Methods for the detection, treatment, and prevention of neurodegeneration

Abstract

In general, the invention provides methods for identifying genes involved in neurodegeneration and therapeutics for treating animals with a neurodegenerative disease. Methods and kits for the detection of compounds which enhance neuroprotection and diagnostic kits for the detection of neurodegenerative diseases are also a part of the invention.

Description

BACKGROUND OF THE INVENTION

The invention relates to methods and reagents for diagnosing, treating, and preventing neurodegeneration.

Loss of neurons by a degenerative process is a major pathological feature of many human neurological disorders. Neuronal cell death can occur as a result of a variety of conditions including traumatic injury, ischemia, neurodegenerative diseases (e.g., Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), stroke, or trauma), or as a normal part of tissue development and maintenance. Several inherited disorders produce late onset neuron loss, each of which is highly specific for particular neural cell types. Nine genes have been cloned that are associated with susceptibility to these various neurological disorders (e.g., Huntington's disease, ataxin, and ALS); however, only in the case of Kennedy's syndrome is the biochemical function of the affected gene, the androgen receptor, understood (La Spada et al., Nature 352: 77-79, 1991). Epileptic seizures and stroke also produce neurodegeneration in humans and rodents.

SUMMARY OF THE INVENTION

In general, the invention features methods for the detection, treatment, and prevention of disorders involving neurodegeneration.

In a first aspect, the invention features a method for identifying a compound to treat or prevent the onset of a neurodegenerative disorder. The method involves contacting a cell that includes a reporter gene operably linked to a cAMP regulatory gene or promoter with a candidate compound and measuring the expression of the reporter gene, where a change in reporter gene expression in response to the candidate compound identifies a compound that is useful to treat or prevent the onset of a neurodegenerative disorder.

In various preferred embodiments of the first aspect of the invention, the cAMP regulatory gene may be an acy-1 gene, an eat-4 gene, an unc-36 gene, or a glutamate receptor-encoding gene. In another preferred embodiment, the change in reporter gene expression is a decrease in expression.

In a second aspect, the invention features a cell for identifying a compound to treat or prevent the onset of a neurodegenerative disorder that includes a reporter gene operably linked to a cAMP regulatory gene or promoter.

In various embodiments of the above aspects, the cell is present in an animal, which may be a nematode (e.g., C. elegans ) or a mammal (e.g., a rodent).

In a third aspect, the invention features a method for treating or preventing the onset of a neurodegenerative disorder in a mammal that includes administering to the mammal a therapeutically effective amount of a compound that decreases a neuronal cAMP level. In a preferred embodiment of this aspect of the invention, the mammal is a human.

In a fourth aspect, the invention features a method for identifying a mammal (for example, a human) having or likely to develop a neurodegenerative disorder which includes determining whether the mammal has an increased level of cellular cAMP in a neuron, where an increased level indicates that the mammal has or is likely to develop a neurodegenerative disorder.

In a fifth aspect, the invention features a method for identifying a mammal (for example, a human) having or likely to develop a neurodegenerative disorder which involves determining whether the mammal has a mutation in a cAMP regulatory gene. In various preferred embodiments of this aspect, the mutation is in an adenylyl cyclase gene (e.g., the acy-1 gene), or in an unc-36 or eat-4 gene. In other preferred embodiments, the mutation is in a gene encoding a Gα s subunit; and the mutation results in an increase in a neuronal cAMP level.

In a preferred embodiment of various aspects of the invention, the neurodegenerative disorder is Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer's disease, stroke, or epilepsy.

In a sixth aspect, the invention features a method for identifying a gene involved in neurodegeneration that involves providing a nematode (for example, C. elegans ) that includes an expression construct that includes a promoter derived from a cAMP regulatory gene operably linked to a reporter gene, isolating a mutant of the nematode exhibiting an altered level of reporter gene expression, and identifying the gene comprising the mutation, wherein the gene is involved in neurodegeneration.

In a seventh aspect, the invention features a method for identifying a gene involved in neurodegeneration that involves providing a nematode (for example, C. elegans ) that includes a glutamate receptor (GluR) promoter operably linked to a gene encoding a GTP-ase defective Gα s subunit, isolating a mutant of the nematode exhibiting a decreased level of paralysis and neurodegeneration, and identifying the gene that includes the mutation, wherein the gene is involved in neurodegeneration.

In an eighth aspect, the invention features a mammalian (for example, a human) EAT-4 polypeptide, and a vector and cell containing the nucleic acid.

In a final aspect, the invention provides a method for identifying a gene involved in neurodegeneration involving the steps of a) providing a cell that includes a cAMP regulatory gene promoter operably linked to a reporter gene; b) introducing into the cell a candidate gene capable of expressing a candidate protein; and c) measuring reporter gene expression in the cell, where an increase in reporter gene expression in the presence of the candidate protein indicates that the candidate gene is involved in neurodegeneration.

In preferred embodiments, the cell is yeast; and the cAMP regulatory gene is an acy-1 gene, an eat-4 gene, an unc-36 gene, or a glutamate receptor-encoding gene.

As used herein, by “protein” or “polypeptide” is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

By “neurodegenerative disorder” is meant a disorder which is characterized by the death or loss of function of neuronal cells, also known as neurons. Neuronal death or loss of function can be associated with a number of diseases and syndromes including, without limitation, stroke, epilepsy, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, and Alzheimer's disease.

By “Gα s -induced toxicity” is meant the neurodegeneration resulting from expression of the GTP-ase defective Gα s protein.

By “reporter gene” is meant any gene which encodes a product whose expression is detectable. A reporter gene product may have one of the following attributes, without restriction: fluorescence (e.g., green fluorescent protein), enzymatic activity (e.g., lacZ), toxicity (e.g., HER-1), or an ability to be specifically bound by a second molecule (e.g., biotin or a detectably labelled antibody).

By “cAMP regulatory gene” is meant any gene whose product regulates or is regulated by cAMP. Exemplary gene products include ACY-1, UNC-36, and EAT-4. Other preferred cAMP regulatory gene products include the ionotropic (cation) glutamate receptors (iGluRs), the Cl − ionotropic glutamate receptors (GluCls), and the metabotropic glutamate receptors (mGluRs).

By “operably linked” is meant that a gene and a regulatory sequence are connected in such a way as to permit expression of the gene product under the control of the regulatory sequence.

By “purified nucleic acid” is meant DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autosomally replicating plasmid or virus; or into the genomic DNA or a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

By a “transgene” is meant a nucleic acid sequence which is inserted by artifice into a cell and becomes a part of the genome of that cell and its progeny. Such a transgene may be partly or entirely heterologous to the cell.

By “mammalian eat-4 polypeptide or mammalian EAT-4” is meant an amino acid sequence derived from a mammalian cell which shares at least 50%, preferably 70%, more preferably 80%, and most preferably 90% amino acid sequence identity with a C. elegans eat-4 amino acid sequence (SEQ ID NO: 1). Preferably, such a polypeptide is capable of at least partially complementing a C. elegans eat-4 mutation.

By “acy-1 polypeptide or ACY-1” is meant an amino acid sequence which is substantially identical to the amino acid sequence provided in FIG. 5 (SEQ ID NO: 2).

By “substantially identical” is meant an amino acid sequence or nucleic acid sequence which shares identity with another of the same class. Preferably, such a sequence is at least 85%, more preferably 90%, and most preferably 95% identical to the sequence described in the references provided herein. For polypeptides, the length of comparison sequences will generally be at least 15 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably at least 35 amino acids. For nucleic acids, the length of comparison sequences will be at least 45 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably at least 105 nucleotides. Identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches similar sequences by assigning degrees of identity to various substitutions, deletions, substitutions, and other modifications.

Other features and advantages of the invention will be apparent from the following detailed description thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are photographs of neuronal cells from young Caenorhabditis elegans larvae co-expressing green fluorescent protein (GFP) with GTP-ase defective rat Gα s as seen morphologically (FIG. 1 A), as well as in bright field optics (FIG. 1 B).

FIG. 2 is a table listing the extent of swelling and cytotoxicity of PVC neurons resulting from the expression of the α s (gf) transgene in various genetic backgrounds. Statistical differences between genotypes were determined by the method of attributable risk described in J. Devore, Probability and statistics for engineering and the sciences (Brooks/Cole, Belmont, ed. second, 1987). Multiple comparisons were compensated for by setting p<0.005 as the threshold for significance.

FIG. 3 is an amino acid sequence of the EAT-4 protein (SEQ ID NO: 1).

FIG. 4 is a schematic diagram showing the genetic and physical map position of the acy-1 gene on the F17C8 cosmid.

FIG. 5 is a set of schematic diagrams of the predicted structures of the acy-1 gene and the GFP fusion protein (KP#107). Positions of the acy-1 mutations nu327, nu343, and nu329 are indicated.

FIG. 6 is an amino acid sequence of the ACY-1 protein (SEQ ID NO: 2). The ACY-1 sequence (top) is shown aligned with the mouse adenylyl cyclase type 9 amino acid sequence (bottom) SEQ ID NO: 3) . Underlined sequences indicate predicted transmembrane domains. Positions of the acy-1mutations nu327, nu343, and nu329 are indicated.

FIGS. 7A and 7B are photographs of GFP-expressing PVC neurons in adult α s (gf) ( FIG. 7A ) and adult α s (gf);acy-1(nu343) ( FIG. 7B ) C. elegans.

FIGS. 8A and 8B are photographs illustrating KP#107 acy-1::gfp fusion gene expression in neurons ( FIG. 8A ) and muscle (FIG. 8 B).

FIGS. 9A and 9B are photographs of PVC neurons from unc-18 L1 larvae as seen with bright field ( FIG. 9A ) and fluorescence ( FIG. 9B ) optics.

FIGS. 10A and 10B are photographs of PVC neurons from unc-18 adults as seen with bright field ( FIG. 10A ) and fluorescence ( FIG. 10B ) optics.

