WO2004078925A2 - Methods and compositions for treating and preventing neurodegenerative diseases - Google Patents

Abstract

Methods and compositions for treating and preventing neurodegenerative diseases. A number of p25/CDK5 substrates were identified, thereby providing a mechanism of p25/CDK5-induced neurodegenerative disease.

Description

METHODS AND COMPOSITIONS FOR TREATING AND PREVENTING NEURODEGENERATIVE DISEASES

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS [01] The present application claims benefit of priority to U.S. Provisional

Patent Application No. 60/451,345, filed February 28, 2003, which is incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

[02] Based upon sequence similarity, Cdk5 is classified as a member of the cyclin dependent kinase family (Lew et al., JBiol Chem 267:13383-13390 (1992); Meyerson et al. , EMBO J 11 :2909-2917 (1992)). However, unlike other cyclin dependent kinases, Cdk5 has not been described as involved in cell cycle regulation. Cdk5 is a serine/threonine kinase, with the active form found in differentiated neurons of the developing and mature brain. Cdk5 activity is dependent upon the association of the Cdk5 protein with a regulatory subunit, one being p35, or a proteolytic fragment of p35, known as p25. The p25 protein has a much longer half-life than that of p35 and is inappropriately localized within the cell. This prolonged presence of p25 and its localization in the cytosol rather than at the plasma membrane result in constitutively active, mislocalized Cdk5 kinase activity (Patrick et al, Nature 402:615-622 (1999)).

[03] Mice lacking either Cdk5 or p35 protein exhibit similar abnormalities in the laminar structure of the cerebral cortex. The migrating neurons in the developing cortex of these mice were not able to by pass the cortical plate neurons to locate beneath the subplate as in normal development (Chae et al, Neuron 18:29-42, 1997; Ohshima et al, Proc Natl Acad Sci USA 93:11173-11178 (1996)).

[04] Unregulated Cdk5 activity, which is associated with p25 instead of p35, has been implicated in the pathology of neurodegenerative disorders, such as Alzheimer's disease (Patrick et al, Nature 402:615-622 (1999); Lee et al, Nature 405:360- 364 (2000); Mandelkow, Nature 402:588-589 (1999)) and amyotrophic lateral sclerosis (ALS) (Nguyen et al, Neuron 30:135-147 (2001)). Cdk5 is also implicated in regulation of numerous other cellular events including neurite extension (Nikolic et al. Genes Developmetit 10:816-825 (1996)), cell adhesion (Kwon et al, Current Biology 10:363-372 (2000)), and axonal transport (Niethammer et al Neuron 28:697-711 (2000); Sasaki et al, Neuron 28:681- 696 (2000)).

[05] Although it has been hypothesized that p25 regulated CDK5 ("p25/CDK5") phosphorylates additional substrates only a handful of substrates have been identified to date. This present invention addresses this and other problems.

BRIEF SUMMARY OF THE INVENTION

[06] This invention provides methods for identifying an agent to treat or prevent a neurodegenerative disease. In some embodiments, the methods comprise contacting the agent to p25/CDK5 and a substrate selected from the group consisting of Stathmin, CRMP2, CRMPl, CRMP4, Tubulin-β2, QCZ8, 14-3-3 ε, 14-3-3 ζ, Lamin A, Lamin B, EF-1 delta, EF-2, p97 ATPase, CAPER, DEAD-Helicase, HSP60, HnRNP K, HnRNP G, Nucleolin, BCAKD kinase, Neurofilament-M, p54nrb, cyt-Dynein (IC2), Peroxiredoxin 2, 3-OA CoA tra, CCT, GM130, p27-Kipl, Cdc25A, Cdc25B, Cdc25C and Weel; and detecting an effect on the phosphorylation of the substrate by p25/CDK5, thereby identifying an agent to treat or prevent a neurodegenerative disease.

[07] In some embodiments, the methods comprise selecting an agent that binds to the substrate, thereby preventing phosphorylation of the substrate. In some embodiments, the methods comprise selecting an agent that binds to p25/CDK5, thereby preventing phosphorylation of the substrate. In some embodiments, the methods comprise selecting an agent that prevents binding of p25/CDK5 to the substrate. In some embodiments, the methods comprise selecting an agent that reduces phosphorylation of the substrate by p25/CDK5 compared to phosphorylation in the absence of the agent. [08] In some embodiments, the method comprises screening a plurality of agents to identify an agent that prevents p25/CDK5 phosphorylation of a substrate.

[09] In some embodiments, the contacting step comprises contacting a cell, the cell comprising p25/CDK5 and the substrate.

[10] In some embodiments, the cell is a neuronal cell. In some embodiments, the cell is selected from the group consisting of a PC 12 cell and a SH-SY5 cell. In some embodiments, the cell comprises a recombinant expression cassette comprising a promoter operably linked to a polynucleotide encoding p25. In some embodiments, the cell comprises an expression cassette comprising a promoter operably linked to a polynucleotide encoding CDK5.

[11] In some embodiments, the detecting step comprises detecting the expression of a gene that is induced by the phosphorylated substrate. In some embodiments, the detection of the expression of the gene is detected by detecting expression of a reporter gene controlled by the promoter of the induced gene.

[12] hi some embodiments, the methods further comprise administering the agent to an animal model for a neurodegenerative disease and determining the effect of the agent on the symptoms of the animal model. In some embodiments, the animal model is an animal model for Alzheimer's disease.

[13] In some embodiments, the methods comprise contacting a neuronal cell with an agent, wherein the cell expresses p25/CDK5 before and/or after the contacting step; detecting viability or apoptosis of the cell contacted by the agent; and selecting an agent that enhances viability of the cell contacted by the agent compared to a cell not contacted by the agent.

[14] In some embodiments, the methods comprise detecting CDK5 phosphorylation of a substrate selected from the group consisting of stathmin, CRMP2, CRMPl, CRMP4, Tubulin-β2, QCZ8, 14-3-3 ε, 14-3-3 ζ, Lamin A, Lamin B, EF-1 delta, EF-2, p97 ATPase, CAPER, DEAD-Helicase, HSP60, HnRNP K, HnRNP G, Nucleolin, BCAKD kinase, Neurofilament-M, p54nrb, cyt-Dynein (IC2), Peroxiredoxin 2, 3-OA CoA tra, CCT, GM130, p27-Kipl, Cdc25A, Cdc25B, Cdc25C and Weel. hi some embodiments, the cell comprises a recombinant expression cassette comprising a promoter operably linked to a polynucleotide encoding p25/CDK5.

[15] In some embodiments, expression of p25/CDK5 is controlled by an inducible promoter. In some embodiments, p25/CDK5 is expressed from a viral vector. In some embodiments, the viral vector is a retroviral, lentiviral or an adenoviral vector.

[16] hi some embodiments, the methods further comprise administering the agent to an animal model for a neurodegenerative disease and determining the effect of the agent on the symptoms of the animal model. In some embodiments, the animal model is an animal model for Alzheimer's disease.

[17] hi some embodiments, the method comprising contacting an agent to a polypeptide selected from the group consisting of stathmin, CRMP2, CRMPl, CRMP4, Tubulin-β2, QCZ8, 14-3-3 ε, 14-3-3 ζ, Lamin A, Lamin B, EF-1 delta, EF-2, p97 ATPase, CAPER, DEAD-Helicase, HSP60, HnRNP K, HnRNP G, Nucleolin, BCAKD kinase, Neurofilament-M, p54nrb, cyt-Dynein (IC2), Peroxiredoxin 2, 3-OA CoA tra, CCT, GM130, p27-Kipl, Cdc25A, Cdc25B, Cdc25C and Weel; and detecting an effect on the activity of the polypeptide. [18] In some embodiments, the methods comprise selecting an agent that binds to the polypeptide. In some embodiments, the methods comprise selecting an agent that inhibits the activity of the polypeptide. In some embodiments, the contacting step comprises contacting the agent to a cell comprising an expression cassette encoding the polypeptide. [19] In some embodiments, the cell is a neuronal cell. In some embodiments, the cell comprises a recombinant expression cassette comprising a promoter operably linked to a polynucleotide encoding p25 and CDK5.

