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WO2004037281A1 - Procedes de neuroprotection - Google Patents

Procedes de neuroprotection Download PDF

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Publication number
WO2004037281A1
WO2004037281A1 PCT/CA2003/001626 CA0301626W WO2004037281A1 WO 2004037281 A1 WO2004037281 A1 WO 2004037281A1 CA 0301626 W CA0301626 W CA 0301626W WO 2004037281 A1 WO2004037281 A1 WO 2004037281A1
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WIPO (PCT)
Prior art keywords
cntf
peptide
biologically
variant
active fragment
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PCT/CA2003/001626
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English (en)
Inventor
Mark A. Ozog
John Bechberger
Christian Naus
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The University Of British Columbia
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Priority to AU2003275846A priority Critical patent/AU2003275846A1/en
Publication of WO2004037281A1 publication Critical patent/WO2004037281A1/fr

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the invention relates, in part, to methods for neuroprotection.
  • Acute and chronic neurodegeneration in the central nervous system is believed to result from a general process termed "excitotoxicity.”," which is mediated by the action of glutamate.
  • Glutamate is a negatively charged amino acid used for excitatory synaptic transmission in the mammalian nervous system. Although the concentration of glutamate can reach the millimolar range in nerve terminals (Waelsch, H., 1951), its extracellular concentration is maintained at a low level to prevent neurotoxicity. As early as 1957, Lucas and Newhouse
  • glutamate can be toxic to neurons if presented at a high concentration. Later, Olney and Sharpe (1969) implemented the term "excitotoxicity" to describe the cytotoxic effect that glutamate (and other such excitatory amino acids) has on neurons when applied at high dosages. Physiologically, high levels of glutamate can be achieved by excessive release, inhibition of uptake, or both. Normally, a low concentration of extracellular glutamate is maintained by both neurons and astrocytes. Neurons store glutamate in intracellular stores and regulate its release (Reagan, R.F., 1994).
  • Such therapies include glutamate release • . _ inhibitors, glutamate receptor antagonists, Ca 2+ channel blockers, GABA receptor agonists, gangliosides, neurotrophic factors, calpain inhibitors, caspase inhibitors, free radical scavengers, irnmuno- and cell metabolism modulators.
  • Gap junctions are proteineous channels that directly link the cytosol of adjacent cells and allow intercellular passage of molecules of M ⁇ ⁇ 1200 (Kumar and Gilula, 1996).
  • the structural unit of the gap junction is the connexon which itself consists of protein subunits termed connexins (Cxs)(Kumar and Gilula, 1996). At least eight different Cxs have been identified in the CNS. The connexins (Cxs) are designated according to relative molecular mass and numerous isoforms have been identified, including at least eight within the CNS (Dermietzel and Spray, 1993; Rozental et al., 2000; Willecke et al, 2002).
  • Cx43 the major Cxn expressed by astrocytes
  • astrocytes have also been reported to be in neurons (Dermietzel and Spray, 1993; Nadarajah et al., 1996; Alvarez-Maubecin et al, 2000; Nedergaard, 1994; Micevych and Abelson, 1991; Micevych et al, 1996).
  • Astrocytes play a supportive role for neurons by providing crucial metabolites, maintaining favourable extracellular levels of neurotransmitters and ions, and helping constitute the blood-brain barrier (Hatten and Mason, 1986).
  • astrocytes also have the ability to readily divide. Normal astrocytes are highly coupled by gap junctions and express primarily Cx43 and to a lesser extent Cxs 26, 30, 31.1 and 40 (Dermietzel et al, 2000).
  • gap junctions can be altered by short-term and long-term regulation (reviewed by Giaume and McCarthy, 1996; Rouach and Giaume, 2001).
  • Short-term regulation occurs within milliseconds to minutes and is the result of a change in the biophysical properties of the channel.
  • Such channel gating is usually caused by post-translational processing such as phosphorylation or channel blocking.
  • Long-term regulation occurs over hours or days and consists of the formation or disappearance of gap junction channels as a result of changes to gene transcription and post-translational processes. While numerous agents have been identified to modulate short-term gap junction regulation (reviewed by Dhein, 1998), long-term regulators have not been extensively studied.
  • Gap junctional coupling can be impaired/blocked by administering various agents including carbenoxolone (3 -hydroxy- l l-oxoolean-12-en-30-oicacid 3 hemisuccinate; CBX) and 18 ⁇ -glycyrrhetinic acid (3-hydroxy-ll-oxo-18,20-olean-12-en-29-oic acid; AGA).
  • CBX is a water-soluble synthetic gap junction blocker and has an inactive analog, glycyrrhizic acid (GZA), which is a derivative of Glycyrrhiza glabra.
  • GZA glycyrrhizic acid
  • AGA is also derived from C glabra and also blocks gap junctions; the exact mechanism to which AGA and CBX block the junctions has not yet been deciphered. Previous studies have reported that these blocking agents have no effect on cell viability if presented at concentrations less than 100 ⁇ M.
  • Cx43 is upregulated in astrocytes treated with transforming growth factor- ⁇ l (Robe et al, 2000) but downregulated by both interleukin (IL)-l ⁇ (John et al, 1999) and transforming growth factor- ⁇ 3 (Reuss et al., 1998).
  • Fibroblast growth factor-2 has opposing effects on cells of the CNS, increasing Cx43 expression in neurons (SiuYi et al, 2001) but decreasing this gap junction component in astrocytes (Reuss et al., 1998; Reuss et al, 2000).
  • Prior research suggests that Cx43 is up-regulated following a CNS insult (Enkvist and McCarthy, 1994; Hossain et al, 1994).
  • Gap junctions could have a direct protective effect, allowing the astrocytic syncytium to buffer extracellular space from cytotoxic levels of metabolites and ions (such as glutamate and K + ).
  • gap junctions could promote the viability of energy- exhausted cells by allowing essential molecules (e.g. ATP and glucose) to move into areas of high demand.
  • essential molecules e.g. ATP and glucose
  • gap junctions could permit the movement of potentially toxic metabolites into cells that are already compromised by the insult.
  • Ciliary neurotrophic factor is a cytokine normally found within the cytosol of astrocytes. CNTF was originally identified, purified and cloned based upon its ability to support the in viiro survival of parasympathetic neurons (Barbin et al, 1984; Lin et al, 1989; Masiakowski et al., 1991; Stockli et al, 1989).
  • CNTF is a member of the IL-6 family that includes IL-6, IL-11, oncostatin M, cardiotrophin-1 and leukemia inhibitory factor (LIF)(Ip and Yancopoulos, 1992; Lin et al, 1989; Patterson, 1992; Stockli et al, 1989); all of these family members share the signaling receptor system of glycoprotein 130 (gpl30) and LIF receptor- ⁇ (LIFR ⁇ ) to initiate intracellular signaling cascades.
  • LIF leukemia inhibitory factor
  • the expression of CNTF is almost exclusively restricted to nervous tissue, where it is normally produced by astrocytes in the CNS and Schwann cells in the peripheral nervous system (Stockli et al, 1991).
  • CNTF is considered a "brain injury" cytokine based on the findings that: 1) astrocytes express and retain CNTF as a cytosolic protein which is only released following brain disturbances (Stockli et al, 1989; Stockli et al, 1991; Rudge et al., 1994a; Rudge et al.,
  • the CNTF specific receptor, CNTFR-alpha or CNTFR ⁇ is located on neurons (Ip et al, 1993a; Kahn et al, 1997; Maclennan et al, 1996) and post-insulted (reactive) astrocytes (Ip et al., 1993b; Rudge et al, 1995); and 3) expression of both CNTF and CNTFR ⁇ is upregulated following disturbances to the CNS (Asada et al., 1995; Asada et al, 1996; Duberley et al, 1995; Ip et al, 1993b; Lee et al, 1997; Lin et al, 1998; Nieto-Sampedro et al, 1982; Park et al, 2000; Rudge et al, 1994a; Wen et al, 1995a).
  • CNTFR ⁇ Upon release by injured astrocytes, CNTF binds to CNTFR ⁇ and subsequently recruits gpl30 and LIFR ⁇ through which a cascade of several signal transduction pathways is initiated.
  • CNTFR ⁇ can be cleaved (i.e., by PLC) from expressing cells and, as a soluble form, can bind CNTF and activate cellular pathways.
  • CNTF may bind its receptor (CNTFR ⁇ ; soluble or membrane bound form) and subsequently activate a cascade of signal transduction pathways.
  • CNTFR ⁇ soluble or membrane bound form
  • the predominant pathways activated by CNTF are the mitogen-activated protein kinase/extracellular signal- regulated kinase (MAPK/ERK) pathway and the j anus kinases/signal transducers of activated transcription (JAK/STAT) pathway (reviewed by Monville et al, 2001). Both MAPK/ERK and JAK/STAT pathways lead to direct transcriptional activation of various genes (Symes et al, 1994).
  • CNTFR ⁇ lacks ⁇ ransmembrane and cytoplasmic domains and is instead anchored to the cell surface by a glycosyl-phosphotidylinositol linkage (Davis, et al. 1991). This linkage can be cleaved by phospholipases and release the receptor as a soluble protein. Although the soluble receptor is functional, it requires CNTF to be released into the extracellular space by dying or compromised cells to which it can bind and subsequently activate the ubiquitously expressed ⁇ components of the receptor. Davis, et al. (1993b) demonstrated that administration of both the soluble receptor and CNTF to various hematopoietic cell lines mimicked the effect of leukemia inhibitory factor (LIF) on these cells.
  • LIF leukemia inhibitory factor
  • the hematopoietic cell lines were not normally responsive to CNTF alone and did not express the receptor. However, only hematopoietic cell lines expressing gpl30 and the ⁇ receptor for LIF responded to the combination of CNTF and its soluble receptor. The authors speculated that CNTFR ⁇ may participate in interactions between the nervous system and hematopoietic and other systems. Similarly, combination of the receptor and CNTF has been shown to promote macrophage chemotaxis in a manner similar to interleukin 6 (Kobayashi, H. & Mizisin, A.P., 2000).
