WO1994016721A1 - Methods of treatment using ciliary neurotrophic factor - Google Patents

Methods of treatment using ciliary neurotrophic factor Download PDF

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WO1994016721A1
WO1994016721A1 PCT/US1994/001057 US9401057W WO9416721A1 WO 1994016721 A1 WO1994016721 A1 WO 1994016721A1 US 9401057 W US9401057 W US 9401057W WO 9416721 A1 WO9416721 A1 WO 9416721A1
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cntf
cntfrα
neurons
expression
mrna
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PCT/US1994/001057
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French (fr)
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Nancy Y. Ip
Stanley Wiegand
John Rudge
Beth Friedman
George D. Yancopoulos
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Regeneron Pharmaceuticals, Inc.
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Priority to AU61300/94A priority Critical patent/AU6130094A/en
Publication of WO1994016721A1 publication Critical patent/WO1994016721A1/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to methods of treatment of neurological disorders comprising administering ciliary neurotrophic factor (CNTF) to a patient in need of such treatment.
  • CNTF ciliary neurotrophic factor
  • the present invention provides for methods of treating corticospinal neuron disorders and disorders that involve the subependymal zone.
  • CNTF Ciliary neurotrophic factor
  • CNTFR ⁇ CNTF binding protein
  • CNTF and IL-6 are distantly related members of a family of factors that also includes leukemia inhibitory factor (LIF) /cholinergic differen- tiation factor (CDF) (Yamamori et al., 1989, Science J245:1412-1416) and oncostatin M (OSM) (Bazan, 1991, Neuron 2:197-208; Rose and Bruce, 1991, Proc. Natl. Acad. Sci. ⁇ 8 . :8641-8645) and that all of these factors utilize multi-component receptors involving shared signal transducing components (Gearing et al. , 1992,
  • LIF leukemia inhibitory factor
  • CDF cholinergic differen- tiation factor
  • OSM oncostatin M
  • CNTF and LIF/CDF appear to share an additional gpl30-related receptor component (hereon referred to as LIFR ⁇ ) , which was initially identified as a LIF binding protein (Gearing et al., 1991, EMBO J. 3 ⁇ :2839-2848).
  • LIFR ⁇ additional gpl30-related receptor component
  • gpl30 and LIFR/3 together comprise a functional LIF receptor complex (Gearing et al. , 1992, Science 255:1434-1437) , and that addition of the CNTFR ⁇ component to this complex is sufficient to convert a functional LIF receptor into a functional CNTF receptor.
  • CNTFR ⁇ appears to be the only component of the CNTF receptor complex that uniquely characterizes cells bearing functional CNTF receptors.
  • the present invention relates to methods of treatment of neurological disorders comprising administering CNTF to a patient in need of such treatment.
  • CNTFR ⁇ CNTF receptor subunit
  • studies revealed high levels of CNTR ⁇ expression during embryonic develop ⁇ ment in neuronal precursors in both the central and 5 peripheral nervous systems.
  • in situ hybridization revealed that CNTFR ⁇ was prominently expressed in a variety of areas of the central nervous system, particularly those associated with motor function, including the corticospinal neurons.
  • the present invention is also based on the discovery that CNTF levels abruptly increased follow ⁇ ing nervous system trauma, and remained elevated for at least twenty days. During the same time period, levels of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 were found to decrease. This data indicates that CNTF may be used as a diagnostic marker of nervous system injury.
  • BDNF brain-derived neurotrophic factor
  • the present invention provides for methods of treating corticospinal neuron disorders, such as those caused by cerebral infarc- tion, hemorrhage, tumor, trauma or infection and which may result in spasticity. Further embodiments provide for methods of treating disorders involving the subependymal zone, including those arising in prema ⁇ ture infants that may result in cerebral palsy.
  • N-terminal signal sequence and C-terminal hydrophobic sequence directing GPI-linkage are underlined.
  • panel B alignments between rat and human forms of both CNTFR ⁇ and IL6R ⁇ are shown to demonstrate remarkable evolutionary conservation of CNTFR ⁇ as compared to IL6R ⁇ ; asterisks signify identities of lower sequence with upper sequence.
  • FIG. 1 Expression of CNTF and its receptor components (including CNTFR ⁇ , gpl30 and LIFR ⁇ ) examined by northern analysis in various tissues from adult rat. Transcripts corresponding to CNTF, CNTFR ⁇ , gpl30 and LIFR3 indicated by the arrows, while migra ⁇ tion of ribosomal bands is indicated on the left. Thal./hypothal. , thalamus and hypothalamus; SCG, superior cervical ganglion; DRG, dorsal root ganglion; Hippo, neurons, hippocampal neurons obtained from E18 rat hippocampal neuron-enriched cultures. Note that cerebellar RNA used for probing with gpl30 and LIFR/3 was partially degraded. Figure 3.
  • Figure 4 In situ localization of CNTFR ⁇ mRNA to motor neurons in adult (A and B) and PI (C) spinal cord.
  • A. Upper panel, line tracing of a coronal section (corresponding to those used in mid and lower panels) of adult rat spinal cord: DH, dorsal horn; VH, ventral horn; LT, lateral tier motor column; MT, medial tier motor column; CG, central gray; DMG, dorsal medial gray; IG, intermediate gray.
  • Mid-panel dark field photomicrographs showing localization of CNTFR ⁇ mRNA to selective areas of ventral horn with antisense (AS) probe. Lower panel, no specific signal was obtained with sense (S) probe.
  • B. High power view of mid-panel from (A) showing localization of CNTFR ⁇ mRNA to motor neurons of adult rat spinal cord.
  • FIG. 5 In situ localization of CNTFR ⁇ mRNA in coronal sections of adult rat brain.
  • A-C Low-power magnification views of successi ⁇ vely more caudal coronal sections of adult brain, hybridized to CNTFR ⁇ antisense probe.
  • Arrows indicate prominent CNTFR ⁇ mRNA detected in subependymal (Se) layer of the lateral ventricle, layer V of cortex (V) , subiculum (Sb) , substantia nigra reticulate (SNR) , nucleus of the trapezoid body (Tz) , and trigeminal nucleus (TN) .
  • A Northern analysis of CNTF receptor component expression in tissues isolated from various stages of development (E9-P19) , including whole embryo (E) , placenta (P) , head (H) , body (B) , or spinal cord (Sp. cord) . Positions of CNTFR ⁇ , gpl30 and LIFR/3 trans ⁇ cripts indicated by arrows on the right, and ribosomal bands indicated on the left. Total RNA prepared from adult brain (A.BR.) was used as a control.
  • B Northern analysis of CNTFR ⁇ in spinal cord isolated from various stages of development, from embryonic day 14 (E14) to adult (AD) .
  • A&B Low-power magnification views of Ell (A) and E15 (B) embryo sections hybridized to CNTFR ⁇ antisense (AS) or sense (S) probes. High-power magnification views of the bracketed areas on left and right sides in B are shown in C and D, respectively.
  • FIG. 8 Neurotrophic activity in extracts from lesioned tissues. Tissue from lesioned cortex and hippocampus was collected at 3, 7, 10, and 20 days following lesion and extracts prepared as described in materials and methods. The soluble fraction from a 10,000 x g spin of each extract was assayed for neurotrophic activity on E8 chick ciliary ganglion neurons (A) or E8 chick dorsal root ganglion neurons (B) . Activity was expressed as trophic units per mg tissue.
  • FIG. 9 Expression of CNTF and CNTF receptor in lesioned tissues.
  • A. Total RNA was isolated from lesioned tissues at various times (1, 3, 7, 10 & 20 days) post-lesion as described in Experimental Procedures. Corres ⁇ ponding tissues from unoperated control or sha - operated control (sham) animals were used as controls. L, tissue contralateral to the lesion side, R, tissue ipsilateral to the lesion side, Cx, cortex, Hp, hippocampus. Migration of the ribosomal bands were indicated on the left side. The transcript size for CNTF was 1.2kB.
  • Figure 10 Expression of BDNF and NT-3 in lesioned tissues.
  • L hippocampal tissues contralateral to the lesion side
  • R hippocampal tissues ipsilateral to the lesion side.
  • Quantitation of the BDNF (A) and NT-3 (B) signals were expressed relative to sham-operated control levels.
  • Figure 11 In situ hybridization of CNTF and glial fibrillary acidic protein (GFAP) immunohistoche- mistry.
  • GFAP glial fibrillary acidic protein
  • a higher power photomicrograph (F, 25x) illustrates the disposition of hybridizing elements at the wound margin (gf, gelfoam).
  • An adjacent section immunostained for GFAP is shown in E (25x) . Note that strongly reactive cells comprising the astroglial
  • “scar” are distributed at the wound margin in much the same pattern as cell expressing CNTF mRNA.
  • the present invention relates to methods of treatment of neurological disorders comprising administering a therapeutically effective amount of CNTF to a patient in need of such treatment.
  • CNTF refers to a ciliary neuro ⁇ trophic protein having a sequence substantially as set forth in Figure 12 which may be obtained and purified using any method known in the art, including, but not limited to, those described in Sendtner et al. U. S. Serial No. 07/731,847 or Masiakowski et al., 1991, J. Neurochem. __!_.1003-1012.
  • Neurological disorders which may, according to the present invention, be treated using CNTF and diagnostic methods are discussed more fully below. 5.1. METHODS OF TREATMENT
  • the present invention is based, at least in part, on the results of experiments described in Section 6, infra f which identified targets, in the nervous system, for CNTF action. Such targets were recognized by their expression of a receptor subunit specific for CNTF binding, namely, the CNTFR ⁇ molecule.
  • CNTFR ⁇ was expressed in a diversity of locations in the central and peripheral nervous systems.
  • particularly prominent expression of CNTFR ⁇ was observed in layer V of the neocortex, the hippocampal formation (dentate gyrus, Ammon-s horn and subiculum) , the thalamus (in parti ⁇ cular the anterior thalamic nuclei) , the subependymal zone of the lateral ventricle, the substantia nigra pars reticulate, cerebellum, pontine nuclei, nucleus of the trapezoid body, motor nucleus of the trigeminal complex, and facial nucleus. Accordingly, CNTF may be used in the treatment of disorders involving these regions.