[20] In some embodiments, the methods further comprise determining the effect of the agent on viability or apoptosis of neuronal cells that express p25/CDK5. In some embodiments, the methods further comprise administering the agent to an animal model for a neurodegenerative disease and determining the effect of the agent on the symptoms of the animal model. In some embodiments, the animal model is an animal model for Alzheimer's disease.

[21] In some embodiments, the methods comprise contacting an agent to a mixture comprising Cdc2 and p25/CDK5, and detecting an effect on the activity of Cdc2 induced by p25/CDK5; thereby identifying an agent that inhibits kinase activity of Cdc2 in a neuronal cell. In some embodiments, activity of Cdc2 induced by p25/CDK5 is reduced in the presence of the agent compared to in the absence of the agent. In some embodiments, the method comprises screening a plurality of agents to identify an agent that prevents p25/CDK5 activation of Cdc2.

[22] In some embodiments, the Cdc2 and p25/CDK5 are expressed in a cell, i some embodiments, the cell is a neuronal cell.

[23] In some embodiments, the methods comprise selecting an agent that inhibits apoptosis in the cell, hi some embodiments, expression of p25/CDK5 is controlled by an inducible promoter. In some embodiments, p25/CDK5 is expressed from a viral vector. In some embodiments, the viral vector is a retroviral, lentiviral or an adenoviral vector.

[24] The present invention also provides methods of identifying an agent that inhibits CDK5 activation of a substrate, hi some embodiments, the methods comprise providing an addressable collection of reporter cells, which reporter cells produce an output in response to a CDK5 inhibitor and which cells express p25/CDK5; introducing polynucleotides into the cells such that different polynucleotides are introduced into different cells for parallel screening, wherein the polypeptides encode CDK5 substrates selected from the group consisting of Stathmin, CRMP2, CRMPl, CRMP4, Tubulin-β2, QCZ8, 14-3-3 ε, 14-3-3 ζ, Lamin A, Lamin B, EF-1 delta, EF-2, p97 ATPase, CAPER, DEAD-Helicase, HSP60, HnRNP K, HnRNP G, Nucleolin, BCAKD kinase, Neurofilament-M, p54nrb, cyt- Dynein (IC2), Peroxiredoxin 2, 3-OA CoA tra, CCT, GM130, p27-Kipl, Cdc25A, Cdc25B, Cdc25C and Weel; and observing the effect of expression of the polynucleotides on the output response, thereby identifying an agent that inhibits CDK5 activation of a substrate. [25] In some embodiments, the cells are arrayed in wells and the cells in each well contain an introduced polynucleotide encoding a different substrate, hi some embodiments, the cells are neuronal cells. In some embodiments, the neuronal cells are selected from the group consisting of PC12 cells and SH-SY5 cells. In some embodiments, expression of p25/CDK5 in the absence of the CDK5 inhibitor induces apoptosis in the cells and the output response is cell viability or apoptosis. h some embodiments, expression of p25/CDK5 is controlled by an inducible promoter. In some embodiments, p25/CDK5 is expressed from a viral vector. In some embodiments, the viral vector is a retroviral, lentiviral or an adenoviral vector. [26] The present invention provides methods of treating or preventing a neurodegenerative disease. For example, the methods comprise treating an individual in need thereof with the agent identified by any of the methods described herein. In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's Disease, Niemann-Pick disease type 3, Amyotrophic Lateral Sclerosis (ALS), Parkinson's Disease and Downs's Syndrome.

[27] The invention also provides CDK5 kinase comprising an F80G mutation, wherein this mutated protein phosphorylates substrates using N⁶(Phenethyl) ATP.

[28] The invention also provides polynucleotides encoding a CDK5 polypeptide, the CDK5 polypeptide comprising an F80G mutation, wherein the polypeptide phosphorylates substrates using N⁶(Phenethyl) ATP. DEFINITIONS

[29] The term "p25/CDK5" is used throughout the application to describe p25-regulated CDK5. This term is to be read to indicate that p25 regulates CDK5 activity. Thus, for example, the statement that "p25/CDK5 is introduced into a cell" is intended to mean that either both p25 and CDK5 are introduced into the cell or only p25 is introduced into the cell such that the introduced p25 regulates endogenous CDK5 activity in the cell.

[30] "Modulators" are used herein to refer to molecules that inhibit or enhance the ability of p25/CDK5 to interact with (e.g., bind to and/or phosphorylate) its substrates. Substrates for p25/CDK5 include, e.g., Stathmin, CRMP2, CRMPl, CRMP4, Tubulin-β2, QCZ8, 14-3-3 ε, 14-3-3 ζ, Lamin A, Lamin B, EF-1 delta, EF-2, p97 ATPase, CAPER, DEAD-Helicase, HSP60, HnRNP K, HnRNP G, Nucleolin, BCAKD kinase, Neurofilament-M, p54nrb, cyt-Dynein (IC2), Peroxiredoxin 2, 3-OA CoA tra, CCT, GM130, p27-Kipl, Cdc25A, Cdc25B, Cdc25C and Weel. Inhibitors are agents that partially or totally block either the interaction of p25/CDK5 and at least one of its substrates or partially or completely block the kinase activity of CDK5. Activators are agents that increase the interaction of p25/CDK5 and at least one of its substrates. Modulators include naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like. Samples or assays comprising p25/CDK5 and a substrate as described herein are treated with a potential modulator are compared to control samples without the modulator to examine the extent of effect. Control samples (not treated with modulators) are assigned a relative activity value of 100%. Inhibition of a p25/CDK5-substrate interaction is achieved when the interaction (e.g., binding, phosphorylation, etc.) compared to the control is less than about 80%, optionally 50%) or 25, 10%, 5% or 1%. Activation of the polypeptide is achieved when the interaction compared to the control is at least 110%, optionally 150%, optionally 200, 300%, 400%, 500%, or 1000-3000% or more.

[31] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol Chem. 260:2605-2608 (1985); and Cassol et al (1992); Rossolini et al, Mol Cell. Probes 8:91-98 (1994)).

[32] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.

[33] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.

[34] Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[35] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequences. Two sequences are substantially identical if the two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. The invention provides polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides, respectively, exemplified herein (e.g., p25, CDK5, Stathmin, CRMP2, CRMPl, CRMP4, Tubulin-β2, QCZ8, 14-3-3 ε, 14-3-3 ζ, Lamin A, Lamin B, EF-1 delta, EF-2, ρ97 ATPase, CAPER, DEAD-Helicase, HSP60, HnRNP K, HnRNP G, Nucleolin, BCAKD kinase, Neurofilament-M, p54nrb, cyt- Dynein (IC2), Peroxiredoxin 2, 3-OA CoA tra, CCT, GM130, p27-Kipl, Cdc25A, Cdc25B, Cdc25C, Cdc2 or Weel). Optionally, the identity exists over a region that is at least about 50 nucleotides or amino acids in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides or amino acids in length.

[36] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[37] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat 'I. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al, Current Protocols in Molecular Biology (1995 supplement)).

[38] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[39] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[40] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

[41] Figure 1 illustrates a general strategy for identifying p25/CDK5 substrates.

[42] Figures 2A-B list p25/CDK5 substrates.

[43] Figure 3 illustrates the current theory of the molecular mechanisms leading to development of Alzheimer's Disease (AD).

[44] Figure 4 illustrates a mechanism for development of neurodegenerative disease triggered by p25/CDK5 -induced resumption of the cell cycle.

[45] Figure 5 illustrates results of p25/CDK5 phosphorylation of CRMP 112 in in vitro kinase assays.

[46] Figure 6 illustrates results of p25/CDK5 phosphorylation of Stathmin in in vitro kinase assays. [47] Figure 7 illustrates results of p25/CDK5 phosphorylation of p27-kipl in in vitro kinase assays.