  • This invention arises from the discovery that co-administration of a CNTF peptide and at a peptide that acts as a receptor for CNTF provides a neuroprotective effect on CNS tissue exceeding the neuroprotective effect of CNTF alone. These effects appear to be brought about by the action of a combination of the receptor and CNTF on mature or un-reactive astrocytes, or alternatively, neoplastic cells of astrocytic origin.
  • Neuroprotection provided by this invention includes protection from damage resulting from neural injury or insult and from neurotoxicity, including excitotoxicity.
  • neuroprotection provided by this invention will be useful in the treatment of acute and chronic neurodegenerative disorders that may involve excitotoxicity, for example glutamate excitotoxicity, including stroke/ischemia, trauma, epilepsy, Huntington's Disease, amyotrophic lateral sclerosis and hypoglycemic encephalopathy.
  • Neuroprotection provided by this invention may be brought about upon injured or diseased tissue or in a preventative fashion during or prior to events expected to lead to a neural insult.
  • the invention provides methods for providing neuroprotection; for inhibiting cell degeneration or cell death; for treatment or prophylaxis of a neurodegenerative disease; or for ameliorating the cytotoxic effect of a compound (for example, a excitatory amino acid such as glutamate; a toxin; or a prophylactic or therapeutic compound that exerts a cytotoxic side effect) in a subject in need thereof, by administering to the subject an effective amount of a CNTF peptide or a biologically-active fragment or variant thereof, or a nucleic acid molecule encoding the CNTF peptide or a biologically-active fragment or variant thereof; and a peptide that acts as a receptor for CNTF or a biologically-active fragment or variant thereof, or a nucleic acid molecule encoding a peptide that acts as a receptor for CNTF or a biologically- active fragment or variant thereof.
  • the methods of the invention include protection against excitotoxicity, for example glutamate excito
  • the subject for example, a human, may be suffering from neural insult or injury; or may be suffering from a condition selected from substance abuse, trauma, stroke, ischemia, Huntington's disease, Alzheimer's disease, Parkinson's disease, prion disease, variant Creutzfeld- Jakob disease, amyotrophic or hypoglycemic encephalopathy; or may be undergoing surgery or other intervention.
  • the subject may have a pre-existing condition which would benefit by neuroprotection or the patient may be treated to reduce deleterious effects of a concomitant or subsequent neural injury, such as may occur during surgery or other intervention.
  • the peptide that acts as a receptor for CNTF is soluble (e.g., soluble CNTFR ⁇ ).
  • the peptides are administered as a complex.
  • the invention also provides the use of a peptide having the activity of a CNTF peptide or a biologically-active fragment or variant thereof, and a peptide that acts as a receptor for CNTF or a biologically-active fragment or variant thereof for neuroprotection; for modulation of cell degeneration or cell death; for treatment or prophylaxis of a neurodegenerative disorder; or for ameliorating the cytotoxic effect of a compound.
  • the invention also provides the use of a peptide having the activity of a CNTF peptide or a biologically-active fragment or variant thereof, and a peptide that acts as a receptor for CNTF or a biologically-active fragment or variant thereof for preparation of a medicament for neuroprotection; modulation of cell degeneration or cell death; treatment or prophylaxis of a neurodegenerative disorder; or ameliorating the cytotoxic effect of a compound.
  • compositions including a CNTF peptide or a biologically- active fragment or variant thereof, and a peptide that acts as a receptor for CNTF or a biologically-active fragment or variant thereof for neuroprotection; modulation of cell degeneration or cell death; treatment or prophylaxis of a neurodegenerative disorder; or ameliorating the cytotoxic effect of a compound.
  • the uses of the invention include protection against excitotoxicity, for example glutamate excitotoxicity.
  • the invention provides a method of screening for a neuroprotective compound, by providing a first system including CNTF or a biologically-active fragment or variant thereof; providing a second system including a peptide that acts as a receptor for CNTF or a biologically-active fragment or variant thereof; providing a third system including CNTF or a biologically-active fragment or variant thereof, and a peptide that acts as a receptor for CNTF or a biologically-active fragment or variant thereof; contacting the first and second systems with a test compound; and determining whether the test compound modulates neuroprotection in the first or second system when compared to the third system.
  • the invention also provides a method of screening for a neuroprotective compound, by providing a system including CNTF or a biologically-active fragment or variant thereof, and a peptide that acts as a receptor for CNTF or a biologically-active fragment or variant thereof; contacting the system with a test compound; and determining whether the test compound modulates neuroprotection in the presence or absence of a gap junction blocker.
  • the invention also provides a method of screening for a nucleic acid molecule that modulates neuroprotection, by providing a neuronal or neuronal-associated cell; contacting the neuronal or neuronal-associated cell with CNTF or a biologically-active fragment or variant thereof; a peptide that acts as a receptor for CNTF or a biologically-active fragment or variant thereof; or a combination comprising the CNTF and the peptide that acts as a receptor for CNTF; and determining the level of nucleic acid molecule expression (for example, by a microarray), where a change in the level of nucleic acid molecule expression in the combination, when compared to the level of nucleic acid molecule expression in either CNTF or the CNTF receptor systems alone, is indicative of a nucleic acid molecule that modulates neuroprotection.
  • the neuroprotection may include protection of neuronal cells or neuronal-associated cells from injury or degeneration or increasing the expression level of a connexin (e.g. Cx43).
  • the system(s) may include neuronal or neuronal associated cells.
  • this invention also provides a method for treatment of a CNS tumor in a patient in need thereof, comprising administering to the patient an effective amount of the aforementioned complex or a pharmaceutical composition comprising the aforementioned complex.
  • This separate aspect also provides the use of a complex of a peptide having the activity of CNTF and a peptide having the function of a receptor for CNTF the treatment of neoplastic disorders.
  • This separate aspect also provides the use of the aforementioned complex for preparation of medicaments for treatment of neoplastic disorders.
  • This anti-neoplastic aspect of the invention is distinct from the neuroprotection aspect.
  • the separate aspect of this invention also provides a method for determining the susceptibility of a neoplastic cell to suppression by the aforementioned complex, comprising determining whether the cell expresses CNTF.
  • This method may further comprise determining whether the cell expresses CNTFR ⁇ .
  • the method may also further comprise isolating and culturing neoplastic cells to be tested, and may further comprise obtaining a sample of tissue from a subject comprising such cells to be tested.
  • the determining may be by any means for detecting the presence of, or quantification of expression, including detection and/or measurement of mRNA or detection and/or quantification of expressed protein.
  • Such detection and/or quantification may involve nucleic acid probing and detection of nucleic acids hybridized to a suitable probe or amplification of target nucleic acids using suitable primers.
  • Detection and/or quantification of expressed protein may involve immunological procedures whereby an antibody to the protein is employed in procedures known in the art.
  • Medicaments and pharmaceutical compositions for use in this invention will comprise one or more peptides having the activity of CNTF and one or more peptides capable of functioning as a CNTF receptor.
  • the peptide functioning as the receptor is a soluble peptide capable of binding CNTF.
  • Pharmaceutical compositions and medicaments of this invention may also comprise one or more pharmaceutically acceptable diluents, carriers, excipients or the like including those known in the art for formulation and facilitation of delivery of peptides to target cells or tissues, including CNS tissue.
  • neurodegenerative disorder is meant a disorder that is characterized by the death or loss of function of neuronal cells, also known as neurons.
  • a neurodegenerative disorder is characterized by the death or loss of function of neuronal- associated cells, such as astrocytes or other glial cells. The cell death may occur by apoptosis or by necrosis.
  • a neurogenerative disorder includes, without limitation, substance abuse, trauma, stroke, ischemia, Huntington's disease, Alzheimer's disease, Parkinson's disease, prion diseases, such as variant Creutzfeld- Jakob, amyotrophic lateral sclerosis (ALS), olivopontocerebellar atrophy, epilepsy, seizures, and hypoglycemic encephalopathy.
  • a neurodegenerative disorder also includes any disorder or condition that would benefit from neuroprotection.
  • Neuronal cells such as neurons
  • neuroprotection refers to protection of any nerve tissue, including neurons and neuronal-associated cells (such as astrocytes or other glial cells) from injury or degeneration.
  • the injury or degeneration may lead to cell death or loss of function.
  • the injury or degeneration may result from a general process termed "excitotoxicity,” which is mediated by the action of glutamate.
  • the injury or degeneration may be due to physical injury or trauma; a neurodegenerative disorder, including a hereditary genetic disorder; action of a compound, for example, a toxin or a drug, such as a therapeutic or prophylactic; or any other factor that results in nerve tissue injury or degeneration.
  • the neuroprotection may extend to at least a portion of affected or injured nerve tissue. For example, in stroke, while neurons within the core area of the ischemic insult may be unsalvageable, neurons within the surrounding penumbra have the potential to be rescued, since most neurons within the penumbra do not succumb to the ischemic insult until days following the stroke, and may benefit from the neuroprotective compositions of the invention.
  • the compounds including test compounds, or combinations of the invention, for example, a CNTF/soluble CNTF receptor combination, exhibit any integer between 10% and 90%, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or over 100%, neuroprotection when compared to no treatment, or treatment with either CNTF or soluble CNTF receptor alone.
  • “Modulating” or “modulates” means changing, by either increase or decrease. The increase or decrease may be a change of any integer value between 10% and 90%, or of any integer value between 30% and 60%, or may be over 100%, when compared with a control or reference sample or compound.
  • test compound is any naturally-occurring or artificially-derived chemical compound.
  • Test compounds may include, without limitation, peptides, polypeptides, synthesised organic molecules, naturally occurring organic molecules, and nucleic acid molecules.