  • cortical layer V which contains the corticospinal neurons that give rise to the descending corticospiral tract; the motor nucleus of the trigeminal complex and the facial nucleus; and motor-related areas such as the anterior thalamus; substantia nigra; pontine nuclei and cerebellum.
  • CNTF in treating disorders of motor neurons has been disclosed (Masiakowski et al., Int. Publ. No. O91/04316, Int. Appln. No. PCT/US90/05241) .
  • the present invention provides, in particular, for the treatment of disorders that involve corticospinal neurons in humans, including those caused by infarc ⁇ tion, infection, tumor, hemorrhage, exposure to a toxic agent, nutritional deficiency, degenerative disease, neuronal storage diseases due to inborn metabolic errors or trauma.
  • Disorders which may be treated include, but are not limited to, stroke, Alzheimer's disease, Jakob-Creuzfeld disease.
  • Additional embodiments of the invention relate to the discovery that the subependymal zone of the lateral ventricle is a target for CNTF action.
  • the subependymal zone in the mouse but not in the human, contains a population of cells that are rapidly dividing.
  • CNTFR ⁇ expression in embryonic rats was studied, a comparable region, namely, the neuroepithelial lining of the brain vesicles, was found to express high levels of CNTFR ⁇ mRNA.
  • CNTF may be used to promote survival and/or proliferation of subependymal neurons in human patients in need of such treatment, including patients suffering cerebral infarction, infection, tumor, hemorrhage, exposure to a toxic agent, nutritional deficiency, degenerative disease, developmental disorder, or trauma. It may be particularly desirable to administer CNTF to premature infants, in which such treatment may result in diminution or prevention of cerebral palsy or mental retardation.
  • CNTF appears to play a role in nervous system injury. Accordingly, it may be used to promote the survival of neural grafts.
  • Pharmaceutical compositions for use according to the invention include CNTF comprised in a liquid, solid, or semi-solid solution.
  • a pharmaceutical composition may comprise CNTF in an aqueous solution, such as sterile water, saline, phosphate buffer, or dextrose solution.
  • CNTF may be comprised in a solid (e.g. wax) or semi-solid (e.g. gelatinous) formulation that may be implanted into a patient in need of such treatment.
  • CNTF may be supplied to a patient via a cellular implant.
  • CNTF may be administered via any appropriate route, including, but not limited to, subcutaneous, intravenous, intrathecal, intramuscular, intra-nasal, intra-arterial, etc. In order to effectively deliver CNTF to affected structures however, it may be desirable to administer the CNTF intrathecally.
  • the preferred dose of CNTF may be that dose that achieves a concentration in blood or cerebrospinal fluid which is equal to or greater than the saturating concentrations of CNTF in cell culture, which is about 1 nanogram per ml.
  • an anti-pyretic compound such as aspirin, acetaminophen, or non-steroidal anti-inflammatory drug may allow a patient to receive a higher dose by controlling side effects.
  • CNTF may be administered at a frequency that ameliorates the patient's clinical condition but is tolerated.
  • CNTF in a dose as set forth above, may preferably be administered between about 1-7 times, preferably 3 times, per week or by continuous infusion. More frequent dosing may be performed if the patient's clinical condition so requires and the patient does not exhibit substantial side-effects.
  • a PCR-labeled CNTF probe 600 bp
  • a random-hexamer labelled CNTFR ⁇ probe 400 or 800 bp
  • the human LIFR/3 probe used was generated by polymerase chain reaction from human placental cDNA (Gearing et al. , 1991, EMBO J. .10:2839-2848) .
  • IL6R ⁇ maintains less than 55% identity between human and rodent forms (Yamasaki et 0 al., 1988, Science 241:825-828; Baumann et al. , 1990, J. Biol. Chem. 265:19853-19862)
  • GM-CSF ⁇ component maintains less than 35% identity between human and rodent forms (Park et al., 1992, Proc. Natl. Acad. Sci. .89:4295-4299)
  • ra t CNTFR ⁇ was 94% identical
  • Figure IB contrasts the conservation maintained between rodent and human forms of CNTFR ⁇ with that of IL6R ⁇ ) .
  • RNA isolated from sources containing presumed targets of CNTF action including sympathetic ganglia, dorsal root sensory ganglia, retina, cultured hippocampal neurons and spinal cord) all expressed abundant levels of CNTFR ⁇ transcripts ( Figure 2A & C) .
  • An antisense probe for rat CNTFR ⁇ revealed significant CNTFR ⁇ expression speci ⁇ fically within the sympathetic neurons of superior cervical ganglion (SCG) ( Figure 3A) , sensory neurons of dorsal root ganglion (DRG) ( Figure 3B) , and parasympathetic neurons of ciliary ganglion ( Figure 3C) ; the majority, if not all, of the neurons in these ganglia expressed CNTFR ⁇ . Adjacent sections hybrid- ized to sense strand detected no signal ( Figure 3A, B & C) .
  • the prominent CNTFR ⁇ expression detected within neurons of the facial and trigeminal nuclei, and within the neurons of cortical layer V, deserves special mention.
  • the facial nucleus contains lower motor neurons that can be rescued by CNTF from retro ⁇ grade degeneration following facial nerve transection in newborn rats (Sendtner et al., 1990, Nature 345:440-441) , and our findings corroborate the sugges ⁇ tion that CNTF acts directly on this motor neuron population.
  • the trigeminal nucleus also contains lower motor neurons, and cortical layer V contains the upper motor neurons that give rise to the descending corticospinal tract, providing the direct cortical input for motor function.
  • CNTFR ⁇ expression in the adult brain suggests that CNTF might act on several different neuronal popula ⁇ tions, the prominent expression of CNTFR ⁇ mRNA in both upper and lower motor neurons, as well as in motor- related brain areas such as anterior thalamus, substantia nigra, pontine nuclei and cerebellum, is consistent with a unique role for CNTF in maintaining motor system function.
  • CNTFR ⁇ in heart and intestinal structures may argue for an effect of CNTF on non-neuronal cells early in development, the observed patterns are also consistent with CNTFR ⁇ expression marking neural crest derived precursors which are invading these structures at this time of development; in fact, patterns within embryonic intestine are consistent with CNTFR ⁇ expres ⁇ sion within the precursors of the developing enteric nervous system.
  • CNTFR ⁇ is quite widely expressed throughout the adult peripheral and central nervous systems, with most of the expression localized to neuronal cells; no significant hybrid ⁇ izing signals were observed in non-neuronal cells including satellite cells in DRG, astrocytes or oligodendrocytes in CNS white matter.
  • CNTFR ⁇ in the nervous system, its expression appears to be an especially prominent and universal marker of both upper and lower motor neurons in the newborn as well as in the adult.
  • both upper and lower classes of motor neurons degenerate in amyotrophic lateral sclerosis (Hughes, 1982, "Human Motor Neuron Diseases", L.P. Rowland, ed. , Raven Press, N.Y. pp.
  • CNTFR ⁇ the most surprising aspect of the current study concerns the high level expression of CNTFR ⁇ during early development, apparently by mitotically active neuronal precursors in both the neuroepithelium of the central nervous system and in neural crest derived progenitors in the peripheral nervous system; . these embryonic localizations are apparently also mirrored by CNTFR ⁇ expression in mitotically active subependymal cells in the adult CNS.
  • the ability of CNTF to induce growth inhibition and/or differentia ⁇ tion of certain neuronal and glial progenitors (Lillien et al., 1988, Neuron 1:485-494; Ernsberger et al., 1989, Neuron 2 .
  • CNTF may play a crucial and widespread role in the growth control and maturation of neuronal precursors.
  • mice Female Sprague-Dawley rats (approx. 200g) were anesthetized with chloral hydrate-pentobarbital solu ⁇ tion and placed in a sterotaxic instrument. Using aseptic surgical technique, a wide craniotomy was made (approximately 12 mm 2 ) on the right side exposing the dorsal surface of the brain from Bregma AP-2.5 to -5.5 and ML + 1mm to + 5mm. The dura was incised and approximately 25-30mm 3 volume of tissue comprising a portion of the dorsal hippocampus and the overlying cortex was removed by aspiration. The wound cavity was filled with gelfoam and skin incision closed with wound clips.
  • mice were perfused transcardially with heparinized saline and buffered 4% paraformaldehyde.
  • the brains were removed and post-fixed overnight at 4°C, and then transferred to a 30% sucrose -0.1 M phosphate buffer solution.
  • Frozen sections (30 ⁇ m) through the lesion area were cut in the coronal plane on a sliding microtome.
  • IMMUNOHISTOCHEMISTRY Series of sections taken at 180 ⁇ m intervals through the levels of the lesion were immunostained using rabbit polyclonal antibodies raised against Glial Fibrillary Acidic Protein (GFAP, 1:10,000 - DAKO) . Immunostaining was carried out on free- floating sections as described in Watson et al., 1986, Peptides 2:155-159. Briefly, sections were incubated in the primary antibodies overnight at 4°C. The tissue was then washed and the secondary antibody (biotinylated goat anti-rabbit, Vector Laboratories) was applied at a dilution of 1:1500 for one hour at room temperature.
  • GFAP Glial Fibrillary Acidic Protein
  • RNA ISOLATION AND ANALYSIS Total RNA was prepared by the lithium chloride precipitation method as described in Auffray and Rougeon, 1980, Eur. J. Biocem. 107:303-314.
  • RNA Five micrograms of RNA were electrophoresed on a formal ⁇ dehyde agarose gel and transferred to a nylon membrane (MSI) .
  • MSI formal ⁇ dehyde agarose gel
  • a rat CNTF cDNA probe containing the 600 bp- coding region was labelled by the polymerase chain reaction. Hybridization conditions were as described in Maisonpierre et al., 1990, Science 247:1446-1451. Band intensity was quantitated with a laser densito- meter (Pharmacia LKB) .
