of transport within the CNS. The wounds were closed and the animals allowed to recover. Approximately 24 hours later, the animals were sacrificed, the brains removed, and specific areas of the CNS excised and counted for 1 minute in a gamma counter. In other experiments, the animals were perfused, the brains removed, sectioned and processed for film and emulsion autoradiography. Hippocampal injections were centered-in the dentate gyrus/CA4-hilar region. Striatal injections were located centrally in the rostral caudate-putamen.
15.1.2. [125-I]-LABELLED NEUROTHROPHIN PREPARATION
Neurotrophin-3 was radioiodinated as described supra in Example Section 14.1.2.
15.2. RESULTS AND DISCUSSION
Experiments involving microdissections and counts (Table 4) indicate that, as previously
demonstrated for NGF, NT-3 is retrogradely transported from the hippocampus to the neurons of the medial septum/diagonal band, although proportionately many fewer counts were transported for NT-3 than NGF. Film and emulsion autoradiographic experiments (Table 5) showed that labeling associated with both neurotrophic factors was well localized to magnocellular neurons of the medial septum and diagonal band, cells which are known to provide the cholinergic input to hippocampus. Magnocellular neurons were very densely labeled and quite numerous in NGF-injected animals compared to animals injected with NT-3. However, NT-3 was found also to be transported by a population of smaller neurons within the medial septum/diagonal band.
Labeling associated with transported NGF was localized exclusively to neurons that were
immunoreactive for both choline acetyl transferase (CAT) and the low affinity NGF receptor (LNGFR), confirming the identity of these cells as cholinergic. Like NGF, NT-3 was also transported by large
cholinergic neurons in the medial septum/diagonal band. However, the smaller cells which transported NT-3, but not NGF, were immunonegative for the above cholinergic markers. This observation indicates that the medial septum/diagonal band nuclei contain a population of non-cholinergic neurons which may be responsive to NT-3, but not NGF.
Experiments involving microdissections and counts (Table 4) indicate that both NGF and NT-3 are transported from the striatum to the ventral
mesencephalon, the region of the brain which contains the substantia nigra. This observation is novel with respect to both trophic factors. Examination of film autoradiograms support the above conclusion, clearly showing circumscribed areas of increased density overlying the substantia nigra. However, the pattern of distribution of NGF and NT-3 associated label is clearly different within this structure. In
experiments where NGF was injected, silver grains were diffusely distributed over the pars reticulate of the substantia nigra indicating anterograde transport of this trophic factor. Following NT-3 injections, aggregations of silver grains were located over neuronal cell bodies in the pars compacta, indicating that NT-3, but not NGF, was retrogradely transported by these cells. Virtually all cells which transported
NT-3 were immunoreactive for tyrosine hydroxylase, a marker for the dopaminergic neurons of the pars compacta.
Examination of film and emulsion autoradiograms clearly show that both NGF and NT-3 are transported to regions of the brain not previously suspected to contain neurons sensitive to these trophic factors.
For NGF, these novel areas appear to be rather limited. At present, in addition to the areas noted above, we have found transport for NGF only to the supramammillary nucleus of the hypothalamus after hippocampal injections.
On the other hand, NT-3 was transported to several additional sites in the rat forebrain. As is the case for NGF, we have demonstrated transport of radiolabelled NT-3 to the supramammillary nucleus, but here the pattern of labeling is more intense than is apparent for NGF. Additionally, NT-3 was transported to a number of brain regions which do not appear to transport NGF at all. After injection into the dentate gyrus of the hippocampus, a few labeled cells were present bilaterally within the CA4/hilus
(Table 5). Retrogradely labeled cells were also apparent within the dorsal thalamus (intralaminar and parafasicular/posterior nuclei) after injections into the striatum. TABLE 4
COUNTS OF RADIOIODINATED NT-3 and NGF
IN SPECIFIC BRAIN REGIONS
HIPPOCAMPAL INJECTIONS
Trophic HPC ( r ) HPC(l) MS/DB(r) MS/DB(l) Cerebellum Factor
NGF
T-9 401721 1106 2381 1835 136
NT-3
T-16 739701 557 193 37 14
STRIATAL INJECTIONS
Trophic Striatum Striatum V. Mes. V. Mes. Cerebellum
Factor (r) ( r) (r) (l)
NGF
T-24 588004 269 2041 94 64
T-25 652932 131 1868 137 35
NT-3
T-18 909235 67 1398 80 73
T-21 810140 133 1328 50 99
HPC, dorsal hippocampus; MS/OB, medial septum/diagonal band of Broca; V. Mes, ventral mesencephalon; r, right side; l, left side. All injections (striatum and hippocampus) were made on the right side of the brain. Numbers with T prefix in the trophic factor column are animal code numbers. All other numbers represent cpm in the brain area indicated.
TABLE 5
RETROGRADE NEURONAL LABELLING-HIPPOCAMPAL
INJECTIONS OF RADIOIODINATED NGF AND NT-3
Brain Area NGF NT-3
Basal Forebrain
Medial Septum ++++ ++
Diagonal Band (v) ++++ ++
Diagonal Band (h) ++ +
Basal Nuc. Meynert + - Hippocampus
Hilus/CA 4 - ++
CA 1 - -
CA 2 - -
CA 3 - ±
Dentate Gyrus ? ?
Subiculum - - Hilus/CA 4 - +
(contra)
Other
Supramam. ++ +++
Nucleus
Data reported above is derived from 6 emulsion
autoradiographic experiments. Pluses represent relative numbers of cells labeled in a given area, from "++++" indicating many labeled cells to "+" indicating a few. Minus signs indicates that no labeled cells were observed. A question mark indicates areas that were difficult to evaluate given their proximity to the injection site.
Various publications are cited herein that are hereby incorporated by reference in their entirety.

