WO2008066744A2

WO2008066744A2 - Methods and compositions for treating and preventing spinal cord injury and other neuronal disease or injury

Info

Publication number: WO2008066744A2
Application number: PCT/US2007/024280
Authority: WO
Inventors: Azad Bonni; Judith Stegmueller
Original assignee: President And Fellows Of Harvard College
Priority date: 2006-11-22
Filing date: 2007-11-20
Publication date: 2008-06-05
Also published as: WO2008066744A3; WO2008066744A9

Abstract

The present invention provides methods for reducing spinal cord injuries and othe neuronal disorders by stimulating axonal growth and/or regeneration. Such growth or regeneration is stimulated by contacting injured or diseased tissue with an agent that increases the level or activity of SnoN.

Description

METHODS AND COMPOSITIONS FOR TREATING AND PREVENTING SPINAL CORD INJURY AND OTHER NEURONAL DISEASE OR INJURY

Background of the Invention Spinal cord injuries that involve trauma typically stem from a sudden strike to the spine that fractures, dislocates, crushes or compresses one or more of the vertebras. Additional damage usually occurs over days or weeks because of bleeding, swelling, inflammation and fluid accumulation in and around the spinal cord. Non-traumatic spinal cord injury may be caused by arthritis, cancer, blood vessel damage or bleeding, inflammation or infections, or disk degeneration of the spine.

Whether the cause is traumatic or non-traumatic, the damage affects the nerve fibers passing through the injured area and may impair part or all of the corresponding muscles and nerves below the injury site. Spinal injuries occur most frequently in the neck (cervical) and lower back (thoracic and lumbar) areas. A thoracic or lumbar injury can affect leg, bowel and bladder control, and sexual function. A cervical injury may affect breathing as well as movements of the upper and lower limbs.

Although spinal cord injuries were almost always fatal, better methods exist today to manage the pain associated with such injuries and allow injured patients to cope with their disabilities. In spite of such medical advances, treatment modalities that promote nerve cell regeneration and improve the function of the nerves that remain after a spinal cord injury are still required.

Summary of the Invention

The invention provides methods for inducing axonal growth and regeneration. These methods are therefore useful to treat spinal cord injuries that result, for example, from trauma (e.g., fall) or from conditions such as cancer, inflammation, arthritis, or infections. The methods are also useful to treat other pathological conditions that are characterized by axonal degeneration or impairment such as multiple sclerosis and peripheral neuropathies.

One method of inducing axonal growth and regeneration involves contacting a neuronal cell (e.g., cerebellar granule neuron) with an agent that reduces the level or activity of the E3 ubiquitin ligase-anaphase promoting complex (Cdhl-APC). For example, the agent increases phosphorylation of Cdhl at amino acid position Serine 40, serine 151, serine 163, or threonine 121 of the naturally-occurring Cdhl polypeptide. The agent reduces the ubiquitination of Skp2, Tome-1, SnoN or Pax 6. Optionally, the agent is a polypeptide that is substantially identical to the naturally-occurring Emil, MAD2B, or ubiquilinl polypeptides. Axonal growth and regeneration is also be induced by contacting a cell with an agent that increases the level or activity of the transcriptional factor SnoN, an agent that reduces the level or activity of Smad proteins (e.g., Smad 2 and Smad 3), or both. For example, the Smad protein inhibitor is an RNAi molecule. Alternatively, the agent is an antisense composition (USPN 6,037,142). In yet another example, the Smad inhibitory compound binds to Smad2 or Smad3 (e.g., to a MHl or MH2 domain), thereby inhibiting its activity. Optionally, the agent that increases the level or activity of SnoN is used in combination with an agent that reduces the level or activity of Cdhl-APC.

In all foregoing aspects of the invention, agents that induce axonal growth and regeneration include small molecule inhibitors and dominant-interfering polypeptides (e.g., a dominant-interfering APC subunit polypeptide such as APCl 1C73A). A small molecule inhibitor is a compound that is less than 2000 daltons in mass. The molecular mass of the inhibitory compounds is preferably less than 1000 daltons, more preferably less than 600 daltons, e.g., the compound is less than 500 daltons, 400 daltons, 300 daltons, 200 daltons, or 100 daltons. Peptide inhibitors are also useful. For example, the peptide is at least 8, 10, 20, 30, 40 residues in length and augments phosphorylation of Cdhl, increases the level or activity of the transcriptional factor SnoN, or reduces the level or activity of Smad proteins. If desired, a second therapeutic regimen is also be used.

The invention also provides methods for identifying a candidate compound for inducing axonal growth and regeneration. These methods involve the steps of: (a) contacting a cell expressing a Cdhl or APC subunit APC2 gene with a candidate compound; and (b) measuring Cdhl or APC2 gene expression or protein activity in the cell. A candidate compound that reduces the expression or the activity of Cdhl or APC2 relative to such expression or activity in a cell that has not been contacted with the candidate compound is useful for inducing axonal growth and regeneration. For example, the compound increases phosphorylation of Cdhl. Optionally, the Cdhl or APC2 is a fusion gene and the Cdhl- or APC2 expressing cell is a mammalian cell (e.g., a rodent or human cell). In other embodiments, step (b) involves the measurement of the level of Cdhl or APC2 mRNA or protein. Optionally, the biological activity of Cdhl or APC is determined by measuring the level of the transcriptional factor SnoN.

Alternatively, the method involves the steps of: (a) contacting a Cdhl or APC2 protein with a candidate compound; and (b) determining whether the candidate compound binds the Cdhl or APC APC2 protein and/or reduces Cdhl or ACP2 activity. Compounds that bind and reduce such activity are identified as compounds useful for inducing axonal growth and regeneration. These compounds are also useful for treating or reducing spinal cord injuries. In yet another screening approach, a method for identifying a candidate compound for reducing or preventing neural cell apoptosis involves the steps of: (a) contacting a Cdhl protein (e.g., human Cdhl protein) with a candidate compound; and (b) determining whether the candidate compound reduces binding of Cdhl to APC2. The candidate compound is first contacted with Cdhl, APC2, or is simultaneously contacted with both proteins or fragments thereof. Candidate compounds that reduce such binding are useful for inducing axonal growth and regeneration, and are thereby useful for treating or reducing spinal cord injuries.

Another method for identifying a candidate compound for inducing axonal growth and regeneration involves the steps of: (a) contacting a cell expressing a SnoN gene with a candidate compound; and (b) measuring SnoN gene expression or protein activity in the cell. A candidate compound that increases the expression or the activity of SnoN relative to such expression or activity in a cell that has not been contacted with the candidate compound is useful for inducing axonal growth and regeneration. Optionally, the SnoN gene is a fusion gene and the SnoN-expressing cell is a mammalian cell (e.g., a rodent or human cell). In other embodiments, step (b) involves the measurement of the level of SnoN mRNA or protein. Optionally, the biological activity of SnoN is determined by measuring the level of Smad proteins.

Yet another method involves the steps of: (a) contacting a SnoN protein with a candidate compound; and (b) determining whether the candidate compound binds the SnoN protein and/or reduces SnoN activity. Compounds that bind and increase such activity are identified as compounds useful for inducing axonal growth and regeneration. In all foregoing aspects of the invention, candidate compounds identified as being useful for inducing axonal growth and regeneration are useful to treat, reduce, or prevent spinal cord injuries. By "reduce the expression or activity of Cdhl -APC" is meant to reduce the level or biological activity of the Cdhl -APC relative to such level or activity in an untreated control. The level or activity is preferably reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to an untreated control. An agent that reduces the expression or activity of Cdhl -APC reduces the expression or activity of Cdhl, APC2, or both. Alternatively, since SnoN is a key downstream target of Cdhl-APC, a reduction in the biological activity of Cdhl-APC is optionally an increase in the expression or activity of SnoN. For example, expression or activity of SnoN is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or even greater than 100%, relative to an untreated control, thereby inducing axonal growth and regeneration. As used herein, the term "activity" with respect to Cdhl-APC includes any activity which is inherent to the naturally occurring Cdhl-APC. Thus, the term "Cdhl-APC activity" includes any activities of molecules involved in Cdhl-APC signaling, such as inhibition of the transcriptional corepressor SnoN. By "increasing axonal growth and regeneration" is meant to increase the length or size of a neuronal axon, or alternatively, to increase the number of neuronal axons.

Preferably, such increase is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% relative to an untreated control, as is measured by any standard technique. By increasing axonal growth and regeneration, spinal cord injuries are treated, reduced, or even prevented such that any of the conditions or symptoms associated with the spinal cord injury before or after it has occurred are ameliorated. Such injuries may result from a physical injury or from a disorder such as cancer, inflammation, or an infection. As compared with an equivalent untreated control, such reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique.

As used herein, by "Cdhl -Anaphase Promoting Complex " is meant the complex that is formed between the APC subunits and the Cdhl polypeptide, which is involved in various signaling pathways such as axonal growth and regeneration. Cdhl polypeptides are substantially identical to the naturally occurring Cdhl polypeptides (e.g., accession numbers NP_062731 (mouse) and NP 057347 (human), the sequences of which are hereby incorporated by reference) and SnoN polypeptides are substantially identical to the naturally- occurring SnoN polypeptide (e.g., accession numbers AAB65848 (mouse) and NP_005405.1 (human), hereby incorporated by reference).

An, Cdhl, or SnoN fusion gene is a construct that contains a portion or the entire, Cdhl, or SnoN promoter and/or all or part of an, Cdhl, or SnoN coding region operably linked to a second, heterologous nucleic acid sequence. In preferred embodiments, the second, heterologous nucleic acid sequence is a reporter gene, that is, a gene whose expression may be assayed; reporter genes include, without limitation, those encoding glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), green fluorescent protein (GFP), alkaline phosphatase, and beta-galactosidase. By "purified antibody" is meant antibody which is at least 60%, by weight, free from proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, antibody. A purified antibody may be obtained, for example, by affinity chromatography using recombinantly-produced protein or conserved motif peptides and standard techniques. An antibody specifically binds an antigen if it recognizes and binds an antigen or antigenic domain but does not substantially recognize and bind other non-antigen molecules in a sample, e.g., a biological sample, that naturally includes protein or domains of a target protein. Neutralizing antibodies interfere with any of the biological activity of a polypeptide (e.g., the ability to increase axonal growth or regeneration). The neutralizing antibody reduces the biological activity of a polypeptide by, preferably 50%, more preferably by 70%, and most preferably by 90% or more.

By "substantially identical," when referring to a protein or polypeptide, is meant a protein or polypeptide exhibiting at least 75%, but preferably 85%, more preferably 90%, most preferably 95%, or even 99% identity to a reference amino acid sequence. For proteins or polypeptides, the length of comparison sequences will generally be at least 20 amino acids, preferably at least 30 amino acids, more preferably at least 40 amino acids, and most preferably 50 amino acids or the full length protein or polypeptide. Nucleic acids that encode such "substantially identical" proteins or polypeptides constitute an example of "substantially identical" nucleic acids; it is recognized that the nucleic acids include any sequence, due to the degeneracy of the genetic code, that encodes those proteins or polypeptides. In addition, a "substantially identical" nucleic acid sequence also includes a polynucleotide that hybridizes to a reference nucleic acid molecule under high stringency conditions.

By "high stringency conditions" is meant any set of conditions that are characterized by high temperature and low ionic strength and allow hybridization comparable with those resulting from the use of a DNA probe of at least 40 nucleotides in length, in a buffer containing 0.5 M NaHPO₄, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at a temperature of 65 ⁰C, or a buffer containing 48% formamide, 4.8XSSC, 0.2 M Tris-Cl, pH 7.6, IX Denhardt's solution, 10% dextran sulfate, and 0.1% SDS, at a temperature of 42 ⁰C. Other conditions for high stringency hybridization, such as for PCR, Northern, Southern, or in situ hybridization, DNA sequencing, etc., are well known by those skilled in the art of molecular biology. See, e.g., F. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1998, hereby incorporated by reference. By "substantially pure" is meant a nucleic acid, polypeptide, or other molecule that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. For example, a substantially pure polypeptide may be obtained by extraction from a natural source, by expression of a recombinant nucleic acid in a cell that does not normally express that protein, or by chemical synthesis.