[48] Figure 8 illustrates results of p25/CDK5 phosphorylation of Cdc25A/C in in vitro kinase assays.

[49] Figure 9 illustrates results of experiments demonstrating that p25 expression leads to increased CDK1 activity and decreased p27 levels.

DETAILED DESCRIPTION

J. Introduction

[50] The present invention is based in part on the surprising discovery of a number of substrates of p25-regulated CDK5. Specifically, the following proteins are phosphorylated in brains by p25/CDK5: Stathmin, CRMP2, CRMPl, CRMP4, Tubulin-β2, QCZ8, 14-3-3 ε, 14-3-3 ζ, Lamin A, Lamin B, EF-1 delta, EF-2, p97 ATPase, CAPER, DEAD-Helicase, HSP60, HnRNP K, HnRNP G, Nucleolin, BCAKD kinase, Neurofilament- M, p54nrb, cyt-Dynein (IC2), Peroxiredoxin 2, 3-OA CoA tra, CCT, GM130, p27-Kipl, Cdc25 A, Cdc25B, Cdc25C and Weel . These proteins were not previously lαiown to be phosphorylated by CDK5. By illuminating the mechanism by which aberrant regulation of CDK5 leads to neurodegenerative diseases, this discovery allows for novel methods for identifying agents that inhibit either phosphorylation of the substrates by CDK5 as well as agents that inhibit the activity of the substrates themselves. Agents identified by these methods are useful for treating neurodegenerative diseases, such as Alzheimer's disease, ALS, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Down Syndrome, Hallervorden-Spatz disease, prion diseases, argyrophilic grain dementia, cortocobasal degeneration, dementia pugilistica, diffuse neurofibrillary tangles, Gerstmann- Straussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Jakob- Creutzfeldt disease, Niemann-Pick disease type 3, progressive supranuclear palsy, subacute sclerosing panencephalitis, Spinocerebellar Ataxias, Huntington's disease, Pick's disease, spinal and bulbar muscular atrophy, dentatorubral-palhdoluysian atrophy, or steatohepatitis. [51] This invention relies on routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al. , eds., 1994)).

II. IDENTIFICATION OF AGENTS THAT MODULATE p25/CDK5-SUBSTRATE INTERACTIONS

[52] Agents that modulate interactions of p25/CHK5 with the substrates described herein are useful for preventing and treating neurodegenerative diseases. By

"interactions", it is meant p25/CDK5 binding to, or phosphorylation of, its substrates. The administration to a subject of a therapeutic amount of a modulator of the invention, and in particular, an inhibitor of p25/CDK5-substrate interaction, is useful to treat a number of neurodegenerative diseases including, e.g., Parkinson's disease, Alzheimer's disease, ALS, dementia with Lewy bodies, multiple system atrophy, Down Syndrome, Hallervorden-Spatz disease, prion diseases, argyrophilic grain dementia, cortocobasal degeneration, dementia pugilistica, diffuse neurofibrillary tangles, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Jakob-Creutzfeldt disease, Niemann- Pick disease type 3, progressive supranuclear palsy, subacute sclerosing panencephalitis, Spinocerebellar Ataxias, Huntington's disease, Pick's disease, spinal and bulbar muscular atrophy, dentatorubral-palhdoluysian atrophy, and steatohepatitis.

[53] Preliminary screens to identify potential modulators can be conducted by screening for agents capable of binding to p25/CDK5 or to a substrate such as Stathmin, CRMP2, CRMPl, CRMP4, Tubulin-β2, QCZ8, 14-3-3 ε, 14-3-3 ζ, Lamin A, Lamin B, EF-1 delta, EF-2, p97 ATPase, CAPER, DEAD-Helicase, HSP60, HnRNP K, HnRNP G, Nucleolin, BCAKD kinase, Neurofilament-M, p54nrb, cyt-Dynein (IC2), Peroxiredoxin 2, 3- OA CoA tra, CCT, GM130, p27-Kipl, Cdc25A, Cdc25B, Cdc25C and Weel. In some embodiments, binding assays involve contacting p25/CDK5 or a substrate with one or more test agents and allowing sufficient time for the motif and test agents to form a binding complex. Any binding complexes formed can be detected using any of a number of established analytical techniques. Protein binding assays include, but are not limited to, methods that measure co-precipitation or co-migration on non-denaturing SDS- polyacrylamide gels, and co-migration on Western blots (see, e.g., Bennet, J.P. and Yamamura, H.I. (1985) "Neurotransmitter, Hormone or Drug Receptor Binding Methods," in Neurotransmitter Receptor Binding (Yamamura, H. I., et al, eds.), pp. 61-89). Other binding assays involve the use of mass spectrometry or NMR techniques to identify molecules bound to a polypeptide of the invention or displacement of labeled substrates. The polypeptides of the invention utilized in such assays can be naturally expressed, cloned or synthesized. [54] In addition, mammalian or yeast two-hybrid approaches (see, e.g.,

Bartel, P.L. et. al. Methods Enzymol, 254:241 (1995)) can be used to identify polypeptides or other molecules that interact or bind when expressed together in a host cell.

[55] Agents can also be directly selected for their ability to prevent binding of p25/CDK5 to a p25/CDK5 substrate, hi this variation, binding assays are performed to detect an agent that interferes with binding between p25/CDK5 and a substrate. In some embodiments, a polypeptide comprising p25/CDK5 or a substrate is labeled and the quantity of label is used to determine the ability of the agent to compete or interfere with the p25/CDK5-substrate interaction.

[56] In some embodiments, p25/CDK5 or a substrate or fragment thereof is immobilized on a solid support. For example, in some embodiments, a polypeptide comprising a substrate is immobilized on a solid support (e.g., a micro titer dish) and incubated with p25/CDK5 in the presence or absence of one or more test compounds. Unbound p25/CDK5 is washed away and the remaining bound p25/CDK5 is quantified. Agents that reduce the amount of bound p25/CDK5 (i.e., that specifically compete with p25/CDK5 for binding) are then selected for further testing. Alternatively, the ability of p25/CDK5 to phosphorylate a substrate can also be tested. In these embodiments, agents are selected that modulate (e.g., reduce or inhibit) phosphorylation of the substrate (e.g., in in vitro kinase assays). [57] An alternative preliminary screen involves contacting an agent to a cell and expressing p25, or p25 and CDK5, in a cell and selecting an agent that blocks apoptosis of the cell. This screen involves use of a cell, such as a neuronal cell, in which p25/CDK5 induces apoptosis. Exemplary neuronal cells include, e.g., PC12 or SH-SY5 cells. This screen allows one to identify those agents that interfere with p25/CDK5-induced apoptosis. Expression of p25 and/or p25 and CDK5 can be mediated by any method known to those of skill in the art. For example, a viral expression vector, such as a retroviral (e.g., VTP3), lentiviral or adenoviral vector controlling expression of the protein can be introduced into the cell. Alternatively, the cells can contain a stably integrated expression cassette encoding the protein(s), for example wherein the promoter is mducible and is induced when triggering of apoptosis is desired.

[58] A variety of assays for determining cell viability or apoptosis are well known in the art. Such methods include light microscopy for determining the presence of one or more morphological characteristics of apoptosis, such as condensed or rounded morphology, shrinking and blebbing of the cytoplasm, preservation of structure of cellular organelles including mitochondria, and condensation and margination of chromatin. Apoptosis can also be measured using terminal deoxytransferase-mediated (TdT) dUTP biotin nick end-labeling (TUNEL) (Gavriel et al, J. Cell Biol. 119:493 (1992); Gorczyca et al, Int. J Oncol. 1:639 (1992)). APOPTAG (ONCOR, Inc.; Gaithersburg Md.), PhiPhiLux® (Oncolmmunin, Inc.) and the "Homogeneous Caspases Assay" (Roche Molecular

Biochemicals) are commercially available kits for identification of apoptotic cells. In addition, apoptosis can be assayed by detecting nucleosomal DNA fragments using agarose gel electrophoresis (Gong et al, Anal Biochem. 218:314 (1994)). Apoptotic or anti- apoptotic activity also can be detected and quantified by determining an altered mitochondrial to nuclear DNA ratio as described in Tepper et al, Anal. Biochem. 203:127 (1992) and Tepper and Studzinski, J. Cell Biochem. 52:352 (1993). One skilled in the art understands that these, or other assays for apoptotic or anti-apoptotic activity, can be performed using routine methodology. Viability screens can be conducted in a high throughput format using alamar blue. Alternatively, commercially available kits measuring caspase activity can be used to run similar screens aimed at apoptosis detection.