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein is based upon genetic studies of the nematode, Caenorhabditis elegans. Constitutive activation of the GTP-binding protein Gα s was found to induce neurodegeneration. A screen for mutations that blocked Gα s -induced killing identified a gene, acy-1, which encodes a protein that is highly similar (40% identical) to mammalian adenylyl cyclases, indicating that Gα s -induced neurotoxicity is likely mediated by changes in cyclic adenosine monophosphate (cAMP) levels. This discovery enables methods and reagents for diagnosing and treating neurodegeneration.

Gα s -induced neurotoxicity

Although neurodegeneration is a major feature in a variety of human neurological disorders, relatively little is known about the signal transduction pathways that lead to neurotoxicity, nor how these pathways could be manipulated to protect against neuron loss in these diseases. Two critical questions in the pathogenesis of human neurodegenerative disorders are (1) what factors predispose particular neurons to undergo degeneration and (2) what is the biochemical mechanism of degeneration. A genetic model for excitotoxicity in the nematode Caenorhabditis elegans was developed to address these questions.

In particular, a rat cDNA encoding a GTPase-defective (Q227L) Gα s subunit, hereafter referred to as α s (gf), was expressed in C. elegans neurons using the glr-1 glutamate receptor (GluR) promoter. The expression vector, KP#20, was constructed by inserting into a derivative of the C. elegans glr-1 expression vector CX#1 (as described in Chalfie et al., Science 263: 802-805, 1994), a 1.5 kb NcoI-XhoI fragment encoding a GTPase defective (Q227L, KP#20) mutant rat Gα s cDNA. C. elegans transgenic for α s (gf) were prepared by microinjecting the KP#20 expression construct together with a glr-1::gfp plasmid (the KP#6 vector) using lin-15 (Huang et al., Mol. Biol. Cell. 5, 395-412, 1994) as a transformation marker. A stable line carrying glr-1 expression constructs for both GFP and the GTPase defective Gα s (nuIs5) was isolated following 3500 rads of γ-irradiation. The glr-1 promoter was chosen because it is highly expressed, and because glr-1-expressing cells control locomotion, an easily assayed behavior. The glr-1 promoter is expressed in 17 classes of neurons, including the interneurons (AVB, PVD, AVA, and AVD) required for locomotion. The glr-1 expressing neurons are as follows: AVG, AVJ, DVC, PVC, PVQ, RIG, RIS, RMD, RMEL/R, SMD, URY, as well as the six ASH synaptic targets AIB, AVA, AVB, AVD, AVE, and RIM (Hart et al., Nature 378: 82-85, 1995; Maricq et al., Nature 378: 78-81, 1995).

Since Gα s was co-expressed with the green fluorescent protein (GFP) of Aequorea (Chalfie et al., supra), examination of the morphology of Gα s -expressing cells was possible. Transgenic glr-1::α s (gf) animals were found to be paralyzed. As shown in FIGS. 1A and 1B , a subset of the Gα s -expressing neurons in young larvae swelled to several times their normal diameter. The swelling was apparent by the morphology of GFP expressing cells ( FIG. 1A ) and by their appearance in bright field optics ( FIG. 1B ) as enlarged, apparently vacuolated cells often with an intact nucleus. The interneurons AVE and AVD were swollen compared to neighboring unaffected cells which have been marked in FIGS. 1A and 1B with asterisks. 88% of the PVC neurons swelled, 5% of RIG neurons swelled, and none of the URY cells swelled in first stage (LI) glr-1::α s (gf) larvae. The neurotoxicity occurred in two phases; subsequent to swelling, the swollen cells eventually disappeared, presumably because the cells had died. In glr-1::α s (gf) animals, 89% of the PVC neurons degenerated, as summarized in the table in FIG. 2 . Other glr-1 expressing cells degenerated at lower frequencies, including AVA, AVD, AVE, AVG, PVQ, RIG, and SMD. Expression of a constitutively active rat Gα s cDNA was found to cause neurotoxicity in C. elegans. Characterization of the neurodegenerative phenotype in the resulting glr-1::α s (gf) was made as follows: Swollen or missing cells were identified by examining the morphology of GFP-expressing cells. Gα s -induced neurotoxicity in various genetic backgrounds was quantitated as the number of swollen PVC neurons in L1 larvae, and the percentage of PVC neurons that were missing or swollen in adults hermaphrodites. These results suggested that exaggerated Gα s signaling killed neurons.

The phenotype of Gα s -induced neurotoxicity was identical to the neurotoxicity due to excessive signaling by the excitatory neurotransmitter glutamate, which has been termed excitotoxicity. Excitotoxic neuron loss occurs in two phases. First, acute neuron loss is associated with swelling of cell bodies and is dependent on extracellular ionic conditions. Cell swelling is the consequence of depolarization of membrane potential by excitotoxic agonists, which leads to the influx of Na + and Cl 31 ions, and water (Olney, Adv. Exp. Med. Biol. 203: 631-645, 1986; Choi, J. Neurosci. 7: 369-379, 1987; Choi, Neuron 1: 623-634, 1988). Second, delayed neuron loss in excitotoxicity is not dependent on the extracellular ionic conditions, but is correlated with elevations of intracellular Ca 2+ and chronic activation of immediate early genes (e.g., fos and jun) (Smeyne et al., Nature 363: 166-169, 1993. Hence, Gα s -induced neurotoxicity is most likely excitotoxicity.

Neurons Differed Greatly in Their Susceptibility to Gα s -induced Toxicity

The mec-7 gene product, MEC-7 tubulin, is abundantly expressed in 5 neurons, called touch cells, that sense light touch to the worm's body (Savage et al., Genes Dev. 3: 870-81, 1989; Hamelin et al., EMBO 11: 2885-2893, 1992; Mitani et al., Development 119: 773-783, 1993). To further investigate the specificity of Gα s -induced toxicity, α s (gf) was expressed in C. elegans utilizing the mec-7 promoter. The mec-7::α s (gf) expression plasmid (KP#7) was constructed by ligating the 1.5 kb NcoI-XhoI Gα s (Q227L) into the mec-7 expression vector pPD52.102. C. elegans transgenic for the mec-7::α s (gf) expression plasmid were prepared by microinjecting the KP#7 expression construct together with a mec7::gfp plasmid using lin-15 (Huang et al., supra) as a transformation marker. A stable line carrying mec-7 expression constructs for both GFP and the GTPase defective Gα s (nuls5) was isolated following γ-irradiation.

C. elegans expressing the mec-7::α s (gf) transgene were found to be indistinguishable from wild type animals, having no obvious defect in touch sensitivity nor in the morphology of the touch cells. Hence, the effects of Gα s on neural activity and on neurotoxicity were cell type specific.

Mutations that Blocked Gα s -induced Neurotoxicity

Both the glr-1 and the mec-7 expression constructs supported the notion that the effects of Gα s on neural activity and on neurotoxicity were cell type specific. Since the mec-7 promoter is very highly expressed in the touch neurons (Savage et al., supra; Hamelin et al., supra; Mitani et al., supra), the results also suggested that the cell type specificity could not be overcome by high levels of Gα s expression. To identify the targets of Gα s , mutations that block Gα s -induced paralysis and neurotoxicity were isolated by identifying mutations isolated from the F2 self-progeny of EMS mutagenized (5 μ1/ml) hermaphrodites that restored normal locomotion rates to α s (gf) homozygotes. Candidate suppressor mutants (7500 hapliod genomes) were subsequently screened for reduction of Gα s -induced swelling in L1 larvae which led to the isolation of 3 semidominant mutations which blocked Gα s -induced paralysis and neurotoxicity

Mutations in acy-1 Blocked Gα s -induced Neurotoxicity

In two factor mapping experiments, the three mutations that blocked Gα s -induced neurotoxicity were all found to be linked to dpy-17. Three factor mapping placed these mutations between emb-5 and dpy-17: (nu327 dpy-7) 37/37 unc-32; (nu329 dpy-17) 16/16 unc-32; (nu343 dpy-17) 4/4 unc-32; unc-79 (6/14) MJ#NEC2 (5/14) nu329 (3/14) dpy-17; emb-5 (1/16) nu327 (15/16) dpy-17. As illustrated in the schematic diagram of FIG. 4 , two of the three mutations were mapped to a 1.5 cM genetic interval between MJ#NEC2 and dpy-17 on the F17C8 cosmid. The cosmid was then microinjected into acy-1(nu327); nuIs5 animals, and transgenic lines were isolated using goa-1::gfp (KP#13) (Segalat, et al., Science 267, 1648-1651, 1995) as a transformation marker. Four independent lines carrying a cosmid from this interval (F17C8) were obtained, two of which corrected the mutant phenotype of acy-1(nu327) animals, i.e., they had increased degeneration of the PVC neurons. This is shown on Table 1.

TABLE 1
Transgenes containing the F17C8 cosmid rescue the acy-1(nu327) mutant
phenotype
genotype                         % PVC degeneration
acy-1(nu327);α                                                                        s                                                                    (gf) 12
acy-1(nu327);α                                                                        s                                                                    (gf); nuEX(F17C8) 75
α                                                                        s                                                                    (gf) 88

In addition, FIG. 5 shows that all three alleles corresponded to mutations in the predicted exons of the gene F17C8.1, one of two predicted adenylyl cyclase genes in the C. elegans genome database. This adenylyl cyclase gene has been named acy-1. Furthermore, FIG. 6 shows the results of a Genbank database scan for sequences related to acy-1 (SEQ ID NO: 2). The amino acid sequence of ACY-1 was found to be 40% identical at the amino acid level to mouse adenylyl cylase type 9. It is unclear why the acy-1 mutations were partially dominant. Analysis of the molecular nature of the mutations suggested that they were simple loss of function mutations. For example, nu329 and nu343 were predicted to disrupt pre-mRNA splicing. Indeed, as is shown in FIGS. 7A and 7B , the GFP-expressing PVC neurons which were typically missing in α s (gf) adult transgenic worms ( FIG. 7A ) were present in α s (gf);acy-1(nu343) (FIG. 7 B). Thus, it is possible that α s (gf) animals were highly sensitive to changes in cAMP levels. Overall, the results suggested that Gα s neurotoxicity was mediated by changes in intracellular cAMP.