The term "isolated DNA" is meant DNA that is free of the genes which, in the naturally occurring genome of the organism from which the given DNA is derived, flank the DNA. Thus, the term "isolated DNA" encompasses, for example, cDNA, cloned genomic DNA, and synthetic DNA.

By "an effective amount" is meant an amount of a compound, alone or in a combination, required to increase axonal growth or regeneration or to treat, reduce or prevent a spinal cord injury in a mammal. The effective amount of active compound(s) varies depending upon the route of administration, age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen.

A candidate compound is a chemical, be it naturally-occurring or artificially-derived that is tested using screening methods described herein to identify synapse modulating activity. Candidate compounds include, for example, peptides, polypeptides, synthetic organic molecules, naturally occurring organic molecules, nucleic acid molecules, peptide nucleic acid molecules, and components and derivatives thereof.

The term "pharmaceutical composition" is meant any composition, which contains at least one therapeutically or biologically active agent and is suitable for administration to the patient. Any of these formulations can be prepared by well-known and accepted methods of the art. See, for example, Remington: The Science and Practice of Pharmacy, 20^th edition, (ed. A. R. Gennaro), Mack Publishing Co., Easton, Pa., 2000.

The present invention provides significant advantages over standard therapies for treatment, prevention, and reduction, or alternatively, the alleviation of one or more symptoms associated with a spinal cord injury. In addition, because the methods specifically target cerebellar neuronal cells, side effects associated with broad-based drug approaches are minimized. In addition, the candidate compound screening methods provided by this invention allow for the identification of novel therapeutics that modify the injury process, rather than merely mitigating the symptoms. In addition to promoting axonal regeneration, inhibition of Cdhl-APC is useful in treating or reducing the severity of symptoms associated with stroke, neurodegeneration (e.g., Alzheimer's disease or other cognitive disorders), and epilepsy (e.g., by regulating hyperexcitability). Inhibition of the complex also has a beneficial effect on synaptic inhibition and leads to stronger synapses.

Cited publications including sequences defined by GENBANK™ accession numbers are incorporated herein by reference. Other features, objects, and advantages of the invention will be apparent from the description of the drawings.

Brief Description of the Figures

FIGURE IA is a diagram of gene constructs and an immunoblot. Constructs containing GFP-Cdhl-Res and modified versions having the NLS or NES sequences are shown. Silent mutations that render Cdhl-Res resistant to RNAi are indicated in red. NES and NLS refer to the nuclear exclusion and nuclear localization sequence, respectively. Lysates of COS cells transfected with the U6 or U6/cdhl plasmid together with a plasmid encoding FLAG-Cdhl or FLAG-Cdhl-Res were immunoblotted using a FLAG antibody and an antibody against 14-3-3 protein to serve as an internal control for loading. Cdhl RNAi induced the knockdown of Cdhl encoded by wild type cDNA but failed to effectively induce knockdown of Cdhl-Res. FIGURE IB is a graph showing the axonal length per neuron. Primary cerebellar granule neurons were transfected eight hours after plating (P6+0DIV) with the U6 or U6/cdhl plasmid and the GFP, GFP-Cdhl, or GFP-Cdhl-Res expression plasmid together with the DsRed and BCI-XL expression plasmids. Neurons were kept in media supplemented with insulin. Three days later, cultures were subjected to immunocytochemistry using a polyclonal DsRed antibody and total axonal length was measured. Cdhl knockdown in granule neurons significantly increased axon length as compared to control U6-transfected neurons (ρ<0.001, ANOVA), and in the background of Cdhl RNAi, GFP-Cdhl-Res but not GFP-Cdhl significantly reduced axon length when compared to control U6-transfected neurons (p<0.001, ANOVA). Total number of neurons measured = 447. FIGURE 1C is a series of photographs showing immunofiuorescent stains. 293T cells

(left panels) and granule neurons (right panels) were transfected with the GFP, GFP-Cdhl- Res, GFP-NES-Cdhl-Res, or GFP-NLS-Cdhl-Res expression plasmid. Cultures were subjected to immunocytochemistry using a monoclonal GFP antibody. FIGURE ID is a graph showing axon length per neuron. Granule neurons were transfected at P6+0DIV with the U6/cdhl plasmid together with the GFP, GFP-Cdhl-Res, GFP-NES-Cdhl-Res, or GFP-NLS-Cdhl-Res plasmid and the DsRed and BCI-XL expression plasmids. Neurons were analyzed three days later as in FIGURE IB. In the background of Cdh 1 RNAi, total axonal length of GFP-Cdh 1 -NES-Res- but not of GFP-Cdh 1 -NLS-Res- expressing neurons was significantly greater than in GFP-Cdhl -Res-expressing neurons (p<0.001, ANOVA). Total number of neurons measured = 519.

FIGURE 2A is a series of immunoblots. In the left panel, lysates of granule neurons prepared from P6 rat pups and placed in culture for the number of indicated days were immunoblotted using a polyclonal SnoN antibody. In the right panel, lysates of granule neurons were subjected to immunoprecipitation with the SnoN or an antibody against actin followed by immunoblotting with the SnoN antibody.

FIGURE 2B is a series immunofluorescent stains. Granule neurons (P6+2DIV) were subjected to immunocytochemistry using the SnoN antibody and the DNA dye bisbenzimide (Hoechst 33258).

FIGURE 2C is a series of photographs. Sagittal sections of cerebella from postnatal rat pups at indicated ages were subjected to immunohistochemistry using the SnoN antibody. Cell nuclei were stained with the DNA dye bisbenzimide (Hoechst 33258). The external granule layer (EGL), molecular layer (ML), and internal granule layer (IGL) are indicated. Asterisks indicate Purkinje cells. Scale bar equals 100 μm.

FIGURE 3 A is a series of immunofluorescent stains. Granule neurons (P6+2DIV) were transfected with the U6 or the U6/snon plasmid together with an expression plasmid encoding farnesylated GFP. Three days later, cultures were subjected to immunocytochemical analysis using the GFP and SnoN antibodies. The arrowhead points to a U6-transfected GFP-positive neuron that is also SnoN positive. The arrow points to a neuron, transfected with the U6/snon plasmid as indicated by GFP expression, that is SnoNnegative. Merged images are shown in third column. Quantification revealed that approximately 70% of the control U6-transfected neurons and only 30% of SnoN hpRNA expressing neurons are SnoN-positive. Scale bar equals 20 μm. FIGURE 3B is a graph showing axonal length per neuron. Granule neurons

(P6+0DIV) were transfected with the U6, U6/snon (mouse), or U6/snon-h (human) plasmid together with the GFP and BCI-XL expression plasmids. Neurons were cultured in media supplements with calf serum for three days, subjected to immunocytochemistry using the

GFP antibody and analyzed as in FIGURE IB. Axon length in mouse SnoN hpRNA (U6/snon)- but not human SnoN hpRNA (U6/snon-h)-expressing neurons was significantly reduced as compared to control U6-transfected neurons (p<0.001, ANOVA). Total number of neurons measured = 378.

FIGURE 3 C is a graph showing axonal length per neuron. Granule neurons (P6+0DIV) were transfected with the control U6 or U6/snon plasmid together with an expression plasmid encoding SnoN-Res or its control vector (pCMV5) and the GFP and BcI- XL expression plasmids. Neurons were cultured in media supplemented with calf serum. Three days after transfection, the cultures were analyzed as in Figure 3B. In the background of SnoN RNAi expression of SnoN-Res (U6/snon + SnoN-Res) significantly increased axonal length as compared to vector-transfected (U6/snon + pCMV5) neurons (p<0.05, ANOVA). Total number of neurons measured = 241.

FIGURE 3D is a graph showing axonal length per neuron. Granule neurons (P6+0DIV) were transfected with the U6 or U6/snon plasmid together with the GFP and BcI- XL expression plasmids. Neurons were cultured in BME supplemented with calf serum for indicated days and analyzed as in FIGURE 3B. SnoN knockdown in granule neurons significantly reduced axonal length at 3 days in vitro and at subsequent days as compared to control U6-transfected neurons (p<0.005, ANOVA). Total number of neurons measured = 716.

FIGURES 3E and 3F are graphs showing axonal length per neuron. Granule neurons (P6+0DIV) were transfected with the U6 or U6/snon plasmid together with the GFP and BcI- XL expression plasmids. Neurons were cultured in BME supplemented with insulin (FIGURE 3E) or calf serum together with membrane depolarizing concentrations of KCl (25 mM) (FIGURE 3F). Three days after transfection, cultures were analyzed as in Figure 3B. Images of representative transfected neurons in BME + insulin are shown in FIGURE 3E. Scale bar equals 50 μm. SnoN knockdown significantly reduced axonal length as compared to the corresponding control U6-transfected neurons in granule neuron cultures in the presence of insulin (p<0.005, ANOVA, total number of neurons measured = 110) or serum and membrane depolarizing concentrations of KCl (p<0.005, ANOVA, total number of neurons measured = 165). FIGURE 3G is a graph showing axonal length per neuron. Granule neurons

(P6+0DIV) were transfected in suspension with the control U6-cmvGFP or the U6/snon- cmvGFP plasmid together with an expression plasmid encoding BCI-XL and placed on top of cerebellar slices from P9 rat pups. Slices were fixed three days later, subjected to immunohistochemistry with the GFP antibody and analyzed using SPOT software. SnoN knockdown significantly reduced axon length when compared to control U6-transfected neurons (p<0.005, ANOVA). Total number of neurons measured = 375.

FIGURES 4A-4C are graphs showing axonal length per neuron. In FIGURE 4A, granule neurons (P6+0DIV) were transfected with the control U6, U6/cdhl, U6/snon plasmid or both U6/cdhl and U6/snon plasmids together with the GFP and Bcl-XLplasmids. Three days later, cultures were analyzed as in Figure 3B. Simultaneous knockdown of Cdhl and SnoN in granule neurons significantly reduces axonal length as compared to control U6- and U6/cdhl -transfected neurons (pO.OOl and p<0.05 respectively, ANOVA). Total number of neurons measured = 521. In FIGURE 4B, granule neurons (P6+0DIV) were transfected with an expression plasmid encoding wild type SnoN, mutant D-box SnoN or the control vector pCMV5 together with the GFP and BCI-XL expression plasmids. Three days later, cultures were analyzed as in FIGURE 3B. Mutant D-box SnoN expression in granule neurons but not the expression of wild type SnoN significantly increased axonal length as compared to control U6-tranfected neurons (p<0.05, ANOVA). Total number of neurons measured = 328. In FIGURE 4C, granule neurons (P6+0DIV) were transfected as in Figure 4B and analyzed at the indicated times as in FIGURE 3B. Expression of mutant D-box SnoN in granule neurons significantly increased axonal growth at day 4 as compared to expression of wild type SnoN or control U6-transfected neurons (p<0.001, ANOVA). Total number of neurons measured = 423. FIGURE 5A is a diagram showing the in vivo electroporation method. Plasmids are injected into the cerebella of anaesthetized P3 rat pups, pups are subjected to electric pulses. Transfected cerebella are analyzed five days later by immunohistochemistry.

FIGURE 5B is a series of immunohistochemical photographs. The control U6- cmvGFP or the U6/snon-cmvGFP plasmid together with the BCI-XL expression plasmid were injected into the cerebellum of P3 rat pups. Five days later at P8, cerebella were isolated from rat pups and 10 μm coronal sections of the cerebella were subjected to immunohistochemistry using the GFP antibody. Representative images of newly generated neurons that extend axons in the EGL. Arrows point to transfected newly generated neurons. Scale bar equals 50 μm. FIGURE 5C is a series of photographs. Coronal sections of cerebella from pups were subjected to in vivo electroporation as described in FIGURE 5B. The external granule layer (EGL), molecular layer (ML) and internal granule layer (IGL) are indicated. Scale bar equals 100 μm in large panels and 50 μm in small panels. FIGURE 5D is a graph quantifying parallel fiber formation. Transfected granule neurons in the IGL were counted in consecutive sections of the U6-cmvGFP or U6/snon- cmvGFP-transfected cerebella. Axons in the molecular layer (see methods). Graph indicates percentage of granule neurons that were associated with parallel fibers. Parallel fiber formation in U6/snon-expressing granule neurons is significantly reduced as compared to control U6-transfected neurons (p<0.001, ANOVA). Total number of neurons measured = 476.