[59] The agents tested in the methods of the invention can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid or lipid. In some embodiments, nucleic acid libraries (e.g., cDNA libraries) are expressed in transgenic melanopsin knockout animals or their cells. Alternatively, test compounds will be small organic molecules or peptides. Essentially any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions are used. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma- Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) and the like. Generally, the compounds to be tested are present in the range

[60] In some embodiments, high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator compounds). Such "combinatorial chemical libraries" or "ligand libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.

[61] A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. [62] Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al, Nature 354:84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al, Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al, J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al, J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al, J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al, Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al, J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083), antibody libraries (see, e.g., Vaughn et al, Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al, Science, 274:1520-1522 (1996) and U.S. Patent 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent 5,569,588; thiazolidinones and metathiazanones, U.S. Patent

5,549,974; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent 5,506,337; benzodiazepines, 5,288,514, and the like).

[63] Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433 A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, MO, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

[64] Samples or assays that are treated with a potential inhibitors or p25/CDK5 substrate interactions (e.g., a "test compound") are compared to control samples without the test compound, to examine the extent of, e.g., protein-protein binding or phosphorylation of substrates. Control samples (untreated with agents) are assigned a relative activity value of 100. Inhibition of binding is achieved when the activity value relative to the control is about 90%, optionally 50%, optionally 25-0%>. [65] Agents that are initially identified by any of the foregoing screening methods can be further tested to validate the apparent activity. Modulators that are selected for further study can be tested for an effect on prevention and treatment of neurodegenerative disease.

[66] For example, the effect of the compound can be assessed in animals. In addition, transgenic animals expressing human polypeptides of the invention can be used to further validate drug candidates. Exemplary animal model systems include those described in, e.g., Wong et al, Nat. Neurosci. 5(7):633-639 (2002), Janus et al, Physiol Behav. 73(5):873-86 (2001); Lee et al Neurology. 56(11 Suppl 4):S26-30 (2001); Zoghbi et al, Annu Rev. Neurosci. 23:217-47 (2002); Dawson, Cell 101(2):115-8 (2000); Betarbet et al. Bioessays 24(4):308-18 (2002); and Morgan D, et al, Nature 412: 660 (2001).

[67] In the high throughput assays of the invention, it is possible to screen up to several thousand different potential modulators in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 or more different compounds are possible using the integrated systems of the invention, h addition, microfluidic approaches to reagent manipulation can be used.

[68] In another embodiment, p25/CDK5 or p25 is expressed in a cell under conditions that induces apoptosis or other outputs that are detectable. Prior to p25/CDK5 expression, the cells are provided in an addressable collection and a polynucleotide is introduced into each cell such that different polynucleotides are introduced into different cells in the collection. p25/CDK5 is then expressed and the effect on the cell, if any, is determined compared to controls or other cells in the collection. In some embodiments, the polynucleotides encode at least one p25/CDK5 substrates selected from the group consisting of Stathmin, CRMP2, CRMPl, CRMP4, Tubulin-β2, QCZ8, 14-3-3 ε, 14-3-3 ζ, Lamin A, Lamin B, EF-1 delta, EF-2, p97 ATPase, CAPER, DEAD-Helicase, HSP60, HnRNP K, HnRNP G, Nucleolin, BCAKD kinase, Neurofilament-M, p54nrb, cyt-Dynein (IC2), Peroxiredoxin 2, 3-OA CoA tra, CCT, GM130, p27-Kiρl, Cdc25A, Cdc25B, Cdc25C, Cdc2 or Weel . In some embodiments, members of a cDNA library (e.g., in an expression vector) are introduced into the cells. In some cases, the effect of different expression levels of the polynucleotides (e.g., as regulated by different promoters) is tested. In some cases, polynucleotides are selected that prevent or modulate p25/CDK5-induced apoptosis.

[69] In some embodiments, the cells are arrayed in wells and the cells in each well contain an introduced polynucleotide encoding a different substrate. In some embodiments, the cells are neuronal cells (e.g., PC12 cells or SH-SY5 cells). In some embodiments, p25/CDK5 is expressed from a viral vector, e.g., a lentiviral vector.

[70] Control reactions that measure the interaction in the absence a potential modulator can be optional, as the assays are generally highly uniform. Such optional control reactions are appropriate and increase the reliability of the assay. Accordingly, in one embodiment, the methods of the invention include such a control reaction. For each of the assay formats described, "no modulator" control reactions that do not include a modulator provide a background level of binding activity.

III. PURIFICA TION OF PROTEINS OF THE INVENTION

[71] Either naturally occurring or recombinant polypeptides of the invention can be purified for use in the assays of the invention. Naturally-occurring polypeptides of the invention can be purified from any source. Recombinant polypeptides can be purified from any suitable expression system.

[72] The polypeptides of the invention may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Patent No. 4,673,641; Ausubel et al, supra; and Sambrook et al, supra).

[73] A number of procedures can be employed when recombinant polypeptides are being purified. For example, proteins having established molecular adhesion properties (e.g., poly-histidine) can be reversibly fused to a polypeptide of the invention. With the appropriate ligand, either protein can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein may be then removed by enzymatic activity. Finally polypeptides can be purified using immunoaffinity columns.

A. Purification of Proteins from Recombinant Bacteria

[74] When recombinant proteins are expressed by the transformed bacteria in large amounts, typically after promoter induction, although expression can be constitutive, the proteins may form insoluble aggregates. There are several protocols that are suitable for purification of protein inclusion bodies. For example, purification of aggregate proteins (hereinafter referred to as inclusion bodies) typically involves the extraction, separation and/or purification of inclusion bodies by disruption of bacterial cells typically, but not limited to, by incubation in a buffer of about 100-150 μg/ml lysozyme and 0.1% Nonidet P40, a non-ionic detergent. The cell suspension can be ground using a Polytron grinder (Brinkman Instruments, Westbury, NY). Alternatively, the cells can be sonicated on ice. Alternate methods of lysing bacteria are described in Ausubel et al. and Sambrook et al, both supra, and will be apparent to those of skill in the art.

[75] The cell suspension is generally centrifuged and the pellet containing the inclusion bodies resuspended in buffer that does not dissolve but washes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, 150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may be necessary to repeat the wash step to remove as much cellular debris as possible. The remaining pellet of inclusion bodies may be resuspended in an appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mM NaCl). Other appropriate buffers will be apparent to those of skill in the art. , [76] Following the washing step, the inclusion bodies are solubilized by the addition of a solvent that is both a strong hydrogen acceptor and a strong hydrogen donor (or a combination of solvents each having one of these properties). The proteins that formed the inclusion bodies may then be renatured by dilution or dialysis with a compatible buffer. Suitable solvents include, but are not limited to, urea (from about 4 M to about 8 M), formamide (at least about 80%, volume/volume basis), and guanidine hydrochloride (from about 4 M to about 8 M). Some solvents that are capable of solubilizing aggregate-forming proteins, such as SDS (sodium dodecyl sulfate) and 70% formic acid, are inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity. Although guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturation may occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing re-formation of the immunologically and/or biologically active protein of interest. After solubilization, the protein can be separated from other bacterial proteins by standard separation techniques. [77] Alternatively, it is possible to purify proteins from bacteria periplasm.