Physiological Function of ACY-1

To determine the physiological function of ACY-1, an analysis of acy-1 expression was carried out. A deleted derivative (KP#106) of the cosmid F17C8 was isolated by digesting with AfiII and re-ligating. KP#106 contained the entire 8.35 kb acy-1 genomic region together with the 5.2 kb 5′ and 4.9 kb 3′ flanking sequences. An acy-1::gfp expression vector (KP#107) was constructed by PCR amplification of a 1.7 kb fragment containing the GFP coding region and the unc-54 transcription terminator from pPD95.75, followed by ligation of this fragment into the unique Asp718 site in KP#106, creating a fusion protein containing the first 6 exons of acy-1 fused to GFP. The ACY-1::GFP fusion protein contained 6 predicted transmembrane domains of ACY-1, and was therefore membrane localized. Transgenic animals carrying KP#107 were isolated by microinjection using lin-15 (Huang et al., supra) as a transformation marker. Expressing cells were identified based on their morphology and nuclear positions.

The expression pattern of acy-1 was determined by analyzing the GFP reporter construct. As is shown in FIGS. 8A and 8B , the acy-1::gfp fusion protein was expressed in virtually all neurons ( FIG. 8A ) and body muscles (FIG. 8 B). In FIG. 8A , ACY-1 expression in the two ventral rows of body muscles (arrows) and in the ventral cord neurons and neuropile (lines) is shown. In FIG. 8B , expression of ACY-1 in the vulva muscles (arrow heads) is shown. Nearly all of the 302 neurons in adult C. elegans appeared to express ACY-1. Cell bodies were identified based upon the bright fluorescence in the intracellular membranes (which are presumably the endoplasmic reticulum of Golgi apparatus). ACY-1 did not appear to be expressed in non-neural tissues or in the pharynx. These results indicated that the ACY-1 adenylyl cyclase is likely to participate in many neural signaling pathways. Therefore, we expected that acy-1 mutants would have defects in behavior or development. Consistent with this notion is that mutations which inactivated the C. elegans Gα s subunit (GSA-1) were found to be homozygous lethal. Surprisingly, we observed that acy-1 homozygotes were nearly indistinguishable from wild type animals. This result suggested that the essential function of GSA-1 was mediated by some other adenylyl cyclase. Alternatively, acy-1 and other adenylyl cyclases could act redundantly in the essential GSA-1 pathways.

Activated Gα s Induced Neurotoxicity by Excitotoxicity

Several previously identified genes were considered good candidates for mediating the toxic effects of Gα s . Two cyclic nucleotide gated ion channel (CNGC) subunit genes tax-2 and tax-4 (Coburn and Bargmann, Neuron 17: 695-706, 1996; Komatsu et al., Neuron 17: 707-718, 1996) are not expressed in glr-1 expressing cells and hence are unlikely targets. The mec-6, unc-8, and deg-1 genes have been previously implicated in neurodegeneration (Chalfie and Wolinksy, Nature 345: 410-416 (1990); Driscoll and Chalfie, Nature 349: 588-593 ,1991; Shreffler et al., Genetics 139: 1261-1272, 1995; Tavernarakis et al., Neuron 18: 107-119, 1997), and the DEG-1 and UNC-8 proteins are similar to mammalian epithelial sodium channel subunits (ENaC), which are potently activated by cAMP-dependent protein kinase (PKA) (Sariban-Sohraby et al., J. Biol. Chem. 263: 13875-13879, 1988; Oh et al., Am. J. Physiol. 265: C85-C91, 1993; Bubien et al., J. Biol. Chem. 269: 17780-17783, 1994). The unc-2, unc-36, and egl-19 genes encode subunits of voltage-dependent Ca 2+ -channels (Schafer and Kenyon, Nature 375: 73-78, 1995) which are likely to be regulated by PKA (Curtis and Catterall, Proc. Nati. Acad. Sci. USA 82: 2528-2532, 1985) and have also been implicated in neurodegeneration. The glr-1 gene encodes an ionotropic GluR (Hart et al., supra; Maricq et al., supra). GluRs have been implicated in neurotoxicity in mammals (Olney, Adv. Exp. Med. Biol. 203, 631-645, 1986; Choi, J., Neurosci. 7: 369-379, 1987; and Choi, Neuron 1, 623-634, 1988), and PKA augments the response of mammalian neurons to glutamatergic agonists (Greengard et al., Science 253: 1135-1138, 1991).

To examine the above genes for a possible role in Gα s induced toxicity, the neurodegenerative phenotype was characterized as described above. As shown in FIG. 2 , of the candidate genes, only the unc-36 mutation significantly reduced Gα s -induced cytotoxicity. Interestingly, the unc-36 mutation had no effect on cell swelling. Since UNC-36 Ca 2+ channels were required for cytotoxicity, these results suggested that Gα s cytotoxicity was mediated in part by either Ca 2+ influx or depolarization of the affected cells. All other candidate genes had no effect on either neuron swelling or deaths in glr-1::α s (gf) animals. Our results do not exclude the possibility that these other candidate PKA targets also play a role in Gα s -induced toxicity. For example, more than one type of channel may be capable of mediating the toxic effects of Gα s in which case neurotoxicity would be prevented only in multiply mutant animals.

The glr-1 Mutation was Unlikely to Completely Abolish Glutamate Signaling in vivo

Given its role in excitotoxicity in mammals, the requirement of endogenous glutamate signaling for Gα s neurotoxicity was tested. Although the glr-1 mutation was not neuroprotective, it was possible that cAMP toxicity was mediated by exaggerated responses to endogenous glutamate. The C. elegans genome sequence (currently ˜70% complete) predicted six additional ionotropic GluR subunits; therefore, the glr-1 mutation was unlikely to completely abolish glutamate signaling in vivo.

Eat-4 Mutant Alleles Eliminated ASH-mediated Touch Sensitivity

Prior work had shown that ASH sensory neurons mediated an aversive response to three distinct stimuli (nose touch, osmotic shock, and volatile repellents), and that the ASH-mediated touch response required functional GLR-1 glutamate receptors in synaptic targets of ASH (Hart et al., supra; Maricq et al., supra; Kaplan and Horovitz, Proc. Natl. Acad. Sci. (USA) 90, 2227-2231, 1993; Troemel et al., Cell 83, 207-218,1995). Hence, genes required for ASH sensory responses were tested for their ability to perturb glutamate signaling.

We screened 11,000 mutagenized haploid genomes for animals that failed to respond to nose touch. Mutants isolated were subjected to a series of secondary screens, including dye-filling of the amphid sensory neurons, and responsiveness to osmotic shock and volatile repellents. Seven alleles of eat-4 were isolated in this screen, all of which were normal for dye-filling but were defective for all three ASH sensory behaviors. The amino acid sequence of the EAT-4 is shown on FIG. 3 . ASH-mediated sensory responses to nose touch, osmotic shock, and volatile repellents were compared in wild type and eat-4, as has been previously described (Hart et al., supra; Maricq et al, supra; Kaplan and Horovitz, supra; Troemel et al., supra). Briefly, for nose touch, animals were tested 10 times each with a positive response being scored when animals either halted forward movement or initiated backward movement following the stimulus. For osmotic avoidance, 50-60 animals were placed in 1 cm rings formed with 8 M glycerol, and the number of animals that escaped the ring after 9 minutes were counted. For volatile avoidance, an eyelash was dipped in 1-octanol and held near an animal's nose, and responses were quantitated by recording the length of time that elapsed before the animals reversed locomotion.

All seven eat-4 strains isolated had similar behavioral defects. In particular, as is portrayed in Table 2, eat-4 strains had severe defects in the ASH-mediated touch, osmosensory, and volatile repellent responses.

TABLE 2
Role of eat-4 in ASH sensory responses
			Osmotic	Volatile
		Nose Touch	Avoidance	Avoidance
	Genotype:	(% Respond)	(% Escape)	(seconds)
	wild type	86 +/− 3	2 +/− 1	2.9 +/− 0.9
	eat-4(ky5)	1 +/− 1	75 +/− 6	9.9 +/− 1.6
	eat-4(n2474)	2 +/− 1	54 +/− 6	9.6 +/− 1.5

Errors indicate standard error of the mean in all cases. The number of animals and trials for each genotype were as follows: for nose touch, 10 animals and 100 trials; for osmotic avoidance, 60 animals and 5 trials; and for volatile avoidance, 25 animals and 25 trials.

Eat-4 Mutations Reduced Gα s -induced Cytotoxicity but Not Cell Swelling

The eat-4 gene was initially identified in screens for mutations that disrupted eating behavior (Avery, Genetics 133: 897-917, 1993). The eat-4 eating defect was caused by elimination of a glutamate-induced inhibitory synaptic signal (mediated by the M3 motor neuron), which could be observed in extracellular recordings of pharyngeal muscle activity (Raizen et al., Neuron 12, 483-495, 1994). Given the results described herein, the eating defects and the ASH sensory defects could both be explained by an underlying defect in glutamate signaling.

To investigate this possibility, neurodegenerative phenotypes were examined as described above. In these experiments, eat-4 mutations were found to be neuroprotective. The mutations significantly reduced Gα s -induced cytotoxicity but had no apparent effect on cell swelling, as indicated in FIG. 2 . In addition to reducing cytotoxicity, the eat-4 mutations also dramatically improved the locomotion rate of α s (gf) animals. These results suggested that Gα s neurotoxicity was at least partially mediated by endogenous glutamate signaling.