FIGURES 6A and 6B are photographs of immunoblots. Cerebellar granule neurons, hippocampal and cortical neurons were lysed at the indicated day in vitro after plating (DIV) and subjected to immunoblotting using an antibody that recognizes Smad2 and Smad3 and an antibody to 14-3-3, the latter to serve as loading control.

FIGURE 6C is a photograph showing the results of immunofluorescence analysis. Cultured granule neurons (DIV2) were subjected to immunofluorescence analysis using the Smad2/3 antibody (left panel) and the DNA dye bisbenzimide (right panel). FIGURE 6D is a photograph of an immunoblot. Granule neurons were subjected to subcellular fractionation. Nuclear (N) and cytoplasmic (C) fractions were immunoblotted using the Smad2/3, SnoN and 14-3-3 antibodies. Asterisk indicates non-specific band.

FIGURE 6E is a photograph of an immunoblot. Granule neuron lysates were subjected to immunoprecipitation using the SnoN antibody followed by immunoblotting with the Smad2/3 antibody or reverse co-immunoprecipitation. Double asterisks indicate heavy chain IgGs.

FIGURE 7A is a photograph showing the results of immunocytochemical analysis. Granule neurons were transfected 8 hours after plating with the Smad2 RNAi or control U6 plasmid together with the GFP expression plasmid and maintained in media supplemented with insulin. Three days after transfection, neurons were fixed and subjected to immunocytochemistry using an antibody to GFP. Images of control U6-transfected and U6/smad2 -transfected neurons, with asterisks indicating cell bodies and arrowheads indicating axons. Scale bar equals lOOμm.

FIGURE 7B is a bar graph showing axonal length in Smad2 knockdown neurons as compared to control U6-transfected neurons. Axonal length was measured in GFP-positive transfected neurons using SPOT software. A total of 268 neurons were measured.

FIGURE 7C is a photograph showing the results of an immunoblot. 293T cells were transfected with the Smad2 RNAi plasmid or control U6 plasmid together with an expression plasmid encoding Smad2 using wild type cDNA (Smad2-WT) or an RNAi-resistant cDNA (Smad2-Rescue). Lysates were immunoblotted using the Smad2/3 and 14-3-3 antibodies, the latter to serve as loading control.

FIGURE 7D is a bar graph demonstrating axonal length in Smad2 knockdown and Smad2 -Rescue neurons as compared to U6-transfected neurons. Granule neurons transfected with the Smad2 RNAi or control U6 plasmid together with pcDNA3 vector, Smad2-WT or Smad2 -Rescue expression plasmid were analyzed. A total of 297 neurons were measured.

FIGURE 8A is a bar graph illustrating axonal length in Smad2 knockdown and SnoN knockdown neurons. Neurons transfected with the control U6, U6/smad2, or U6/snon RNAi plasmid or both U6/smad2 and U6/snon plasmids were maintained in media supplemented with 10% calf serum and membrane depolarizing concentrations of KCl and analyzed as in Figure 7B. A total of 438 neurons were measured.

FIGURE 8B is a bar graph demonstrating axonal length in neurons transfected with the control U6, U6/cdhl , or U6/smad2 RNAi plasmid or both U6/cdhl and U6/smad2 plasmids. The neurons were placed in media supplemented with insulin and analyzed as in Figure 7B. A total of 429 neurons were measured.

FIGURE 9A is a photograph of an immunoblot. Granule neurons were subjected to subcellular fractionation and analyzed with immunoblotting using an antibody that recognizes Smad2 specifically when phosphorylated at Serines 465 and 467 as well as the SnoN and 14- 3-3 antibodies. Asterisk indicates non-specific band. FIGURE 9B is a bar graph demonstrating axonal growth in neurons transfected with the control pCMV5 vector, Smad6 or Smad7 expression plasmids. Neurons were placed in media supplemented with 10% calf serum and analyzed as in Figure 7B. A total of 247 and 305 neurons were measured, respectively.

FIGURE 9C are photographs of immunoblots. Lysates of granule neurons exposed to SB431542 and SB505124 at the indicated concentrations for 48 hours were immunoblotted with the SnoN, phosphoS465/467-Smad2, Smad2/3, and 14-3-3 antibodies.

FIGURE 9D is a bar graph illustrating axonal length in granule neurons transfected with the GFP expression plasmid at DIVO and placed in media supplemented with 10% calf serum. Neurons were treated with SB431542 or its vehicle (DMSO) starting at DIVl for 48 hours and analyzed as in Figure 7B. A total of 170 neurons were measured.

FIGURE 9E is a series of images of control vehicle and SB431542-treated neurons. Asterisks indicate cell bodies and arrowheads indicate axons. Scale bar equals lOOμm.

FIGUIRE 1OA is a series of images of neurons plated on polyornithine or myelin- coated coverslips (13.3 μg/ml) and transfected with the control U6 or U6/smad2 RNAi plasmid were analyzed at DIV3 as in Figure 7A. Asterisks and arrowheads indicate cell bodies and axons, respectively. Scale bar equals lOOμm.

FIGURE 1OB is a bar chart quantifying the results shown in Figure 1OA analyzed as in Figure 7B. A total 130 of neurons were measured.

Detailed Description of the Invention

The growth of axons is critical to the establishment of neuronal connectivity and normal wiring of the developing nervous system. Axon growth and guidance cues act on neurons via cell-surface receptors that couple attractive and repulsive extrinsic signals to the cytoskeletal machinery of the axon growth cone. Cell-intrinsic mechanisms also play a role in the control of axonal morphogenesis. The mechanisms governing axonal morphogenesis was explored. The present invention is based on the discovery that the ubiquitin ligase Cdhl- anaphase promoting complex (Cdhl-APC) reduces the activity of the transcriptional co- repressor SnoN (Nomura et al., Nucleic Acids Res. 17:5489 (1989); Pearson-White, S., ibid. 21:4632 (1993); Crittenden, R., 25:2930 (1997)), thereby reducing the growth of axons in the mammalian brain. Cdhl-APC is a multisubunit E3 ubiquitin ligase that promotes the ubiquitination and consequent degradation of B-type cyclins and other proteins in dividing cells and thereby ensures the proper transitions of the cell cycle. The regulatory subunit Cdhl has the dual function of stimulating the APC ubiquitin ligase activity and targeting

APC to its substrates. Substrate recognition by Cdhl is dependent on specific peptide motifs, including the D-box and the KEN-box, that are present in APC substrates. In the present study, knockdown of SnoN in primary granule neurons by RNAi significantly reduced axon length. Importantly, SnoN knockdown suppressed the Cdhl RNAi-induced phenotype of enhanced axonal growth. Conversely, expression of a SnoN protein in which the Cdhl recognition destruction box (D-box) was mutated robustly stimulated the growth of axons. Furthermore, the in vivo knockdown of SnoN by electroporation in the developing rat cerebellum profoundly impaired the development of granule neuron parallel fibers in the developing cerebellar cortex. These findings uncover an essential function for the transcriptional co-repressor SnoN in axonal growth in the mammalian brain. In addition, the data indicates that by acting in the nucleus via regulation of SnoN-dependent programs of gene expression, Cdhl-APC plays a pivotal cell-intrinsic role in the control of axonal morphogenesis. Accordingly, the methods and compositions provided herein are useful for inducing axonal growth and regeneration by administering to a subject in need thereof an agent that reduces the expression or activity of Cdhl-APC in neuronal cells. For example, the subject is administered an agent that increases the level or activity of the transcriptional factor SnoN. Agents that induce axonal growth and differentiation are useful to treat, prevent, or reduce spinal cord injuries. Methods for identifying compounds that are useful for inducing axonal growth or regeneration are also described herein.

The experiments described herein were performed using the following Materials and Methods.

Cerebellar granule neuron culture and transfections Granule neurons were prepared from isolated cerebella of P6 Long-Evans rat pups.

Neurons were plated on polyornithine-coated glass coverslips and cultured in BME supplemented with 10% calf serum, 25 mM KCl, glutamine, penicillin and streptomycin. Neurons were transfected either eight hours after plating or at 2DIV with a modified calcium phosphate method as described, and placed in BME supplemented with glucose, 1% glutamine/penicillin/streptomycin and either insulin, calf serum, or calf serum together with membrane depolarizing concentrations of KCl (25mM). One day after culture preparation or transfection, neurons were treated with AraC to prevent proliferation of glial cells. To rule out the possibility that the effects of RNAi or protein expression on axonal length were due to any effect of these manipulations on cell survival, the anti-apoptotic protein Bcl-XLwas co- expressed in all experiments. The expression of BCI-XL itself has little or no effects on axonal length.

Morphometry

Analysis of the morphology of axons of the cerebellar granule neurons in vitro and in the slice overlay assay was carried out by capturing images of the neurons in a blinded manner using a Nikon eclipse TE2000 epifluorescence microscope. Measurements of axons were performed using SPOT imaging software as described (Gaudilliere et al., Neuron 41 : 229-241, 2004).

In vivo electroporation

In vivo electroporation was performed as described (Konishi et al., Science 303: 1026-1030, 2004). Briefly, the U6-cmvGFP or U6/snon-cmvGFP plasmid together with the BCI-XL expression plasmid were diluted in PBS/0.3% fast green (3-4 μl with 4 μg/μl of U6 plasmids and 1 μg/μl of BCI-XL plasmid) and injected into the cerebellum of P3 Sprague- Dawley rat pups. The pups were subjected to 5 electric pulses of 160 mV with 950ms intervals. To analyze the parallel fiber in the in vivo electroporation experiments, the identity of transfected granule neurons in the EGL was confirmed based on the small size of the nuclei, as determined by staining with the DNA dye bisbenzimide (Hoechst 33258) and MEF2 immunoreactivity, a marker of granule neurons in the IGL. Transfected granule neurons were counted in consecutive sections of individual cerebella. Parallel fibers were counted in a restricted area of consecutive sections to prevent recounting.

Slice overlay assay Slice overlay assay was performed as described (Konishi et al., supra). Briefly, cerebellar slices from P8 or P9 rat pups were prepared using a Mclllwain Tissue Chopper. 400 μm slices were cultured on 0.4 μm membranes using medium-air- interface method (MEM/, 25 raM HEPES, 25% horse serum, 6.5mg/ml D-glucose, ImVlOOmI PSG) for 24 hours at 36°C/5% CCh before coculture with granule neurons. Granule neurons were isolated from P6 rats as described and transfected 3 hours later in suspension (2.5x10 cells/2 ml

DMEM) using a modified calcium phosphate method with the control U6 plasmid that also contained an expression cassette encoding GFP (U6-cmvGFP) or the U6/snon-cmvGFP together with an expression plasmid encoding BCI-XL. Transfection reaction was terminated by adding a large volume of DMEM. Cells were pelleted and then plated on top of thecerebellar slices and cocultured for 3 days. Slices were then subjected to immunostaining using the GFP antibody. Slice integrity was assessed using the DNA dye bisbenzimide (Hoechst 33258).

Therapeutic Agents As described herein, an axonal inducer is any agent that increases axonal growth or regeneration and includes any agent that reduces the level or activity of Cdhl-APC, any agent that increases the level or activity of SnoN, or both in a cell relative to a control cell. The control cell is a cell that has not been treated with the axonal inducer. The expression or activity of a polypeptide is determined by any standard method in the art, including those described herein. For example, the levels or activity of a polypeptide is measured by Western blot analysis, immunohistochemistry, ELISA, and Northern Blot analysis. The activity level of Cdhl is also measured by assessing phosphorylation levels at various amino acid sites. Alternatively, the biological activity of Cdhl-APC is measured by assessing the expression or activity of any of the molecules involved in Cdhl-APC signaling, such as SnoN. Axonal inducers include polypeptides, polynucleotides, small molecule antagonists, or siRNA.