Where the protein is exported into the periplasm of the bacteria, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to those of skill in the art (see, Ausubel et al, supra). To isolate recombinant proteins from the periplasm, the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose. To lyse the cells, the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO₄ and kept in an ice bath for approximately 10 minutes. The cell suspension is centrifuged and the supernatant decanted and saved. The recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art. B. Standard Protein Separation Techniques For Purifying Proteins

1. Solubility Fractionation

[78] Often as an initial step, and if the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest. The preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations. A typical protocol is to add saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20- 30%). This will precipitate the most hydrophobic proteins. The precipitate is discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt removed if necessary, through either dialysis or diafiltration. Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures.

2. Size Differential Filtration

[79] Based on a calculated molecular weight, a protein of greater and lesser size can be isolated using ultrafiltration through membranes of different pore sizes (for example, Ainicon or Millipore membranes). As a first step, the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of the protein of interest. The retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest. The recombinant protein will pass through the membrane into the filtrate. The filtrate can then be chromatographed as described below.

3. Column Chromatography

[80] The proteins of interest can also be separated from other proteins on the basis of their size, net surface charge, hydrophobicity and affinity for ligands. In addition, antibodies raised against proteins can be conjugated to column matrices and the proteins immunopurified. All of these methods are well lαiown in the art.

[81] Immunoaffmity chromatography using antibodies raised to a variety of affinity tags such as hemagglutinin (HA), FLAG, Xpress, Myc, hexahistidine (His), glutathione S transferase (GST) and the like can be used to purify polypeptides. The His tag will also act as a chelating agent for certain metals (e.g., Ni) and thus the metals can also be used to purify His-containing polypeptides. After purification, the tag is optionally removed by specific proteolytic cleavage.

[82] It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech).

IV. ADMINISTRATION AND PHARMACEUTICAL COMPOSITIONS

[83] Agents that reduce or inhibit p25/CDK5-substrate interactions can be administered directly to the mammalian subject. Administration is by any of the routes normally used for introducing a modulator compound into ultimate contact with the tissue to be treated and is well known to those of skill in the art.

[84] The pharmaceutical compositions of the invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington 's Pharmaceutical Sciences, 17^u ed. 1985)).

[85] Formulations suitable for administration include aqueous and non- aqueous solutions, isotonic sterile solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, orally, nasally, topically, intravenously, intraperitoneally, intrathecally or into the eye (e.g., by eye drop or injection). The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. The modulators can also be administered as part of a prepared food or drug.

[86] The dose administered to a patient, in the context of the present invention should be sufficient to induce a beneficial response in the subject over time, i.e., to prevent or treat a neurodegenerative disease. The optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific modulator employed, the age, body weight, physical activity, and diet of the patient, and on a possible combination with other drug. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound or vector in a particular subject.

[87] hi determining the effective amount of the modulator to be administered a physician may evaluate circulating plasma levels of the modulator, modulator toxicity, and the production of anti-modulator antibodies, hi general, the dose equivalent of a modulator is from about 1 ng/kg to 10 mg/kg for a typical subject.

[88] For administration, modulators of the present invention can be administered at a rate determined by the LD-50 of the modulator, and the side-effects of the modulator at various concentrations, as applied to the mass and overall health of the subject. Administration can be accomplished via single or divided doses.

[89] The modulators of the invention may be used alone or in conjunction with other agents that are known to be beneficial in treating or preventing neurodegenerative disease. The modulators of the invention and an other agent may be coadministered, either in concomitant therapy or in a fixed combination, or they may be administered at separate times.

V. DETECTING NEUROLOGICAL DISORDERS

[90] The invention also provides methods for detecting neurological disorders. The methods include, e.g., (a) obtaining a biological sample from a subject suspected of having a neurological disorder, (b) determining in the biological sample phosphorylation of a p25/CDK5 substrate selected from the group consisting of Stathmin, CRMP2, CRMPl, CRMP4, Tubulin-β2, QCZ8, 14-3-3 ε, 14-3-3 ζ, Lamin A, Lamin B, EF-1 delta, EF-2, p97 ATPase, CAPER, DEAD-Helicase, HSP60, HnRNP K, HnRNP G, Nucleolin, BCAKD kinase, Neurofilament-M, p54nrb, cyt-Dynein (IC2), Peroxiredoxin 2, 3- OA CoA tra, CCT, GM130, p27-Kipl, Cdc25A, Cdc25B, Cdc25C and Weel, and (c) comparing the phosphorylation with the phosphorylation of the sequence in a control sample. An increase or stimulation of phosphorylation or the decrease when compared to a control sample indicates the presence of a neurological disorder.

[91] For example, the biological sample for this assay may be obtained from a biopsy of the brain which contains neurons or, in some embodiments, from a blood sample. The control sample may be obtained from the brain or blood of a subject without a neurological disorder. In an embodiment of the detecting methods, the biological, test, and control samples are obtained from organisms including mouse and human. Those biological, test, and control samples which are extracted from organisms can be obtained from tissues including brain, specifically neurons, and/or blood. In another embodiment, the biological, test and control samples are extracted from a cell culture.

EXAMPLES

[92] The following examples are offered to illustrate, but not to limit the claimed invention.

[93] Using a chemical-genetic method, we have identified potential substrates of CDK5 in extracts of brains and neuronal cell lines. Knowledge of these substrates allowed us to predict how CDK5 mediates neurodevelopment and neurodegenerative diseases such as Alzheimer's Disease (AD) and Amyotrophic Lateral Sclerosis (ALS). A modified CDK5, which can use a synthetic substrate to phosphorylate proteins was used to identify substrates of p25-regulated CDK5. See, Figure 1.

[94] CDK5 was mutated at position 80 to replace a phenylalanine residue with glycine (F80G) to create an additional hydrophobic pocket near the ATP binding site. This mutation allowed CDK5 mutant to use the ATP analog N⁶(Phenethyl)ATP, unlike any other kinase. Creation of this specific enzyme-substrate pair then allowed us to label only the substrates of CDK5/p25 in cell lysate using γ³²P-N⁶(Phenethyl)ATP. Only the direct substrates of CDK5 were labeled with a radioactive phosphate.

[95] The complex mixture of proteins present in labeled cell lysates was resolved by 2D electrophoresis. The resulting 2D gels were exposed to a film sensitive to radioactivity and this film and the gel were aligned to find protein spots which were labeled and visible by coomassie staining. We found that fractionating lysates (cellular compartment fractionation, anion exchange, cation exchange, size exclusion) provided enough material for identification of the individual proteins. The desired spots are cut out from the gel, digested into peptides and identification of the labeled protein is conducted by MALDI or ESI mass spectrometry.

[96] Proteins that were specifically phosphorylated by F80G CDK5 and identified in the screen are listed in Figures 2A-B. CDK5/p25 substrates identified in mouse brain lysate using 2D gels and MS with a few exceptions fall into 4 broad categories: 1. Proteins lαiown to be involved in neuronal functions, and especially neurodevelopment, include CRMPl, CRMP2, CRMP4, 14-3-3ε and 14-3-3ζ. 2. Proteins disregulated or hyperphosphorylated/misfolded (NFT) in Alzheimer's disease include CRMP2, rubulin-β2, 14-3-3ε, 14-3-3ζ, EF-2, Nucleolin, NF-M and Peroxiredoxin 2.

3. Proteins that are substrates of cdc2 include Stathmin, Lamin B, EF-1 delta, p97 ATPase, Nucleolin, Peroxiredoxin 2 and GM 130.

4. Proteins that are substrates of CDK5 include NF-M.

[97] Figure 3 illustrates some aspects of the current state of understanding regarding the molecular mechanism of Alzheimer's Disease. As described below, we propose a different mechanism for development of neurodegenerative disease. [98] Without intending to be limited to a specific theory of action, it is believed that CDK5/p25 is responsible for cell cycle re-entry or aberrant phosphorylation of cell cycle related proteins and subsequent degeneration in neurons, including in Alzheimer's disease. See, Figure 4. As evidence for this mechanism, we note that:

1) CDK5 has the same substrate specificity as most CDKs: *S/TPXK/R. It is most similar to that of CDK2 and CDC2, which play key roles in S, G2 and M phases.