Apoptosis Was Not Required for Gα s Neurotoxicity

Apoptosis is a naturally occurring process thought to play a critical role in the developing animal and is characterized morphologically by condensation of the chromatin followed by shrinkage of the cell body. Biochemically, DNA laddering, the degradation of nuclear DNA into oligonucleosomal fragments, is the hallmark of apoptosis. DNA laddering precedes cell death. Apoptosis is most likely dependent upon the activation of a cell death pathway. The best defined genetic pathway of cell death is in C. elegans where both effector (ced-3 and ced-4) and repressor (ced-9) genes have been isolated. Similar genes have been identified in mammals. Whether excitotoxic death occurs by apoptosis or by necrosis has remained controversial. This uncertainty is primarily due to lack of genetic control of the apoptosis pathway in the previously described models for excitotoxicity.

In our experiments, we found that a mutation in the ced-3 gene, which encodes an ICE protease and is required for apoptosis (Ellis and Horovitz, Cell 44: 817-829, 1986; Yuan et al., Cell 75: 641-652, 1993), had no effect on Gα s -induced swelling or killing (see FIG. 2 ). Thus, apoptosis was not required for Gα s -induced killing. However, in α s (gs);unc-18 double mutants, a significant fraction of PVC neurons had a highly condensed morphology, and these PVC neuron corpses appeared to be engulfed by surrounding hypodermal cells, both of which are characteristic of apoptotic deaths (Ellis et al., Ann. Rev. Cell Biol. 7: 663-698, 1991). As shown in FIGS. 9A and 9B , in unc-18 L1 larvae, 13% of the PVC neurons exhibit the condensed morphologies characteristic of programmed cell deaths, which was apparent in both bright field ( FIG. 9A ) and fluorescence ( FIG. 9B ) optics. In unc-18 adults, 25% of the PVC neurons exhibited condensed morphologies and appeared to have been engulfed by surrounding hypodermal cells in the tail, as shown in FIG. 10A (bright field optics) and FIG. 10B (fluorescence optics). (Note that the position of the indicated cell body in FIG. 10B is much further posterior than in FIG. 7 B). Gα s neurotoxicity was concluded to be, in part, mediated by synaptic input to the dying cells, since unc-18 mutations impair synaptic vesicle exocytosis (Gengyo-Ando, et al., Neuron 11: 703-711, 1993; Hata et al., Nature 366: 347-351, 1993). Furthermore, these results suggest that Gα s neurotoxicity occurs via two independent mechanisms. Synaptic input promotes an excitotoxic pattern of cell deaths; however, when synaptic input is impaired an apoptotic pattern emerges.

Screens for Compounds that Inhibit cAMP-based Neurodegeneration

As described herein, constitutive activation of the GTP-binding protein Gα s induces a neurodegeneration phenotype that shares several properties with excitotoxic neuron loss in mammals. First, neuron loss occurs in two phases, whereby affected cells undergo a swelling response in young larvae, and subsequently die sometime during larval development. Second, neurons differ greatly in their susceptibility to Gα s -induced toxicity, ranging from 0-88% of cells affected. Third, a mutation that impairs the function of voltage-dependent calcium channels and one that reduces glutamate neurotransmission are neuroprotective.

The acy-1 gene was identified in a screen for mutations that blocked G s -induced killing and has been positionally cloned. The predicted ACY-1 protein (SEQ ID NO: 2) is highly similar (40% identical) to a mammalian adenylyl cyclase. Most consistent with this result is that Gα s -induced neurotoxicity is mediated by changes in cyclic adenosine monophosphate (cAMP) levels. Mutations that prevent programmed cell death, also known as apoptosis, do not prevent Gα s -induced neurotoxicity; however, when synaptic transmission was impaired (by an unc-18 mutation), a subset of the deaths appear to become apoptotic. These experiments suggested that excitotoxicity normally occurs by both apoptosis and a second cytotoxic pathway. Given these results, screens for compounds that inhibit cAMP signaling may be carried out to identify drugs that alter cAMP-based neurodegeneration and to provide therapies to ameliorate these disorders in humans and other mammals. These assays may be carried out in vivo or in vitro, and a number of exemplary assays now follow.

a) C. elegans assays

The microscopic nematode, C. elegans, is a useful model for studying neurodegeneration because it allows researchers to observe changes in neuronal cells within the living organisms over the three days required for a C. elegans to develop from a single cell zygote to a mature adult. The biology of the C. elegans nervous system, which includes 302 neurons, has been well documented. Furthermore, there are several similarities between the C. elegans and human nervous systems. For example, many of the C. elegans neurotransmitters are the same as human neurotransmitters. In addition, many C. elegans genes used both inside and outside of the nervous system have counterparts in mammals.

To identify a compound capable of inhibiting cAMP-based neurotoxicity, candidate compounds are screened for an ability to alter cAMP levels using a C. elegans strain carrying a reporter transgene operably linked to a promoter of a gene that is either (i) regulated by cAMP or (ii) involved in cAMP regulation. Exemplary promoters include the acy-1, unc-36, and eat-4 promoters. Other desirable promoters include any promoter from a nematode glutamate receptor (GluR) gene; such genes are listed, for example, in Table 3.

TABLE 3
C. elegans glutamate signalling genes
	Gene Product	Genetic locus
	iGluRs:	C06E1.4	glr-1
		CO6A8.8
		BO280.12
		F41B4.4
		C43H6
		K10D3.1
		ZC196.c
	GluCls:	GluCLα1
		GluCLβ1
		ZC317.3
		T10G3
	mGluRs:	ZC506.4	mgr-1
		F45H11

Once constructed, a transgenic C. elegans strain carrying such a reporter gene is treated with a candidate compound, or any number of compounds in combination, and animals are screened for alterations in cAMP levels as reflected by alterations in the levels of reporter gene expression.

Useful reporter genes are those whose expression is detectable, preferably, using simple and rapid techniques. Preferable reporter genes include, without limitation, green fluorescent protein (gfp), spectrally shifted green fluorescent proteins (Rizzuto et al., Curr. Biol. 6:183-188, 1996; Heim and Tsien, Curr. Biol. 6:178-182, 1996); lacZ, her1 (Perry et al., Gen. and Dev. 7(2): 216-228, 1993), and mec4 (dominant) (Maricq et al., supra). Expression levels of these reporter genes may be directly measured by a variety of techniques known in the art. For example, if the reporter protein is a toxin (e.g., MEC4), the expression level may be detected by measuring or observing cell viability. The expression level of a reporter protein with enzymatic activity (e.g., lacZ) may be quantitated using colorimetric substrates (e.g., 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal)). And reporter gene products such as GFP may be screened directly by visual inspection.

If desired, reporter proteins may be fusion proteins that incorporate portions of the sequences involved in cAMP regulation, for example, the ACY-1, UNC-36, or EAT-4 sequences. These fusion proteins are generated using nucleotide sequences and methods known in the art and described herein.

In one particular embodiment, such compound screens are carried out using rapid, high through-put assays. For example, transgenic C. elegans animals carrying acy-1::gfp reporter constructs are utilized. The animals are distributed into 96-well microtiter dishes such that there is one animal per well. Candidate compounds are then individually or combinatorially added to the wells and assessed for an ability to reduce GFP expression as a means to test for an ability to inhibit cAMP-based neurodegeneration. GFP assays may be carried out by any means, but are preferably monitored using a microtiter plate fluorescence reader.

In an alternative compound screen, the reporter protein need not be GFP. For example, the transgenic animal may carry a lacZ reporter gene and be distributed into microtiter wells as described above. Following compound administration, transgenic animals are subjected to standard β-galactosidase activity assays described in the art (see, for example, Ausubel et al., supra). The Promega β-gactosidase enzyme assay system with reporter gene lysis buffer kit (Catalog # E2000) may be employed in this rapid high throughput 96 well assay system. By this method, reporter lysis buffer is added to each well. The C. elegans extracts are then incubated with the buffer and the o-nitrophenyl-β-D-glactopyranoside (OPTG) substrate provided in the kit. Optical density of the plate is then measured on a microtiter plate reader. Again, a reduced level of lacZ activity in a compound-treated well as compared to an untreated well indicates that the compound has an ability to inhibit cAMP-based neurodegeneration.

In addition, a variety of methods may be used in combination to screen for compounds capable of inhibiting cAMP-based neurodegeneration. For example, a C. elegans carrying two different expression constructs (e.g., the acy-1 promoter operably linked to gfp and the glr-1 promoter operably linked to lacZ) may be used to screen for a compound capable of inhibiting cAMP-based neurodegeneration by assaying for a reduction in the expression of both the acy-1 and eat-4 genes. In this assay, preferred compounds are capable of reducing the expression levels of both GFP and lacZ. However, a decrease in expression of one reporter gene (e.g., gfp), but not the other reporter gene (e.g., lacZ) identifies compounds capable of targeting particular components in a neurodegenerative pathway (in this case, the acy-1 gene). Such compounds may be useful for treating particular types of neurodegenerative disorders.

In addition, nematode screens for compounds capable of inhibiting cAMP-based neurodegeneration may be based upon both neuroprotection and reporter gene expression. By this approach, for example, a transgenic glr-1::α s (gf) C. elegans is transformed with a second worm marker (e.g., the acy-1::gfp expression vector). Compound-treated glr-1::α s (gf); acy-1::gfp double transgenic animals are then screened, for example, for improved locomotion (i.e., a compound affecting the glr-1 gene), reduction of GFP expression (i.e., a compound affecting the acy-1 gene), or both (i.e., a compound affecting both the glr-1 and acy-1 genes) as compared to untreated glr-1::α s (gf); acy-1::gfp double trangenic animals. Again, gene-specific compounds may be useful for treating neurodegenerative disorders involving specific genes.