Optionally, the axonal inducer is a dominant interfering protein or a nucleic acid encoding a dominant interfering protein that interferes with the biological activity of a polypeptide. For example, a dominant negative Cdhl is used to interfere with the activity of Cdhl-APC. Alternatively, a dominant active SnoN is used to increase the activity of SnoN in a cell. A dominant interfering protein is any amino acid molecule having a sequence that has at least 50%, 70%, 80%, 90%, 95%, or even 99% sequence identity to at least 10, 20, 35, 50, 100, or more than 150 amino acids of the wild type protein to which the dominant interfering protein corresponds. For example, a dominant active SnoN has mutation such that it is constitutively active in a cell. Alternatively, the axonal inducer is a Cdhl dominant negative protein having a mutation such that it can no longer be phosphorylated and such that the activity of Cdhl-APC is reduced.

The dominant negative or dominant active protein is administered as an expression vector. The expression vector may be a non-viral vector or a viral vector (e.g., recombinant retrovirus, recombinant lentivirus, recombinant adeno-associated virus, or a recombinant adenoviral vector). Alternatively, the dominant negative protein is directly administered as a recombinant protein systemically or to the infected area using, for example, microinjection techniques. The axonal inducer is an antisense molecule, an RNA interference (siRNA) molecule, a small molecule antagonist that targets (the subunit APC2) or Cdhl expression or activity, or a small molecule agonist that increases SnoN level or activity. By the term "siRNA" is meant a double stranded RNA molecule which prevents translation of a target mRNA. Standard techniques of introducing siRNA into a cell are used, including those in which DNA is a template from which an siRNA RNA is transcribed. The siRNA includes a sense Cdhl or nucleic acid sequence, an anti-sense Cdhl or nucleic acid sequence or both. Optionally, the siRNA is constructed such that a single transcript has both the sense and complementary antisense sequences from the target gene, e.g., a hairpin. Binding of the siRNA to a Cdhl or transcript in the target cell results in a reduction in Cdhl or production by the cell. The length of the oligonucleotide is at least 10 nucleotides and may be as long as the naturally- occurring Cdhl or transcript. Preferably, the oligonucleotide is 19-25 nucleotides in length. Most preferably, the oligonucleotide is less than 75, 50 , 25 nucleotides in length.

Small molecules includes, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic and inorganic compounds (including heterorganic and organomettallic compounds) having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 2,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

The preferred dose of the axonal inducer is a biologically active dose. A biologically active dose is a dose that will increase axonal growth or regeneration or that will treat, reduce, or prevent a spinal cord injury.

Optionally, the subject is administered one or more additional therapeutic regiments. The additional therapeutic regimens are administered prior to, concomitantly, or subsequent to administration of the axonal inducer. For example, the axonal inducer and the additional agent are administered in separate formulations within at least 1, 2, 4, 6, 10, 12, 18, or more than 24 hours apart. Optionally, the additional agent is formulated together with the axonal inducer. When the additional agent is present in a different composition, different routes of administration may be used. The agent is administered at doses known to be effective for such agent for treating, reducing, or preventing a spinal cord injury.

Concentrations of the axonal inducer and the additional agent depends upon different factors, including means of administration, target site, physiological state of the mammal, and other medication administered. Thus treatment dosages may be titrated to optimize safety and efficacy and is within the skill of an artisan. Determination of the proper dosage and administration regime for a particular situation is within the skill of the art.

Therapeutic Administration

The invention includes administering to a subject a composition that includes a compound that induces axonal growth or regeneration (referred to herein as an "axonal inducer" or "therapeutic compound").

An effective amount of a therapeutic compound is preferably from about 0.1 mg/kg to about 150 mg/kg. Effective doses vary, as recognized by those skilled in the art, depending on route of administration, excipient usage, and coadministration with other therapeutic treatments including use of other agents or therapeutic agents for treating, preventing or alleviating a symptom of a spinal cord injury. A therapeutic regimen is carried out by identifying a mammal, e.g., a human patient suffering from (or at risk of developing) a spinal cord injury, using standard methods.

The pharmaceutical compound is administered to such an individual using methods known in the art. Preferably, the compound is administered orally, rectally, nasally, topically or parenterally, e.g., subcutaneously, intraperitoneally, intramuscularly, and intravenously. The compound is administered prophylactically, or after the detection of a spinal cord injury. The compound is optionally formulated as a component of a cocktail of therapeutic drugs to treat the injury. Examples of formulations suitable for parenteral administration include aqueous solutions of the active agent in an isotonic saline solution, a 5% glucose solution, or another standard pharmaceutically acceptable excipient. Standard solubilizing agents such as PVP or cyclodextrins are also utilized as pharmaceutical excipients for delivery of the therapeutic compounds.

The therapeutic compounds described herein are formulated into compositions for other routes of administration utilizing conventional methods. For example, the axonal inducer is formulated in a capsule or a tablet for oral administration. Capsules may contain any standard pharmaceutically acceptable materials such as gelatin or cellulose. Tablets may be formulated in accordance with conventional procedures by compressing mixtures of a therapeutic compound with a solid carrier and a lubricant. Examples of solid carriers include starch and sugar bentonite. The compound is administered in the form of a hard shell tablet or a capsule containing a binder, e.g., lactose or mannitol, a conventional filler, and a tableting agent. Other formulations include an ointment, suppository, paste, spray, patch, cream, gel, resorbable sponge, or foam. Such formulations are produced using methods well known in the art.

Where the therapeutic compound is a nucleic acid encoding a protein, the Therapeutic nucleic acid is administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular (e.g., by use of a retroviral vector, by direct injection, by use of microparticle bombardment, by coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (See, e.g., Joliot, et al, 1991. Proc Natl Acad Sci USA 88:1864-1868), and the like. A nucleic acid therapeutic is introduced intracellularly and incorporated within host cell DNA or remain episomal.

For local administration of DNA, standard gene therapy vectors used. Such vectors include viral vectors, including those derived from replication-defective hepatitis viruses (e.g., HBV and HCV), retroviruses (see, e.g., WO 89/07136; Rosenberg et al., 1990, N. Eng. J. Med. 323(9):570-578), adenovirus (see, e.g., Morsey et al., 1993, J. Cell. Biochem., Supp. 17E,), adeno-associated virus (Kotin et al., 1990, Proc. Natl. Acad. Sci. USA 87:2211-2215,), replication defective herpes simplex viruses (HSV; Lu et al., 1992, Abstract, page 66, Abstracts of the Meeting on Gene Therapy, Sept. 22-26, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York), and any modified versions of these vectors. The invention may utilize any other delivery system which accomplishes in vivo transfer of nucleic acids into eucaryotic cells. For example, the nucleic acids may be packaged into liposomes, e.g., cationic liposomes (Lipofectin), receptor-mediated delivery systems, non-viral nucleic acid- based vectors, erythrocyte ghosts, or microspheres (e.g., microparticles; see, e.g., U.S. Patent No. 4,789,734; U.S. Patent No. 4,925,673; U.S. Patent No. 3,625,214; Gregoriadis, 1979, Drug Carriers in Biology and Medicine, pp. 287-341 (Academic Press,). Naked DNA may also be administered.

DNA for gene therapy can be administered to patients parenterally, e.g., intravenously, subcutaneously, intramuscularly, and intraperitoneally. DNA or an inducing agent is administered in a pharmaceutically acceptable carrier, i.e., a biologically compatible vehicle which is suitable for administration to an animal e.g., physiological saline. A therapeutically effective amount is an amount which is capable of producing a medically desirable result, e.g., an increase in the level or activity of SnoN in a treated animal. Such an amount can be determined by one of ordinary skill in the art. As is well known in the medical arts, dosage for any given patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosages may vary, but a preferred dosage for intravenous administration of DNA is approximately 10⁶ to 10²² copies of the DNA molecule.

Typically, plasmids are administered to a mammal in an amount of about 1 nanogram to about 5000 micrograms of DNA. Desirably, compositions contain about 5 nanograms to 1000 micrograms of DNA, 10 nanograms to 800 micrograms of DNA, 0.1 micrograms to 500 micrograms of DNA, 1 microgram to 350 micrograms of DNA, 25 micrograms to 250 micrograms of DNA, or 100 micrograms to 200 micrograms of DNA. Alternatively, administration of recombinant adenoviral vectors encoding the axonal inducer into a mammal may be administered at a concentration of at least 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, or 10¹¹ plaque forming unit (pfu). Gene products encoding the axonal inducer are administered to the patient intravenously in a pharmaceutically acceptable carrier such as physiological saline. Standard methods for intracellular delivery of peptides can be used, e.g. packaged in liposomes. Such methods are well known to those of ordinary skill in the art. It is expected that an intravenous dosage of approximately 1 to 100 moles of the polypeptide of the invention would be administered per kg of body weight per day. The compositions of the invention are useful for parenteral administration, such as intravenous, subcutaneous, intramuscular, and intraperitoneal.

Axonal inducerss are effective upon direct contact of the compound with the affected tissue or may alternatively be administered systemically (e.g., intravenously, rectally or orally). The axonal inducer may be administered intravenously or intrathecally (i.e., by direct infusion into the cerebrospinal fluid). For local administration, the compound is injected or infused directly into brain or other CNS tissue or a compound-impregnated wafer or resorbable sponge is placed in direct contact with CNS tissue. The compound or mixture of compounds is slowly released in vivo by diffusion of the drug from the wafer and erosion of the polymer matrix. Alternatively, the compound is infused into the brain or cerebrospinal fluid using standard methods. For example, a burr hole ring with a catheter for use as an injection port is positioned to engage the skull at a burr hole drilled into the skull. A fluid reservoir connected to the catheter is accessed by a needle or stylet inserted through a septum positioned over the top of the burr hole ring. A catheter assembly (described, for example, in U.S. Patent No. 5,954,687) provides a fluid flow path suitable for the transfer of fluids to or from selected location at, near or within the brain to allow administration of the drug over a period of time.

One in the art will understand that the patients treated according to the invention may have been subjected to the tests to diagnose a subject as having a spinal cord injury or may have been identified, without examination, as one at high risk due to the presence of one or more risk factors (e.g., genetic predisposition). Reduction of spinal injury symptoms may also include, but are not limited to, alleviation of symptoms (e.g., headaches, pain, and inflammation), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, and amelioration or palliation of the disease state. Treatment may occur at home with close supervision by the health care provider, or may occur in a health care facility. Screening Assays

Screening methods are carried out to identify compounds that reduce the expression or activity of Cdhl-APC or increase the level or activity of SnoN. Useful compounds are identified by detecting an attenuation of the expression or activity of any of the molecules involved in Cdh-1-APC signaling. Given its ability to increase axonal growth or regeneration, a compound identified using the present screening methods may be used, for example, as a therapeutic agent to treat, reduce, or prevent a spinal cord injury, or alternatively, to alleviate one or more symptoms associated with such an injury.

A number of methods are available for carrying out such screening assays. According to one approach, candidate compounds are added at varying concentrations to the culture medium of cells expressing SnoN. Gene expression of SnoN is then measured, for example, by standard Northern blot analysis, using any appropriate fragment prepared from the nucleic acid molecule of SnoN as a hybridization probe or by real time PCR with appropriate primers. The level of gene expression in the presence of the candidate compound is compared to the level measured in a control culture medium lacking the candidate molecule. If desired, the effect of candidate compounds may, in the alternative, be measured at the level of SnoN polypeptide using the same general approach and standard immunological techniques, such as Western blotting or immunoprecipitation with an antibody specific to SnoN for example. For example, immunoassays may be used to detect or monitor the level of SnoN. Polyclonal or monoclonal antibodies which are capable of binding to SnoN may be used in any standard immunoassay format (e.g., ELISA or RIA assay) to measure the levels of SnoN. SnoN can also be measured using mass spectroscopy, high performance liquid chromatography, spectrophotometric or fluorometric techniques, or combinations thereof.