Claims

WHAT IS CLAIMED IS:
1. A method for treating a disease or disorder of the nervous system by augmenting the survival, growth or differentiation of nervous system cells that express the NT-3 receptor, comprising contacting the nervous system cells with NT-3 in an amount effective to promote the survival, growth or differentiation of the nervous system cells.
2. The method of Claim 1 in which the disease or disorder involves nervous system cells of the anterior nucleus of the olfactory bulb.
3. The method of Claim 1 in which the disease or disorder involves nervous system cells of the superficial layers of the neocortex.
4. The method of Claim 1 in which the disease or disorder involves nervous system cells of the nucleus of the lateral olfactory tract.
5. The method of Claim 1 in which the disease or disorder involves nervous system cells of the dentate gyrus.
6. The method of Claim 1 in which the disease or disorder involves nervous system cells of the CA1, CA3 or CA4 of the hippocampus.
7. The method of Claim 1 in which the disease or disorder involves nervous system cells of the caudate-putamen.
8. The method of Claim 1 in which the disease or disorder involves nervous system cells that are sensory neurons.
9. The method of Claim 8 in which the sensory neurons are located in the cervical spinal cord.
10. The method of Claim 8 in which the sensory neurons are located in the lumbar spinal cord.
11. The method of Claim 1 in which the disorder is Alzheimer's disease. Pick's disease, or Jacob-Creuzfeldt disease.
12. The method of Claim 1 in which the disorder is caused by a brain lesion.
13. The method of Claim 1 in which the disorder is Huntington's disease, or Parkinson-plus syndromes.
14. A method for treating a disease or disorder of the nervous system by inhibiting the survival, growth or differentiation of nervous system cells that express the NT-3 receptor, comprising contacting native NT-3 with
neutralizing anti-NT-3 antibody in an amount effective to inhibit the survival, growth or differentiation of the nervous system cells.
15. A method for treating a disease or disorder of the nervous system by inhibiting the survival, growth or differentiation of nervous system cells that express the NT-3 receptor, comprising delivering to the cellular source of native NT-3 production, an antisense or ribozyme molecule in an amount effective to inhibit expression of native NT-3.
16. The method of Claim 14 or 15 in which the disease or disorder involves nervous system cells of the anterior nucleus of the olfactory bulb.
17. The method of Claim 14 or 15 in which the disease or disorder involves nervous system cells of the superficial layers of the neocortex.
18. The method of Claim 14 or 15 in which the disease or disorder involves nervous system cells of the nucleus of the lateral olfactory tract.
19. The method of Claim 14 or 15 in which the disease or disorder involves nervous system cells of the dentate gyrus.
20. The method of Claim 14 or 15 in which the disease or disorder involves nervous system cells of the CA1, CA3 or CA4 of the hippocampus.
21. The method of Claim 14 or 15 in which the disease or disorder involves nervous system cells of the caudate-putamen.
22. The method of Claim 14 or 15 in which the disease or disorder is a neoplasm.
23. The method of Claim 22 in which the neoplasm is a malignant tumor.
24. A method for analyzing the distribution of NT-3 receptors in neurological tissue, comprising
(a) contacting the neurological tissue with a detectable compound that specifically binds to the NT-3 receptor; and (b) determining the binding of the detectable NT-3 compound to the neurological tissue in which binding indicates the presence of NT-3 receptors in the neurological tissue.
25. The method of Claim 24 in which the detectable compound is NT-3 labeled with a radioisotope, a radioopaque compound, a fluor or an enzyme.
26. The method of Claim 24 in which the detectable compound is an antibody labeled with a
radioisotope, a radioopaque compound, a fluor or an enzyme.
27. The method of Claim 24 which is conducted in vitro on a biopsy or autopsy specimen.
28. The method of Claim 24 which is conducted in vivo.
29. A method for analyzing the expression of NT-3 in neurological tissue, comprising
(a) contacting the neurological tissue with a detectable compound that specifically binds to NT-3; and
(b) determining the binding of the detectable compound to the neurological tissue in which binding indicates the expression of NT-3 by the neurological tissue.
30. The method of Claim 29 in which the detectable compound is an antibody labeled with a
radioisotope, a radioopaque compound, a fluor or an enzyme.
31. The method of Claim 29 which is conducted in vitro on a biopsy or autopsy specimen.
32. The method of Claim 29 which is conducted in vivo.
PCT/US1992/009652 1991-11-12 1992-11-12 Therapeutic and diagnostic methods based on tissue specific nt-3 expression and receptor binding Ceased WO1993009798A1 (en)

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CN110812532A (en) * 2019-08-20 2020-02-21 中山大学 A method for constructing a tissue engineering scaffold that targets and promotes the connection of the corticospinal tract to repair spinal cord injury

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WO1998016242A1 (en) * 1996-10-16 1998-04-23 Regeneron Pharmaceuticals, Inc. Method of enhancing the delivery of growth factors
WO1998021245A1 (en) * 1996-11-08 1998-05-22 Roche Diagnostics Gmbh Highly affine antibody against human bdnf, method for the production and use thereof
EP1050758A1 (en) * 1999-05-03 2000-11-08 Evotec BioSystems AG Methods of diagnosing or treating neuropsychiatric diseases on basis of increased cerebrospinal fluid levels of neurotrophin 3
WO2000067032A1 (en) * 1999-05-03 2000-11-09 Evotec Biosystems Ag Methods of diagnosing or treating depression
WO2001064247A3 (en) * 2000-02-29 2002-02-14 Cephalon Inc Method of treating cancer with anti-neurotrophin agents
US6548062B2 (en) 2000-02-29 2003-04-15 Cephalon, Inc. Method of treating cancer with anti-neurotrophin agents

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