  • a test compound can "compete" with a known compound or combination such as a CNTF/soluble CNTF receptor combination by, for example, interfering with upregulation of connexin protein or mRNA expression levels, e.g., Cx43, Cxs 26, Cxs 30, Cxs 31.1, or Cxs 40 levels, by interfering with the action of gap junction blockers, or by interfering with any biological response induced by the known combination, such as neuroprotection.
  • a test compound can exhibit any value between 10% and 200%), or over 500%, modulation when compared to a CNTF/soluble CNTF receptor combination, or other reference compound.
  • a test compound may exhibit at least any positive or negative integer from 10% to 200% modulation, or at least any positive or negative integer from 30% to 150% modulation, or at least any positive or negative integer from 60% to 100% modulation, or any positive or negative integer over 100% modulation.
  • a compound that is a negative modulator will in general decrease modulation relative to a known compound, while a compound that is a positive modulator will in general increase modulation relative to a known compound.
  • contacting is meant to submit an animal, cell, lysate, extract, molecule derived from a cell, or synthetic molecule to a test compound.
  • determining is meant analysing the effect of a test compound on the test system.
  • the means for analysing may include, without limitation, polymerase chain reaction, intercellular coupling assays, immunological assays (e.g, immunoprecipitation, immunofluorescence, ELISA, Western blotting), ultrastructural analysis, histological analysis, kinase assays, cell death assays, Northern or Southern blotting, gene array assays, animal models, or any other methods described herein or known to those skilled in the art.
  • immunological assays e.g, immunoprecipitation, immunofluorescence, ELISA, Western blotting
  • ultrastructural analysis e.g, histological analysis, kinase assays, cell death assays, Northern or Southern blotting, gene array assays, animal models, or any other methods described herein or known to those skilled in the art.
  • nucleic acid or “nucleic acid molecule” encompass both RNA (plus and minus strands) and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA.
  • the nucleic acid may be double-stranded or single-stranded. Where single-stranded, the nucleic acid may be the sense strand or the antisense strand.
  • a nucleic acid molecule may be any chain of two or more covalently bonded nucleotides, including naturally occurring or non-naturally occurring nucleotides, or nucleotide analogs or derivatives.
  • RNA is meant a sequence of two or more covalently bonded, naturally occurring or modified ribonucleotides.
  • RNA is phosphorothioate RNA.
  • DNA is meant a sequence of two or more covalently bonded, naturally occurring or modified deoxyribonucleotides.
  • cDNA is meant complementary or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase).
  • a cDNA clone means a duplex DNA sequence complementary to an RNA molecule of interest, carried in a cloning vector.
  • nucleic acids e.g., DNA or RNA
  • nucleic acids e.g., DNA or RNA
  • each nucleotide in a nucleic acid molecule need not form a matched Watson-Crick base pair with a nucleotide in an opposing complementary strand to form a duplex.
  • a nucleic acid molecule is "complementary" to another nucleic acid molecule if it hybridizes, under conditions of high stringency, with the second nucleic acid molecule.
  • the level of nucleic acid molecule expression may be measured by any technique known in the art, for example, by quantification of hybridization signals.
  • a change in the level of nucleic acid molecule expression may a change of any value between 10% and 200%, for example a change of 10%, 30%, 50%, 100%, or 150%, or over 500%, when compared to a reference system or compound.
  • a "microarray” generally refers to a high density nucleic acid molecule array, which may for example be used to monitor the presence or level of expression of a large number of genes or for detecting sequence variations, mutations and polymorphisms. Microarrays generally require a solid support (for example, nylon, glass, ceramic, plastic, etc.) to which the nucleic acid molecules are attached in a specified 2-dimensional arrangement, such that the pattern of hybridization to a probe is easily determinable.
  • a solid support for example, nylon, glass, ceramic, plastic, etc.
  • the invention provides, in part, methods for neuroprotection using a "Complex" or a combination of a CNTF peptide or a biologically-active fragment or variant thereof, and a peptide that as a receptor for CNTF, e.g., CNTFR-alpha, or a biologically-active fragment or variant thereof.
  • the Complex can increase cellular communication among CNS cells, and therefore increases the resilience of CNS cells to insults or injury.
  • Neuroprotection using the Complex or combination of the invention is more effective than that achieved in the absence of treatment (i.e., without applying exogenous agents) or by treatment with CNTF alone, or with soluble CNTF receptor alone.
  • Compounds for use in this invention include, without limitation, a peptide having the activity of CNTF; a peptide having the activity or function of a CNTF receptor, e.g., CNTFR- alpha; and combinations thereof.
  • Narious peptides and sequences of peptides having the activity of C ⁇ TF are known in the art and additional variants may be prepared by persons of skill in the art or as described herein. Examples of prior literature describing C ⁇ TF and homologous peptides include WO 91/04316 and United States Patents 4,997,929, 5,011,914 and 5,426,177.
  • Narious peptides and sequences of peptides having the activity of a C ⁇ TF receptor, including its soluble portion, are known in the art and additional variants may be prepared by persons of skill in the art or as described herein.
  • Examples of prior literature describing C ⁇ TF receptors, including their soluble portions and homologous peptides include Davis, et al. (1991); and United States Patents 5,426,177 and 5,849,897.
  • a soluble peptide having the activity of a CNTF receptor may be obtained commercially, for example, from R&D Systems, Minneapolis, MN, USA.
  • a soluble peptide having the activity of a CNTF receptor may also be obtained by, for example, cleaving the membrane linkage of a CNTF receptor using a phospholipase, or by recombinant techniques whereby a peptide having the activity of a CNTF receptor is mutated so that it is no longer capable of associating with membranes.
  • a peptide having the activity of a CNTF receptor is capable of binding CNTF.
  • a peptide having the activity of a CNTF receptor is also capable of binding gpl30 and/or the LIF receptor.
  • Antibodies to the receptor are known, including those described in United States Patent 5,892,003.
  • the CNTF and/or CNTF receptor may be human, or may be derived from any other animal, such as mouse, rat, rabbit, chicken, pig, or monkey.
  • the invention excludes polypeptide compounds composed of a fusion between CNTF and a CNTF receptor, for example, a full length CNTF receptor.
  • the invention includes polypeptides including CNTF fused with a soluble CNTF receptor.
  • a "protein,” “peptide” or “polypeptide” is any chain of two or more amino acids, including naturally occurring or non-naturally occurring amino acids or amino acid analogues, regardless of post-translational modification (e.g., glycosylation or phosphorylation).
  • amino acid sequence may include peptides or proteins that have abnormal linkages, cross links and end caps, non-peptidyl bonds or alternative modifying groups. Such modified peptides are also within the scope of the invention.
  • modifying group is intended to include structures that are directly attached to the peptidic structure (e.g., by covalent coupling), as well as those that are indirectly attached to the peptidic structure (e.g., by a stable non-covalent association or by covalent coupling to additional amino acid residues, or mimetics, analogues or derivatives thereof, which may flank the core peptidic structure).
  • the modifying group can be coupled to the amino-terminus or carboxy-terminus of a peptidic structure, or to a peptidic or peptidomimetic region flanking the core domain.
  • the modifying group can be coupled to a side chain of at least one amino acid residue of a peptidic structure, or to a peptidic or peptido- mimetic region flanking the core domain (e.g., through the epsilon amino group of a lysyl residue(s), through the carboxyl group of an aspartic acid residue(s) or a glutamic acid residue(s), through a hydroxy group of a tyrosyl residue(s), a serine residue(s) or a threonine residue(s) or other suitable reactive group on an amino acid side chain).
  • Modifying groups covalently coupled to the peptidic structure can be attached by means and using methods well known in the art for linking chemical structures, including, for example, amide, alkylamino, carbamate or urea bonds.
  • a "biologically-active fragment" of CNTF or a peptide having the activity or function of a CNTF receptor, e.g., CNTFR-alpha includes an amino acid sequence found in a naturally-occurring CNTF or a soluble CNTF receptor peptide that is capable of neuroprotection as described herein or known to those of ordinary skill in the art.
  • a "variant" of CNTF or a peptide having the activity or function of a CNTF receptor includes a modification, for example, by deletion, addition, or substitution, of an amino acid sequence found in a naturally-occurring CNTF or a soluble CNTF receptor peptide that is capable of neuroprotection as described herein or known to those of ordinary skill in the art.
  • Compounds can be prepared by, for example, replacing, deleting, or inserting an amino acid residue at any position of CNTF or a peptide having the activity or function of a CNTF receptor, e.g., CNTFR-alpha, with other conservative amino acid residues, i.e., residues having similar physical, biological, or chemical properties, and screening for the ability of the compound to modulate neuroprotection.
  • a CNTF receptor e.g., CNTFR-alpha
  • polypeptides of the present invention also extend to biologically equivalent peptides that differ from a portion of the sequence of the polypeptides of the present invention by conservative amino acid substitutions.
  • substitutions refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution can be made without substantial loss of the relevant function.
  • substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing.
  • amino acids means those L-amino acids commonly found in naturally occurring proteins, D-amino acids and such amino acids when they have been modified. Accordingly, amino acids of the invention may include, for example: 2-Aminoadipic acid; 3-Aminoadipic acid; beta-Alanine; beta-Aminopropionic acid; 2-Aminobutyric acid; 4- Aminobutyric acid; piperidinic acid; 6-Aminocaproic acid; 2-Aminoheptanoic acid; 2- Aminoisobutyric acid; 3-Aminoisobutyric acid; 2-Aminopimelic acid; 2,4 Diaminobutyric acid; Desmosine; 2,2'-Diaminopimelic acid; 2,3-Diaminopropionic acid; N-Ethylglycine; N- Ethylasparagine; Hydroxylysine; allo-Hydroxylysine; 3-Hydroxyproline; 4-Hydr
  • conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydrophilicity value (e.g., within a value of plus or minus 2.0, or plus or minus 1.5, or plus or minus 1.0, or plus or minus 0.5), where the following may be an amino acid having a hydropathic index of about -1.6 such as Tyr (-1.3) or Pro (-1.6) are assigned to amino acid residues (as detailed in United States Patent No.