As a specific example, mammalian cells (e.g., rodent cells) that express a nucleic acid encoding SnoN are cultured in the presence of a candidate compound (e.g., a peptide, polypeptide, synthetic organic molecule, naturally occurring organic molecule, nucleic acid molecule, or component thereof). Cells may either endogenously express SnoN or may alternatively be genetically engineered by any standard technique known in the art (e.g., transfection and viral infection) to overexpress SnoN. The expression level of SnoN is measured in these cells by means of Western blot analysis and subsequently compared to the level of expression of the same protein in control cells that have not been contacted by the candidate compound. A compound which promotes an increase in the level of SnoN activity as a result of increasing its synthesis or biological activity is considered useful in the invention. Alternatively, the screening methods of the invention may be used to identify candidate compounds that increase axonal growth or regeneration by reducing the biological activity or expression of Cdhl by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative to an untreated control. As an example, a candidate compound may be tested for its ability to reduce Cdhl activity in cells that naturally express Cdhl , after transfection with cDNA for Cdhl, or in cell-free solutions containing Cdhl, as described further below. The effect of a candidate compound on the binding or activation of Cdhl can be tested by radioactive and non-radioactive binding assays, competition assays, and receptor signaling assays. As a specific example, a candidate compound may be contacted with two proteins, the first protein being a polypeptide substantially identical to Cdhl and the second protein being APC (i.e., a protein that binds the Cdhl polypeptide under conditions that allow binding). According to this particular screening method, the interaction between these two proteins is measured following the addition of a candidate compound. A decrease in the binding of Cdh 1 to APC following the addition of the candidate compound (relative to such binding in the absence of the compound) identifies the candidate compound as having the ability to inhibit the interaction between the two proteins, and thereby having the ability to increase axonal growth or regeneration. The screening assay of the invention may be carried out, for example, in a cell-free system or using a yeast two-hybrid system. If desired, one of the proteins or the candidate compound may be immobilized on a support as described above or may have a detectable group.

Alternatively, or in addition, candidate compounds may be screened for those which specifically bind to and thereby inhibit Cdhl or APC, for example. The efficacy of such a candidate compound is dependent upon its ability to interact with Cdhl or APC. Such an interaction can be readily assayed using any number of standard binding techniques and functional assays. For example, a candidate compound may be tested in vitro for interaction and binding with Cdhl or APC and its ability to modulate axonal growth or regeneration may be assayed by any standard assays (e.g., those described herein).

For example, a candidate compound that binds to APC may be identified using a chromatography-based technique. For example, a recombinant APC may be purified by standard techniques from cells engineered to express APC (e.g., those described above) and may be immobilized on a column. Alternatively, the naturally-occurring APC may be immobilized on a column. A solution of candidate compounds is then passed through the column, and a compound specific for APC2 is identified on the basis of its ability to bind to APC and be immobilized on the column. To isolate the compound, the column is washed to remove non-specifϊcally bound molecules, and the compound of interest is then released from the column and collected. Compounds isolated by this method (or any other appropriate method) may, if desired, be further purified (e.g., by high performance liquid chromatography).

Screening for new inhibitors and optimization of lead compounds may be assessed, for example, by assessing their ability to modulate the level or activity of Cdhl, (APC subunit 2), or SnoN using standard techniques. In addition, these candidate compounds may be tested for their ability to increase axonal growth or regeneration (e.g., as described herein). Compounds isolated by this approach may also be used, for example, as therapeutics to treat, reduce, or prevent a spinal cord injury, or alternatively, to alleviate one or more symptoms associated with such injuries. Compounds which are identified as binding to Cdhl, APC, or SnoN with an affinity constant less than or equal to 10 mM are considered particularly useful in the invention. Ubiquitination assays are used to identify candidate compounds for inducing axonal growth and regeneration. In this cell free assay, the activity of Cdhl -APC is proportional to the degree of ubiquitination of a known in vitro translated substrate (Cyclin B). Candidate compounds are added to the in vitro ubiquitination reaction. Compounds that reduce the activity of Cdhl -APC are identified by a reduced or impaired ubiquitination of the substrate. In another example, a dual Luciferase-based assay is used to identify compounds that increase the amount of SnoN protein. Renilla is fused to wild type SnoN or non-degradable SnoN Dbox mutant (DBM) and expressed in neuronal cells such as cerebellar granule neurons. Candidate compounds are added to the media. A compound that inhibits Cdhl- APC activity leads to higher amounts of wild type SnoN as compared to mock treated neurons. In addition, the amount of wild type SnoN is comparably high or higher than the non-degradable SnoN DBM.

Compounds that increase SnoN activity are also screened using a Dual Luciferase assay. Constructs that express luciferase under the promoter of a SnoN target that controls axon growth are transfected into neuronal cells such as cerebellar granule neurons. Candidate compounds are added to the media. Compounds that block Cdhl -APC (or alternatively activate SnoN) enhance SnoN activity and result in the predicted readout of the luciferase activity as compared to mock treated neurons. Another screening assay involves identification of candidate compounds using an in vitro axon outgrowth assay. Wild type and mutant neuronal cells such as cerebellar granule neurons, in which GFP expression is driven by the actin promoter are co-cultured. 0.14% of the neurons will be expressing GFP and be monitored for axonal growth. The candidate compound or vehicle is added to the medium and the axon length will be determined in treated and mock-treated GFP-expressing neurons. This method screens for candidate compounds that reduce Cdhl-APC activity or induce SnoN activity and result in enhanced axon length. Alternatively, mixed populations of cortical or hippocampal neurons may be used. In addition to dissociated neuronal cultures, tissue explants (DRG, cerebellar explants) can be used in neurite/axon growth assay.

Potential therapeutic agents include organic molecules, peptides, peptide mimetics, polypeptides, and antibodies that bind to a nucleic acid sequence or polypeptide that encodes Cdhl, APC2, or SnoN and thereby modulate their activity. Potential agents also include small molecules that bind to and occupy the binding site of such polypeptides thereby preventing or allowing binding to cellular binding molecules, such that normal biological activity is modulated.

This invention is based in part on the experiments described in the following examples. These examples are provided to illustrate the invention and should not be construed as limiting.

Example 1: Cdhl-APC acts in the nucleus to control axonal growth

To characterize the mechanism by which Cdhl-APC controls axonal growth, the subcellular site of action of Cdhl-APC in neurons was determined. A large fraction of Cdhl and the APC core protein Cdc27 reside in the nucleus. Consistent with these results, the ubiquitin ligase activity of Cdhl-APC in neurons was also found to be predominantly but not exclusively present in the nuclear fraction. These results raise the important question of whether Cdhl-APC operates in the nucleus or the cytoplasm to control axonal growth.

Structure-function analyses of Cdhl in the inhibition of axon growth were performed. Since the overexpression of Cdhl in neurons failed to alter axon growth, an RNAi-resistant form of Cdhl (Cdhl -Res) was tested for its ability to rescue the Cdhl knockdown-induced axonal phenotype in neurons and in turn, allow Cdhl structure-function analyses in this background. To perform the rescue experiment, an expression plasmid encoding a GFP- Cdhl fusion protein was constructed using wild type Cdhl cDNA and a plasmid encoding an

RNAi-resistant form of Cdhl (GFP-Cdhl-Res, FIGURE IA). Immunoblotting revealed that Cdhl RNAi induced the knockdown of Cdhl, encoded by wild type Cdhl cDNA, but failed to effectively reduce the expression of Cdhl -Res (FIGURE IA). Cerebellar granule neurons were next transfected with the Cdhl RNAi plasmid U6/cdhl encoding Cdhl hairpin RNAs (hpRNAs) or the control U6 plasmid together with a DsRed expression plasmid (FIGURE IB). In the background of Cdhl RNAi, GFP-Cdhl or GFP-Cdhl-Res was also expressed. GFP-Cdhl-Res, but not

GFP-Cdhl, reversed the Cdhl knockdown-induced enhancement of axonal growth (FIGURE IB). These experiments indicate that the axonal phenotype upon Cdhl RNAi is the result of specific knockdown of Cdhl. To determine if the localization of Cdhl is of importance for Cdhl -APC function in neurons, GFP-Cdhl-Res was targeted to the nucleus or the cytoplasm (FIGURE 1C). The GFP-Cdhl-Res expression plasmid was modified by appending a nuclear exclusion sequence (NES) or a nuclear localization sequence (NLS) to the N-terminus of Cdhl -Res. The expression of GFP-NES-Cdhl-Res and GFP-NLS-Cdhl-Res was assessed both in 293T cells and granule neurons. The localization of GFP-NLS-Cdhl-Res was predominantly nuclear in 293T cells and in primary neurons. By contrast, GFP-NES-Cdhl-Res was excluded from the nucleus (FIGURE 1C).

The ability of Cdhl -Rescue mutants to reverse the increase in axonal length triggered by Cdhl knockdown was next examined (FIGURE ID). Granule neurons were transfected with the Cdhl RNAi plasmid together with the control GFP or one of the GFP-Cdhl-Res series of plasmids and the DsRed expression vector. Neurons were analyzed three days after transfection. Analysis of axon length revealed that GFP-Cdhl-Res and GFP-NLS-Cdhl-Res behaved in a very similar manner, reversing the Cdhl RNAi -induced enhancement of axonal growth (Fig. ID). In contrast, GFP-NES-Cdhl-Res failed to reverse the Cdhl RNAi axonal phenotype, and axons remained at a comparable length as control axons (Fig. ID). Together, these results suggest that the localization of Cdhl in the nucleus is required for Cdhl -APC inhibition of axon growth.

Example 2: The Cdhl -APC target SnoN promotes axonal growth The importance of the nuclear localization of Cdhl -APC in the control of axonal growth indicates that the targets of Cdhl -APC reside in the nucleus. To identify potential targets of neuronal Cdhl -APC that might regulate axonal growth, a candidate approach was taken and targets of APC in proliferating cells that control transcription were considered.

Almost all APC substrates in dividing cells have functions in anaphase, including motor spindle and kinetochore proteins, and in the regulation of DNA replication. The transcriptional corepressor SnoN however is the sole identified substrate of Cdhl-APC in proliferating cells that directly regulates transcription. Whether SnoN is expressed in neurons and whether SnoN might control axonal growth downstream of Cdhl-APC was next examined.

The expression of SnoN in granule neurons in the cerebellum was characterized. Immunoblotting of lysates from primary cerebellar granule neurons with an antibody to SnoN revealed the expression of two alternatively spliced forms of SnoN (FIGURE 2A). Immunoprecipitation of lysates from granule neurons followed by immunoblotting with the SnoN antibody demonstrated the specificity of the two bands representing two SnoN isoforms (FIGURE 2A). Indirect immunocytochemistry revealed a nuclear staining pattern of SnoN in granule neurons (FIGURE 2B).

By immunohistochemical analysis of the developing rat cerebellar cortex, SnoN was found to be expressed in both granule neurons and Purkinje cells (FIGURE 2C). Within the granule cell lineage, SnoN appeared to be absent or expressed at low levels in granule cell precursors and newly generated neurons in the external granule layer (EGL) (FIGURE 2C). However, granule neurons in the internal granule layer (IGL) displayed robust expression of SnoN, which persisted from P6 to Pl 3 (FIGURE 2C). The pattern of SnoN expression in granule neurons temporally correlates with axon growth in these neurons during brain development.