2) Degradation of p35 into p25 provides CDK5 with a different cellular localization (cytosol and nucleus).

3) A substrate-binding domain common to cyclin Ds is conserved in p25.

4) Common CDK regulation mechanisms seem not to affect CDK5/p25 since CDK5 normally operates in post-mitotic cells.

[99] Collapsin Response Mediator Protein (CRMP, aka DHP, ULIP, UNC, TOAD) was studied in more detail. CRMP has 5 known family members of 60-70kDa phosphoproteins and is expressed in the nervous system, mostly during development. The protein is involved in neuronal differentiation and axonal guidance. CRMP-1 is involved in inhibition of tumor cell invasion. CRMP-2 plays a role in axon induction and microtubule dynamics regulation. CRMP-2 is multiply phosphorylated in neurofibrillary tangles (NFTs) that develop in Alzheimer's patients. The level of multiply phosphorylated CRMP-2 is doubled in AD brain's cytosol.

[100] CRMP-1 and CRMP-2 were cloned, expressed and purified as GST- tagged proteins. CRMP-1 was expressed as a fragment of the full length protein. As illustrated in Figure 5, in vitro kinase assays confirmed that CDK5 phosphorylates GST- CRMP-1 and GST-CRMP-2. [101] Another p25/CDK5 substrate, Stathmin (aka Oncoprotein 18, prosolin) is a 19kDa cytoplasmic phosphoprotein. Phosphorylation of Stathmin sites can be targeted by PKA (SI 6, S63), CaMK TV (SI 6) and CDKs (S25, S38). Stathmin is highly expressed in proliferating cells and neurons and plays a central role in microtubule dynamics. Unphosphorylated Stathmin promotes microtubule destabilization. Sequential phosphorylations of Stathmin allow for the downregulation of its activity. As illustrated in Figure 6, in vitro kinase assays confirm that CDK5 phosphorylates stathmin.

[102] Without intending to be limited to a particular theory of action, it is believed that CDK5/p25 promotes cell cycle re-entry by phosphorylation of p27, CDC25 and Weel.

[103] p27 inhibits CDKs through binding and its absence leads to cell proliferation. Phosphorylation on T187 (a cdc 2/cyc B site) promotes its ubiquitination and rapid degradation. As illustrated in Figure 7, CDK5/p25 efficiently phosphorylates p27 on T187 in vitro. [104] The phosphatases Cdc25 A, B and C activate CDKs by the dephosphorylation of T14 and Y15. hi turn, their phosphorylation by active CDKs enhance their dephosphorylation abilities. As illustrated in Figure 8, CDK5/p25 efficiently phosphorylates Cdc25A/C in vitro.

[105] Weel phosphorylates CDKs on Y15, leading to their downregulation. Active CDKs phosphorylate Weel in its regulatory N-terminal domain, thereby inhibiting it.

[106] We also demonstrated that p25 expression leads to increased CDK1 activity and decreased p27 levels in PC 12 neuronal cells. See, Figure 9.

[107] Thus, reducing or eliminating the phosphorylation of p27, a Cdc25 phosphatase or Weel can be used to treat or prevent neurodegenerative disease.

MATERIALS AND METHODS

Cloning, expression and purification of CDK5, CDK5(F80G) and p25.

[108] CDK5 and p25 were cloned into the pGEX-2T bacterial expression system. The mutant CDK5 protein (F80G) construct was made from CDK5 in pGEX-2T bacterial expression system using the Quickchange™ protocol from Qiagen according to the manufacturer's instructions (see sequence listing for the sequences of all mutants).

[109] Mutant CDK5 cDNA was transformed in BL21-Gold Competent Cells™ from Stratagene (#230130) according to the manufacturer's instructions. An overnight culture of BL21-Gold cells containing one of the GST-tagged plasmids described above was diluted 1 :50 in Superbroth™ medium (Q-Biogene, #3010-032) supplemented with 50 mg/L carbenicillin (Sigma, #C1389). After it had grown to an OD = 0.6, synthesis of the GST-H-Ras protein was induced by the addition of either 10 μM (for CDK5 and CDK5(F80G)) or 100 μM (for p25) isopropyl-/?-D-thiogalactopyranoside (Promega, #PRV3951) and by further incubation at 30°C for 12h. The cells expressing CDK5 (or

CDK5(F80G)) and p25 were then mixed, centrifuged at 3,500g for 15 min at 4°C, and the cell pellet was frozen at -80°C for lh. The bacterial cell pellet was resuspended in lysis buffer (20 mM HEPES pH 7.5, 75 mM KCl, 25 mM MgCl₂, 5 mM DTT, 0.1 mM EDTA, 0.05% Triton X-100, 5 mM Benzamidine, 10 mg/L Aprotinin, 10 mg/L Antipain, 10 mg/L Pepstatin, 10 mg/L Leupeptin and 1 mM Phenyhnethylsulfonyl chloride), chilled on ice and sonicated

(Sonics, Vibracell) for 3x20 sec. The resulting lysate was centrifuged at 30,000g for 30 min at 4°C, and the supernatant was added to glutathione sepharose beads (Pharmacia, #274574- 01) for a 30 min incubation at 4°C on a rotating wheel. The beads were washed with lysis buffer once, and then with wash buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT) three times. The beads were further washed using 1 mM glutathione and 10 mM glutathione solutions in wash buffer. The protein complex was batch eluted using 20 mM glutathione in wash buffer after a 30 min incubation at 4°C on a rotating wheel, concentrated and dialysed overnight against dialysis buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM DTT, 0.1 mM EDTA). Protein concentration was determined by a Bradford assay and the protein purity was assessed by gel electrophoresis. Alternatively, the protein complex could be further purified by treatment of the washed beads with thrombin (Haematologic Technologies Inc., #HCT-0020) at 1/5000 dilution in cleavage buffer (20 mM Tris-HCl pH 8.5, lOOmM NaCl, 1 mM CaCl₂) for 4 h at 4°C. The supernatant was run through a column containing glutathione sepharose beads (0.5 mL) and benzamidine sepharose beads (0.1 mL), concentrated and dialysed overnight against dialysis buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM DTT, 0.1 mM EDTA and 1 mM benzamidine). Alternatively, the CDK5(F80G)/p25 complex was prepared from Sf9 insect cells using the baculovirus Bac-to- Bac expression system (Invitrogen) according to the manufacturer's instructions. The only modification was to mix the cells expressing CDK5 (or CDK5(F80G)) and p25 before the lysis. Proteins kept at 4°C conserved their activity for several weeks but purified kinase complexes were freshly prepared for all substrate labeling experiments. Preparation of [γ-³²P] N⁶(PhenethyI)ATP

[110] [γ-³²P] N⁶ (Phenethyl) ATP was synthesized as described before (Shah et al Chem Biol. 9(l):35-47 (2002)).

In vitro kinase assay

[111] To measure the enzymatic activity of the kinase, purified protein was mixed with kinase buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 10 mM MgCl₂) and 100 μM peptide containing an optimal CDK5 phosphorylation motif. Kinase reactions were initiated by the addition of 0.5 μCi of [γ-³²P] ATP or [γ-³²P] N⁶ (Phenethyl) ATP and were incubated for 30 min. at room temperature (RT). Reaction mixtures were spotted on DE-81 paper (Whatman) and subsequently washed with 10% acetic acid, 0.5% phosphoric acid, and acetone before drying. Radioactivity of the dried filter paper was determined by scintillation counter (Beckman) with Ecoscint H counting solution (National Diagnostics).