  • conservative amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydropathic index (e.g., within a value of plus or minus 2.0, or plus or minus 1.5, or plus or minus 1.0, or plus or minus 0.5).
  • each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: He (+4.5); Nal (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gin (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).
  • conservative amino acid substitutions may be made using publicly available families of similarity matrices (Altschul, S.F. 1991. "Amino acid substitution matrices from an information theoretic perspective.” Journal of Molecular Biology, 219: 555- 665; Dayhoff, M.O., Schwartz, R.M., Orcutt, B.C. 1978. "A model of evolutionary change in proteins.” In “Atlas of Protein Sequence and Structure” 5(3) M.O.
  • conservative amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, Nal, Leu, He, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral: Gly, Ser, Thr, Cys, Asn, Gin, Tyr.
  • Conservative amino acid changes can include the substitution of an L-amino acid by the corresponding D-amino acid, by a conservative D-amino acid, or by a naturally-occurring, non- genetically encoded form of amino acid, as well as a conservative substitution of an L-amino acid.
  • Naturally-occurring non-genetically encoded amino acids include beta-alanine, 3-amino- propionic acid, 2,3-diamino propionic acid, alpha-aminoisobutyric acid, 4-amino-butyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine, citrulline, t-butylalanine, t- butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine, norvaline, 2- napthylalanine, pyridylalanine, 3-benzothienyl alanine, 4-chlorophenylalanine, 2- fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4- tetrahydro-isoquinoline-3-carboxylix acid, beta-2-thienylalan
  • conservative amino acid changes include changes based on considerations of hydrophilicity or hydrophobicity, size or volume, or charge.
  • Amino acids can be generally characterized as hydrophobic or hydrophilic, depending primarily on the properties of the amino acid side chain.
  • a hydrophobic amino acid exhibits a hydrophobicity of greater than zero, and a hydrophilic amino acid exhibits a hydrophilicity of less than zero, based on the normalized consensus hydrophobicity scale of Eisenberg et al(J. Mol. Bio. 179:125-142, 184).
  • hydrophobic amino acids include Gly, Ala, Phe, Nal, Leu, He, Pro, Met and Trp, and genetically encoded hydrophilic amino acids include Thr, His, Glu, Gin, Asp, Arg, Ser, and Lys.
  • ⁇ on-genetically encoded hydrophobic amino acids include t-butylalanine, while non-genetically encoded hydrophilic amino acids include citrulline and homocysteine.
  • Hydrophobic or hydrophilic amino acids can be further subdivided based on the characteristics of their side chains.
  • an aromatic amino acid is a hydrophobic amino acid with a side chain containing at least one aromatic or heteroaromatic ring, which may contain one or more substituents such as -OH, -SH, -C ⁇ , -F, -CI, -Br, -I, - ⁇ O 2 , -NO, - NH 2 , -NHR, -NRR, -C(O)R, -C(O)OH, -C(O)OR, -C(O)NH 2 , -C(O)NHR, -C(O)NRR, etc., where R is independently (C ⁇ -C 6 ) alkyl, substituted (C ⁇ -C 6 ) alkyl, (C ⁇ -C 6 ) alkenyl, substituted (C ⁇ -C 6 ) alkenyl, (C ⁇ -C 6 ) alkynyl
  • Aromatic amino acids include Phe, Tyr, and Trp, while non-genetically encoded aromatic amino acids include phenylglycine, 2-napthylalanine, beta-2- thienylalanine, 1, 2,3 ,4-tetrahydro-isoquinoline-3 -carboxylic acid, 4-chlorophenylalanine, 2- fluorophenylalanine3 -fluorophenylalanine, and 4-fluorophenylalanine.
  • An apolar amino acid is a hydrophobic amino acid with a side chain that is uncharged at physiological pH and which has bonds in which a pair of electrons shared in common by two atoms is generally held equally by each of the two atoms (i.e., the side chain is not polar).
  • Genetically encoded apolar amino acids include Gly, Leu, Nal, He, Ala, and Met, while non- genetically encoded apolar amino acids include cyclohexylalanine.
  • Apolar amino acids can be further subdivided to include aliphatic amino acids, which is a hydrophobic amino acid having an aliphatic hydrocarbon side chain.
  • Genetically encoded aliphatic amino acids include Ala, Leu, Nal, and He, while non-genetically encoded aliphatic amino acids include norleucine.
  • a polar amino acid is a hydrophilic amino acid with a side chain that is uncharged at physiological pH, but which has one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms.
  • Genetically encoded polar amino acids include Ser, Thr, Asn, and Gin, while non-genetically encoded polar amino acids include citrulline, ⁇ -acetyl lysine, and methionine sulfoxide.
  • An acidic amino acid is a hydrophilic amino acid with a side chain pKa value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include Asp and Glu. A basic amino acid is a hydrophilic amino acid with a side chain pKa value of greater than 7. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion. Genetically encoded basic amino acids include Arg, Lys, and His, while non-genetically encoded basic amino acids include the non-cyclic amino acids ornithine, 2,3,-diaminopropionic acid, 2,4-diaminobutyric acid, and homoarginine.
  • amino acids can be classified based on known behaviour and or characteristic chemical, physical, or biological properties based on specified assays or as compared with previously identified amino acids.
  • Amino acids can also include bifunctional moieties having amino acidlike side chains.
  • Conservative changes can also include the substitution of a chemically derivatised moiety for a non-derivatised residue, by for example, reaction of a functional side group of an amino acid.
  • substitutions can include compounds whose free amino groups have been derivatised to amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t- butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
  • free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides, and side chains can be derivatized to form O-acyl or O-alkyl derivatives for free hydroxyl groups or N-im-benzylhistidine for the imidazole nitrogen of histidine.
  • Peptide analogues also include amino acids that have been chemically altered, for example, by methylation, by amidation of the C-terminal amino acid by an alkylamine such as ethylamine, ethanolamine, or ethylene diamine, or acylation or methylation of an amino acid side chain (such as acylation of the epsilon amino group of lysine).
  • a substituted amide for example, groups of the formula -C(O)-NR, where R is (C ⁇ -C 6 ) alkyl, (C ⁇ -
  • the compound can be covalently linked, for example, by polymerisation or conjugation, to form homopolymers or heteropolymers.
  • Spacers and linkers typically composed of small neutral molecules, such as amino acids that are uncharged under physiological conditions, can be used. Linkages can be achieved in a number of ways. For example, cysteine residues can be added at the peptide termini, and multiple peptides can be covalently bonded by controlled oxidation. Alternatively, heterobifunctional agents, such as disulfide/amide forming agents or thioether/amide forming agents can be used.
  • the compound can also be linked to a another compound that can modulate a neuroprotective response.
  • the compound can also be constrained, for example, by having cyclic portions.
  • Peptides or peptide analogues can be synthesised by standard chemical techniques, for example, by automated synthesis using solution or solid phase synthesis methodology.
  • test extracts or compounds are not critical to the method(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available.
  • libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceanographic Institute (Ft. Pierce, FL, USA), and PharmaMar, MA, USA.
  • natural and synthetically produced libraries of, for example, candidate polypeptides are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods.
  • any library or compound is readily modified using standard chemical, physical, or biochemical methods.
  • Compounds identified as being of therapeutic, prophylactic, diagnostic, or other value may be subsequently analyzed using a murine stroke model, or any other animal model suitable for neuroprotection studies, such as animal models of neurodegenerative disease (see, for example, Jankowsky JL et al, 2002, Transgenic mouse models of neurodegenerative disease: opportunities for therapeutic development, Curr Neurol Neurosci Rep. Sep;2(5):457-64; Hodgson et al. (1999) Neuron 23, 181-192).
  • compositions for example, nucleic acid molecules, small molecules, peptides, or peptide analogues
  • a liposome for example, an adjuvant, or any pharmaceutically acceptable carrier
  • treatment with a compound according to the invention may be combined with more traditional and existing therapies for providing neuroprotection, or for treating or prevention a neurodegenerative disorder.
  • the CNTF peptide, or biologically-active fragment or variant thereof may be administered in the same formulation as the soluble peptide having the activity or function of a CNTF receptor, e.g., CNTFR-alpha, or biologically-active fragment or variant thereof, or may be administered as separate formulations. If administered in the same formulation, in some embodiments, the CNTF peptide, or biologically-active fragment or variant thereof, and the soluble peptide having the activity or function of a CNTF receptor, e.g., CNTFR-alpha, or biologically-active fragment or variant thereof, may be administered as a complex, for example, as a covalently bound complex, or a non-covalently bound complex.
  • a "complex" refers to a combination in which the relevant ingredients are capable interacting, such as binding of CNTF to a soluble peptide or compound having the activity or function of a CNTF receptor.
  • compositions may be employed to provide suitable formulations or compositions to administer the compounds to subjects suffering from or presymptomatic for a neurodegenerative disorder, or in need of neuroprotection.
  • Any appropriate route of administration may be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration.
  • Therapeutic formulations maybe in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
  • the compounds of the invention are delivered directly or indirectly to CNS tissue.
  • Methods for formulation and delivery of peptides to CNS tissue are known, including methods for administering CNTF to the brain as described in United States Patent application 10/073,658 published June 13, 2002.
  • Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • parenteral delivery systems for modulatory compounds include ethylene- vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • the compounds are administered to an individual in an amount sufficient to stop or slow neurodegeneration or to provide neuroprotection, depending on the disorder.