To study the function of SnoN in granule neurons, RNAi interference was used to acutely knockdown SnoN (FIGURE 3A). A plasmid encoding SnoN hpRNAs (U6/snon) that induces the knockdown of mouse SnoN was used. The SnoN hpRNAs target a sequence in murine SnoN mRNA that is identical in rat SnoN (NCBI reference, XM 226979). Consistent with this observation, expression of the SnoN hpRNAs in primary rat cerebellar granule neurons led to the efficient knockdown of endogenous SnoN as determined by immunocytochemical analyses (FIGURE 3A). Whereas 70% of control U6-transfected neurons displayed robust SnoN immunoreactivity, only 30% of SnoN hpRNA-expressing neurons had SnoN immunoreactivity (FIGURE 3A). SnoN knockdown in granule neurons led to a striking axonal phenotype. Granule neurons in which SnoN RNAi was triggered had significantly shorter axons than control U6- transfected neurons (FIGURE 3B), indicating that SnoN is required for the normal development of axons. Whether the SnoN RNAi-induced axonal phenotype is the result of specific knockdown of SnoN was next examined. A plasmid encoding human SnoN hpRNAs (U6/snon-h) which contain 3 nucleotide mismatches with the rodent SnoN hpRNAs was used. Expression of human SnoN hpRNAs in granule neurons failed to reduce axon length (FIGURE 3B). Second, a rescue experiment in the background of SnoN RNAi was performed (FIGURE 3C). For these experiments, a plasmid encoding human SnoN harboring additional silent mutations in its cDNA was used (SnoN-Res). These mutations were designed to render the cDNA resistant to RNAi. The effect of SnoN-Res expression on axonal growth was determined in the background of SnoN knockdown. The expression of SnoN-Res triggered a significant increase in axonal length, restoring total axonal length to 70% of control U6-transfected granule neurons (FIGURE 3C). Taken together, these results indicate that the SnoN RNAi-triggered reduction in axonal length is the result of specific knockdown of SnoN rather that off-target effects of SnoN RNAi or non-specific activation of the RNAi machinery. To assess the nature of SnoN function in the development of axons, neurons were transfected with the control U6 or the U6/snon plasmid at a time when they begin to extend axons and measured the length of axons in cohorts of neurons each day for four days, beginning one day after transfection (FIGURE 3D). In control U6-transfected granule neurons, axons increased significantly in length from day 2 to day 5 after transfection. However, upon SnoN knockdown the axonal growth curve was significantly reduced in slope (FIGURE 3D). Axons were two-fold shorter in neurons with SnoN knockdown as compared to control U6-transfected neurons at day 5 (FIGURE 3D). These results indicate that a key function of the transcriptional corepressor SnoN in neurons is to promote the growth of axons. The effect of extrinsic culture conditions on SnoN during axonal growth of granule neurons was next determined. Specifically, the effect of SnoN knockdown in granule neurons that were exposed to the growth factor insulin, serum, or serum together with membrane depolarization was next assessed. Under each of these conditions, SnoN knockdown significantly reduced total axonal length in granule neurons (FIGURES 3B, 3E and 3F). These results indicate that SnoN promotes axonal growth independently of the extrinsic conditions in which neurons were cultured. Having identified a cell-autonomous function for SnoN in axonal growth in primary dissociated cultures of granule neurons, the role of SnoN during axonal growth in the context of the cerebellar cortex was determined

(FIGURE 3G). To address this question, cerebellar slice overlay assays were used. P6 neurons that were transfected with a plasmid encoding both SnoN hpRNAs and GFP bicistronically (U6/snon-cmvGFP) or the control U6-cmvGFP plasmid were used on top of cerebellar slices prepared from P9 rat pups. Three days later, slices were subjected to immunohistochemistry. Granule neurons in which SnoN RNAi was triggered had a significant reduction in total axonal length as compared to the control U6 plasmid-transfected neurons (FIGURE 3G). These results indicate that SnoN promotes axonal growth in granule neurons in the tissue environment of the cerebellar cortex.

Example 3: SnoN acts downstream of neuronal Cdhl-APC in the control of axon growth To determine if Cdhl-APC and its substrate SnoN act in a linear pathway to regulate axonal growth, an epistatic analysis of the effects of Cdhl and SnoN knockdown on axonal length was performed (FIGURE 4A). Primary granule neurons were transfected with the control U6, U6/cdhl, or U6/snon, or both U6/cdhl and U6/snon plasmids. Cdhl knockdown significantly stimulated an increase in total axonal length, while SnoN knockdown significantly inhibited axon growth compared to control neurons (FIGURE 4A). The simultaneous induction of Cdhl and SnoN RNAi led to an axonal phenotype that was identical to that of SnoN knockdown resulting in significantly shorter axons as compared to Cdhl hpRNA-expressing neurons or as compared to control U6-transfected neurons (FIGURE 4A). SnoN knockdown completely suppressed the Cdhl RNAi-induced axonal growth phenotype. These findings indicate that Cdhl-APC and SnoN operate in a linear pathway, with SnoN acting downstream of Cdhl-APC in the control of axonal growth.

To corroborate the finding that SnoN acts downstream of Cdhl-APC in the control of axon growth, structure-function analyses of SnoN were performed in neurons (FIGURE 4B). SnoN contains a conserved Cdhl recognition D-box peptide motif, whose mutation renders SnoN resistant to APC-mediated ubiquitination. The axonal length was measured in granule neurons in which wild type SnoN or a SnoN protein with a mutated D-box (mutD-box SnoN) was expressed. Expression of wild type SnoN in granule neurons had little effect on axon length (FIGURE 4B). In contrast, neurons in which mutD-box SnoN was expressed, had a significant increase in total axonal length as compared to wild type SnoN-expressing neurons or control vector-transfected neurons (FIGURE 4B). When axonal length was measured for several days after transfection, mutD-box SnoN but not wild type SnoN significantly enhanced the rate of axonal growth as compared to control transfected neurons (FIGURE 4C). Thus, mutating the Cdhl recognition D-box in SnoN unmasks SnoN's ability to promote axonal growth. Taken together with the Cdhl and SnoN knockdown epistasis analysis, these findings indicate that the axon growth promoting function of SnoN is regulated by Cdhl-APC in neurons.

Example 4: SnoN promotes the formation of granule neuron parallel fibers in vivo Following the identification of SnoN as a key Cdhl-APC target protein that promotes axonal growth in primary granule neurons, the in vivo function of SnoN in the cerebellar cortex was next examined. In vivo electroporation method was used to acutely knockdown SnoN in the postnatal cerebellum (FIGURE 5A). The control U6/cmvGFP or the U6/snon- cmvGFP plasmid was injected into the cerebellar cortex of P3 rat pups, who were next subjected to electroporation. Five days after electroporation, the cerebellum was isolated and coronal sections of the cerebellum were subjected to immunohistochemistry with GFP antibodies to allow the assessment of granule neuron parallel fiber axons. GFP-positive granule neurons were present in the cerebellar cortex of electroporated animals. Most of the transfected GFP-positive granule neurons were in the IGL, with a smaller population in the EGL. Examination of the EGL neurons revealed that both the control U6-transfected and SnoN hpRNAs-expressing granule neurons extended robust axons at this stage of development (FIGURE 5B).

Analysis of the IGL granule neurons revealed a striking SnoN knockdown-induced phenotype in parallel fiber formation (FIGURE 5C). The control U6-transfected granule neurons residing in the IGL displayed a normal anatomy including the typical T-shaped axons and association with parallel fibers in the molecular layer (ML). Parallel fibers extended in large numbers in and beyond the region of the transfected IGL granule neurons (FIGURE 5C). By contrast, although granule neurons in which SnoN RNAi was triggered were present in the IGL in similar numbers as control U6-transfected neurons, few of the SnoN hpRNA-expressing neurons were associated with parallel fibers in the ML (FIGURE 5C).

The effect of SnoN knockdown on parallel fibers in the cerebellar cortex was quantified. More than 90% of the control U6-transfected IGL granule neurons were associated with parallel fibers. In contrast, only 30% of the IGL granule neurons in which SnoN RNAi was triggered were associated with parallel fibers (FIGURE 5D). These remaining parallel fibers that were associated with SnoN hpRNA-expressing IGL granule neurons appeared underdeveloped and failed to extend for long distances beyond the area of the transfected IGL neurons (FIGURE 5C). These results indicate that SnoN plays a key role in parallel fiber formation and that SnoN is required in axonal growth of IGL but not EGL granule neurons. Consistent with this observation is the finding that SnoN is robustly expressed in granule neurons in the IGL but not in the EGL (FIGURE 2C). Taken together, these findings indicate that SnoN specifically promotes the elongation or maintenance of granule neuron axons in the cerebellar cortex in vivo at a developmental stage following axonogenesis.

The present studies provide a new mechanism in which the ubiquitin ligase Cdhl- APC controls axonal growth in the mammalian brain. Accordingly, cdhl-APC operates in the nucleus to control axonal growth in granule neurons of the developing cerebellum. The transcriptional corepressor SnoN is a key target of neuronal Cdhl-APC that promotes granule neuron axonal growth and parallel fiber formation in the developing cerebellar cortex. Thus, Cdhl-APC and SnoN form a cell-intrinsic pathway that orchestrates axonal morphogenesis in the mammalian brain. SnoN may exert its function by physically interacting with the TGFβ- regulated transcription factors Smad2 and Smad3 and thereby represses Smad-dependent gene expression.

Example 5: TGFβ-Smad2 signaling regulates the Cdhl-APC/SnoN pathway of axonal morphogenesis

Axon growth is critical to the establishment of neuronal connectivity. The ubiquitin ligase Cdhl-APC and its substrate the transcriptional modulator SnoN form a cell-intrinsic pathway that orchestrates axonal morphogenesis in the mammalian brain. Prior to the invention, how the Cdhl-APC/SnoN pathway is controlled in the nervous system remained unknown. Results described herein demonstrate that the TGFβ-regulated signaling protein Smad2 plays a key role in regulating the Cdhl-APC/SnoN pathway in neurons. Smad2 is expressed in primary granule neurons of the developing rat cerebellar cortex. The Smad signaling pathway is basally activated in neurons. Endogenous Smad2 is phosphorylated, localized in the nucleus, and forms a physical complex with endogenous SnoN in granule neurons. Inhibition of Smad signaling by several distinct approaches, including genetic knockdown of Smad2, stimulates axonal growth. Biochemical evidence and genetic epistasis analyses reveal that Smad2 acts upstream of SnoN in a shared pathway with Cdhl-APC in the control of axonal growth. Remarkably, Smad2 knockdown also overrides the ability of adult rat myelin to inhibit axonal growth. Collectively, the findings described herein define a novel function for Smad2 in regulation of the Cdhl-APC/SnoN cell-intrinsic pathway of axonal morphogenesis, and indicate that inhibition of Smad signaling stimulates axonal growth following injury in the central nervous system.

Axon growth and guidance are essential events in the developing nervous system that ensure the proper connectivity of neurons. A variety of extrinsic cues, including guidance factors and morphogens coordinate the growth and navigation of axons through the complex environment of the nervous system. Neurons also harbor cell-intrinsic programs that govern axonal growth and patterning. In particular, growing evidence supports an important role for transcription factors and the ubiquitin proteasome system in axonal development.

The E3 ubiquitin ligase Cdhl -anaphase promoting complex (Cdhl-APC) is a key regulator of axonal morphogenesis in the mammalian brain. Known as an essential component of the cell cycle in dividing cells, Cdhl-APC ligase activity in postmitotic neurons coordinates the growth and spatial development of axons. The transcriptional regulator SnoN is a critical substrate of Cdhl-APC in neurons. Gain-and loss-of-function analyses indicate that SnoN enhances axonal growth. Cdhl-APC stimulates the ubiquitination and subsequent degradation of SnoN in neurons. Genetic epistasis experiments reveal that Cdhl-APC and SnoN function in a shared pathway whereby Cdhl- APC limits axonal growth by inhibiting SnoN. Thus, Cdhl-APC and SnoN comprise a cell- intrinsic pathway that orchestrates axonal growth by regulating transcription. Identification of the transcriptional protein Id2 as another crucial substrate of neuronal Cdhl-APC in the control of axon growth confirms the observation that neuronal Cdhl-APC operates in the nucleus as a critical regulator of axonal morphogenesis.

Prior to the invention described herein, a major question that remained to be addressed was how the Cdhl-APC cell-intrinsic pathway of axonal development is regulated in neurons. An important clue is provided by the finding that SnoN acts as a substrate of neuronal Cdhl-APC. SnoN function is intimately linked to TGFβ-Smad signaling in dividing cells. SnoN associates with the TGFβ-regulated transcription factors Smad2 and Smad3 and thereby modulates TGFβ-dependent transcription. Conversely, TGFβsignaling regulates SnoN activity in a Smad2/3 -dependent manner. Manipulation of the Smad signaling pathway through functional interactions with SnoN exerts important biological effects in postmitotic neurons.

TGFβligand binding induces the heterodimeric association of TGFβtype I and II serine/threonine receptor kinases. Activated TGFβtype I receptor recruits and phosphorylates Smad2 and Smad3 that in turn associate with the co-Smad Smad4. Complexed Smads translocate to the nucleus where they regulate transcription of TGFβ-responsive genes. SnoN binding to Smad2/3 enhances or represses TGFβ-induced Smad-dependent transcription in a cell-type specific manner. Thus, SnoN is a versatile transcriptional regulator that modulates Smad signaling. The ability of SnoN to modulate Smad2/3- dependent transcription is consistent with the model that SnoN acts upstream of Smad2/3 in the TGFβsignaling pathway.