Preparation of cell lysates and fractionations

[112] A mouse brain was cut in small pieces and added to a 15 mL conical tube containing 5 mL of lysis buffer (20 mM HEPES pH 7.5, 1 mM DTT, 2 mM EDTA, 10 mM EGTA, 1% Triton X-100, 5 mM Benzamidine, 10 mg/L Aprotinin, 10 mg/L Antipain, 10 mg/L Pepstatin, 10 mg/L Leupeptin and 1 mM Phenyhnethylsulfonyl chloride, 1 mM Na₃V04 and 100 nM Calyculin A) and the mixture was vortexed for 15 sec. After transfer to a dounce glass, homogenization of the lysate was achieved by 20 strokes using the loose piston and a further 20 strokes using the tight piston. The lysate was then centrifuged first at 4,000g for 25 min at 4°C and then at 50,000g for 30 min at RT. The supernatant was either directly frozen at -80°C or dialyzed overnight at 4°C against storage buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 1 mM DTT, 0.2 mM EDTA) and then frozen at -80°C.

[113] For anion-exchange fractionation, whole lysate (0.9 mL) was applied to a Resource Q column (Pharmacia). The elution was conducted at 1 mL/min with a gradient of 100% buffer A (20 mM Tris-HCl pH 7.5) to 100% buffer B (20 mM Tris-HCl pH 7.5, 1 M KCl) over 20 minutes. [114] For fractionation of whole lysate using cation-exchange chromatography, whole lysate was dialyzed against lysate buffer (50 mM PIPES pH 6.8, 0.2 mM MgCl₂, 1 mM EGTA) and loaded onto the phosphocellulose column (Whatman) equilibrated with the same buffer. After collecting the flowthrough (F.T.), the column was eluted with a linear gradient of 100% lysate buffer A (as above) and buffer B (lysate buffer + 1 M KCl).

[115] For in vitro labeling of proteins from whole cell lysate or from fractionated lysate, 10 to 50 μg of lysate was added into kinase buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 10 mM MgCl ). Varying concentrations of ATP, 50 μM to 5 mM, were then added to the reaction and an incubation was carried out for 10 min. at RT. The kinase complex was subsequently added and the kinase reactions were initiated immediately by adding 0.5μCi of [γ-³²P] ATP or [γ-³²P] N⁶(Phenethyl)ATP. Phosphatase inhibitors Calyculin A and Na₃VO₄ were added at the same time and the reactions were incubated for 15 min. at RT. Reactions were terminated by adding SDS sample buffer (62.5mM Tris-HCl pH 6.8, 10% glycerol, 1% SDS, 5% 2-mercaptoethanl, 0.02% Bromophenol blue) and boiled for 5 minutes, labeled proteins were separated on 8-12% gradient SDS-PAGE gel and were transferred to Immobilon-P nylon membrane (Millipore) by electroblotting. For autoradiography, the membrane was exposed to the Phospho screen overnight and visualized by Storm860 (Molecular Dynamics).

2D-gel electrophoresis and MS spectrometry

[116] To prepare a labeled sample for 2D gel electrophoresis, kinase reactions with whole cell lysate or fractionated lysate were carried out as described above with a total protein amount ranging from 200 to 1,000 μg. After incubation for 15 min. at RT, reactions were collected into fresh screw cap tube and then mixed with 10X nuclease solution (50 mM MgCl₂, 100 mM Tris-HCl (pH 7.0), 500 μg/ml RNaseA, 1000 μg/ml DNasel). After keeping on ice for 10 min., SDS was added to the samples to a final concentration of 5%>. The samples were boiled for 1 min. and then dialyzed overnight at 4°C against lyophihzation buffer (2 mM Tris-HCl pH 7.5, 2 mM NaCl, 0.1% SDS). The dialyzed samples were quickly frozen using liquid N₂, lyophilized overnight and resuspended in SDS boiling buffer (IX: 5% SDS, 5% 2-mercaptoethanol, 10% glycerol, 60 mM Tris-HCl pH 6.8). 2D gel electrophoreses of labeled samples were carried out by Kendrick Labs (Madison, Wisconsin) with 2% pH 4-10 ampholines (Gallard-Schlesinger Industries, Inc., Garden City, NY) at 9,600 volt-hrs. One μg of an IEF internal standard, tropomyosin, was added to each sample. Ten percent SDS slab gel electrophoresis was carried out for about 4 hr at 12.5 niA/gel. The gels were strained with Coomassie Brilliant Blue R-250, dried between sheets of cellophane, and exposed to Kodak X-OMAT AR film. [117] Gel spots were manually excised and automatically processed for peptide mapping experiments using a Micromass MassPREP Station in conjunction with manufacturer specified protocols. Briefly, gel spots were destained (2x with 50% acetonitrile/ 50 mM ammonium bicarbonate), dehydrated (acetonitrile), reduced (DTT), alkylated (iodoacetamide), and digested with sequencing grade trypsin overnight. The resulting peptides were extracted with 30 μL of 1% formic acid, and 2 μL was mixed with 1.5 μL of a 10 mg/ml solution of c-cyano-4-hydroxycinnamic acid and spotted directly onto MALDI target plates. MALDI mass spectra were obtained automatically using a Micromass M@LDI-R TOF MS, with ACTH fragment 18-39 as a Lockmass reference. Protein identification searches were performed using MASCOT (Matrix Sciences). Alternatively, the solution was passed through a Zip-tip (Millipore), and eluted in 2 μL directly onto MALDI target plates before analysis.

[118] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS:

L A method for identifying an agent to treat or prevent a neurodegenerative disease, the method comprising, contacting the agent to p25/CDK5 and a substrate selected from the group consisting of Stathmin, CRMP2, CRMPl, CRMP4, Tubulin-β2, QCZ8, 14-3-3 ε, 14-3-3 ζ, Lamin A, Lamin B, EF-1 delta, EF-2, p97 ATPase, CAPER, DEAD-Helicase, HSP60, HnRNP K, HnRNP G, Nucleolin, BCAKD kinase, Neurofilament-M, p54nrb, cyt-Dynein (IC2), Peroxiredoxin 2, 3-OA CoA tra, CCT, GM130, p27-Kipl, Cdc25A, Cdc25B, Cdc25C, and Weel; and detecting an effect on the phosphorylation of the substrate by p25/CDK5, thereby identifying an agent to treat or prevent a neurodegenerative disease.

2. The method of claim 1, comprising selecting an agent that binds to the substrate, thereby preventing phosphorylation of the substrate.

3. The method of claim 1, comprising selecting an agent that binds to p25/CDK5, thereby preventing phosphorylation of the substrate.

4. The method of claim 1, comprising selecting an agent that prevents binding of p25/CDK5 to the substrate.

5. The method of claim 1, comprising selecting an agent that reduces phosphorylation of the substrate by p25/CDK5 compared to phosphorylation in the absence of the agent.

6. The method of claim 1, wherein the method comprises screening a plurality of agents to identify an agent that prevents p25/CDK5 phosphorylation of a substrate.

7. The method of claim 1, wherein the contacting step comprises contacting a cell, the cell comprising p25/CDK5 and the substrate.

8. The method of claim 7, wherein the cell is a neuronal cell.

9. The method of claim 7, wherein the cell is selected from the group consisting of a PC12 cell and a SH-SY5 cell.

10. The method of claim 7, wherein the cell comprises a recombinant expression cassette comprising a promoter operably linked to a polynucleotide encoding p25.

11. The method of claim 7, wherein the cell comprises an expression cassette comprising a promoter operably linked to a polynucleotide encoding CDK5.

12. The method of claim 7, wherein the detecting step comprises detecting the expression of a gene that is induced by the phosphorylated substrate.

13. The method of claim 12, wherein the detection of the expression of the gene is detected by detecting expression of a reporter gene controlled by the promoter of the induced gene.

14. The method of claim 1, further comprising administering the agent to an animal model for a neurodegenerative disease and determining the effect of the agent on the symptoms of the animal model.

15. The method of claim 14, wherein the animal model is an animal model for Alzheimer's disease.

16. A method of treating or preventing a neurodegenerative disease, the method comprising, treating an individual in need thereof with the agent identified in claim 1.