In yet another approach, compounds which affect neurodegenerative signals generated by a mammalian glutamate receptor may also be employed in a C. elegans screen. A large number of mammalian glutamate receptors (GluRs) have been previously described, and a comprehensive list of these proteins may be found in Hollmann and Heinemann (Ann. Rev. Neurosci. 17: 31-108, 1994). To carry out such a screen, the coding regions of one or more of these genes are inserted into a C. elegans expression vector such that the expression of the gene product is directed by the glr-1 gene promoter. This construct is microinjected into a glr::α s (gf) transgenic C. elegans. Since only a subset of the glr-1 expressing neurons die in such animals, any additional cell death (as measured, for example, by increased paralysis, neuronal swelling, or neurodegeneration) may be attributed to mammalian GluR expression. Candidate compounds are then administered to these animals, and differences observed in compound-treated animals versus untreated animals are used to identify a compound having an ability to affect mammalian GluR signalling. Again, compounds identified by this assay are useful for treating neurodegenerative disorders in a mammal.

b) Mammalian Cell Assays

Mammalian cells carrying a reporter gene operably linked to the promoter of a gene either regulated by cAMP or involved in cAMP regulation, for example, the mammalian homologues of the acy-1, unc-36, or eat-4 genes, may also be used to screen for compounds that inhibit cAMP-based neurodegeneration. In one particular example, the promoter of the murine adenylyl cyclase type 9 encoding gene may be used to direct the expression of a reporter (e.g., GFP) in a mammalian expression vector. This vector is transfected into a mammalian cell by any of a number of different transfection methods well known in the art (e.g., electroporation, CaPO 4 precipitation, or DEAE-Dextran). Preferably, the mammalian cell is a mouse neuronal cell line, for example, a PC12 cell line. Candidate compounds are added to the culture medium of the transfected cells, and the level of expression of the reporter gene is measured and compared to a control, untreated cell line. A reduced level of reporter gene expression in a compound-treated cell line indicates that the compound has an ability to inhabit cAMP-based neurodegeneration in mammalian cells.

In addition, such a mammalian cell line may be transfected with more than one reporter gene operably linked to more than one cAMP regulatory gene promoter. For example, a mammalian cell transfected with a gfp-adenylyl cyclase type 9 construct may be doubly transfected with a construct comprising a mammalian unc-36 promoter operably linked to a second reporter (e.g., luciferase). Following addition of candidate compounds to the culture medium of doubly transfected cells, GFP expression is analyzed, for example, by flow cytometric analysis of half of the compound-treated cell population, and the remaining half is assayed for luciferase activity using known methods (e.g., the luciferase assay kit commercially available from Promega). By comparing GFP and luciferase expression levels to those in untreated cells, a compound capable of altering cAMP-based neurodegeneration is identified. Furthermore, a compound capable of affecting, for example, the murine adenylyl cyclase type 9 gene but not the mammalian unc-36 gene may also be isolated. Such a compound may be useful for treating specific types of neurodegenerative disorders in mammals.

Alternatively, mammalian cells which endogenously express homologues of C. elegans genes involved in cAMP regulation or regulated by cAMP may be used to identify compounds capable of altering cAMP-induced neurodegeneration. According to this method, following administration of a candidate compound, endogenous gene expression is measured by any of a variety of nucleic acid or immunological based assays including, without limitation, Northern blot, Western blot, and ELISA analyses. Compounds affecting endogenous gene expression levels as compared to untreated cells are useful for treating cAMP-based neurodegeneration.

c) Animal Models

A number of animal models exist for the study of neurodegenerative disorders and find use in the screening methods described herein. For example, such models may serve as a system in which to screen candidate compounds being tested de novo for an ability to alter cAMP-based neurodegeneration or as a secondary screen for testing compounds isolated in a C. elegans, yeast, or mammalian cell culture assay (for example, those assays described herein). Candidate compounds may be administered to animals prior to neurological damage to assay for an ability to prevent cAMP-based neurodegeneration. Alternatively, candidate compounds may be assessed for an ability to treat cAMP-based neurodegeneration following neurological insult. Animal models may also serve to determine the dosage requirement for an effective compound.

Particularly useful animal models include, without limitation, Parkinson's disease (PD) rat models, which are established by injecting the catecholamine-specific neurotoxin, 6-hydroxydopamine (6-OHDA), into the medial forebrain bundle or the substantia nigra pars compact to achieve a rapid degeneration of the nigrostriatal pathway, or into the striatum to achieve progressive degeneration, as has been described (see, for example Gerlach and Riederer, J. Neural. Transm. 103 (8-9): 987-1041, 1996; Bernard et al., J. Comp. Neurol. 368 (4): 553-568, 1996; Asada et al., Ex. Neurol. 139 (2): 173-187, 1996). Alternatively, rats may be rendered “epileptic” (i.e., induced to suffer brain seizures which often result in neuronal cell death) by administration of a variety of compounds including, for example, intravenous injection of bicuculline (Blennow et al., J. Cereb. Blood Flow Metab. 5: 439-445, 1995) or daily application of low intensity electrical stimulation. Finally, neuronal cell death which often results from stroke-induced ischemia may be mimicked by the 4-vessel occlusion experimental model described by Pulsinelli et al. (Ann. Neurol. 11: 491-498, 1982) and Francis and Pulsinelli (Brain Res. 243: 271-278, 1982).

d) Candidate inhibitors of cAMP-based neurodegeneration

A number of compounds have been shown to affect cAMP levels, and these provide good candidates for inhibitors of neurodegeneration. Such compounds are commercially available (e.g., from Research Biochemicals International) and include, without limitation, agonists of receptors that couple to Gi and inhibit adenylyl cyclases. Alpha 2 adrenergic receptor agonists (including B-HT 920 diHCl and Xylazine HCl), opioid delta receptor agonists (including [D-Ala2, D-Leu5]-enkephalin and [D-Pen2,5]-enkephalin), and D2 dopaminc receptor agonists (including bromocriptine methane sulfonate and Quinelorane 2HCl) all inhibit adenylyl cylcases and may be assessed in screens described herein for an ability to inhibit cAMP-based neurodegeneration.

Therapeutics for Treating Human Neurodegenerative Disorders

A number of human neurological disorders are characterized by a loss of neurons through a degenerative process. Compounds isolated as described above based on their effect on cAMP levels are useful in treating these disorders. In addition, drugs known to lower cAMP levels are also useful therapeutics for treating, preventing, or slowing neurodegeneration. In particular, disorders that may be treated using such compounds include, without limitation, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis (ALS), Alzheimer's disease, multiple sclerosis, epilepsy, and stroke.

Compounds that alter cAMP levels may be administered by any appropriate route. For example, administration may be parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, by suppositories, or by oral administration. Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.

Methods well known in the art for making formulations are found, for example, in “Remington's Pharmaceutical Sciences.” Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

Dosage is determined by standard techniques and is dependent, for example, upon the weight of the mammal and the type or extent of disorder being treated.

Diagnostics for Neurodegenerative Disorders

To determine whether an individual either has or is likely to develop a neurodegenerative disorder, that individual is screened for mutations in genes which are either involved in regulation of cAMP, or are regulated by cAMP, for example, genes encoding the adenylyl cyclases, G proteins, or human homologues of UNC-36 or EAT-4 proteins described herein. Such assays may be carried out by any standard technique including, without limitation, methods involving sequencing or mismatch binding or cleaving assays. In one particular example, a nucleic acid sample derived from the neuronal cells of an individual may be isolated (for example, by PCR amplification), and a cAMP regulatory gene (or a portion thereof) subjected to rapid sequence analysis by automated sequencing techniques using primers generated from sequences described herein and in the art.

Alternatively, an individual who either has or is likely to develop a neurodegenerative disorder may be screened for altered expression of adenylyl cyclases, G proteins, or the human homologues of UNC-36 or EAT-4 proteins, or for an increased level of cellular cAMP, particularly in neuronal cells. Such assays may be carried out, for example, using any standard nucleic acid-based assay (e.g., Northern blot analysis) or immunological assay (e.g., enzyme-linked immunosorbent assay (ELISA)), preferably in a high through-put assay format. In one particular example, neuronal cells obtained from an individual being screened for a neurodegenerative disorder may be isolated and analyzed for the expression of adenylyl cyclases, G proteins, and the human homologues of UNC-36 and EAT-4 proteins by ELISAs using fluorophore-tagged antibodies directed toward these proteins as probes. Individuals incapable of expressing certain proteins may be identified by rapidly assessing the results of these ELISAs in a microtiter plate format.

In particular examples, candidate human genes, for example, those involved in cAMP regulation, are examined for genetic linkage to hereditable forms of neurodegeneration found in humans or, as a model system, mice. These genetic linkages are assessed using standard methods known in the art, and, upon identification of a linkage with neurodegeneration, diagnostic mutation detection is conducted as described herein. Listed in Table 4 are exemplary candidate human genes likely involved in neurodegeneration.

TABLE 4
Candidate Human Neurodegeneration Genes
Class of Protein     Gene Product
Phosphodiesterases   PDE4A
                     PDE4B
                     PDE6G
                     PDE7A
G alpha subunits     GNAS1
                     GNAI1
                     GNAI2
                     GNAI3
                     Golf
Protein Phosphatases PPP1CB
                     PPP1CC
                     PPP2CA
                     PPP2R4
                     PPP2R5A
                     PPP2R5C
                     PPP2R5D
                     PPP2R5E
                     PPP3CA
                     PPP3CB
                     PPP3R1

Methods for Isolating Genes Involved in Neurodegeneration a) C. elegans Screens

Additional genes involved in neurodegeneration may be isolated using the methods described herein. For example, a gene involved in neurodegeneration may be isolated by inducing paralysis and neurodegeneration in C. elegans. This is accomplished, for example, by generating a nematode strain carrying a constitutively active (GTP-ase defective) Gα s subunit gene operably linked to a glutamate receptor (GluR) promoter, such as glr-1. The transgenic C. elegans is screened for gene mutations which restore locomotion and reduce neurodegeneracy (cytotoxicity genes) or which reduce Gα s -induced neuronal cell swelling (swelling genes).

I. Gα s -associated Cytotoxicitv Genes

To isolate a Gα s -associated cytotoxicity gene, glr-1::α s (gf) transgenic nematodes are mutagenized, for example, with EMS or γ-irradiation, and then screened for mutants with both improved locomotion and increased survival of the Gα s expressing neurons. If desired, these mutants may be genetically mapped and placed into complementation groups. The genes identified in these mutants may then be positionally cloned.