SnoN protein turnover is also regulated by TGFβsignaling pathway in a Smad2/3- dependent manner. TGFβ-activated Smad2 and Smad3 act as scaffolding molecules to recruit SnoN to Cdhl-APC leading to the ubiquitination and subsequent degradation of SnoN. These data support a mechanism in which Smad2 and Smad3 act upstream of SnoN in TGFβsignaling. The intimate relationship of SnoN with Smad signaling raises the question of whether and how the Smad signaling regulates the Cdhl-APC/SnoN cell-intrinsic pathway of axonal morphogenesis in neurons.

The present invention describes a critical role for Smad signaling in regulating the function of the Cdhl-APC/SnoN pathway in axonal growth. Smad2 is expressed and associates with SnoN in granule neurons of the cerebellar cortex. Inhibition of endogenous Smad signaling in primary granule neurons by several distinct approaches including genetic knockdown of Smad2 by RNAi stimulates axonal growth, indicating that Smad signaling inhibits axonal growth. Smad2 acts together with Cdhl -APC in a shared pathway upstream of SnoN in the control of axonal growth. Smad2 knockdown also overrides myelin-inhibition of axonal growth. These findings implicate the Smad pathway in axon growth control and indicate that this pathway holds potential for therapeutic approaches to enhance the intrinsic ability of neurons to extend axons following injury and disease.

The following materials and methods were used to generate the data described in Example 5.

Plasmids and reagents

The U6/smad2 and pcDNA3/Smad2 plasmids were kindly provided by Daniel Bernard (Bernard, 2004). To generate the pcDNA3/Smad2-Rescue expression plasmid, silent mutations in Smad2 cDNA were introduced using QuikChange site directed mutagenesis kit (Stratagene, USA). The U6/cdhl , U6/snon plasmids are described (Stegmuller et al., 2006 Neuron 50:389-400). The pCMV5/Smad6 and pCMV5/Smad7 expression plasmids were a gift from Shirin Bonni. SB431542 and SB505124 were purchased from Sigma. The SMAD2 RNAi construct was directed against the following sequence from mouse SMAD2 exon 3: 5'-

GGACTGAGTACAGC AAATACGG-3' (SEQ ID NO: 1) (Bernard D, 2004 Molecular Endocrinology, 18(3):606-623; incorporated herein by reference).

Cerebellar granule neuron culture and transfections

Granule neurons were isolated from postnatal Long-Evans rat cerebellum (P6) as described previously (Konishi et al., 2002 MoI Cell 9:1005-1016). Neurons were plated on polyornithine-coated glass coverslips and kept in BME supplemented with either 10 μg/ml insulin and 2 mM glutamine, penicillin and streptomycin, or with 10% calf serum and 2 mM glutamine, penicillin and streptomycin, or with 10% calf serum (Hyclone Laboratories, UT), 25 mM KCl and 2 mM glutamine, penicillin and streptomycin (referred to as full media). Neurons were treated with the mitosis inhibitor cytosine-β-D-arabinofuranoside (lOμM) when kept in serum-supplemented media to inhibit proliferation of non-neuronal cells. Neurons were transfected 8 hours after plating using the calcium-phosphate method with indicated plasmids together with a GFP expression plasmid to visualize transfected neurons. To rule out the possibility that the effects of RNAi or protein expression on axonal length were due to any effect of these manipulations on cell survival, the anti-apoptotic protein BcI- xL was co-expressed in all experiments. The expression of Bcl-xL itself has little or no effects on axonal length (Konishi et al., 2004 Science 303:1026-1030). Neurons were fixed in paraformaldehyde after three days in vitro and subjected to immunocytochemistry using a GFP antibody (Molecular probes).

Axon growth assay and morphometry Axonal growth and morphometry was done as described (Konishi et al., 2004 Science

303:1026-1030; Stegmuller et al., 2006 Neuron 50:389-400). Transfected neurons images of GFP-positive neurons were captured in a blinded manner using a Nikon Eclipse TE2000 epifluorescence microscope. Axonal growth was analyzed by measuring the length of axons using SPOT software.

Western blot and subcellular fractionation analyses

Granule neurons were harvested at indicated DIV and lysates were analyzed by SDS- PAGE followed by Western blotting using the Smad2/3 (BD), phosphoS465/467-Smad2 (Calbiochem), SnoN antibodies (Santa Cruz), or 14-3-3beta antibody (Santa Cruz). For subcellular fractionation, granule neurons were scraped into detergent-free buffer A (IO mM Hepes pH7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, protease inhibitors) and mechanically disrupted using a 2 ml dounce. Nuclei were spun down (50Og, 4°C, Eppendorf table centrifuge), and supernatant was collected as the cytoplasmic fraction. Nuclei were subjected to one wash in 0.1% NP40 supplemented buffer A. Nuclei were then lysed in buffer B (20 mM Hepes pH7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, protease inhibitors) and pelleted (maximum speed, 4⁰C, Eppendorf table centrifuge). Supernatant was collected as the nuclear fraction.

Isolation of myelin

Myelin was prepared according to Norton and Poduslo (Norton and Poduslo, 1973 J Neurochem 21 :749-757). Briefly, brains from Long Evans adult rats were homogenized in 10.5 % sucrose and subjected to a series of 10.5%/30% sucrose gradient ultra-centrifugation steps alternating with non-gradient ultra-centrifugations. Purified myelin was resuspended in water and integrity of myelin proteins was determined by SDS-PAGE followed by Coomassie staining. Glass coverslips, pre-coated with polyorhnithine, were coated overnight at 4°C with 13.3 μg/ml myelin diluted in PBS.

SnoN function in axonal growth

To investigate the mechanistic basis of SnoN function in axonal growth in the mammalian brain, the role of SnoN-interacting protein in neurons was characterized. In dividing cells, SnoN associates with the transcription factors Smad2 and Smad3 and thus plays a critical role in the TGFβ-Smad signaling pathway (Sun et al., 1999 Proc Natl Acad Sci USA 96: 12442-12447; Liu et al., 2001 Cytokine Growth Factor Rev 12: 1-8). It was hypothesized that the Smad proteins play a role in the regulation of axonal growth.

The expression of Smad2 and Smad3 in granule neurons of the developing rat cerebellar cortex was characterized. Immunoblotting with a widely used antibody to the related proteins Smad2 and Smad3 revealed that Smad2 was highly expressed in cerebellar granule neurons (Figure 6A). Smad2 was identified based on co-migration of the immunoreactive band in lysates of neurons with transfected Smad2 but not Smad3 in cells. Smad2 was also expressed in cerebral cortical and hippocampal neurons, suggesting that this protein is widely expressed in the brain (Figure 6B). Immunofluorescence analysis revealed Smad2 immunoreactivity in granule neurons (Figure 6C). These results demonstrate that Smad2 is expressed in postmitotic neurons.

To determine if Smad2 acts in concert with SnoN to regulate axonal growth and if that is linked to the function of SnoN as a key target of Cdhl-APC in the control of axonal growth, it was first determined if SnoN interacts with Smad2 in neurons. Both immuofluorescence and fractionation studies showed that a large portion of Smad2 is localized in the nucleus, where SnoN resides (Figures 6C, 6D). In coimmunoprecipitation analyses, in which SnoN was immunoprecipitated followed by Smad2 immunoblotting, endogenous Smad2 associated with endogenous SnoN (Figure 6E). The reciprocal approach of immunoprecipitating Smad2 followed by SnoN immunoblotting confirmed the interaction (Figure 6E). Together, these results suggest that endogenous SnoN forms a physical complex with endogenous Smad2 in neurons.

Smad2 function in the regulation of axonal growth was characterized. A plasmid- based method of RNAi to acutely knockdown Smad2 in cerebellar granule neurons was used (Gaudilliere et al., 2002 J Biol Chem 277:46442-46446). Primary granule neurons were transfected with the U6 or U6/smad2 plasmid together with a GFP expression plasmid, and three days after transfection axonal length of transfected GFP-positive neurons was measured. Axonal length was robustly increased in Smad2 knockdown neurons as compared to control U6-transfected neurons (Figure 7A). Quantitation of these results revealed that Smad2 RNAi increased axonal length by nearly 50% (t-test, pO.OOOl) (Figure 7B). These results suggest that Smad2 inhibits axonal growth.

To determine if the Smad2 RNAi-induced phenotype is due to specific knockdown of Smad2 and not due to off-target effects of RNAi, a rescue experiment was performed. A Smad2 rescue (Smad2-Rescue) expression construct that harbors multiple silent mutations in the targeting region was designed. While Smad2 RNAi induced the efficient knockdown of Smad2 encoded by wild type cDNA (Smad2-WT), Smad2 RNAi failed to induce knockdown of Smad2 -Rescue (Figure 7C). The ability of Smad2-Rescue to reverse Smad2-mediated enhancement of axonal growth was determined. Granule neurons were transfected with the control U6 or the Smad2 RNAi plasmid together with Smad2 WT or Smad2-Rescue expression plasmid. Expression of Smad2 encoded by wild type cDNA (Smad2-WT) had little effect on the ability of Smad2 RNAi to increase axonal length (Figure 7D). By contrast, expression of Smad2-Rescue significantly reduced axonal length in the background of Smad2 RNAi (ANOVA, p<0.0001). These findings indicate that Smad2 RNAi-enhancement of axonal growth is the result of specific knockdown of Smad2 in neurons.

The interaction of Smad2 with SnoN plays a dual role in TGFβsignaling, serving to modulate Smad2-dependent transcription or alternatively to allow the Smad2-recruited ubiquitin ligase Cdhl-APC to target SnoN for ubiquitination and consequent degradation

(Stroschein et al., 1999 Science 286:771-774; Stroschein et al., 2001 Genes Dev 15:2822- 2836; Wan et al., 2001 MoI Cell 8:1027-1039; He et al., 2003 J Biol Chem 278:30540-30547; Sarker et al., 2005 J Biol Chem 280:13037-13046; Hsu et al., 2006 J Biol Chem 281:33008- 33018). Due to the dual function of the Smad2/SnoN interaction, the functional relationship of Smad2 and SnoN in neurons using epistasis analyses was characterized. The effect of the simultaneous knockdown of both SnoN and Smad2 was compared to the effect of knockdown of SnoN or Smad2 alone on axonal length in granule neurons. While Smad2 RNAi enhanced and SnoN RNAi reduced axonal length, simultaneous knockdown of Smad2 and SnoN led to the appearance of short axons, resembling the SnoN knockdown phenotype (Figure 8A) (ANOVA, p=0.0633). Thus, SnoN knockdown suppressed the ability of Smad2 RNAi to stimulate axonal growth. These results suggest that Smad2 acts upstream of SnoN in the control of axonal morphogenesis in neurons.

Since the ubiquitin ligase Cdhl-APC also acts upstream of SnoN in neurons and owing to Smad2's function in recruiting Cdhl-APC to its substrate SnoN (Stroschein et al., 2001 Genes Dev 15:2822-2836; Wan et al., 2001 MoI Cell 8:1027-1039; Stegmuller et al., 2006 Neuron 50:389-400), the role of Smad2 in the Cdhl-APC pathway of axonal growth was determined. The effect of Smad2 RNAi or Cdhl RNAi alone or together on axonal length in granule neurons was measured. Axonal length was increased in Cdhl knockdown as well as Smad2 knockdown neurons (Figure 8B) (ANOVA, p<0.001). Simultaneous knockdown of both Cdhl and Smad2 in granule neurons did not result in additive effects of axonal length when compared to individual knockdown of Cdhl and Smad2 (Figure 8B). These results indicate that Cdhl and Smad2 operate in a shared pathway, whereby Smad2 and Cdhl-APC both act upstream of SnoN in neurons to regulate axonal growth.