  • rat CNTF The coding region of rat CNTF (approx. 600 bp) was subcloned into the EcoRl site of Bluescript (Strategene) . Transcription of the antisense strand of the coding region was performed using a standard transcription protocol (Promega) and 5' [a- 35 S]-CTP (New England Nuclear, specific activity of 1,300 Ci/mmol) . Similarly, a riboprobe encoding the sense strand was used for controls. Coronal brain sections (8 ⁇ m) were thaw-mounted onto gelatin/polylysine-coated glass slides (Fisher) , fixed with 4% paraformaldehyde and acetylated prior to hybridization. Hybridization and washing conditions (Friedman et al., 1992, Neuron 1:295-305) .
  • Frozen tissue samples were thawed, homogenized in PBS and centrifuged at 10,000 x g for 15 minutes. Assays were carried out in 96 well microtiter culture plates (Costar A/2) which were coated with 100 ⁇ g/ml polyornithine (2 hrs. room temp.) followed by 10 ⁇ g/ml laminin (overnight at 37°C) and stored at 4°C. Prior to assay the plates were washed and DME plus 10% fetal calf serum added. Samples were serially diluted and an equal volume of neuronal cell suspension added to the wells.
  • Embryonic day 8 chick ciliary ganglia, dorsal root ganglia or nodose ganglia were dissected, dissociated and preplated on tissue culture plastic for 2 hrs. to remove non-neuronal cells (Varon et al., 1972, "Methods and Techniques of Neurosciences", R.Fried (ed.), Dekker, New York. pp. 203-229).
  • the supernatants, enriched to 80-95% neurons, were collected, the neurons counted and approximately 3000 neurons were seeded per A/2 well. After 40 hrs.
  • MTT mitochondria in living cells convert MTT [3- (4,4-diomethyl thiazol 2-yl)-2,5-diphenyl tetrazolium bromide] into an insoluble formazan product
  • MTT was added to the wells to a final concentration of 0.5 mg/ml and the plates incubated for a further 8 hrs.
  • NGF was purified according to the method of Darling and Shooter 1984, "Methods of Cell Culture", G. Sato et al. (eds.), New York, Alan R. Liss, pp.79-83, human recombinant basic FGF was purchased from Boehringer Mannheim (1123 149) and CNTF used was purified according to Masiakowski et al., 1991, J. Neurochem. 57:1003-1012. 7 .2 . RESULTS
  • Aspiration lesions were made of the dorsal hippocampus and overlying cortex in adult rats.
  • Neurotrophic activity in tissue bordering the wound site was determined by assaying tissue extracts on cultures of sensory and parasympathetic neurons of embryonic chicks (Varon, 1975, Exp. Neurol. 48:93- 134) .
  • An increase in survival promoting activity on ciliary neurons was observed in extracts of either cortical or hippocampal tissues bordering the lesion (Figure 8A) . This increase was apparent by 3 days after surgery, and remained elevated up to 20 days, the longest time point examined.
  • dissociated dorsal root ganglion cells ( Figure 8B) or nodose ganglion cells from embryonic chicks the lesioned tissue extracts also showed survival promoting activity. This activity was not blocked by a neutralizing antibody specific for NGF.
  • FGF and CNTF are known to exhibit survival promoting effects on chick ciliary ganglion neurons in culture.
  • passage of the lesion extract (obtained at 20 days post-lesion) through a heparin column did not diminish bioactivity, while it did block survival effects of exogenous bFGF ( Figure 8C) .
  • CNTF mRNA was increased to a maximal level of 5-fold 10 days after the lesion declining to 3-fold by 20 days.
  • No significant change in CNTF mRNA was observed in hippocampal or cortical tissues contralateral to the lesion site ( Figure 9B) .
  • Sham surgical procedures produced only a slight, but detectable increase in the level of CNTF mRNA in the underlying tissues.
  • Expression of CNTF receptor mRNA was also examined. However, no appreciable change in the expression of CNTF receptor was observed in either contralateral or lesioned tissues at the time points examined ( Figure 9C) .
  • the profile for the distribution of cells expressing CNTF mRNA corresponds to the distribution of reactive astrocytes at the wound site.
  • cells most strongly immunopositive for GFAP were concentrated at the margin of the wound, and decreased in number and staining intensity with distance from the lesion ( Figure 10E) .
  • Reactive astrocytes were also visible within the gel foam, accounting for the CNTF hybridization signal that we have observed in cells within the wound cavity.
  • Trophic activity on dorsal root ganglion neurons was also present in tissue extracts, but this activity was not blocked by addition of a neutralizing antibody to NGF.
  • gene expression for the neurotrophins BDNF and NT-3 decreased slightly at the time points examined.
  • Northern analysis has revealed moderately high levels of CNTF mRNA in optic nerve and olfactory bulb with substantially lower levels detectable in other brain regions (Ip et al., 1991, Soc. Neurosci. Abs. r7:1121; Stockli et al., 1991, J. Cell. Biol.
  • CNTF has been localized to subpopulations of astrocytes (Stockli et al. , 1991, J. Cell. Biol. 115:447-459) .
  • Our data reveals that the levels of CNTF mRNA in cerebral cortex and hippocampus are barely detectable unless they are subjected to traumatic injury. This raises the question as to what cells are responsible for this marked upregulation of CNTF in the CNS in vivo.
  • Within one day after trau ⁇ matic injury there is a marked influx of polymorpho- nuclear leucocytes into the wound site.
  • astrocytes By comparing neurotrophic factor mRNA levels in primary cultures of astrocytes, fibroblasts and macrophages, we have found that astrocytes alone possess the capacity to express CNTF mRNA at levels 65 fold higher than the levels in normal rat brain. In comparison, peritoneal macro- phages show no detectable CNTF mRNA and fibroblasts show only very low levels when the contaminating astrocyte signal is subtracted. Thus, the specific upregulation of CNTF by astrocytes suggests that these cells have more in common with reactive astrocytes than normal astrocytes in vivo .

Abstract

The present invention relates to methods of treatment of neurological disorders comprising administering ciliary neurotrophic factor (CNTF) to a patient in need of such treatment, as well as to methods of diagnosing structural damage to the nervous system comprising detecting increased levels of CNTF in a patient's tissue or body fluid. In non-limiting embodiments, the present invention provides for methods of treating upper motor neuron disorders and disorders that involve the subependymal zone, and for methods of diagnosing nervous system damage comprising detecting increased levels of CNTF in a patient's cerebrospinal fluid.

Description

METHODS OF TREATMENT USING CILIARY NEUROTROPHIC FACTOR
1. INTRODUCTION
The present invention relates to methods of treatment of neurological disorders comprising administering ciliary neurotrophic factor (CNTF) to a patient in need of such treatment. In non-limiting embodiments, the present invention provides for methods of treating corticospinal neuron disorders and disorders that involve the subependymal zone.
2. BACKGROUND OF THE INVENTION
2.1. TARGETS FOR CILIARY NEUROTROPHIC FACTOR Ciliary neurotrophic factor (CNTF) was identi¬ fied, purified and cloned based on its ability to sustain the survival of parasympathetic neurons derived from embryonic chick ciliary ganglia (Adler et al., 1979, Science 204: 1434-1436; Lin et al. , 1989, Science 246:1023-1025; Stockli et al., 1989, Nature 342:920-923) . CNTF subsequently has been found to support the survival of motor neurons (Arakawa et al. , 1990, J. Neurosci. .10.: 3507-3515; Sendtner et al., 1990, Nature 345:440-441; Oppenheim et al., 1991, Science 251: 1616-1618) , sympathetic neurons (Blottner et al., 1989, Neurosci. Lett. 105: 316-320; Saadat, 1989, J. Cell Bol. 108:1807-1816) , sensory neurons (Skaper and Varon, 1986, Brain Res. 389:39-46) , hippocampal neurons (Ip et al., 1991, J. Neurosci. .11:3124-3134), retinal neurons and septal as well as neostriata neurons (Hagg et al., 1992, Neuron 8.:145- 158) , in vitro and/or in vivo .
Despite these findings, questions remain as to whether the observed actions of CNTF result from direct effects on the neurons under investigation, or ■whether they are indirectly mediated via other neurons or cell types present in the experimental paradigms utilized (e.g. see Hagg et al., 1992, Neuron 8.:145- 158) .