II. Gα s -associated Swelling Genes

To isolate a Gα s -associated swelling gene, glr-1::α s (gf) transgenic nematodes are mutagenized, for example, with EMS or γ-irradiation. First stage larvae are then isolated and screened by fluorescence microscopy (as described herein) for mutants which show a reduced incidence of swelling of Gα s -expressing neurons. These mutants may be genetically mapped and positionally cloned.

III. Other Genes

The transgenic animals developed to identify compounds that inhibit cAMP-based neurodegeneration may also be used to identify additional genes involved in neurodegeneration. For example, C. elegans doubly transgenic for acy-1::gfp; glr- 1::αs (gf) may be mutagenized (for example, with EMS or γ-irradiation) and then analyzed for restored locomotion and reduced neurodegeneration (i.e., for a mutation in a gene which affects the glr-1 promoter) or a reduced level of GFP expression (i.e., for a mutation in a gene which affects the acy-1 promoter), or both (i.e., a mutation in a gene which affects both acy-1 and glr-1 gene promoters).

In an alternative approach, physiological stresses, such as ischemia, due to, for example, the interruption of available oxygen, may be administered to EMS or γ-irradiated worms to induce neurodegeneration. Mutants which resist ischemia-induced neurodegeneration may then be isolated and characterized to identify the neuroprotective mutant gene.

A gene involved in neurodegeneration may be cloned and sequenced by standard methods (see, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1994 and Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2 d ed.), CSH Press, 1989). If desired, a protein product from this gene may then be produced, for example, by inserting the cloned gene into an expression vector and introducing this vector into bacterial or eukaryotic cells to produce recombinant proteins. Techniques for such manipulations are disclosed in Sambrook et al., supra, and are well known in the art. Genes involved in neurodegeneration or their protein products may be used in any of the screening or diagnostic assays described herein.

b) Yeast Screens

Another approach to identify genes involved in neurodegeneration utilizes yeast carrying a reporter gene operably linked to a promoter from a cAMP regulatory gene, and, preferably, a mammalian cAMP regulatory gene. The reporter construct is stably introduced into yeast by any standard method. A cDNA library (preferably, from a mammalian cell) is then introduced into the yeast carrying the reporter construct, and yeast colonies exhibiting an increased level of reporter gene expression (e.g., lacZ reporter yeast with increased blue colony color on X-Gal) are identified. Such yeast carry a cDNA capable of binding to the cAMP promoter and are therefor good candidates for a gene involved in cAMP-based neurodegeneration. If desired, the promoter sequences from the newly isolated gene may also be used to generate reporter cells (e.g., reporter yeast or transgenic C. elegans ) to identify additional genes involved in cAMP-based neurodegeneration.

Moreover, this yeast system may be used to screen for compounds which inhibit the ability of the cDNA to induce reporter gene expression. Such compounds provide good candidates for therapeutics for treating cAMP-based neurodegeneration.

Mammalian Genes Involved in Neurodegeneration

a) Mammalian eat-4 genes

Any of a variety of procedures well known in the art may be utilized to clone the mammalian homologues of the nematode eat-4 gene, and one so skilled will routinely adapt one of these methods in order to obtain the desired gene.

One such method for obtaining a mammalian gene sequence is to use an oligonucleotide probe generated by the C. elegans eat-4 gene sequence to screen a mammalian cDNA or genomic DNA library for sequences which hybridize to the probe. Hybridization techniques are well known to the skilled artisan, and are described, for example, in Ausubel et al., supra, and Sambrook et al., supra. cDNA or genomic DNA library preparation is also well known in the art. A large number of prepared nucleic acid libraries are also commercially available. The oligonucleotide probes are readily designed using the sequences described herein and standard techniques. The oligonucleotide probes may be based upon the sequence of either strand of DNA encoding the eat-4 gene product (SEQ ID NO: 1). Exemplary oligonucleotide probes are degenerate probes (i.e., a mixture of all possible coding sequences for the EAT-4 protein).

If desired, the cloned gene may be inserted into an expression vector and introduced into bacterial or eukaryotic cells to produce the mammalian EAT-4 protein. Techniques for such manipulations are disclosed, for example, in Sambrook et al., supra. The mammalian eat-4 gene or gene product may be used in the neurodegeneration screening or diagnostic assays described herein.

b) eat-4 related C. elegans genes

Genes related to eat-4 may be isolated by methods similar to those described above. For example, a cosmid library from C. elegans may be screened with the degenerate oligonucleotide probes described above under low stringency hybridization conditions to isolate eat-4 related C. elegans genes. Oligonucleotide probes may be prepared from these gene sequences and may be used to screen mammalian nucleic acid libraries for hybridizing sequences, thus, identifying mammalian homologues of these eat-4 related genes.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the appended claims.

Other embodiments are within the claims.