Identification of a novel function for Smad2 as an inhibitor of axon growth raised the question of the role of the TGFβ-regulated Smad signaling pathway in the control of axonal development. Smad2 activation was monitored in granule neurons upon exposure to TGFβl. In cycling cells, TGFβligand binding triggers the phosphorylation of Smad2 at the key regulatory sites Serines 465 and 467 leading to the consequent turnover of SnoN (Stroschein et al., 2001 Genes Dev 15:2822-2836; Wan et al., 2001 MoI Cell 8:1027-1039; Bonni et al., 2001 Nat Cell Biol 3:587-595). Surprisingly, Smad2 was basally phosphorylated at Serines 465 and 467 in neurons, and exposure of neurons to TGFβl did not further stimulate the Smad2 phosphorylation. Consistent with these results, in subcellular fractionation analyses, Smad2 phosphorylated at Serines S465 and 467 was localized in the nucleus (Figure 9A). These results indicate that Smad signaling is constitutively active in neurons. Corroborating these findings are results in transgenic mice engineered to express a TGFβ-induced Smad- responsive luciferase reporter demonstrating that high basal activity of TGFβsignaling is present in the brain (Luo et al., 2006 Proc Natl Acad Sci USA 103:18326-18331).

After identifying a high basal level of Smad2 activation in neurons, the effect of manipulation of endogenous TGFβ-Smad signaling in neurons was determined. The proteins Smadό and Smad7 inhibit the Smad signaling pathway primarily at the level of TGFβ receptors (Hayashi et al., 1997 Cell 89:1165-1173; Imamura et al., 1997 Nature 389:622- 626). The effect of Smadό or Smad7 on axonal growth in granule neurons was tested. Expression of Smadό or Smad7 in neurons robustly increased axonal length (Figure 9B) (t- test, p<0.001). These results suggest that inhibition of Smad signaling stimulates axonal growth.

The biochemical and biological response of neurons to small molecule inhibitors of TGFβreceptors, SB431542 and SB505124 was characterized (Inman et al., 2002 MoI Pharmacol 62:65-74; DaCosta Byfield et al., 2004 MoI Pharmacol 65:744-752). Exposure of granule neurons to both small molecule inhibitors led to loss of Smad2 phosphorylation at Serines 465 and 467 (Figure 9C). These results indicate that small molecule inhibitors of TGFβreceptors inactivate Smad signaling in neurons. Together with the reduction in Smad2 phosphorylation, SnoN levels were significantly increased in neurons upon exposure to SB431542 or SB505124 (Figure 9C). These results indicate that activation of Smad2 signaling promotes SnoN turnover, consistent with the model that activated Smad2 recruits SnoN to Cdhl-APC leading to the subsequent degradation of SnoN (Stroschein et al., 2001 Genes Dev 15:2822-2836).

To test the idea that TGFβ-Smad signaling inhibits axonal growth, axon length was measured in neurons exposed to the small molecule inhibitor SB431542. Neurons were transfected 8 hours after plating and treated for 48 hours with 20μM of the inhibitor or the vehicle (DMSO). Axonal length was significantly increased upon exposure to SB431542 (Figures 9D (t-test, pO.Ol), 9E). These findings indicate that constitutive TGFβ-Smad signaling suppresses axon growth in neurons.

The Smad pathway plays a role in the control of axonal growth in granule neurons of the developing rat cerebellum and this pathway is involved in the ability of neurons to grow axons under conditions that axons encounter following injury in the nervous system. Myelin proteins are thought to play an important role in the inhibition of axonal growth in the injured central nervous system (Caroni and Schwab, 1988 Neuron 1:85-96; Filbin, 2003 Nat Rev Neurosci 4:703-713). Inhibition of Cdhl-APC overrides the ability of myelin to suppress axonal growth (Konishi et al., 2004 Science 303: 1026-1030). As this study demonstrated that the TGFβ-Smad2 pathway acts together with Cdhl-APC to inhibit axonal growth, it was hypothesized that Smad2 knockdown might also stimulate axon growth upon myelin inhibition. Granule neurons were plated on myelin or control substrate and transfected with the Smad2 RNAi or control U6 plasmid together with the GFP expression plasmid. As expected, myelin significantly inhibited axonal growth (ANOVA, p<0.001) (Figures 1OA, 10B). Strikingly, Smad2 knockdown robustly increased axonal length as compared to control-transfected neurons on myelin (ANOVA, p<0.0005) (Figures 1OA, 10B). These results suggest that inhibition of Smad2 overcomes myelin-inhibition of axonal growth. Collectively, these data support the conclusion that Smad signaling engages the Cdhl- APC/SnoN pathway to limit the intrinsic ability of neurons to extend axons. By regulating the Cdhl-APC/SnoN pathway, Smad signaling plays an role in the failure of CNS neurons to regenerate. Thus, inhibition of Smad signaling is useful in the treatment of CNS injury and disease.

Described herein is a novel function for the Smad signaling pathway in control of axonal morphogenesis. Genetic knockdown of Smad2 by RNAi in primary neurons stimulated axonal growth. Likewise, expression of the inhibitory Smad protein Smadό or Smad7 significantly increased axonal length. Smad signaling influences the growth of axons by regulating the Cdhl-APC/SnoN cell-intrinsic pathway of axonal morphogenesis. Endogenous SnoN associates with endogenous Smad2 in neurons. Epistasis analyses revealed that Smad2 acts together with the ubiquitin ligase Cdhl-APC upstream of the transcriptional modulator SnoN to control axonal growth. Exposure of neurons to small molecule inhibitors of TGFβreceptors inactivated Smad2 phosphorylation and concomitantly increased the levels of SnoN protein. Consistent with these results, the small molecule inhibitors stimulated the growth of axons in granule neurons. Genetic knockdown of Smad2 overrides the ability of adult rat brain myelin to suppress axonal growth. Collectively, the findings indicate that the TGFβ-Smad signaling pathway plays a critical role in regulating axonal development and represent targets for drugs for enhancing recovery in the nervous system following injury or disease.

Identification of a novel function for the Smad proteins in the control of axonal growth in mammalian CNS neurons bears several significant ramifications. Cdhl-APC/SnoN pathway is a pivotal cell-intrinsic regulator of axonal morphogenesis. Both biochemical and genetic lines of evidence support the conclusion that in neurons Smad2 act together with Cdhl-APC upstream of SnoN in the control of axonal growth. These findings indicate that TGFβ-Smad signaling plays a key role in regulating the Cdhl-APC/SnoN cell-intrinsic pathway in neurons.

Aside from SnoN, the SCF scaffold protein Skp2 and the signaling protein HEFl have been identified as substrates of Cdhl-APC that are targeted for degradation by the proteasome in response to TGFβsignaling in dividing cells. Like SnoN, the degradation of HEFl and Skp2 is enhanced in the presence of activated Smad3. Skp2 and HEFl may function downstream of Smad signaling and the Cdhl-APC/SnoN pathway in controlling axonal growth in CNS neurons.

Consistent with the role of Smad signaling in regulating the Cdhl-APC/SnoN pathway in neurons, inhibition of Smad signaling endows neurons with the ability to grow axons on the substrate myelin. Since myelin is thought to play an important role in inhibiting axonal regeneration in the injured central nervous system, these results indicate that components of the TGFβ-Smad signaling pathway represent targets for drugs that stimulate axonal regeneration following injury and disease in the central nervous system. TGFβsignaling contributes to central nervous system development and its role is increasingly appreciated in neurological diseases. TGFβs are thought to have prosurvival effects in distinct populations of neurons. Mounting evidence implicates an important neuroprotective role of TGFβs in Alzheimer's disease and ischemia. Thus, TGFβs may have beneficial impact on neurons after brain injury. Among the TGFβ superfamily, TGFβl, TGFβ2 and their receptors are upregulated at the site of injury. TGFβsignaling engages an intrinsic mechanism of axon growth inhibition. Thus, in the setting of injury, while TGFβ signaling may have pro-survival effects on neurons, it might simultaneously counteract axon regeneration by suppressing intrinsic programs of axonal growth.

Other Embodiments

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

What is claimed is:

1. A method of inducing axonal growth or regeneration by contacting a neuronal cell with an agent that increases the level or activity of SnoN.

2. The method of claim 1, wherein said neuronal cell is a cerebellar granule cell.

3. The method of claim 1, wherein said agent reduces the level or activity of Smad 2 or Smad 3.

4. The method of claim 1, wherein said agent is a small molecule inhibitor or an RNA interfering molecule.

5. A method of inducing axonal growth or regeneration by contacting a neuronal cell with an agent that reduces the level or activity of the ubiquitin ligase-anaphase promoting complex (Cdhl-APC), wherein said agent increases the level of phosphorylation of Cdhl at amino acid position Serine 40, serine 151, serine 163, or threonine 121.

6. The method of claim 5, wherein said neuronal cell is a cerebellar granule neuron.

7. The method of claim 5, wherein said agent increases the level or activity of SnoN.

8. The method of claim 5, wherein said agent is a small molecule inhibitor or an RNA interfering molecule.

9. A method of treating spinal cord injury in a mammal in need thereof by administering to said mammal an agent that reduces the level or activity of the E3 ubiquitin ligase-anaphase promoting complex (Cdhl-APC).

10. The method of claim 9, wherein said agent reduces the level or activity of the ubiquitin ligase-anaphase promoting complex (Cdhl-APC) in cerebellar granule neurons.

11. The method of claim 9, wherein said agent is a dominant interfering APC subunit protein (e.g. APCl 1, APC2).

12. The method of claim 9, wherein said agent increases phosphorylation of Cdhl at amino acid position Serine 40, serine 151, serine 163, or threonine 121.

13. The method of claim 9, wherein agent reduces the ubiquitination 1 of Tome-1 ,

SnoN or Pax 6.

14. The method of claim 9, wherein said agent increase the level or activity of SnoN or Pax6.

15. The method of claim 9, wherein said agent is substantially identical to Emil, MAD2B, or ubiquilinl .

16. The method of claim 9, wherein said mammal is further administered a second therapeutic regimen.

17. A method for identifying a candidate compound for inducing axonal growth and regeneration, said method comprising: (a) contacting a cell expressing a Cdhl gene or an APC2 gene with a candidate compound and (b) measuring Cdhl or APC2 gene expression or protein activity in said cell, wherein a decrease in the level of said expression or said activity in the presence of said compound compared to that in the absence of said compound indicates that said compound induces axonal growth or regeneration.

18. The method of claim 17, wherein said candidate compound reduces binding of Cdhl to APC core.

19. The method of claim 17, wherein said Cdhl or APC2 gene is a construct encoding a polypeptide, said polypeptide comprising a Cdhl or APC2 gene and a heterologous gene.

20. The method of claim 17, wherein step (b) comprises measuring expression of

APC2or Cdhl mRNA or protein.

21. The method of claim 17, wherein step (b) comprises measuring the level or expression of SnoN in said cell, wherein an increase in the level of SnoN expression or activity in the presence of said compound compared to that in the absence of said compound indicates that said compound induces axonal growth or regeneration.

22. The method of claim 17, wherein said cell is a mammalian cell.

23. The method of claim 22, wherein said cell is a rodent or human cell.

24. The method of claim 17, wherein said cell is a neuronal cell.

25. The method of claim 24, wherein said neuronal cell is a cerebellar granule neuron.

26. A method for identifying a candidate compound for inducing axonal growth and regeneration, said method comprising: (a) contacting a cell expressing a SnoN gene with a candidate compound and (b) measuring SnoN gene expression or protein activity in said cell, wherein an increase in the level of said expression or said activity in the presence of said compound compared to that in the absence of said compound indicates that said compound induces axonal growth or regeneration.

27. The method of claim 26, wherein said SnoN gene is a construct encoding a polypeptide, said polypeptide comprising a SnoN gene and a heterologous gene.

28. The method of claim 26, wherein step (b) comprises measuring expression of SnoN mRNA or protein.

29. The method of claim 26, wherein said cell is a mammalian cell.

30. The method of claim 29, wherein said cell is a rodent or human cell.

31. The method of claim 26, wherein said cell is a neuronal cell.

32. The method of claim 31, wherein said neuronal cell is a cerebellar granule neuron.

33. A method for identifying a candidate compound for inducing axonal growth or regeneration, said method comprising: (a) contacting a Cdhl, APC2, or SnoN protein with a candidate compound; and (b) determining whether said candidate compound binds to said a Cdhl, APC2, or SnoN protein, wherein binding of said compound to said a Cdhl, APC2 or SnoN protein indicates that said candidate compound induces axonal growth or regeneration.

34. The method of claim 33, wherein said agent reduces binding of APC subunits to Cdhl.

35. The method of claim 33, wherein said protein is a human protein.