17. The method of claim 16, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's Disease, Amyotrophic Lateral Sclerosis (ALS), Parkinson's Disease, Niemann-Pick disease type 3, and Downs's Syndrome.

18. A method of identifying an agent for treating or preventing a neurodegenerative disease, the method comprising contacting a neuronal cell with an agent, wherein the cell expresses p25/CDK5 before and/or after the contacting step; detecting viability or apoptosis of the cell contacted by the agent; and selecting an agent that enhances viability of the cell contacted by the agent compared to a cell not contacted by the agent.

19. The method of claim 18, comprising detecting CDK5 phosphorylation of a substrate selected from the group consisting of Stathmin, CRMP2, CRMP 1 , CRMP4, Tubulin-β2, QCZ8, 14-3-3 ε, 14-3-3 ζ, Lamin A, Lamin B, EF-1 delta, EF-2, ρ97 ATPase, CAPER, DEAD-Helicase, HSP60, HnRNP K, HnRNP G, Nucleolin, BCAKD kinase, Neurofilament-M, p54nrb, cyt-Dynein (IC2), Peroxiredoxin 2, 3-OA CoA tra, CCT, GM130, ρ27-Kipl, Cdc25A, Cdc25B, Cdc25C, and Weel.

20. The method of claim 18, wherein the cell comprises a recombinant expression cassette comprising a promoter operably linked to a polynucleotide encoding p25/CDK5.

21 . The method of claim 18, wherein expression of p25/CDK5 is controlled by an inducible promoter.

22. The method of claim 18, wherein p25/CDK5 is expressed from a viral vector.

23. The method of claim 22, wherein the viral vector is a lentiviral vector.

24. The method of claim 22, wherein the viral vector is a retroviral vector.

25. The method of claim 22, wherein the viral vector is an adenoviral vector.

26. The method of claim 18, further comprising administering the agent to an animal model for a neurodegenerative disease and determining the effect of the agent on the symptoms of the animal model.

27. The method of claim 26, wherein the animal model is an animal model for Alzheimer's disease.

28. A method of treating or preventing a neurodegenerative disease, the method comprising, treating an individual in need thereof with the agent identified in claim 18.

29. The method of claim 28, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's Disease, Amyotrophic Lateral Sclerosis (ALS), Parkinson's Disease, Niemann-Pick disease type 3, and Downs's Syndrome.

30. A method of identifying an agent for treating or preventing a neurodegenerative disease, the method comprising contacting an agent to a polypeptide selected from the group consisting of Stathmin, CRMP2, CRMPl, CRMP4, Tubulin-β2, QCZ8, 14-3-3 ε, 14-3-3 ζ, Lamin A, Lamin B, EF-1 delta, EF-2, p97 ATPase, CAPER, DEAD-Helicase, HSP60, HnRNP K, HnRNP G, Nucleolin, BCAKD kinase, Neurofilament- M, p54nrb, cyt-Dynein (IC2), Peroxiredoxin 2, 3-OA CoA tra, CCT, GM130, p27-Kiρl, Cdc25 A, Cdc25B, Cdc25C, and Weel ; and detecting an effect on the activity of the polypeptide.

31. The method of claim 30, comprising selecting an agent that binds to the polypeptide.

32. The method of claim 30, comprising selecting an agent that inhibits the activity of the polypeptide.

33. The method of claim 30, wherein the contacting step comprises contacting the agent to a cell comprising an expression cassette encoding the polypeptide.

34. The method of claim 33, wherein the cell is a neuronal cell.

35. The method of claim 33 , wherein the cell comprises a recombinant expression cassette comprising a promoter operably linked to a polynucleotide encoding p25/CDK5.

36. The method of claim 30, further comprising determining the effect of the agent on viability or apoptosis of neuronal cells that express p25/CDK5.

37. The method of claim 30, further comprising administering the agent to an animal model for a neurodegenerative disease and determining the effect of the agent on the symptoms of the animal model.

38. The method of claim 37, wherein the animal model is an animal model for Alzheimer's disease.

39. A method of treating or preventing a neurodegenerative disease, the method comprising administering the agent identified in claim 30 to an individual in need thereof.

40. The method of claim 39, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's Disease, Amyo trophic Lateral Sclerosis (ALS), Parkinson's Disease, Niemann-Pick disease type 3, and Downs's Syndrome.

41. A method of identifying an agent that inhibits CDK5 activation of a substrate, the method comprising providing an addressable collection of reporter cells, which reporter cells produce an output in response to a CDK5 inhibitor and which cells express p25/CDK5; introducing polynucleotides into the cells such that different polynucleotides are introduced into different cells for parallel screening, wherein the polypeptides encode CDK5 substrates selected from the group consisting of Stathmin, CRMP2, CRMP 1 , CRMP4, Tubulin-β2, QCZ8, 14-3-3 ε, 14-3-3 ζ, Lamin A, Lamin B, EF-1 delta, EF-2, ρ97 ATPase, CAPER, DEAD-Helicase, HSP60, HnRNP K, HnRNP G, Nucleolin, BCAKD kinase, Neurofilament-M, p54nrb, cyt-Dynein (IC2), Peroxiredoxin 2 3-OA CoA tra, CCT, GM130, p27-Kipl, Cdc25A, Cdc25B, Cdc25C, and Weel ; and observing the effect of expression of the polynucleotides on the output response, thereby identifying an agent that inhibits CDK5 activation of a substrate.

42. The method of claim 41 , wherein the cells are arrayed in wells and the cells in each well contain an introduced polynucleotide encoding a different substrate.

43. The method of claim 41, wherein the cells are neuronal cells.

44. The method of claim 43, wherein the neuronal cells are selected from the group consisting of PC12 cells and SH-SY5 cells.

45. The method of claim 41 , wherein expression of p25/CDK5 in the absence of the CDK5 inhibitor induces apoptosis in the cells and the output response is apoptosis or cell viability.

46 . The method of claim 41, wherein expression of p25/CDK5 is controlled by an inducible promoter.

47. The method of claim 41 , wherein p25/CDK5 is expressed from a viral vector.

48. The method of claim 47, wherein the viral vector is a lentiviral vector.

49. The method of claim 47, wherein the viral vector is a retroviral vector.

50. The method of claim 47, wherein the viral vector is an adenoviral vector.

51. A method of identifying an agent for treating or preventing a neurodegenerative disease, the method comprising contacting an agent to a mixture comprising Cdc2 and p25/CDK5, and detecting an effect on the activity of Cdc2 induced by p25/CDK5; thereby identifying an agent that inhibits kinase activity of Cdc2 in a neuronal cell.

52. The method of claim 51 , wherein activity of Cdc2 induced by p25/CDK5 is reduced in the presence of the agent compared to in the absence of the agent.

53. The method of claim 51 , wherein the method comprises screening a plurality of agents to identify an agent that prevents p25/CDK5 activation of Cdc2.

54. The method of claim 51 , wherein the Cdc2 and p25/CDK5 are expressed in a cell.

55. The method of claim 54, wherein the cell is a neuronal cell.

56. The method of claim 54, comprising selecting an agent that inhibits apoptosis in the cell.

57 . The method of claim 54, wherein expression of p25/CDK5 is controlled by an inducible promoter.

58. The method of claim 54, wherein p25/CDK5 is expressed from a viral vector.

59. The method of claim 58, wherein the viral vector is a lentiviral, retroviral or adenoviral vector.

60. A method of treating or preventing a neurodegenerative disease, the method comprising administering the agent identified in claim 51 to an individual in need thereof.

61. The method of claim 60, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's Disease, Amyofrophic Lateral Sclerosis (ALS), Parkinson's Disease, Niemann-Pick disease type 3, and Down's Syndrome.

62. A CDK5 polypeptide comprising an F80G mutation, wherein the polypeptide phosphorylates substrates using N⁶(Phenethyl) ATP.

63. A polynucleotide encoding a CDK5 polypeptide, the CDK5 polypeptide comprising an F80G mutation, wherein the polypeptide phosphorylates substrates using N (Phenethyl) ATP.