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Phe Ala Ile Ser Gly Phe Asn Val Asn His Leu Asp Ile 420 425 430Ala Pro Arg Tyr Ala Ala Ile Leu Met Gly Phe Ser Asn Gly Ile Gly 435 440 445Thr Leu Ala Gly Leu Thr Cys Pro Phe Val Thr Glu Ala Phe Thr Ala 450 455 460His Ser Lys His Gly Trp Thr Ser Val Phe Leu Leu Ala Ser Leu Ile465 470 475 480His Phe Thr Gly Val Thr Phe Tyr Ala Val Tyr Ala Ser Gly Glu Leu 485 490 495Gln Glu Trp Ala Glu Pro Lys Glu Glu Glu Glu Trp Ser Asn Lys Glu 500 505 510Leu Val Asn Lys Thr Gly Ile Asn Gly Thr Gly Tyr Gly Ala Ala Glu 515 520 525Thr Thr Phe Thr Gln Leu Pro Ala Gly Val Asp Ser Ser Tyr Gln Ala 530 535 540Gln Ala Ala Pro Ala Pro Gly Thr Asn Pro Phe Ala Ser Ala Trp Asp545 550 555 560Glu His Gly Ser Ser Gly Val Val Glu Asn Pro His Tyr Gln Gln Trp 565 570 575 2 1253 PRT Caenorhabditis elegans 2Met Asp Asp Asp Val Gly Glu Arg Thr Pro Ala Leu Gly Gly Ser Cys 1 5 10 15Gly Pro Ser Val Arg Ala His Ser Ser Ser Pro Arg Arg Val Pro Leu 20 25 30Phe Glu Arg Ala Ser Ala Arg Trp Trp Asn Pro Gln Phe Arg Ser Ala 35 40 45Thr Leu Glu Ala Gln Tyr Trp Lys Cys Ser Phe Ser Gln Leu Arg Asp 50 55 60Arg Phe Arg Ser Gly Leu Ile Tyr Ile Ala Val Val Ile Ala Ala Trp65 70 75 80Thr Leu Tyr Leu Ala Leu Phe Asp Arg Thr Phe Ile Gln His Trp Ile 85 90 95Val Ser Leu Cys Leu Cys Ala Ile Ile Phe Ala Met Phe Ala Phe Thr 100 105 110Ala Cys Ala Ala Gln Tyr Gln Arg Phe Tyr Met Pro Thr Ser Phe Leu 115 120 125Cys Thr Phe Leu Ile Cys Leu Val Thr Leu Leu Ile Phe Ser Ala Glu 130 135 140Asn Gln Ala Ala Phe Met Thr Pro Val Ala Ser Leu Ala Thr Ser Phe145 150 155 160Gln Val Val Leu Leu Ile Tyr Thr Val Ile Pro Leu Pro Leu Tyr Leu 165 170 175Cys Ile Leu Ile Gly Ile Ile Tyr Ser Ile Leu Phe Glu Ile Leu Asn 180 185 190Lys Asn Lys Ile Gly Leu Glu Glu Ala Gly Tyr Ile Lys Leu Val Leu 195 200 205His Ala Gly Val His Leu Leu Gly Val His Leu Phe Ile Leu Thr Gln 210 215 220Val Arg Gln Arg Lys Thr Phe Leu Lys Val Gly Gln Ser Met Leu Ala225 230 235 240Arg Lys Asp Leu Glu Leu Glu Thr Gln Phe Lys Asp His Met Ile Gln 245 250 255Ser Val Met Pro Lys Lys Val Ala Asp Glu Leu Leu Lys Asp Ala Ser 260 265 270Glu Leu Arg Arg Pro Ser Ala Ser Asn Asp Ser Asn Cys Arg Thr Ser 275 280 285Asn Ala Thr Gln Val Asp Gln Pro Leu Ala Lys Met Val Pro Glu Tyr 290 295 300Arg Lys Phe Arg Pro Phe Thr Met Asn Leu Met Thr Asn Val Ser Ile305 310 315 320Leu Phe Ala Asp Ile Ala Gly Phe Thr Lys Met Ser Ser Asn Lys Ser 325 330 335Ala Asp Glu Leu Val Asn Leu Leu Asn Asp Leu Phe Gly Arg Phe Asp 340 345 350Thr Leu Cys Arg Leu Arg Gly Leu Glu Lys Ile Ser Thr Leu Gly Asp 355 360 365Cys Tyr Tyr Cys Val Ala Gly Cys Pro Glu Pro Cys Asp Asp His Ala 370 375 380Cys Arg Thr Val Glu Met Gly Leu Asp Met Ile Val Ala Ile Arg Gln385 390 395 400Phe Asp Ile Asp Arg Gly Gln Glu Val Asn Met Arg Val Gly Ile His 405 410 415Thr Gly Lys Val Met Cys Gly Met Val Gly Thr Lys Arg Phe Lys Phe 420 425 430Asp Val Phe Ser Asn Asp Val Thr Leu Ala Asn Glu Met Glu Ser Ser 435 440 445Gly Val Ala Gly Arg Val His Val Ser Glu Ala Thr Ala Lys Leu Leu 450 455 460Lys Gly Leu Tyr Glu Ile Glu Glu Gly Pro Asp Tyr Asp Gly Pro Leu465 470 475 480Arg Met Gln Val Gln Gly Thr Glu Arg Arg Val Lys Pro Glu Ser Met 485 490 495Lys Thr Phe Phe Ile Lys Gly Arg Ile Asn Asp Gly Val Glu Glu Glu 500 505 510Val Met Gln Val Gln Glu Val Glu Ser Leu His Ser Gln Lys Ser Ser 515 520 525Lys Lys Ser Thr Leu Lys Gln Lys Trp Ala Glu Lys Leu Lys Met Asn 530 535 540His Thr Asn Ser Tyr Pro Met Arg Ala Ala Ala Arg Glu Gly Gly Gly545 550 555 560Ser Leu Arg Ile Lys Leu Ala Glu Arg Asn Arg Ser Thr Gln Leu Leu 565 570 575Pro Lys Glu Ser Asn Ser Ile Cys Ile Met Glu Asp Asn Arg Lys Ser 580 585 590Ala Ser Leu Gln Ala Leu Ala Thr Asn Asn Phe Asn Gly Ser Asn Thr 595 600 605Asp Thr Asn Asn Thr Tyr Ser Glu Arg Gly Val Ala Gly Ser Val Ser 610 615 620Lys Lys Ser Val Ala Gly Ser Glu Ser Asn Ser Ile Lys Gly Ser Arg625 630 635 640Ser Ser Gly Leu Gln Leu Ser Leu Gln Asp Gly Asn Ser Asp Leu Asn 645 650 655Ser Val Gly Gly Leu Asp Thr Ala Ile Ser His His His Asn Ala Ala 660 665 670Ser Leu Thr Arg Phe Asp Thr Asp Asn Asn Phe Asp Gln Arg Leu Ala 675 680 685Met Val Ile Gly Gln Gly Glu Gly Gly Phe Asp Lys Gly Phe Trp Asn 690 695 700His His Asp Ser Leu Asn Lys Trp Thr Leu Arg Phe Asn Glu Lys Asp705 710 715 720Val Glu Glu Glu Tyr Arg Ala His Phe Val Asp Ser Ser Glu Arg Tyr 725 730 735Thr Ala Ser Lys Lys Gly His Val Glu Arg His Lys Asp Leu Met Glu 740 745 750Gln Gly Gly Glu Lys Asp Gly Ile Thr Gly Ser Thr Val Asn Lys Tyr 755 760 765Arg Tyr Ser Gly Val Phe Ile Asp Ile Ile Val Ala Thr Leu Ile Phe 770 775 780Val Ile Ser Gly Ala Val Ala Ile Met Ser Val Arg Pro Phe Pro Leu785 790 795 800Ser Leu Ile Ala Tyr Phe Pro Phe Ala Ala Ala Ile Leu Ile Leu Thr 805 810 815Ile Val Leu Ile Gly Leu Pro Leu Leu Ala Arg Lys Lys Ser Phe Gln 820 825 830Cys Ala Asn Gln Trp Met Pro Arg His Leu Ile Gly Leu Leu Leu Ile 835 840 845Phe Leu Pro Ile Gly Val Ala Ile Cys Ile Met Pro Leu Cys Gln Ser 850 855 860Gly Asp Cys Ala Asn Val Ile Leu Asn Tyr Arg Leu Ala Phe Ser Tyr865 870 875 880Val Thr Ile Leu Ala Ile Phe Ala His Cys Asn Phe Ser Gln Leu Ala 885 890 895Ala Trp Pro Lys Thr Thr Ala Ala Val Phe Ile Gly Leu Leu His Ile 900 905 910Ala Gly Val Phe Tyr Cys Glu Phe Asn Leu Lys His Leu Val Glu Glu 915 920 925Gln Asp Thr Cys Asn Val Thr Ala Ile Met Ile Pro Pro Ile Arg Lys 930 935 940Gly Leu Asn Tyr Thr Ile Ala Leu Asn Ser Thr Ser Ala Arg Thr Leu945 950 955 960Ser Gln Asp Phe Gly Ser Pro Leu Phe Ile Trp Glu Leu Leu Leu Asp 965 970 975Val Ile Leu Ser Ile Val Leu Val Ala Phe Leu Asn Tyr Gln Phe Glu 980 985 990Thr Ala Phe Arg Met Ser Phe Phe Gly Asp Val Gln Ala Arg Arg Asp 995 1000 1005Thr Glu Arg Met Gln Ile Val Arg Asp Gln Ala Asp Trp Leu Leu Asn 1010 1015 1020Asn Val Ile Pro Ala His Ala Val Glu Ser Leu Lys Thr Asp Thr Lys1025 1030 1035 104Tyr Ser Glu Asn His Glu Thr Val Gly Val Leu Phe Ala Ser Ile Thr 1045 1050 1055Asn Trp Asn Asp Met Tyr Glu Glu Asn Phe Glu Gly Gly Arg Glu Phe 1060 1065 1070Leu Arg Val Leu Asn Glu Val Ile Gly Asp Phe Asp Glu Leu Leu Asp 1075 1080 1085Arg Pro Asp Phe Thr His Ile Glu Lys Ile Lys Thr Ile Gly Pro Ala 1090 1095 1100Tyr Met Ala Ala Ser Gly Leu Asn Pro Glu Arg Lys Lys Asn Met Leu1105 1110 1115 112His Pro Lys Glu His Leu Tyr Gln Met Val Asp Phe Ala Leu Ala Val 1125 1130 1135Gln His Val Leu Ser Val Phe Asn Glu Asp Leu Leu Asn Phe Asp Phe 1140 1145 1150Val Cys Lys Leu Gly Leu Asn Ile Gly Pro Val Thr Ala Gly Val Ile 1155 1160 1165Gly Thr Thr Lys Leu Tyr Tyr Asp Ile Trp Gly Asp Thr Val Asn Ile 1170 1175 1180Ala Ser Arg Met Tyr Ser Thr Gly Val Leu Asn Arg Ile Gln Val Ser1185 1190 1195 120Gln His Thr Arg Glu Tyr Leu Leu Asp Arg Tyr Glu Phe Glu Phe Arg 1205 1210 1215Asp His Ile Glu Val Lys Gly Ile Asp Gly Gly Met Asp Thr Tyr Leu 1220 1225 1230Leu Val Gly Arg Lys Gly Asp Gly Ile Pro Pro Ser Ile Lys Asp Asn 1235 1240 1245Gln Glu Asp Glu Phe 1250 3 1305 PRT Mus musculus 3Met Ala Ser Ser Pro His Gln Gln Leu Leu His His His Ser Thr Glu 1 5 10 15Val Ser Cys Asp Ser Ser Gly Asp Ser Asn Ser Val Arg Val Lys Ile 20 25 30Asn Pro Lys Gln Leu Ser Ser Asn Thr His Pro Lys His Cys Lys Tyr 35 40 45Ser Ile Ser Ser Ser Cys Ser Ser Ser Gly Asp Ser Gly Gly Leu Pro 50 55 60Arg Arg Val Gly Gly Gly Gly Arg Leu Arg Arg Gln Lys Lys Leu Pro65 70 75 80Gln Leu Phe Glu Arg Ala Ser Ser Arg Trp Trp Asp Pro Lys Phe Asp 85 90 95Ser Met Asn Leu Glu Glu Ala Cys Leu Glu Arg Cys Phe Pro Gln Thr 100 105 110Gln Arg Arg Phe Arg Tyr Ala Leu Phe Tyr Val Gly Phe Ala Cys Leu 115 120 125Leu Trp Ser Ile Tyr Phe Ala Val His Met Lys Ser Lys Val Ile Val 130 135 140Met Val Val Pro Ala Leu Cys Phe Leu Val Val Cys Val Gly Phe Phe145 150 155 160Leu Phe Thr Phe Thr Lys Leu Tyr Ala Arg His Tyr Ala Trp Thr Ser 165 170 175Leu Ala Leu Thr Leu Leu Val Phe Ala Leu Thr Leu Ala Ala Gln Phe 180 185 190Gln Val Trp Thr Pro Leu Ser Gly Arg Val Asp Ser Ser Asn His Thr 195 200 205Leu Thr Ala Thr Pro Ala Asp Thr Cys Leu Ser Gln Val Gly Ser Phe 210 215 220Ser Ile Cys Ile Glu Val Leu Leu Leu Leu Tyr Thr Val Met Gln Leu225 230 235 240Pro Leu Tyr Leu Ser Leu Phe Leu Gly Val Val Tyr Ser Val Leu Phe 245 250 255Glu Thr Phe Gly Tyr His Phe Arg Asn Glu Asp Cys Tyr Pro Ser Pro 260 265 270Gly Pro Gly Ala Leu His Trp Glu Leu Leu Ser Arg Ala Leu Leu His 275 280 285Val Cys Ile His Ala Ile Gly Ile His Leu Phe Val Met Ser Gln Val 290 295 300Arg Ser Arg Ser Thr Phe Leu Lys Val Gly Gln Ser Ile Met His Gly305 310 315 320Lys Asp Leu Glu Val Glu Lys Ala Leu Lys Glu Arg Met Ile His Ser 325 330 335Val Met Pro Arg Ile Ile Ala Asp Asp Leu Met Lys Gln Gly Asp Glu 340 345 350Glu Ser Glu Asn Ser Val Lys Arg His Ala Thr Ser Ser Pro Lys Asn 355 360 365Arg Lys Lys Lys Ser Ser Ile Gln Lys Ala Pro Ile Ala Phe Arg Pro 370 375 380Phe Lys Met Gln Gln Ile Glu Glu Val Ser Ile Leu Phe Ala Asp Ile385 390 395 400Val Gly Phe Thr Lys Met Ser Ala Asn Lys Ser Ala His Ala Leu Val 405 410 415Gly Leu Leu Asn Asp Leu Phe Gly Arg Phe Asp Arg Leu Cys Glu Gln 420 425 430Thr Lys Cys Glu Lys Ile Ser Thr Leu Gly Asp Cys Tyr Tyr Cys Val 435 440 445Ala Gly Cys Pro Glu Pro Arg Ala Asp His Ala Tyr Cys Cys Ile Glu 450 455 460Met Gly Leu Gly Met Ile Lys Ala Ile Glu Gln Phe Cys Gln Glu Lys465 470 475 480Lys Glu Met Val Asn Met Arg Val Gly Val His Thr Gly Thr Val Leu 485 490 495Cys Gly Ile Leu Gly Met Arg Arg Phe Lys Phe Asp Val Trp Ser Asn 500 505 510Asp Val Asn Leu Ala Asn Leu Met Glu Gln Leu Gly Val Ala Gly Lys 515 520 525Val His Ile Ser Glu Ala Thr Ala Lys Tyr Leu Asp Asp Arg Tyr Glu 530 535 540Met Glu Asp Gly Arg Val Ile Glu Arg Leu Gly Gln Ser Val Val Ala545 550 555 560Asp Gln Leu Lys Gly Leu Lys Thr Tyr Leu Ile Ser Gly Gln Arg Ala 565 570 575Lys Glu Ser His Cys Ser Cys Ala Glu Ala Leu Leu Ser Gly Phe Glu 580 585 590Val Ile Asp Asp Ser Arg Glu Ser Ser Gly Pro Arg Gly Gln Gly Thr 595 600 605Ala Ser Pro Gly Ser Val Ser Asp Leu Ala Gln Thr Val Lys Thr Phe 610 615 620Asp Asn Leu Lys Thr Cys Pro Ser Cys Gly Ile Thr Phe Ala Pro Lys625 630 635 640Ser Glu Ala Gly Ala Glu Gly Gly Thr Val Gln Asn Gly Cys Gln Asp 645 650 655Glu Pro Lys Thr Ser Thr Lys Ala Ser Gly Gly Pro Asn Ser Lys Thr 660 665 670Gln Asn Gly Leu Leu Ser Pro Pro Ala Glu Glu Lys Leu Thr Asn Ser 675 680 685Gln Thr Ser Leu Cys Glu Ile Leu Gln Glu Lys Gly Arg Trp Ala Gly 690 695 700Val Ser Leu Asp Gln Ser Ala Leu Leu Pro Leu Arg Phe Lys Asn Ile705 710 715 720Arg Glu Lys Thr Asp Ala His Phe Val Asp Val Ile Lys Glu Asp Ser 725 730 735Leu Met Lys Asp Tyr Phe Phe Lys Pro Pro Ile Asn Gln Phe Ser Leu 740 745 750Asn Phe Leu Asp Gln Glu Leu Glu Arg Ser Tyr Arg Thr Ser Tyr Gln 755 760 765Glu Glu Val Ile Lys Asn Ser Pro Val Lys Thr Phe Ala Ser Ala Thr 770 775 780Phe Ser Ser Leu Leu Asp Val Phe Leu Ser Thr Thr Val Phe Leu Ile785 790 795 800Leu Ser Ile Thr Cys Phe Leu Lys Tyr Gly Ala Thr Ala Thr Pro Pro 805 810 815Pro Pro Ala Ala Leu Ala Val Phe Gly Ala Asp Leu Leu Leu Glu Val 820 825 830Leu Ser Leu Ile Val Ser Ile Arg Met Val Phe Phe Leu Glu Asp Val 835 840 845Met Thr Cys Thr Lys Trp Leu Leu Glu Trp Ile Ala Gly Trp Leu Pro 850 855 860Arg His Cys Ile Gly Ala Ile Leu Val Ser Leu Pro Ala Leu Ala Val865 870 875 880Tyr Ser His Ile Thr Ser Glu Phe Glu Thr Asn Ile His Val Thr Met 885 890 895Phe Thr Gly Ser Ala Val Leu Val Ala Val Val His Tyr Cys Asn Phe 900 905 910Cys Gln Leu Ser Ser Trp Met Arg Ser Ser Leu Ala Thr Ile Val Gly 915 920 925Ala Gly Leu Leu Leu Leu Leu His Ile Ser Leu Cys Gln Asp Ser Ser 930 935 940Ile Val Met Ser Pro Leu Asp Ser Ala Gln Asn Phe Ser Ala Gln Arg945 950 955 960Asn Pro Cys Asn Ser Ser Val Leu Gln Asp Gly Arg Arg Pro Ala Ser 965 970 975Leu Ile Gly Lys Glu Leu Ile Leu Thr Phe Phe Leu Leu Leu Leu Leu 980 985 990Val Trp Phe Leu Asn Arg Glu Phe Glu Val Ser Tyr Arg Leu His Tyr 995 1000 1005His Gly Asp Val Glu Ala Asp Leu His Arg Thr Lys Ile Gln Ser Met 1010 1015 1020Arg Asp Gln Ala Asp Trp Leu Leu Arg Asn Ile Ile Pro Tyr His Val1025 1030 1035 104Ala Glu Gln Leu Lys Val Ser Gln Thr Tyr Ser Lys Asn His Asp Ser 1045 1050 1055Gly Gly Val Ile Phe Ala Ser Ile Val Asn Phe Ser Glu Phe Tyr Glu 1060 1065 1070Glu Asn Tyr Glu Gly Gly Lys Glu Cys Tyr Arg Val Leu Asn Glu Leu 1075 1080 1085Ile Gly Asp Phe Asp Glu Leu Leu Ser Lys Pro Asp Tyr Asn Ser Ile 1090 1095 1100Glu Lys Ile Lys Thr Ile Gly Ala Thr Tyr Met Ala Ala Ser Gly Leu1105 1110 1115 112Asn Thr Ala Gln Cys Gln Glu Gly Gly His Pro Gln Glu His Leu Arg 1125 1130 1135Ile Leu Phe Glu Phe Ala Lys Glu Met Met Arg Val Val Asp Asp Phe 1140 1145 1150Asn Asn Asn Met Leu Trp Phe Asn Phe Lys Leu Arg Val Gly Phe Asn 1155 1160 1165His Gly Pro Leu Thr Ala Gly Val Ile Gly Thr Thr Lys Leu Leu Tyr 1170 1175 1180Asp Ile Trp Gly Asp Thr Val Asn Ile Ala Ser Arg Met Asp Thr Thr1185 1190 1195 120Gly Val Glu Cys Arg Ile Gln Val Ser Glu Glu Ser Tyr Arg Val Leu 1205 1210 1215Ser Lys Met Gly Tyr Asp Phe Asp Tyr Arg Gly Thr Val Asn Val Lys 1220 1225 1230Gly Lys Gly Gln Met Lys Thr Tyr Leu Tyr Pro Lys Cys Thr Asp Asn 1235 1240 1245Gly Val Val Pro Gln His Gln Leu Ser Ile Ser Pro Asp Ile Arg Val 1250 1255 1260Gln Val Asp Gly Ser Ile Gly Arg Ser Pro Thr Asp Glu Ile Ala Asn1265 1270 1275 128Leu Val Pro Ser Val Gln Tyr Ser Asp Lys Ala Ser Leu Gly Ser Asp 1285 1290 1295Asp Ser Thr Gln Ala Lys Glu Ala Arg 1300 1305