  • An "effective amount" of a compound according to the invention includes a therapeutically effective amount or a prophylactically effective amount.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as neuroprotection, or the inhibition or prevention of neurodegeneration.
  • a therapeutically effective amount of a compound may vary- according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects.
  • a prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as neuroprotection, or the inhibition or prevention of neurodegeneration.
  • a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount.
  • a preferred range for therapeutically or prophylactically effective amounts of a compound may be any integer from 0.1 nM-O.lM, 0.1 nM-0.05M, 0.05 nM-15 ⁇ M or 0.01 nM-10 ⁇ M. It is to be noted that dosage values may vary with the severity of the condition to be alleviated.
  • dosage regimens maybe adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions.
  • Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners.
  • the amount of active compound in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • an immunogenically effective amount of a compound of the invention can be provided, alone or in combination with other compounds, with an immunological adjuvant, for example, Freund's incomplete adjuvant, NSA3, dimethyldioctadecylammonium hydroxide, or aluminum hydroxide.
  • an immunological adjuvant for example, Freund's incomplete adjuvant, NSA3, dimethyldioctadecylammonium hydroxide, or aluminum hydroxide.
  • the compound may also be linked with a carrier molecule, such as bovine serum albumin or keyhole limpet hemocyanin to enhance immunogenicity.
  • Toxicity of the compounds of the invention can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LD100 (the dose lethal to 100% of the population). In some circumstances however, such as in severe disease conditions, it may be necessary to administer substantial excesses of the compositions.
  • C6 glioma cells obtained from American Type Culture Collection, were grown in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS), 10 ⁇ g/ml streptomycin and 10 units/ml penicillin. Twenty-four hours prior to and throughout, all cells were maintained in serum-reduced media (1% FBS). Where noted, cells were exposed to the following agents: vehicle (phosphate buffered saline, PBS), CNTF (20 ng/ml), CNTFR ⁇ (200 ng/ml) or Complex (CNTF + CNTFR ⁇ ).
  • vehicle phosphate buffered saline, PBS
  • CNTF ng/ml
  • CNTFR ⁇ 200 ng/ml
  • Complex CNTF + CNTFR ⁇
  • RT-PCR Reverse transcription- polymerase chain reaction
  • the cDNA product (1 ⁇ l) was mixed with Tris-HCl (pH 8.4; 20 mM), MgCl 2 (1.5 mM), dNTP (200 ⁇ M), Platinum Taq (2 U), and one set of oligonucleotide primers (200 ⁇ M) given in final concentrations in a final volume of 40 ⁇ l.
  • Samples were denatured for 5 min at 95 °C and then amplified for 30 cycles of 94°C/45 sec, 58°C/1 min, and 72°C/1 min. Twenty microlitres of each PCR sample was run on a 1.8 % agarose gel in parallel with a 1.5 kb DNA standard (Invitrogen Corp., Burlington, ON).
  • Cells were grown to 80% confluence and subsequently treated with agents every 24 h (with fresh media changes) for three days. Cells were then washed twice with PBS and fixed for 10 minutes with 70% ethanol containing 0.15 M NaCl. Following blockage of non-specific antibody binding using 10% normal goat serum in PBS for 1 h, cells were incubated with rabbit polyclonal anti-Cx43 antibody (1 :400 dilution, Sigma- Aldrich, Oakville, ON) for 1 h, rinsed with PBS and subsequently incubated with Alexa-Fluor-conjugated goat anti-rabbit IgG secondary antibody (1 :500 dilution, Molecular Probes, Eugene, OR).
  • C6 cells were treated with agents every 24 h (with fresh media changes) for three days, rinsed twice with PBS and scraped off the plates in lysis buffer (RIP A buffer supplemented with protease inhibitors (complete, Mini; Roche, Indianapolis, IN )). DNA in the lysate was sheared using a 22 gauge needle. Total cell lysate was collected following microcentrifugation at 10,000xg for 10 minutes and total protein concentration was determined using the BCA Protein Assay Kit (Pierce-BioLynx, Brockville, ON).
  • the membrane was incubated in anti-Cx43 antibody (1:400 dilution, Sigma- Aldrich) for 1 h, rinsed again with PBS, and then bathed in secondary antibody tagged with horseradish peroxidase (1 :20,000 dilution, CedarLane Lab. Ltd., Hornby, ON). Following three PBS rinses, the membrane was incubated in Supersignal (Pierce-BioLynx) and exposed to X-ray film to visualize antibody binding. To normalize protein loading the membranes were gently stripped of antibodies and immunoblotted for GAPDH (1: 20,000 dilution, CedarLane Lab. Ltd.).
  • Pre-Loading Gap junctional coupling of C6 cells was determined by the pre-loading method as described by Goldberg et al. (1995). Briefly, C6 cells were grown to confluence in 12-well plates. Twenty-four h prior to pre-loading, a media change containing the various agents was performed. The agents were present in all solutions throughout the procedures. Donor C6 cells were preloaded with dye solution (5 ⁇ M calcein-AM (Molecular Probes) and 10 ⁇ M Dil (Sigma- Alderich) in an isotonic (0.3 M) glucose solution) for 20 min in a humidified incubator (37°C, 5% CO 2 /95% air).
  • dye solution 5 ⁇ M calcein-AM (Molecular Probes) and 10 ⁇ M Dil (Sigma- Alderich) in an isotonic (0.3 M) glucose solution
  • donor cells were rinsed twice with glucose solution, trypsinized, suspended in growth media, and seeded onto recipient (unlabeled) cells at 1:500 ratio. After being maintained in the incubator for 3 h, cells were examined with a photomicroscope. Gap junctional communication was assessed by the passage of calcein from donor cells to the underlying recipient cells. Only cells that were coupled to at least one other cell were examined. Growth Curves
  • C6 cells were seeded into 12-well plates at 2 x 10 4 cells/well (designated Day -1). Cells received fresh media containing the various agents every 24 h commencing on Day 0. The number of cells in each well were counted on Days 0-4, 6 and 8 using a haemocytometer and trypan blue as a dilutant.
  • Results were expressed as means ⁇ standard error of the means of four or more independent procedures. Statistical comparisons were performed using one way analysis of variance with a P value of ⁇ 0.05 considered significant.
  • RT-PCR was performed on total RNA. Compared to GAPDH levels, a low amount of CNTF mRNA was present in C6 cells while CNTFR ⁇ mRNA could not be detected. Specificity of the CNTFR ⁇ primers was confirmed on adult mouse brain samples.
  • C6 cell cultures contain a mixed population of cells in regards to protein expression (Roser et al, 1991).
  • a heterogenous population of Cx43 -expressing cells was identified; some cells showed low Cx43 staining (-70% of the population) and others with very low staining (the remaining 30% of the population). While no detectable changes in the ratios of this heterogenous population were induced by CNTF, CNTFR ⁇ or Complex, immunocytochemical analysis revealed that Complex induced an increase in Cx43 compared to vehicle, CNTF and CNTFR ⁇ alone. This increase in Cx43 was detected in both the cytoplasm and at the cell membrane.
  • the C6 glioma cell line expresses the transcript for CNTF but not CNTFR ⁇ .
  • CNTF is administered in the presence of the soluble CNTFR ⁇ , an increase in Cx43 and gap junctional communication is detected in C6 cells, accompanied by a significant decrease in C6 cell growth rate.
  • Positive identification of CNTF mRNA in C6 glioma cells shows that this glioma is astrocytic in origin.
  • examination for CNTF provides an alternative method in determining whether a neoplastic cell has an astrocytic lineage.
  • This identification technique is highly useful, since the vast majority of astrocytomas, including both cell lines and in vivo biopsies, lack glial fibrillary acidic protein expression, a marker commonly used to identify and distinguish astrocytes from other CNS cell types (Deck et al, 1978).
  • C6 glioma cells are not immortalized reactive astrocytes. Although C6 cells lack CNTFR ⁇ , they respond to CNTF if co-administered with CNTFR ⁇ .
  • Cx43 in C6 cells induced by Complex shows that the CNTF signaling pathway can overcome the Cx43 expression deficiency in these cells.
  • Cells were plated onto 60 mm dishes (3 ml cell suspension) or 100 mm dishes (10 ml cell suspension) and maintained in a humidified incubator at 37°C in 95% air/5% CO 2 . Media was changed every 3 days thereafter in addition to shaking the cultures.
  • Astrocytes were treated with either vehicle (PBS), C ⁇ TF (20 ng/ml; R&D Systems), soluble C ⁇ TFR ⁇ (200 ng/ml; R&D Systems), or Complex (20 ng/ml C ⁇ TF + 200 ng/ml C ⁇ TFR ⁇ ).
  • vehicle PBS
  • C ⁇ TF 20 ng/ml; R&D Systems
  • soluble C ⁇ TFR ⁇ 200 ng/ml; R&D Systems
  • Complex 20 ng/ml C ⁇ TF + 200 ng/ml C ⁇ TFR ⁇ .
  • Cx43 protein cells were treated with agents in fresh Media I every 24 hours for a total of 3 days.
  • mR ⁇ A levels cells were treated with agents for 24 hours.
  • STAT3, ERK1 (p44 MAPK) or ERK2 (p42 MAPK) phosphorylation cells received media change and 24 hours later, agents were added for 5, 15 and 60 minutes.
  • the STAT3 and MAPK/ERK pathways were inhibited by administering the pharmacological agents AG490 (50 ⁇ M; Calbiochem)(Hodge et al, 2002) and U0126 (10 ⁇ M; Promega Corp.)(Favata et al, 1998), respectively, 45 minutes prior to adding vehicle or Complex.