Use of an "epitope-tagged" CNTF (Squinto et al., 1991, Neuron 5_:757-766) , allowed for the identifica¬ tion of a CNTF binding protein ("CNTFRα") (Davis et al., 1991, Science 253:59-63) which was almost exclusively expressed within the nervous system. CNTFRα displayed striking homology to one of the two receptor components (IL-6Rα) utilized by a hemopoietic cytokine, interleukin-6 (IL-6) . Subsequent studies have revealed that CNTF and IL-6 are distantly related members of a family of factors that also includes leukemia inhibitory factor (LIF) /cholinergic differen- tiation factor (CDF) (Yamamori et al., 1989, Science J245:1412-1416) and oncostatin M (OSM) (Bazan, 1991, Neuron 2:197-208; Rose and Bruce, 1991, Proc. Natl. Acad. Sci. ^8.:8641-8645) and that all of these factors utilize multi-component receptors involving shared signal transducing components (Gearing et al. , 1992,
Science 255:1434-1437) . In particular, all of these factors utilize receptor complexes that include gpl30 (Gearing et al., 1992, Science 255:1434-1437) , a protein initially identified as the second component of the IL-6 receptor complex that is absolutely required to initiate IL-6 mediated signal transduction (Taga et al., 1989, Cell J58.:573-581; Hibi et al. , 1990, Cell 3.:1149-1157; Murakami et al. , 1991, Proc. Natl. Acad. Sci. U.S.A. 88:11349-113531. CNTF and LIF/CDF (but not IL-6) appear to share an additional gpl30-related receptor component (hereon referred to as LIFRβ) , which was initially identified as a LIF binding protein (Gearing et al., 1991, EMBO J. 3^:2839-2848). Evidence suggests that gpl30 and LIFR/3 together comprise a functional LIF receptor complex (Gearing et al. , 1992, Science 255:1434-1437) , and that addition of the CNTFRα component to this complex is sufficient to convert a functional LIF receptor into a functional CNTF receptor. Thus the broad distributions of both gpl30 and LIFR0 would allow for the widespread actions of LIF throughout the body, while the limited distribution of CNTFRα (predominately to the nervous system) would restrict the actions of CNTF to a subset of all LIF- responsive cells. From an evolutionary standpoint, it is fascinating that generally acting factors with important roles during hemopoiesis share overlapping receptor components with a factor, CNTF, whose actions seem primarily limited to the nervous system. The inclusion of a functional LIF receptor within the CNTF receptor complex explains why CNTF-responding neuronal populations, when examined, generally display indistinguishable (and non-additive) responses to LIF (Rao et al., 1990, Dev. Biol. 139:65-74; Hall and Rao, 1992, TINS 15_:35-37; Martinou et al. , 1992, Neuron 8.:737-744) , but also raises questions whether some previously identified LIF-responsive neuronal popula¬ tions are true physiologic targets for LIF action, or whether CNTF is the relevant ligand involved. Due to the wide distribution of gpl30 and LIFR/3, as well as their involvement in receptor complexes for other cytokines, CNTFRα appears to be the only component of the CNTF receptor complex that uniquely characterizes cells bearing functional CNTF receptors. Thus studies that localize CNTFRα during development and in the adult, according to the present invention, can deli¬ neate potential targets of direct CNTF action, and provide insight into whether previously identified actions of LIF may be attributable to CNTF. 2.2. RESPONSE TO INJURY IN THE CENTRAL NERVOUS SYSTEM
Mechanical or chemical injury to the adult central nervous system (CNS) results in the formation s of an astroglial-mesenchymal scar which seals the wound site and restores the integrity of the parenchyma (Berry et al., 1983, Acta Neurochir.
(Supp.) 22.'-31-53; Clemente, 1955, "Regeneration in the
Central Nervous System", . F. indle (ed.), Charles 0 C. Thomas, Springfield, 111. pp. 147-161; Reier and Houle, 1988, "Advances in Neurology: Functional Recovery in Neurological Disease", S.G. axman (ed.) , Raven Press, New York, pp. 87-138). As the scar is formed, there is a transient increase in the level of 5 neurotrophic activity at the lesion site which promotes the survival of parasympathetic, sympathetic and sensory neurons in culture (Nieto-Sampedro et al. ,
1982, Science 217:860-861; Nieto-Sampedro et al. ,
1983, J. Neurosci. 3.:2219-2229) . The source and 0 identity of the molecules that contribute to the increase in neurotrophic activity have been unknown prior to the present invention.
3. SUMMARY OF THE INVENTION 5 The present invention relates to methods of treatment of neurological disorders comprising administering CNTF to a patient in need of such treatment.
It is based, at least in part, on the identifi- 0 cation of targets for CNTF action by experiments which examined the in vivo localization of a CNTF receptor subunit (CNTFRα) in rats. These studies revealed high levels of CNTRα expression during embryonic develop¬ ment in neuronal precursors in both the central and 5 peripheral nervous systems. Furthermore, in situ hybridization revealed that CNTFRα was prominently expressed in a variety of areas of the central nervous system, particularly those associated with motor function, including the corticospinal neurons. The present invention is also based on the discovery that CNTF levels abruptly increased follow¬ ing nervous system trauma, and remained elevated for at least twenty days. During the same time period, levels of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 were found to decrease. This data indicates that CNTF may be used as a diagnostic marker of nervous system injury.
In various embodiments, the present invention provides for methods of treating corticospinal neuron disorders, such as those caused by cerebral infarc- tion, hemorrhage, tumor, trauma or infection and which may result in spasticity. Further embodiments provide for methods of treating disorders involving the subependymal zone, including those arising in prema¬ ture infants that may result in cerebral palsy.
4. DESCRIPTION OF THE FIGURES
Figure 1. (A) Sequence of rat CNTFRα and (B) remarkable conservation with its human counterpart. In panel A, nucleotide and translated amino acid sequence of rat CNTFRα is depicted; presumed
N-terminal signal sequence and C-terminal hydrophobic sequence directing GPI-linkage are underlined. In panel B, alignments between rat and human forms of both CNTFRα and IL6Rα are shown to demonstrate remarkable evolutionary conservation of CNTFRα as compared to IL6Rα; asterisks signify identities of lower sequence with upper sequence.
Figure 2. Expression of CNTF and its receptor components (including CNTFRα, gpl30 and LIFRβ) examined by northern analysis in various tissues from adult rat. Transcripts corresponding to CNTF, CNTFRα, gpl30 and LIFR3 indicated by the arrows, while migra¬ tion of ribosomal bands is indicated on the left. Thal./hypothal. , thalamus and hypothalamus; SCG, superior cervical ganglion; DRG, dorsal root ganglion; Hippo, neurons, hippocampal neurons obtained from E18 rat hippocampal neuron-enriched cultures. Note that cerebellar RNA used for probing with gpl30 and LIFR/3 was partially degraded. Figure 3. In situ localization of CNTFRα mRNA to neurons in superior cervical ganglia (SCG) , dorsal root ganglia (DRG) and ciliary ganglia (CG) of adult rat; figure shows low-power dark-field photomicro¬ graphs of tissue sections of adult rat SCG (A) , DRG (B) , and CG (C) using antisense (AS) and sense (S) CNTFRα probes.
Figure 4. In situ localization of CNTFRα mRNA to motor neurons in adult (A and B) and PI (C) spinal cord. A. Upper panel, line tracing of a coronal section (corresponding to those used in mid and lower panels) of adult rat spinal cord: DH, dorsal horn; VH, ventral horn; LT, lateral tier motor column; MT, medial tier motor column; CG, central gray; DMG, dorsal medial gray; IG, intermediate gray. Mid-panel, dark field photomicrographs showing localization of CNTFRα mRNA to selective areas of ventral horn with antisense (AS) probe. Lower panel, no specific signal was obtained with sense (S) probe. B. High power view of mid-panel from (A) showing localization of CNTFRα mRNA to motor neurons of adult rat spinal cord.
C. Line tracing of a coronal section (corres¬ ponding to lower panels) of PI rat spinal cord. Lower panels depict dark field photomicrographs showing CNTFR mRNA using antisense (AS) probe and sense (S) control probe.
Figure 5. In situ localization of CNTFRα mRNA in coronal sections of adult rat brain. A-C. Low-power magnification views of successi¬ vely more caudal coronal sections of adult brain, hybridized to CNTFRα antisense probe. Arrows indicate prominent CNTFRα mRNA detected in subependymal (Se) layer of the lateral ventricle, layer V of cortex (V) , subiculum (Sb) , substantia nigra reticulate (SNR) , nucleus of the trapezoid body (Tz) , and trigeminal nucleus (TN) .
D. High-power magnification views showing hybridization of CNTFRα antisense (AS) probe to layer V of cortex (V) and subiculum (Sb) , while the sense (S) probe showed little or no signal.
E. High power view of the motor trigeminal nucleus using antisense (AS) and sense (S) CNTFRα probes. Figure 6. Developmental profile of expression of CNTF receptor components.
A. Northern analysis of CNTF receptor component expression in tissues isolated from various stages of development (E9-P19) , including whole embryo (E) , placenta (P) , head (H) , body (B) , or spinal cord (Sp. cord) . Positions of CNTFRα, gpl30 and LIFR/3 trans¬ cripts indicated by arrows on the right, and ribosomal bands indicated on the left. Total RNA prepared from adult brain (A.BR.) was used as a control. B. Northern analysis of CNTFRα in spinal cord isolated from various stages of development, from embryonic day 14 (E14) to adult (AD) .
C. Northern analysis of CNTFRα at embryonic day 14 (E14) was examined in liver (LIV) and brain (BR) . Figure 7. In situ localization of CNTFRα mRNA in sagittal sections from embryonic day 11 (Ell) and 15 (E15) embryos.
A&B. Low-power magnification views of Ell (A) and E15 (B) embryo sections hybridized to CNTFRα antisense (AS) or sense (S) probes. High-power magnification views of the bracketed areas on left and right sides in B are shown in C and D, respectively.
C. Dark-field photomicrograph showing prominent hybridization of CNTFRα antisense probe to neuroepi- thelial layer of the brain vesicles and developing thalamus in E15 embryo.
D. Dark-field photomicrograph showing prominent hybridization of CNTFRα antisense probe to developing DRG of E15 embryo; hybridization detected in the region between liver and DRGs may correspond to developing enteric neurons in the gut (see text) .
Figure 8. Neurotrophic activity in extracts from lesioned tissues. Tissue from lesioned cortex and hippocampus was collected at 3, 7, 10, and 20 days following lesion and extracts prepared as described in materials and methods. The soluble fraction from a 10,000 x g spin of each extract was assayed for neurotrophic activity on E8 chick ciliary ganglion neurons (A) or E8 chick dorsal root ganglion neurons (B) . Activity was expressed as trophic units per mg tissue. C) - Recombinant rat CNTF (10 ng/ l) , recombinant human FGF (10 ng/ml) and 20 day lesioned hippocampal extract were passed over a heparin column in the presence of 1M NaCl and the eluate assayed for activity on chick ciliary ganglion neurons.
Figure 9. Expression of CNTF and CNTF receptor in lesioned tissues. A. Total RNA was isolated from lesioned tissues at various times (1, 3, 7, 10 & 20 days) post-lesion as described in Experimental Procedures. Corres¬ ponding tissues from unoperated control or sha - operated control (sham) animals were used as controls. L, tissue contralateral to the lesion side, R, tissue ipsilateral to the lesion side, Cx, cortex, Hp, hippocampus. Migration of the ribosomal bands were indicated on the left side. The transcript size for CNTF was 1.2kB.