Claims

1. A method for identifying a gene involved in neurodegeneration, said method comprising the steps of:a) providing a nematode comprising an expression construct, said expression construct comprising a promoter derived from a cAMP regulatory gene selected from the group consisting of an acy-1 gene, an eat-4 gene, an unc-36 gene, and a glutamate receptor-encoding gene, said promoter operably linked to a reporter gene; b) isolating a nematode comprising a mutation on the basis that said nematode exhibits an altered level of reporter gene expression relative to an unmutated nematode comprising said expression construct; and c) identifying said gene comprising said mutation, said gene being involved in neurodegeneration.

2. A method for identifying a gene involved in neurodegeneration, said method comprising the steps of:a) providing a nematode comprising a glutamate receptor (GluR) promoter operably linked to a gene encoding a GTP-ase defective Gαs subunit; b) isolating a mutant of said nematode exhibiting a decreased level of paralysis and neurodegeneration; and c) identifying said gene comprising said mutation, said gene being involved in neurodegeneration.

3. The method of claim 1 or 2, wherein said nematode is C. elegans.

4. The method of claim 1, wherein said cAMP regulatory gene is an acy-1 gene.

5. The method of claim 1, wherein said cAMP regulatory gene is an eat-4 gene.

6. The method of claim 1, wherein said cAMP regulatory gene is an unc-36 gene.

7. The method of claim 1, wherein said cAMP regulatory gene is a glutamate receptor-encoding gene.

8. The method of claim 1, wherein said reporter gene is selected from the group consisting of lacZ, gfp, her-1, and mec-4.

9. The method of claim 7, wherein said glutamate receptor is selected from the group consisting of GLR-1, C06A8.8, B0280.12, F41B4.4, C43H6, K10D3.1, ZC196.C, GluCLα1, GluCLβ1, ZC317.3, T1063, MGR-1, and F45H11.

10. A method for identifying a gene involved in neurodegeneration, said method comprising the steps of:a) providing a nematode comprising an expression construct, said expression construct comprising a promoter derived from a cAMP regulatory gene whose product regulates cAMP, said promoter operably linked to a reporter gene; b) isolating a nematode comprising a mutation on the basis that said nematode exhibits an altered level of reporter gene expression relative to an unmutated nematode comprising said expression construct; and c) identifying said gene comprising said mutation, said gene being involved in neurodegeneration.

11. The method of claim 10, wherein said nematode is C. elegans.

12. The method of claim 10, wherein said reporter gene is selected from the group consisting of lacZ, gfp, her-1, and mec-4.

13. The method of claim 10, wherein said cAMP regulatory gene encodes an adenylyl cyclase.

14. The method of claim 10, wherein said cAMP regulatory gene encodes a G protein alpha subunit.

15. The method of claim 10, wherein said cAMP regulatory gene encodes a phosphodiesterase.

16. The method of claim 10, wherein said cAMP regulatory gene encodes a protein phosphatase.