  • Astrocyte monolayers were rinsed twice with PBS and harvested in radioimmuno- precipitation assay buffer (1% Nonidet P-40, 0.5% ⁇ sodium deoxycholate and 0.1% SDS in PBS) supplemented with protease inhibitors (mini,Complete; Roche) using a rubber policeman. The lysate was sheared using a 22 gauge needle and centrifuged at 10,000 xg for 10 minutes at 4°C. For Cx43 protein analyses, some samples were treated with alkaline phosphatase (calf- intestinal; Roche) to detect total Cx43 protein. Protein concentration of total cell lysate was determined using the BCA Protein Assay Kit (Pierce).
  • Protein samples 50 ⁇ g each
  • molecular weight markers Bio-Rad Lab.
  • SDS-polyacrylamide gel electrophoresis subsequently electro-transferred onto a nitrocellulose membrane for 1 hour at 80 watts.
  • the membrane was immunoblotted using appropriate primary antibodies (Cx43, Sigma; phospho-STAT3 and phospho-p44/42 MAPK, Cell Signaling Tech.) and subsequently incubated in secondary antibodies tagged with horseradish peroxidase (CedarLane Lab. Ltd.).
  • the blots were then incubated in Supersignal (Pierce) and exposed to Kodak X-Omat x-ray film to visualize antibody binding.
  • membranes were stripped of antibodies using 2- ⁇ mercaptoethanol (10 mM), SDS (2%), and Tris (62.5 mM, pH 6.7) for 30 min at 65°C and probed for glyceraldehyde-3 -phosphate dehydrogenase (GAPDH; CedarLane Lab. Ltd.), STAT3 (Cell Signaling Tech.), or ERK1 and ERK2 (Santa Cruz Biotechnology, Inc.).
  • GPDH glyceraldehyde-3 -phosphate dehydrogenase
  • STAT3 Cell Signaling Tech.
  • ERK1 and ERK2 Santa Cruz Biotechnology, Inc.
  • Scrape-Loading/Dye Transfer Assay Intercellular coupling between astrocytes was measured using a modified version of the scrape-loading technique described by el-Fouly et al. (1987). Briefly, media was aspirated from the cultures, cells were bathed in 50 ⁇ L of dye solution (0.1 % carboxyfluorescein and 0.1 % dextran tetramethylrhodamine (Molecular Probes) in PBS) and, subsequently, scraped using a surgical blade.
  • dye solution 0.1 % carboxyfluorescein and 0.1 % dextran tetramethylrhodamine (Molecular Probes) in PBS
  • Cytoplasmic R ⁇ A was isolated from astrocytes using the phenol-chloroform-isoamyl alcohol method outlined by Sambrook et al. (1989). Briefly, cells were washed with PBS and, subsequently, lysed using Lysis Solution (0.2 M ⁇ aCl, 20 mM MgCl 2 , and 20 mM Tris pH 8.8).
  • RT-PCR was performed on the R ⁇ A as for Example A. 3 ⁇ g of R ⁇ A was pretreated with D ⁇ Ase and subsequently reverse transcribed in a thermal cycler. For semiquantitative RT-PCR, 25 cycles were used to amplify the cD ⁇ A to avoid saturation. Primer sets used for amplification are provided in Table I. The amplified cD ⁇ A was run on a 1.8% agarose gel containing ethidium bromide
  • the blots were washed 30 min each with 2X SSC (40°C), IX SSC (40°C), and 0.1X SSC with 0.1% SDS (42°C) and then exposed to Kodak Bio-Max film at -80°C for 6-48 hours using intensifying screens.
  • CNTF and its receptor, CNTFR ⁇ cortical astrocytes matured in vi ⁇ ro was compared to that in the cerebral cortex of adult mice.
  • RT-PCR analysis revealed that astrocytes expressed the transcript for CNTF but did not transcribe mRNA for the receptor subunit CNTFR ⁇ .
  • both CNTF and CNTFR ⁇ were detected at their expected product sizes. No transcripts were observed when the same RNA samples were amplified in the absence of reverse transcriptase.
  • astrocytes matured in culture exhibit a CNTF/CNTFR ⁇ phenotype comparable to astrocytes in an uninjured or non-reactive state.
  • CNTF Complex Increases Cx43 Protein Expression and Intercellular Coupling
  • Cx43 protein Localization of the Cx43 protein was examined by immunocytochemistry following three-day treatment of the astrocytes with vehicle, CNTF, CNTFR ⁇ or Complex.
  • the protein distributed intracellularly as well as to the periphery of the cells.
  • no noticeable difference in either immunostaining intensity or localization of the Cx43 was detected in cells treated with either CNTF or CNTFR ⁇ .
  • astrocytes treated with Complex showed a dramatic increase in Cx43 immunolabeling. This Complex-induced increase in Cx43 could be detected both within the cytoplasm and at the periphery of the cells.
  • MAPK/ERK pathway and the JAK/STAT pathway were employed.
  • Astrocytes were treated with the ERK-activating kinase (MEK)l/2 inhibitor l,4-diamino-2,3-dicyano-l,4bis[2- aminophenylthio]butadiene (U0126) for 45 minutes prior to and throughout the addition of Complex.
  • Phosphorylation of the ERK1/2 normally induced by Complex was inhibited by U0126.
  • astrocytes were pretreated with the JAK2/JAK3 inhibitor ⁇ - cyano-(3,4-dihydroxy)-N-benzylcinnamide (AG490).
  • JAK3 expression is limited to hematopoietic and lymphoid cells (Kawamura et al, 1994; Rane and Reddy, 1994) and has not been shown to be associated with CNTF signaling (Stahl et al, 1994).
  • AG490 inhibited most, but not all, phosphorylation of STAT3 upon addition of Complex.
  • RT-PCR analysis for Cx43 mRNA was performed on astrocytes treated with vehicle or Complex in the presence or absence of the pathway inhibitors, Complex was still capable of inducing an increase in Cx43 mRNA levels in the presence of the inhibitor solvent (DMSO) and in the presence of U0126.
  • DMSO inhibitor solvent
  • the Complex-induced increase in Cx43 mRNA was not observed in cells pretreated with AG490.
  • both CNTF and Complex induce phosphorylation of STAT3 protein, only
  • astrocytes in adult brain or matured in culture express other connexins, including Cx30 (Dahl et al, 1996; Kunzelmann et al, 1999; Nagy et al, 1999).
  • Cx30 Dahl et al, 1996; Kunzelmann et al, 1999; Nagy et al, 1999.
  • mRNA isolated from astrocytes treated with vehicle, CNTF, CNTFR ⁇ , or Complex was analysed by RT-PCR. No change in Cx3 transcript levels could be detected following 24 hour treatment with the agents examined.
  • astrocytes cultured from neonatal mice were matured in vilro for seven weeks prior to the described procedures the described procedures. These astrocytes have a mature, non-reactive phenotype, including expression of Cx30 (Dahl et al, 1996; Nagy et al, 1999; Dermietzel et al, 2000; Ip et al, 1993a; Kordower et al, 1997; Maclennan et al, 1996; Monville et al, 2001; Squinto et al, 1990).
  • Previous studies demonstrated that astrocytes exhibit inconsistent or highly variable responses to CNTF. Different states of reactivity exist for astrocytes both in vivo and in vitro, from normal to reactive.
  • Reactive astrocytes are known to express both mRNA and protein for CNTFR ⁇ (Duberley et al, 1995; Duberley and Johnson, 1996; Ip et al, 1993b; Lee et al, 1997; Rudge et al, 1994a; Rudge et al, 1995). Astrocytes isolated and cultured from neonatal animals and subsequently maintained in culture for a long duration are in a non-reactive state.
  • Example mature murine cortical astrocytes are shown to express CNTF but not CNTFR ⁇ . Only when CNTF was administered in the presence of CNTFR ⁇ was upregulation of both Cx43 mRNA and protein observed. The regulation of Cx43 expression by Complex was dependent on the JAK/STAT pathway but not the MAPK/ERK pathway. The ability of Complex to upregulate Cx expression is selective as the expression of Cx30 is not affected. Although an increase in Cx43 was detected within the cytoplasm, Complex induced an increase in Cx43 mainly at the periphery of the cells.
  • Cx43 becomes phosphorylated, forms a connexon, which subsequently combines with the connexon from an adjacent cell to form the gap junction (reviewed by Lampe and Lau, 2000; Musil and Goodenough, 1991).
  • astrocytes are typically highly coupled, Complex further increased or stabilized functional intercellular communication via gap junction formation.
  • Complex also increases Cx43 and gap junctional coupling in C6 cells as shown in Example A. Therefore, upregulation of Cx43 and intercellular communication by Complex occurs in both gap junction-competent and -deficient cells of the CNS. Upregulation of Cx43 and gap junctions by Complex will circumvent extensive tissue damage in brain injuries, insults and in progressive CNS diseases.
  • CNTF has shown limited neuroprotective effects within the CNS following various insults (Kumon et al., 1996; Ogata et al., 1996; Unoki and LaVail, 1994; Wen et al., 1995b), the limitation now appears to be due to the lack of CNTFR ⁇ on astrocytes.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • F12 fetal bovine serum
  • Neurobasal Medium penicillin-streptomyosin
  • HBSS Hank's balanced salt solution
  • PBS Phosphate Buffered Saline
  • Earle's Balanced Salt Solution were obtained from GIBCO Laboratories (Burlington, ON, Canada).
  • Cytosine arabinoside poly-D-lysine, D- glucose, insulin, transferrin, putrescine-HCl, progesterone, sodium selenite, CBX, GZA, AGA, propidium iodide, Hoechst 33342 dye, glutamate, anti-microtubual associated protein 2 (MAP2) antibody, anti-glial fibrillary acidic protein (GFAP) antibody and lactate dehydrogenase (LDH) Detection kit were products of Sigma (Oakville, ON, Canada). L- [ 3 H]glutamate ([ 3 H]Glu, 38-46 Ci/mmol) was purchased from Amersham Canada (Oakville, ON, Canada).