B. Quantitation of the CNTF signals in (A) was performed for both cortical and hippocampal lesion tissues, and was expressed relative to sham-operated control levels. c. Quantitation of the CNTFR signals was similarly performed for cortical and hippocampal lesion tissues, and was expressed relative to sham- operated controls. Dashed line represents value obtained for tissues from unoperated control animals relative to sham-operated controls.
Figure 10. Expression of BDNF and NT-3 in lesioned tissues.
Total RNA was isolated from lesioned hippocampal tissues at 3 or 7 days post-lesion. L, hippocampal tissues contralateral to the lesion side, R, hippocampal tissues ipsilateral to the lesion side. Quantitation of the BDNF (A) and NT-3 (B) signals were expressed relative to sham-operated control levels. Figure 11. In situ hybridization of CNTF and glial fibrillary acidic protein (GFAP) immunohistoche- mistry.
In situ hybridization using a CNTF antisense probe was carried out in normal (A, C) and lesioned (B,D,F) brains. Film autoradiograms of full coronal sections taken from normal and lesioned brains are shown in figures A and B respectively, illustrating the position and extent of the lesion. Darkfield photomicrographs (12.5x) of the cortex and hippocampus in normal and lesioned brains are depicted in panels C and D respectively. No specific hybridization signal is detectable in normal cortex or hippocampus (C) , but dense hybridization signal is present over numerous, small cells distributed along the borders of the wound (D) . Some cells which express CNTF mRNA are also present within the gelfoam which fills the wound cavity. A higher power photomicrograph (F, 25x) illustrates the disposition of hybridizing elements at the wound margin (gf, gelfoam). An adjacent section immunostained for GFAP is shown in E (25x) . Note that strongly reactive cells comprising the astroglial
"scar" are distributed at the wound margin in much the same pattern as cell expressing CNTF mRNA.
Figure 12. Amino acid sequence of human CNTF.
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods of treatment of neurological disorders comprising administering a therapeutically effective amount of CNTF to a patient in need of such treatment. The term "CNTF", as used herein, refers to a ciliary neuro¬ trophic protein having a sequence substantially as set forth in Figure 12 which may be obtained and purified using any method known in the art, including, but not limited to, those described in Sendtner et al. U. S. Serial No. 07/731,847 or Masiakowski et al., 1991, J. Neurochem. __!_.1003-1012. Neurological disorders which may, according to the present invention, be treated using CNTF and diagnostic methods are discussed more fully below. 5.1. METHODS OF TREATMENT
The present invention is based, at least in part, on the results of experiments described in Section 6, infraf which identified targets, in the nervous system, for CNTF action. Such targets were recognized by their expression of a receptor subunit specific for CNTF binding, namely, the CNTFRα molecule.
It was shown that CNTFRα was expressed in a diversity of locations in the central and peripheral nervous systems. However, particularly prominent expression of CNTFRα was observed in layer V of the neocortex, the hippocampal formation (dentate gyrus, Ammon-s horn and subiculum) , the thalamus (in parti¬ cular the anterior thalamic nuclei) , the subependymal zone of the lateral ventricle, the substantia nigra pars reticulate, cerebellum, pontine nuclei, nucleus of the trapezoid body, motor nucleus of the trigeminal complex, and facial nucleus. Accordingly, CNTF may be used in the treatment of disorders involving these regions.
Importantly, a number of these areas are involved with motor function, including cortical layer V, which contains the corticospinal neurons that give rise to the descending corticospiral tract; the motor nucleus of the trigeminal complex and the facial nucleus; and motor-related areas such as the anterior thalamus; substantia nigra; pontine nuclei and cerebellum.
The use of CNTF in treating disorders of motor neurons has been disclosed (Masiakowski et al., Int. Publ. No. O91/04316, Int. Appln. No. PCT/US90/05241) . The present invention provides, in particular, for the treatment of disorders that involve corticospinal neurons in humans, including those caused by infarc¬ tion, infection, tumor, hemorrhage, exposure to a toxic agent, nutritional deficiency, degenerative disease, neuronal storage diseases due to inborn metabolic errors or trauma. Disorders which may be treated include, but are not limited to, stroke, Alzheimer's disease, Jakob-Creuzfeld disease. Pick's disease, cerebral palsy, Down's syndrome, Huntington's chorea, cortical-basal ganglionic degeneration, Gerstmann-Straussler disease, familial dementia with spastic pareses, Friedereick's ataxia, other spino- cerebellar degenerations, and primary lateral sclerosis.
Additional embodiments of the invention relate to the discovery that the subependymal zone of the lateral ventricle is a target for CNTF action. The subependymal zone, in the mouse but not in the human, contains a population of cells that are rapidly dividing. When CNTFRα expression in embryonic rats was studied, a comparable region, namely, the neuroepithelial lining of the brain vesicles, was found to express high levels of CNTFRα mRNA. These two pieces of converging evidence indicate that CNTF may be used to promote survival and/or proliferation of subependymal neurons in human patients in need of such treatment, including patients suffering cerebral infarction, infection, tumor, hemorrhage, exposure to a toxic agent, nutritional deficiency, degenerative disease, developmental disorder, or trauma. It may be particularly desirable to administer CNTF to premature infants, in which such treatment may result in diminution or prevention of cerebral palsy or mental retardation.
As exemplified in Section 7 infra. CNTF appears to play a role in nervous system injury. Accordingly, it may be used to promote the survival of neural grafts. Pharmaceutical compositions for use according to the invention include CNTF comprised in a liquid, solid, or semi-solid solution. For example, a pharmaceutical composition may comprise CNTF in an aqueous solution, such as sterile water, saline, phosphate buffer, or dextrose solution. Alternati¬ vely, CNTF may be comprised in a solid (e.g. wax) or semi-solid (e.g. gelatinous) formulation that may be implanted into a patient in need of such treatment. In alternate embodiments of the invention, CNTF may be supplied to a patient via a cellular implant.
CNTF may be administered via any appropriate route, including, but not limited to, subcutaneous, intravenous, intrathecal, intramuscular, intra-nasal, intra-arterial, etc. In order to effectively deliver CNTF to affected structures however, it may be desirable to administer the CNTF intrathecally.
The preferred dose of CNTF may be that dose that achieves a concentration in blood or cerebrospinal fluid which is equal to or greater than the saturating concentrations of CNTF in cell culture, which is about 1 nanogram per ml. Further, the co-administration of an anti-pyretic compound, such as aspirin, acetaminophen, or non-steroidal anti-inflammatory drug may allow a patient to receive a higher dose by controlling side effects.
CNTF may be administered at a frequency that ameliorates the patient's clinical condition but is tolerated. For example, CNTF, in a dose as set forth above, may preferably be administered between about 1-7 times, preferably 3 times, per week or by continuous infusion. More frequent dosing may be performed if the patient's clinical condition so requires and the patient does not exhibit substantial side-effects. 6. EXAMPLE: IDENTIFYING TARGETS OF CNTF
ACTION IN THE ADULT AND DURING DEVELOPMENT
6.1. MATERIALS AND METHODS 6.1.1. ISOLATION OF RAT CNTFRα CDNA CLONES
Screening of an adult rat brain cDNA library in the Lambda-ZAP vector (Stratagene, Inc.) using human CNTFRα probes, isolation of rat CNTFRα cDNA phage clones, "zapping" procedures, and subsequent DNA sequence analysis were performed described in Maisonpierre et al., 1990, Science 247: 1446-1451; Maisonpierre et al. , 1991, Genomica JLO:558-568; and Ip et al., 1992, Proc. Natl. Acad. Sci. 89:3060-3064) .
6.1.2. NORTHERN ANALYSIS
Selected tissues were dissected from embryonic or adult Sprague-Dawley rats and RNA was isolated by LiCl precipitation or guanidinium thiocyanate extraction as described in Bothwell et al., 1990, "Methods for cloning and analysis of eukaryotic genes" (Boston, Massachusetts: Jones and Bartlett) and Chomczynski and Sacci, 1987, Anal. Biochem. 162: 156-159. For Northern analysis, a PCR-labeled CNTF probe (600 bp) and a random-hexamer labelled CNTFRα probe (400 or 800 bp) spanning the coding region were used. The human LIFR/3 probe used was generated by polymerase chain reaction from human placental cDNA (Gearing et al. , 1991, EMBO J. .10:2839-2848) .
6.1.3. IN SITU HYBRIDIZATION Two non-overlapping Pstl fragments (400 bp and 800 bp) or EcoRI fragments (600 bp) from the coding region of rat CNTFRα were subcloned into Bluescript (KS+) for the purpose of generating RNA probes. Radiolabelled antisense or sense strand RNA probes for rat CNTFRα was transcribed from linearized plasmids using a transcription kit purchased from Promega.
Specificity of the observed hybridization was confirmed by comparing the non-overlapping antisense
5 probes as well as the sense probes.
Sagittal or coronal cryosections (10 μm thick) of tissues from embryonic or adult rats were thawed, mounted onto poly-lysine coated slides, and kept frozen at -80°C until use. In situ hybridization was 0 performed according to a protocol described in
Friedman et al., 1992, "Regulation of CNTF expression in yelin-related Schwann cells jLn vivo" Neuron,
£:295-305.
6.2. RESULTS 5 6.2.1. CLONING OF RAT CNTFRα AND
REMARKABLE CONSERVATION WITH ITS HUMAN COUNTERPART
In order to explore the localization of CNTFRα in rat, we first molecularly cloned the rat counterpart 0 of the previously identified human CNTFRα. Several cDNA clones encoding rat CNTFRα were obtained by screening a rat brain cDNA library with probes derived from human CNTFRα. Comparison of the CNTFRα sequences between human and rat revealed a remarkably high 5 degree of conservation (Figure 1A) . This degree of conservation makes CNTFRα the most conserved member of the cytokine receptor superfamily described thus far. For example, while IL6Rα maintains less than 55% identity between human and rodent forms (Yamasaki et 0 al., 1988, Science 241:825-828; Baumann et al. , 1990, J. Biol. Chem. 265:19853-19862) , and while the GM-CSFα component maintains less than 35% identity between human and rodent forms (Park et al., 1992, Proc. Natl. Acad. Sci. .89:4295-4299), rat CNTFRα was 94% identical
_ J3c to its human counterpart (Figure IB contrasts the conservation maintained between rodent and human forms of CNTFRα with that of IL6Rα) .