  • Apoptosis Detection System (terminal dUTP nick end labeling, TUNEL, Fluroescein) was from Promega (Madison, WI, USA). Cell strainers (pore size 70 ⁇ m) and 6- well plates were acquired from NWR (Mississauga, ON, Canada). Dil (l,l'-dioctadecyl- 3,3,3',3'-tetramethylindocarbocyaine perchlorate) and calcein acetoxymethyl ester (calcein- AM) were purchased from Molecular Probes (Eugene, OR, USA). Solution I consisted of HBSS supplemented with 10%> FBS.
  • Media I was comprised of Neurobasal Medium and DMEM/F12 (2:3) supplemented with 10% FBS.
  • Media II was prepared with 54 ml Neurobasal Medium, 36 ml DMEM/F12, 50 ⁇ l penicillin-streptomyosin, and 2 ml of N2 Supplements.
  • N2 Supplements (Mazzoni, I.E., et al, 1991) consisted of the following (given in final concentrations in Media II): glucose (0.6%), insulin (10 ⁇ g/ml), transferrin (20 ⁇ g/ml), putrescine-HCl (62 ⁇ M), progesterone (20 nM), and sodium selenite (30 nM).
  • Sodium Hepes Buffer contained (final concentrations) 134 mM NaCl, 5.2 mM KC1, 1.8 mM CaCl 2 , 0.8 mM MgSO 4 , 10 mM glucose, and 20 mM HEPES, pH 7.3.
  • astrocytes were prepared from murine cortices using the method described for Example B. Cortices were dissected from 1 to 2 day old mouse pups, placed into PBS, and subsequently freed of meninges. Cortices were then placed into DMEM supplemented with 10% FBS and were mechanically dissociated using a serological pipette. This cell suspension was then passed through a cell strainer and diluted using DMEM supplemented with 10% FBS and penicillin-streptomyosin at a ratio of 7 ml/cortex. Cells were plated onto 6-well plates (previously coated with poly-D-lysine, 100 ⁇ g/ml) in 2 ml aliquots.
  • Cultures were maintained within a humidifed incubator at 37°C in 95% air/5% CO 2 . Culture media was continuously replaced every three days in addition to shaking the cultures. Astrocytic cultures were maintained for 15-17 days from initial plating prior to use or co- culturing with neurons. Immunocytochemical characterization of cell types present in the final astrocytic cultures revealed that 95-97% of the population were GFAP immunopositive.
  • Neurons were prepared from mouse cortices in a modified method by Mazzoni and Kenigsberg (1991). Cortices were dissected from embryonic mice of gestation age 16 days and placed into HBSS supplemented with 10% FBS. Cortices were then freed of meninges, minced with scalpels and mechanically disrupted by gently triturating through a fire-polished pasteur pipette (bore size of approximately 0.6 mm internal diameter). Cell suspension was passed through a cell strainer, centrifuged, and subsequently resuspended in HBSS and Media 1 (1:1) supplemented with 10% FBS. Cells (1 x 10 6 cells/well) were seeded on top of astrocyte cultures and allowed to settle for 2 hours.
  • Co-cultures consisted of a monolayer of astrocytes to which cortical neurons were seeded on top.
  • Blocking of astrocytic gap junctions was determined by dye coupling using the preloading method as described by Goldberg et al. (1995). Astrocyte cultures were maintained in Media II for 7 days. Narious concentrations of the blocking agents CBX and AGA and the inactive analogue GZA were tested; all agents were added to media 1 hour prior to pre-loading and were present in all solutions.
  • Blocking agents were added to co-cultures 1 hour prior and were present in all solutions throughout the procedure. Co-cultures were rinsed with PBS and then bathed in EBSS with glutamate (1 mM) or sham (vehicle) for 3 hours in a humidified incubator (37°C, 95% air/5% CO 2 ). Cells were subsequently maintained for 24 hours post glutamate/sham exposure in fresh Media II.
  • Glutamate uptake procedures were performed in a manner similar to that described by Sitar et al. (1999). Confluent cultures of astrocytes were switched to Media II for 1 week prior to procedures. Blocking agents were added to cultures 1 hour prior to glutamate uptake and were present in all solutions during transport. Cells were rinsed with Sodium Hepes Buffer and exposed to glutamate solution for 120 seconds. Uptake was terminated by washing cultures with ice-cold Tris-sucrose (pH 7.3) solution. Cells were harvested by osmotic lysis (1 ml water/dish) and mechanical scraping. The radioactive contents of the buffer and cells were measured by liquid scintillation counting. Glutamate uptake was computed based on the specific activity of radiolabeled glutamate in the buffer and taken-up by the cells.
  • a minimal concentration of CBX and AGA required to block astrocytic gap junctions was determined by assaying unlabeled cells receiving calcein from a labeled cell (Dil and calcein). Final concentrations of 0 (vehicle), 1, 10, 25 and 50 ⁇ M of blockers were tested. While vehicle (water) and GZA had no affect on the passage of calcein from donor cells to, and among, recipient cells, concentrations including and exceeding 25 ⁇ M CBX effectively blocked the passage of this dye. A lower concentration of AGA (10 ⁇ M) was required to similarly block astrocytic gap junctions while its vehicle (DMSO) had no effect. From the results obtained in pre-loading, concentrations of 25 ⁇ M CBX and 10 ⁇ M AGA were chosen for use in the remainder of this Example.
  • Glutamate cytotoxicity was analyzed by three mechanisms: LDH release, the inability to exclude propidium iodide, and TUNEL labeling.
  • LDH release When presented with a sham insult, neither vehicle, CBX nor GZA caused the cells to release a significant amount of LDH.
  • glutamate was administered in the presence of vehicle or GZA, no significant LDH release occurred.
  • gap junctions were blocked with CBX, a similar glutamate insult caused the cells to release a significant amount of LDH.
  • Gap Junction Blocker Does Not Affect Astrocytic Glutamate Transporters
  • This Example demonstrates the important role of intercellular communication that allows cells to endure high levels of extracellular glutamate.
  • Glutamate can induce a cytotoxic effect to sensitive cells (i.e. neurons) by one of two mechanisms.
  • glutamate activates ionotrophic receptors which subsequently depolarizes the cell resulting in cell swelling and, potentially, necrosis.
  • glutamate metabotrophic receptor overstimulation may cause the cell to undergo transcriptionally active suicide (apoptosis).
  • apoptosis transcriptionally active suicide
  • PI and TUNEL staining indicated that the dosage of glutamate employed significantly insulted the cells. More specifically, the PI and TUNEL labelling was highly associated with the glutamate-sensitive neurons and labelled very few underlying astrocytes (similar results to Choi (1987)). Astrocytic tolerance to the glutamate insult is likely due to astrocytes lacking NMDA receptors and housing large glycogen stores (a crucial metabolite for energy production).
  • the increased glutamate cytotoxicity in the presence of the gap junction blockers seems directly related to the blockers impeding intercellular gap junctional communication.
  • the blockers alone caused no significant cell death and no significant difference in mortality was determined between vehicle and the inactive blocker in the presence of glutamate.
  • the cell suspension was passed through a cell strainer, centrifuged at 5000 xg for 5 minutes, and resuspended in Hank's Balanced Salt Solution (Invitrogen Corp.) and Media I (Neurobasal Medium and DMEM/F12 (2:3); Invitrogen Corp.) in a 1 :1 ratio supplemented with 10% FBS.
  • Immunocytochemistry Co-cultures were rinsed with phosphate buffered saline (PBS) and fixed with 4% formaldehyde (in PBS) for 10 minutes. Non-specific binding of antibodies was blocked by incubating the cultures with 10% normal goat serum (in PBS). Cultures were then incubated with microtubule associated protein 2 (MAP2) monoclonal antibody and glial fibrillary acidic protein (GFAP) polyclonal antibody (Sigma). Following three washes with PBS, cultures were incubated in Alexa- and fluorescein-conjugated secondary IgG (Molecular Probes) for 1 hour and then mounted with Nectashield mounting medium (Nector). Immunostaining was viewed using a Zeiss Axioskop microscope and images were captured using Northern Exposure, Nersion2 (ImageExperts Inc.).
  • MAP2 microtubule associated protein 2
  • GFAP glial fibrillary acidic protein
  • Co-cultures were rinsed with PBS and then bathed in Earle's Balanced Salt Solution (Invitrogen Corp.) with glutamate (1 mM) or PBS (sham insult) for 3 hours in a humidified incubator (37°C, 95% air/5% CO 2 ). Following the insult, cells were maintained in fresh Media II.
  • Earle's Balanced Salt Solution Invitrogen Corp.
  • glutamate 1 mM
  • PBS sham insult
  • the stained cells were rinsed three times with PBS and mounted using Nectashield mounting medium (Nector). Staining was viewed and images were captured as outlined above.
  • TUNEL-labelling vehicle pretreatment
  • Similar glutamate-induced cytotoxicity as marked by TUNEL-labelling was seen in CNTFR ⁇ -pretreated co-cultures.
  • considerably less number of cells stained with TUNEL was observed in the co-cultures pretreated with CNTF and Complex. Comparisons between groups revealed that only Complex pretreatment significantly reduced the number of TUNEL-positive cells, as compared to vehicle pretreatment.
  • CNTF Glutamate-induced cytotoxicity in neuron and astrocyte co-cultures is reduced by CNTF pretreatment. Since neurons express CNTFR ⁇ , CNTF alone can directly activate those cells and potentially increase their resilience to glutamate excitotoxicity. Skaper et al. (1992) but the extended neuroprotection shown in this Example requires administration of both CNTF and CNTFR ⁇ .
  • CNTF causes a reduction in glutamate-induced cytotoxicity within its own pretreatment group, this difference is not significantly different from vehicle pretreatment under similar conditions.