Human CNTFRα has been shown to utilize an unusual glycosyl phosphatidylinositol linkage (GPI-linkage) to the membrane (Davis et al., 1991, Science 253:69-63) . The analysis here of multiple rat CNTFRα cDNA clones revealed that all encode the same C-terminus as described for human CNTFRα, arguing against additional non-GPI-linked forms of the CNTFRα receptor.
6.2.2. NORTHERN ANALYSIS OF CNTF RECEPTOR COMPONENTS IN ADULT RAT TISSUES
Consistent with the notion that CNTFRα is the component which uniquely characterizes CNTF-responsive populations, Northern analysis revealed that while
LIFR/S and gpl30 transcripts were ubiquitously distri¬ buted in all tissues examined, the expression of CNTFRα in the adult rat was highly restricted to the nervous system (Figure 2A & B) . Many regions of the nervous system examined, including olfactory bulb, cortex, hippocampus, thalamus, midbrain, hindbrain and cerebellum, spinal cord and peripheral nerve, showed abundant levels of CNTFRα transcripts (Figure 2A & B) . Since neuronal cell bodies are not present in peri- pheral nerve, the presence of CNTFRα transcripts in peripheral nerve implies synthesis by non-neuronal elements such as schwann cells or fibroblasts. Outside of the nervous system, significant levels of CNTFRα mRNA were only found in skeletal muscle (Davis et al., 1991, Science 253:59-63) . while low but detectable levels were also found in skin, liver, kidney and testes (Figure 2A & B) . Of particular importance, RNA isolated from sources containing presumed targets of CNTF action (including sympathetic ganglia, dorsal root sensory ganglia, retina, cultured hippocampal neurons and spinal cord) all expressed abundant levels of CNTFRα transcripts (Figure 2A & C) .
6.2.3. IN SITU HYBRIDIZATION TO LOCALIZE CNTFRα-EXPRESSING CELLS IN KNOWN TARGET AREAS OF CNTF ACTION, INCLUDING PERIPHERAL GANGLIA AND SPINAL CORD
To determine whether previously observed actions of CNTF result from direct effects on the neurons under investigation, or whether they are indirectly mediated via other neurons or non-neuronal cell types present in the experimental paradigms utilized, we performed in situ hybridizations to localize CNTFRα- expressing cells in known target areas of CNTF action. We first examined peripheral targets known to be responsive to CNTF, such as sympathetic, sensory and parasympathetic ganglia. An antisense probe for rat CNTFRα revealed significant CNTFRα expression speci¬ fically within the sympathetic neurons of superior cervical ganglion (SCG) (Figure 3A) , sensory neurons of dorsal root ganglion (DRG) (Figure 3B) , and parasympathetic neurons of ciliary ganglion (Figure 3C) ; the majority, if not all, of the neurons in these ganglia expressed CNTFRα. Adjacent sections hybrid- ized to sense strand detected no signal (Figure 3A, B & C) .
Both CNTF and LIF have the ability to promote survival of motor neurons in a variety of experimental paradigms (Sendtner et al. , 1990, Nature 345:440-441; Oppenheim et al., 1991, Science 251:1616-1618) . To determine whether motor neurons are direct targets of CNTF action, we attempted to localize CNTRFα expres¬ sion within the spinal cord. Although occasional neurons within the dorsal horn and elsewhere within the spinal cord gray matter displayed some specific CNTRFα expression, the CNTFRα in situ hybridizations performed on adult spinal cord were remarkable for the prominent signals observed in the ventral horn over large motor neurons of both the medial and lateral motor tiers (Figure 4A & B) . Similarly, in situ hybridizations performed on newborn (PI) spinal cords localized CNTFRα expression to the large motor neurons (Figure 4C) . These are the first data suggesting that spinal cord motor neurons remain responsive to CNTF in the adult, and also indicate that spinal cord motor neurons are direct targets of CNTF action throughout life.
6.2.4. DEFINING ADDITIONAL POTENTIAL TARGETS OF CNTF ACTION IN THE ADULT BRAIN
To define new potential targets of CNTF action, in situ hybridizations for the CNTFRα were performed on the adult brain (Figure 5) . Specific hybridization to neuronal, but not glial, populations were quite generally found throughout the brain, suggesting that CNTF could have actions on many different CNS neuronal populations. Particularly prominent hybridization was found in layer V of the neocortex (Figure 5A & B) , the hippocampal formation (dentate gyrus, Ammon's horn and subiculum) (Figure 5B) , the thalamus (in particular the anterior thalamic nuclei) (Figure 5b) , the subependymal zone of the lateral ventricle (Figure 5A) , the substantia nigra pars reticulate (Figure 5B) , cerebellum (Figure 5C) and within a number of brain- ste nuclei, including the pontine nuclei, the nucleus of the trapezoid body (Figure 5C) , the motor nucleus of the trigeminal complex (Figure 5D) and the facial nucleus. Higher magnification views of cortical and hippocampal regions (Figure 5B) , and of brainstem (Figure 5C) , revealed significant specific hybrid- ization localized to neuronal cell bodies in these areas.
The prominent CNTFRα expression detected within neurons of the facial and trigeminal nuclei, and within the neurons of cortical layer V, deserves special mention. The facial nucleus contains lower motor neurons that can be rescued by CNTF from retro¬ grade degeneration following facial nerve transection in newborn rats (Sendtner et al., 1990, Nature 345:440-441) , and our findings corroborate the sugges¬ tion that CNTF acts directly on this motor neuron population. The trigeminal nucleus also contains lower motor neurons, and cortical layer V contains the upper motor neurons that give rise to the descending corticospinal tract, providing the direct cortical input for motor function. Thus, while our examination of CNTFRα expression in the adult brain suggests that CNTF might act on several different neuronal popula¬ tions, the prominent expression of CNTFRα mRNA in both upper and lower motor neurons, as well as in motor- related brain areas such as anterior thalamus, substantia nigra, pontine nuclei and cerebellum, is consistent with a unique role for CNTF in maintaining motor system function.
6.2.5. CNTFRα IS PROMINENTLY EXPRESSED IN NEURONAL PRECURSORS DURING EMBRYONIC DEVELOPMENT
It is interesting to note that prominent CNTFRα hybridizing signal was observed in the region of subependymal zone of the lateral ventricle which in mouse comprises a population of cells that remain actively dividing, even in the adult (Morshead and van der Kooy, 1992, J. Neurosci. JL2.:249-256) . This finding was consistent with CNTF playing a role for neuronal precursors reminiscent of the roles he o- poietic cytokines play during hemopoiesis. To further pursue this possibility, we examined CNTFRα expression in the embryo. Northern analysis revealed that transcripts for the three CNTF receptor components were all present at high levels as early as embryonic day 11 (Figure 6) . While high levels of expression were observed for LIFRJ and gpl30 in the placenta, CNTFRα expression was absent in the placenta, and was present at high levels only in the embryo proper. in situ hybridization analysis of CNTFRα expres¬ sion during development revealed abundant CNTFRα mRNA in rat embryos, most notably in the neuroepithelial lining of the brain vesicles, as well as in the developing DRGs and spinal cord (Figure 7) ; this pattern of hybridization was apparent in the Ell through E15 embryos examined. Significant hybrid¬ ization was also apparent in the nasal region, in heart structures, and in intestine, with little expression in remaining head and body regions. The CNTFRα distributions defined by the in situ hybrid¬ izations were verified by Northern analysis of selected embryonic structures (Figure 6C) .
CNTFRα expression in the neuroepithelial lining of the ventricles during the time when most of these cells represent dividing neuronal precursors
(Frederiksen and McKay, 1988, J. Neurosci. 8.:1144- 1151) suggests an important role for CNTF in neuronal precursor development and maturation. Similarly, while LIF has previously been shown to support the generation and/or maturation of sensory neurons in cultured precursors derived from embryonic dorsal root ganglia (Murphy et al. , 1991, Proc. Natl. Acad. Sci. 8.:3498-3501) , CNTFRα expression in developing embryonic DRG (prior to the birth of post-mitotic sensory neurons) indicates that CNTF could have a similar role on sensory neuron precursors, and may in fact be the physiologically relevant ligand in this circumstance. Although the expression of CNTFRα in heart and intestinal structures may argue for an effect of CNTF on non-neuronal cells early in development, the observed patterns are also consistent with CNTFRα expression marking neural crest derived precursors which are invading these structures at this time of development; in fact, patterns within embryonic intestine are consistent with CNTFRα expres¬ sion within the precursors of the developing enteric nervous system.
6.3. DISCUSSION The recent cloning of a CNTF binding protein led to studies demonstrating that CNTF utilizes a multi- component receptor complex that includes components shared with the LIF and IL-6 receptor complexes. In this study we have molecularly cloned cDNAs encoding the rat version of the one subunit (CNTFRα) which uniquely characterizes CNTF responsive cells, and found that it is remarkably well conserved with its human counterpart; this degree of conservation between species makes CNTFRα the most highly conserved cytokine receptor identified thus far. We have used probes derived from the cloned rat CNTFRα cDNAs to explore the distribution of CNTFRα-expressing cells. This strategy has identified precise cellular targets of CNTF action in previously defined target tissues, and has also revealed new targets of CNTF action in the adult nervous system as well as in the developing embryo.