  • CTNF CTNF in combination with CNTFR ⁇
  • the "Complex" of CNTF-(soluble)CNTFR ⁇ was administered to mice following induced stroke. Stroke was induced in mice by middle cerebral artery occlusion (MCAO). Occlusion of the blood vessel in mice is a widely used stroke model (Siushansian et al., 2001 ; Nakase et al., 2003). Immediately following the insult (2 hour occlusion in the mouse model), the respective agents (vehicle, CNTF, or Complex) were administered via intraperitoneal injection. Since CNTF administration into both rodents and humans induces weight reduction, the body weights and food intake (provided ad libum) were closely monitored.
  • MCAO middle cerebral artery occlusion
  • infarct core and penumbra
  • TUNEL terminal dUTP nick end labeling
  • a large portion of the body weight reduction can be attributed to the agents inducing loss of both appetite and thirst since food and H 2 O intake were significantly reduced compared to vehicle.
  • the loss in appetite is solely attributed to the effect of Complex and independent of the stroke since Complex injection without stroke caused similar results to that of Complex with insult.
  • the initial concentration of Complex chosen for injections to reduce stroke-induced brain damage was that in which the same concentration of CNTF alone would induce physiological changes (i.e. weight loss; Fantuzzi et al., 1995).
  • concentration of Complex may be titered to determine the amount most effective for neuroprotection in vivo, by for example reducing brain tissue damage without causing substantial side-effects.
  • MCAO insults are performed on knockout mice. While CNTFR ⁇ knockout mice are not viable, mice lacking CNTF expression thrive normally. Using mice in which the CNTF gene has been deleted through homologous recombination (generous gift of Dr. Micheal Sendtner, Universitaat W ⁇ urzburg), the importance of CNTF expression in circumventing brain injury following stroke is assessed. A similar MCAO insult and similar methods to assess brain injury as outlined herein is performed on the CNTF knockout mice and compared to wild-type littermates. Furthermore, in cases where the lack of endogenous CNTF expression leads to increased brain injury in the knockout mice, rescue of brain injury by administration of exogenous CNTF or Complex following the insult is determined.
  • RNA RNA which will be analyzed by, for example, gene arrays.
  • Narious genes are differentially expressed by treatment of cultures with Complex compared to vehicle or C ⁇ TF treatment.
  • the identified differential gene candidates are confirmed by other techniques (e.g. northern blot analysis and/or reverse transcription polymerase chain reactions).
  • Connexin43 a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol 105:2621-2629. Blanc EM, Bruce-Keller AJ, and Mattson MP (1998) Astrocytic gap junctional communication decreases neuronal vulnerability to oxidative stress-induced disruption of Ca2+ homeostasis and cell death. J Neurochem 70:958-970. Blottner D, Bruggemann W, and Unsicker K (1989) Ciliary neurotrophic factor supports target- deprived preganglionic sympathetic spinal cord neurons. Neurosci Lett 105:316-320. Brosnan CF, Scemes E, and Spray DC (2001) Cytokine regulation of gap junction connectivity: an open-and-shut case or changing partners at the Nexus? Am J Paihol
  • GD (1991) The receptor for ciliary neurotrophic factor. Science 253:59-63. Davis, S., T.H. Aldrich, N. Stahl, L. Pan, T. Taga, T. Kishimoto, N.Y. Ip, and G.D.
  • Ciliary neurotrophic factor induces serum amyloid A, hypoglycaemia and anorexia, and potentiates IL-1 induced corticosterone and IL-6 production in mice.
  • TUNEL assay In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note.
  • TGF-beta 1 protects hippocampal neurons against degeneration caused by transient global ischemia. Dose-response relationship and potential neuroprotective mechanisms. Stroke 27:1609-1614. Hertz L. Possible role of neuroglia: a potassium-mediated neuronal—neuroglial—neuronal impulse transmission system. Nature 1965;206:1091-1094 Hirano T, Nakajima K, and Hibi M (1997) Signaling mechanisms through gpl30: a model of the cytokine system. Cytokine Growth Factor Rev 8:241-252. Hodge, D.R., D. Li, S.M. Qi, and W.L. Farrar. 2002. IL-6 induces expression of the Fli-1 proto-oncogene via STAT3. Biochem. Biophys. Res. Commun. 292:287-291.
  • JNeurosci 11:3124-3134 Ip NY, McClain J, Barrezueta NX, Aldrich TH, Pan L, Li Y, Wiegand SJ, Friedman B, Davis S, and Yancopoulos GD (1993a) The alpha component of the CNTF receptor is required for signaling and defines potential CNTF targets in the adult and during development. Neuron 10:89-102. Ip NY, Wiegand SJ, Morse J, and Rudge JS (1993) Injury-induced regulation of ciliary neurotrophic factor mRNA in the adult rat brain. Eur JNeurosci 5:25-33. Ip, N.Y. and G.D. Yancopoulos. 1992. Ciliary neurotrophic factor and its receptor complex.
  • Kandler K Katz LC. Neuronal coupling and uncoupling in the developing nervous system.
  • C ⁇ TF ciliary neurotrophic factor
  • Ciliary neurotrophic factor may activate mature astrocytes via binding with the leukemia inhibitory factor receptor.
  • Nedergaard M (1994) Direct signaling from astrocytes to neurons in cultures of mammalian brain cells. Science 263:1768-1771.
  • Nedergaard M Cooper AJ, Goldman SA. Gap junctions are required for the propagation of spreading depression. J Neurobiol 1995;28:433-444 Negro, A., G. Corona, E. Bigon, I. Martini, C Grandi, S.D. Skaper, and L. Callegaro. 1991.
  • JAK3 a novel JAK kinase associated with terminal differentiation of hematopoietic cells.
  • Fibroblast growth factors-5 and -9 distinctly regulate expression and function of the gap junction protein connexin43 in cultured astroglial cells from different brain regions. Glia 30:231-241. Reuss, B., R. Dermietzel, and K. Unsicker. 1998. Fibroblast growth factor 2 (FGF-2) differentially regulates connexin (ex) 43 expression and function in astroglial cells from distinct brain regions. Glia 22:19-30. Robe PA, Rogister B, Merville MP, and Bours V (2000) Growth regulation of astrocytes and
  • RNA from eukaryotic cells in Molecular Cloning: A Labora ⁇ ory Manual Cold Springs
  • Acidic fibroblast growth factor prevents death of hippocampal CA1 pyramidal cells following ischemia.
  • Gap junctions modulate survival-promoting effects of fibroblast growth factor-2 on cultured midbrain dopaminergic neurons.
  • Tanaka K Tanaka K
  • Tsukahara T Hashimoto N
  • Ogata N Hashimoto N
  • Yonekawa Y Kimura T
  • Taniguchi T (1994) The role of brain-derived neurotrophic factor in transient forebrain ischemia in the rat brain. Neurosurgery 34:323-331. Tsuzuki N, Miyazawa T, Matsumoto K, Nakamura T, and Shima K (2001) Hepatocyte growth factor reduces the infarct volume after transient focal cerebral ischemia in rats. Neurol
  • Zahs KR Heterotypic coupling between glial cells of the mammalian central nervous system.
  • Accession numbers refer to Accession numbers from multiple databases, including GenBank, the European Molecular Biology Laboratory (EMBL), the DNA Database of Japan (DDBJ), or the Genome Sequence Data Base (GSDB), for nucleotide sequences, and including the Protein Information Resource (PIR), SWISSPROT, Protein Research Foundation (PRF), and Protein Data Bank (PDB) (sequences from solved structures), as well as from translations from annotated coding regions from nucleotide sequences in GenBank, EMBL, DDBJ, or RefSeq, for polypeptide sequences. Numeric ranges are inclusive of the numbers defining the range.

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Abstract

L'invention concerne des procédés de neuroprotection au moyen d'un peptide CNTF et d'un peptide qui agit en tant que récepteur du CNTF, par exemple CNTFR-alpha. La combinaison de l'invention est plus efficace que lorsqu'elle est obtenue en l'absence de traitement (i.e. sans application d'agents exogènes) ou par traitement avec CNTF seul, ou avec le récepteur CNTF seul.
PCT/CA2003/001626 2002-10-24 2003-10-24 Procedes de neuroprotection WO2004037281A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5846935A (en) * 1992-10-09 1998-12-08 Regeneron Pharmaceuticals, Inc. Modified ciliary neurotrophic factors
WO2002022149A1 (fr) * 2000-09-14 2002-03-21 Ares Trading S.A. Utilisation d'une chimere de il-6r/il-6 permettant de traiter la maladie d'huntington
WO2002064021A2 (fr) * 2001-02-12 2002-08-22 Baylor College Of Medicine Procedes et compositions pour reguler la formation osseuse par modulation de l'activite ciliaire du facteur neurotrophique

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5846935A (en) * 1992-10-09 1998-12-08 Regeneron Pharmaceuticals, Inc. Modified ciliary neurotrophic factors
US6440702B1 (en) * 1992-10-09 2002-08-27 Regeneron Pharmaceuticals, Inc. Nucleic acids encoding modified ciliary neurotrophic factors
WO2002022149A1 (fr) * 2000-09-14 2002-03-21 Ares Trading S.A. Utilisation d'une chimere de il-6r/il-6 permettant de traiter la maladie d'huntington
WO2002064021A2 (fr) * 2001-02-12 2002-08-22 Baylor College Of Medicine Procedes et compositions pour reguler la formation osseuse par modulation de l'activite ciliaire du facteur neurotrophique

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OZOG MARK A ET AL: "Ciliary neurotrophic factor ( CNTF ) in combination with its soluble receptor (CNTFRalpha) increases connexin43 expression and suppresses growth of C6 glioma cells.", CANCER RESEARCH, (2002 JUN 15) 62 (12) 3544-8. JOURNAL CODE: 2984705R. ISSN: 0008-5472., June 2002 (2002-06-01), XP002232406 *

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