Our studies have revealed that CNTFRα is quite widely expressed throughout the adult peripheral and central nervous systems, with most of the expression localized to neuronal cells; no significant hybrid¬ izing signals were observed in non-neuronal cells including satellite cells in DRG, astrocytes or oligodendrocytes in CNS white matter. Interestingly, despite the rather widespread distribution of CNTFRα in the nervous system, its expression appears to be an especially prominent and universal marker of both upper and lower motor neurons in the newborn as well as in the adult. As both upper and lower classes of motor neurons degenerate in amyotrophic lateral sclerosis (Hughes, 1982, "Human Motor Neuron Diseases", L.P. Rowland, ed. , Raven Press, N.Y. pp. 61-73) , expression of CNTFRα by these cells may not only reflect potentially important shared characteristics of these cells, but may indicate that CNTF can provide trophic support for both classes of these susceptible motor neurons. The universally prominent expression of CNTFRα by all motor neuron populations once again raises an important question - whether CNTF is the long sought after muscle-derived motor neuron trophic factor. While CNTF expression in skeletal muscle is quite low (Stockli et al., 1989, Nature 3_42:920-923) , it is clearly detectable (Figure 2A) , and mechanisms capable of enhancing the trophic action of CNTF may be acti¬ vated in denervated skeletal muscle.
Perhaps the most surprising aspect of the current study concerns the high level expression of CNTFRα during early development, apparently by mitotically active neuronal precursors in both the neuroepithelium of the central nervous system and in neural crest derived progenitors in the peripheral nervous system; . these embryonic localizations are apparently also mirrored by CNTFRα expression in mitotically active subependymal cells in the adult CNS. The ability of CNTF to induce growth inhibition and/or differentia¬ tion of certain neuronal and glial progenitors (Lillien et al., 1988, Neuron 1:485-494; Ernsberger et al., 1989, Neuron 2.:1275-1284) , as well as the important roles of its cytokine relatives during hemopoiesis (Miyajima et al., 1992, Annu. Rev. Immunol. 1J):295-331) indicates that CNTF may play a crucial and widespread role in the growth control and maturation of neuronal precursors.
7. EXAMPLE: INJURY-INDUCED REGULATION OF CNTF mRNA IN THE ADULT RAT BRAIN
7.1. MATERIALS AND METHODS
7.1.1. ANIMALS AND SURGERY
Female Sprague-Dawley rats (approx. 200g) were anesthetized with chloral hydrate-pentobarbital solu¬ tion and placed in a sterotaxic instrument. Using aseptic surgical technique, a wide craniotomy was made (approximately 12 mm2) on the right side exposing the dorsal surface of the brain from Bregma AP-2.5 to -5.5 and ML + 1mm to + 5mm. The dura was incised and approximately 25-30mm3 volume of tissue comprising a portion of the dorsal hippocampus and the overlying cortex was removed by aspiration. The wound cavity was filled with gelfoam and skin incision closed with wound clips. In sham-operated animals, a full cranio¬ tomy was made, but the dura and underlying tissues were not ablated. Animals were allowed to survive for 1, 3, 7, 10 or 20 days post-operatively, at which time they were again anesthetized. The animals were then decapitated, the brains rapidly removed and blocked in the coronal plane. Hippocampal and cortical tissues immediately surrounding the wound site (within 1.5 - 2.0 mm), or the corresponding contralateral tissues, were dissected, snap frozen and stored at -80°C prior to northern analysis or bioassay. For in situ hybrid¬ ization, blocks of brain encompassing the lesion area were frozen in methylbutane and stored at -80°C prior to sectioning. For immunohistochemical studies, animals were perfused transcardially with heparinized saline and buffered 4% paraformaldehyde. The brains were removed and post-fixed overnight at 4°C, and then transferred to a 30% sucrose -0.1 M phosphate buffer solution. Frozen sections (30 μm) through the lesion area were cut in the coronal plane on a sliding microtome.
7.1.2. IMMUNOHISTOCHEMISTRY Series of sections taken at 180 μm intervals through the levels of the lesion were immunostained using rabbit polyclonal antibodies raised against Glial Fibrillary Acidic Protein (GFAP, 1:10,000 - DAKO) . Immunostaining was carried out on free- floating sections as described in Watson et al., 1986, Peptides 2:155-159. Briefly, sections were incubated in the primary antibodies overnight at 4°C. The tissue was then washed and the secondary antibody (biotinylated goat anti-rabbit, Vector Laboratories) was applied at a dilution of 1:1500 for one hour at room temperature. The tissue was then incubated in an avidin-biotinylated horseradish peroxidase (HRP) complex (Vectastain Elite, Vector Laboratories, 1:500 dilution) . The sections were reacted in a solution containing DAB (0.03%), H202 (0.003%) and nickel sulfate (100 mM) . Sections were mounted onto gelatin coated slides, dehydrated in graded alcohols, cleared in xylenes and coverslipped with DPX™. 7.1.3. RNA ISOLATION AND ANALYSIS Total RNA was prepared by the lithium chloride precipitation method as described in Auffray and Rougeon, 1980, Eur. J. Biocem. 107:303-314. Five micrograms of RNA were electrophoresed on a formal¬ dehyde agarose gel and transferred to a nylon membrane (MSI) . A rat CNTF cDNA probe containing the 600 bp- coding region was labelled by the polymerase chain reaction. Hybridization conditions were as described in Maisonpierre et al., 1990, Science 247:1446-1451. Band intensity was quantitated with a laser densito- meter (Pharmacia LKB) .
7.1.4. IN SITU HYBRIDIZATION
The coding region of rat CNTF (approx. 600 bp) was subcloned into the EcoRl site of Bluescript (Strategene) . Transcription of the antisense strand of the coding region was performed using a standard transcription protocol (Promega) and 5' [a-35S]-CTP (New England Nuclear, specific activity of 1,300 Ci/mmol) . Similarly, a riboprobe encoding the sense strand was used for controls. Coronal brain sections (8 μm) were thaw-mounted onto gelatin/polylysine-coated glass slides (Fisher) , fixed with 4% paraformaldehyde and acetylated prior to hybridization. Hybridization and washing conditions (Friedman et al., 1992, Neuron 1:295-305) .
7.1.5. ASSAY FOR NEUROTROPHIC ACTIVITY
Frozen tissue samples were thawed, homogenized in PBS and centrifuged at 10,000 x g for 15 minutes. Assays were carried out in 96 well microtiter culture plates (Costar A/2) which were coated with 100 μg/ml polyornithine (2 hrs. room temp.) followed by 10 μg/ml laminin (overnight at 37°C) and stored at 4°C. Prior to assay the plates were washed and DME plus 10% fetal calf serum added. Samples were serially diluted and an equal volume of neuronal cell suspension added to the wells. Embryonic day 8 chick ciliary ganglia, dorsal root ganglia or nodose ganglia were dissected, dissociated and preplated on tissue culture plastic for 2 hrs. to remove non-neuronal cells (Varon et al., 1972, "Methods and Techniques of Neurosciences", R.Fried (ed.), Dekker, New York. pp. 203-229). The supernatants, enriched to 80-95% neurons, were collected, the neurons counted and approximately 3000 neurons were seeded per A/2 well. After 40 hrs. neuronal viability was determined using the MTT assay in which mitochondria in living cells convert MTT [3- (4,4-diomethyl thiazol 2-yl)-2,5-diphenyl tetrazolium bromide] into an insoluble formazan product (Manthorpe et al., 1986, Brain-Res. 390:191-198; Mosmann, 1983, J. Immunol., Methods. j55_:55-63). MTT was added to the wells to a final concentration of 0.5 mg/ml and the plates incubated for a further 8 hrs. At this time all medium was removed and 120 μl of DMSO added, the formazan solubilized and the product read on a 96 well plate spectrophotometer at a test wavelength of 570 nm and a reference wavelength of 650 nm. In some cases, neurons were fixed with 2% glutaraldehyde and counted on an inverted-stage phase contrast microscope.
In these experiments, NGF was purified according to the method of Darling and Shooter 1984, "Methods of Cell Culture", G. Sato et al. (eds.), New York, Alan R. Liss, pp.79-83, human recombinant basic FGF was purchased from Boehringer Mannheim (1123 149) and CNTF used was purified according to Masiakowski et al., 1991, J. Neurochem. 57:1003-1012. 7 .2 . RESULTS
7.2.1. DETECTION OF NEUROTROPHIC ACTIVITY IN LESION TISSUES
Aspiration lesions were made of the dorsal hippocampus and overlying cortex in adult rats.
Neurotrophic activity in tissue bordering the wound site was determined by assaying tissue extracts on cultures of sensory and parasympathetic neurons of embryonic chicks (Varon, 1975, Exp. Neurol. 48:93- 134) . An increase in survival promoting activity on ciliary neurons was observed in extracts of either cortical or hippocampal tissues bordering the lesion (Figure 8A) . This increase was apparent by 3 days after surgery, and remained elevated up to 20 days, the longest time point examined. When assayed on dissociated dorsal root ganglion cells (Figure 8B) or nodose ganglion cells from embryonic chicks, the lesioned tissue extracts also showed survival promoting activity. This activity was not blocked by a neutralizing antibody specific for NGF.
Both FGF and CNTF are known to exhibit survival promoting effects on chick ciliary ganglion neurons in culture. However, passage of the lesion extract (obtained at 20 days post-lesion) through a heparin column did not diminish bioactivity, while it did block survival effects of exogenous bFGF (Figure 8C) . This suggests that the family of heparin binding growth factors did not contribute appreciably to the neurotrophic activity observed in hippocampal tissues at 20 days post-lesion.
7.2.2. REGULATION OF CNTF EXPRESSION FOLLOWING ASPIRATIVE LESION -
To investigate the possibility that CNTF itself was responsible for the increase in CNTF-like neurotrophic activity at the wound site, the level of CNTF mRNA in lesioned tissues was examined. In non- lesioned cortex and hippocampus, CNTF mRNA levels were very low, near the limit of detectability (Figure 9A) . There was, however, a striking increase in CNTF mRNA after lesion. CNTF mRNA was increased by 7-fold 3 days post-operatively in hippocampal tissues surround¬ ing the lesion site and remained elevated for up to 20 days after lesion (Figure 9B) . In cortical tissues surrounding the lesion site, CNTF mRNA was increased to a maximal level of 5-fold 10 days after the lesion declining to 3-fold by 20 days. No significant change in CNTF mRNA was observed in hippocampal or cortical tissues contralateral to the lesion site (Figure 9B) . Sham surgical procedures produced only a slight, but detectable increase in the level of CNTF mRNA in the underlying tissues. Expression of CNTF receptor mRNA was also examined. However, no appreciable change in the expression of CNTF receptor was observed in either contralateral or lesioned tissues at the time points examined (Figure 9C) .
In addition, we examined mRNA levels for two neurotrophic factors in the neurotrophin family - BDN- and NT-3. In contrast to CNTF, the expression of botl neurotrophins was decreased in lesioned hippocampus (Figure 10) and cortex and when compared to control levels. Our results on BDNF mRNA is consistent with the findings of Lindholm et al., (1992, J. Cell Biol. 117:395-400) who also found no change in the level of BDNF message after traumatic injury to the adult rat CNS. These data suggest that members of the neuro¬ trophin family are not primarily responsible for the increase in neurotrophic activity at the lesion site. 7.2.3. LOCALIZATION OF CNTF mRNA AT LESIONED SITE
Regional dissection of the tissues demonstrated that CNTF mRNA was unregulated only in those tissues immediately bordering the wound site. In tissues as little as 2 mm from the margins of the wound, CNTF mRNA levels were present only at sham-control levels. To examine the precise topography of cellular local¬ ization of CNTF mRNA in intact and lesioned CNS tissues, we have performed in situ hybridization for CNTF. The level of CNTF mRNA hybridization in intact brain was very low (Figure 11A & C) , with no detect¬ able signal present in cortex or hippocampus. Ten days following lesion, CNTF mRNA was markedly increased in regions immediately adjacent to the wound (Figure 11 & D) . Hybridization signal was present over small cells directly bordering the wound site (Figure 11F) , as well as over cells which had migrated into the gel foam pledget that filled the wound cavity.
The distribution of hybridizing cells was distinctly heterogeneous within the tissue bordering the wound cavity. By far, the greatest number of cells expressing CNTF mRNA were found at the margin of the wound, immediately bordering the gelfoam which filled the cavity (Figure 11D & E) . The proportion of cells expressing CNTF mRNA and the intensity of the hybridization signal decreased sharply with distance, such that little or no hybridization above background was detectable beyond 300-400 μm from the wound edge. Thus, the Northern blot analysis which utilized rather larger blocks of tissue surrounding the wound substan¬ tially underestimates the fold increase in CNTF mRNA in individual cells at the site of injury. Results utilizing a CNTF sense control probe revealed no specific hybridization in either intact or lesioned brain. The profile for the distribution of cells expressing CNTF mRNA corresponds to the distribution of reactive astrocytes at the wound site. At 10 days post-lesion, cells most strongly immunopositive for GFAP were concentrated at the margin of the wound, and decreased in number and staining intensity with distance from the lesion (Figure 10E) . Reactive astrocytes were also visible within the gel foam, accounting for the CNTF hybridization signal that we have observed in cells within the wound cavity.
7.3. DISCUSSION
In this study, we have demonstrated that follow- ing CNS injury there is a marked increase in the levels of CNTF mRNA immediately bordering the lesion, but not in areas distant to the wound site. This increase in CNTF gene expression parallels the increase in neurotrophic activity found in extracts of lesioned tissues from both hippocampus and cortex.
Trophic activity on dorsal root ganglion neurons was also present in tissue extracts, but this activity was not blocked by addition of a neutralizing antibody to NGF. In contrast to CNTF, gene expression for the neurotrophins BDNF and NT-3 decreased slightly at the time points examined. Taken together the data suggests that the known members of the neurotrophin family cannot account for the increase in trophic activity present at the wound site. in the normal adult rat CNS, Northern analysis has revealed moderately high levels of CNTF mRNA in optic nerve and olfactory bulb with substantially lower levels detectable in other brain regions (Ip et al., 1991, Soc. Neurosci. Abs. r7:1121; Stockli et al., 1991, J. Cell. Biol. 115:447-459) . In both optic nerve and olfactory bulb, CNTF has been localized to subpopulations of astrocytes (Stockli et al. , 1991, J. Cell. Biol. 115:447-459) . Our data reveals that the levels of CNTF mRNA in cerebral cortex and hippocampus are barely detectable unless they are subjected to traumatic injury. This raises the question as to what cells are responsible for this marked upregulation of CNTF in the CNS in vivo. Within one day after trau¬ matic injury there is a marked influx of polymorpho- nuclear leucocytes into the wound site. These cells mobilize macrophages (monocytes and ameboid microglia) which direct the wound healing response and recruit fibroblasts and astrocytes to form the glial- mesenchy al scar (Beck et al., 1983, J. Neuropathol. Exp. Neurol. 4J2:601-614; Cavanagh, 1970, J. Anat.
106:471-487; Nathan, 1987, J. Clin. Invest. 79:319- 326; Persson, 1976, Virchows Arch B. Cell Pathol. 2 .- 21-37) . It is known that macrophages express and release a wide range of biologically active factors after trauma (Nathan, 1987, J. Clin. Invest. 79:319- 326) such as the cytokines - IL13, IL6, TNFa, LIF and TGF-01 (Lindhol et al., 1992, J.Cell Biol. 117:395- 400; Minami et al. , 1991, Biochem. Biophys. Res. Commun. 176:593-598) . By comparing neurotrophic factor mRNA levels in primary cultures of astrocytes, fibroblasts and macrophages, we have found that astrocytes alone possess the capacity to express CNTF mRNA at levels 65 fold higher than the levels in normal rat brain. In comparison, peritoneal macro- phages show no detectable CNTF mRNA and fibroblasts show only very low levels when the contaminating astrocyte signal is subtracted. Thus, the specific upregulation of CNTF by astrocytes suggests that these cells have more in common with reactive astrocytes than normal astrocytes in vivo . In a similar paradigm of cortical brain injury, basic FGF has been shown to increase 4 hrs after injury and persist for at least 2 weeks (Frautschy et al., 1991, Brain Res. 553:291-299) . The expression of bFGF was increased in ependy al cells of the lateral ventricle as well as in macrophages at the wound site shortly after the injury. After 1 week the reactive astrocytes stained intensely for anti-bFGF. This suggests, at least for bFGF, that the sustained expression at the wound site may reflect a sequential cooperative expression among several distinct cell types rather than expression restricted to a single cell type. However, sustained elevated expression of a factor on the order of three weeks following trauma, even for basic FGF, is most likely due to reactive astrocytes. In this context, it is interesting to note that in our lesion paradigm, basic FGF does not appear to contribute appreciably to the trophic activities observed on DRG and ciliary ganglion neurons twenty days after lesion, as passage of the lesion extract over a heparin column did not result in the loss of trophic activity.
While the level of expression of CNTF is normally very low in most areas of the CNS, this trophic factor has recently been found to act on a variety of CNS cell types including several types of neurons (Arakawa et al., 1990, J. Neurosci. 1_:3507-3515; Ip et al. , 1991b, J. Neurosci. 11:3124-3134; Oppenhei et al., 1991, Science 251:1616-1618; Sendtner et al., 1990, Nature 345:440-441: Larkfors et al. , 1991, IBRO World Congress of Neurosci. Abs. 3.:46) and astrocytes (Lillien et al., 1988, Neuron 2:485-494). The widespread constitutive expression of CNTF receptor on cells in the adult rat brain (Ip et al., 1991a, Soc. Neurosci. Abs. 12:1121), the dramatic upregulation of CNTF around the wound and the lack of a signal peptide (Stockli et al. , 1989, Nature 342:920-923) suggest that the expression of CNTF is actively restricted to the wound site after trauma. This would serve to prevent widespread exposure of undamaged CNS tissues to high levels of this pleiotropic molecule and focus its actions at the site of maximum injury. This focal unpregulation of neurotrophic activity at the wound site correlates well with the enhanced survival of fetal CNS tissues when transplantation into the wound cavity is delayed for several days (Lewis and Cotman, 1980a, Brain Res. __§._307-330; Lewis and Cotman, 1980, J. eurosci. 2.:66-77; Manthorpe et al., 1983, Brain Res. 267:47-56) . Various publications are cited herein which are hereby incorporated by reference in their entirety.

Claims

WHAT IS CLAIMED IS:
1. A method of promoting the survival and/or proliferation of corticospinal neurons in a patient comprising administering, to the patient, an effective amount of a ciliary neurotrophic factor protein having a sequence substantially as depicted in Figure 12.
2. A method of promoting the survival and/or proliferation of neurons located in the subependymal zone in a patient comprising administering, to the patient, an effective amount of a ciliary neurotrophic factor protein having a sequence substantially as depicted in Figure 12.
3. The method of claim 2 in which the patient has suffered a cerebral infarction.
4. The method of claim 2 in which the patient is a premature infant.
5. A purified and isolated protein having a sequence substantially as set forth in Figure 1A.
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WO1991019009A1 (en) * 1990-06-01 1991-12-12 Regeneron Pharmaceuticals, Inc. The ciliary neurotrophic factor receptor
US5141856A (en) * 1989-01-05 1992-08-25 Synergen, Inc. Expression of purified ciliary neurotrophic factor

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US5141856A (en) * 1989-01-05 1992-08-25 Synergen, Inc. Expression of purified ciliary neurotrophic factor
WO1991019009A1 (en) * 1990-06-01 1991-12-12 Regeneron Pharmaceuticals, Inc. The ciliary neurotrophic factor receptor

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EUROPEAN JOURNAL OF BIOCHEMISTRY, Volume 201, issued 1991, A. NEGRO et al., "Cloning and Expression of human Ciliary Neurotrophic Factor", pages 289-294. *
JOURNAL OF NEUROCHEMISTRY, Volume 57, Number 3, issued September 1991, P. MASIAKOWSKI et al., "Recombinant Human and Rat Ciliary Neurotrophic Factors", pages 1003-1012. *
NATURE, Volume 358, issued 06 August 1992, M. SENDTNER et al., "Ciliary Neurotrophic Factor Prevents Degeneration of Motor Neurons in Mouse Mutant Progressive Motor Neuronopathy", pages 502-504. *
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