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Neurodegenerative disorders constitute one of the major challenges of modern medicine. Although these diseases are relatively common and often highly debilitating, the mechanisms responsible for their pathologies are poorly understood, and there are currently no effective preventative therapies. Recently, linkage studies have begun to identify genes underlying heritable forms of the neurodegenerative disorders. While these breakthroughs potentially provide a window into the more common sporadic forms of these disorders, we currently know very little about the functions of these genes and the mechanisms by which their mutational alteration results in neuronal death. One approach to this problem that has great potential is the use of classical genetic analysis in Drosophila to identify the genetic pathways leading to pathology in fly models of these disorders. A large body of work has convincingly demonstrated the great degree to which genes and genetic pathways are conserved between Drosophila and mammals, and recent studies indicate that this conservation of molecular mechanisms also makes Drosophila an excellent system in which to model human neurodegenerative diseases. Below, I describe the disorders we are trying to model in Drosophila, our current progress and future plans. |
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Niemann Pick Type C Disease (NPCD) is a fatal autosomal recessive disorder characterized by progressive widespread neurodegeneration and hepatosplenomegaly. These phenotypes are thought to arise from a primary defect in intracellular trafficking of lipids, including LDL-derived cholesterol, resulting in the accumulation of cholesterol and other lipids in late endosomes and lysosomes. The gene responsible for most cases of NPCD was cloned in 1997 (termed NPC1) and was found to encode a large multi-pass transmembrane protein of unknown function. To gain insight into the normal biological function of NPC1, as well as the pathology underlying NPCD, we are attempting to create a Drosophila model of this disorder. Although Drosophila (like other insects) is a cholesterol auxotroph, previous work indicates that insects package dietary cholesterol into particles closely resembling LDL particles, and that these particles are released into the hemolymph and taken up by tissues through an endocytic mechanism closely resembling that of vertebrates. The Drosophila genome encodes two NPC1 homologs (designated NPC1a and NPC1b) exhibiting 40-43% amino acid identity to the human NPC1 gene product. Over the past several years we have generated mutations in the NPC1b gene using a gene targeting strategy and have identified transposon insertions that moderately attenuate NPC1a gene expression and are currently using these transposon insertions to generate null alleles of this gene. |
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Our current collection of hypomorphic NPC1a mutants are viable and display no discernable phenotype as homozygotes. By contrast, homozygous or hemizygous NPC1b mutants exhibit second-instar larval lethality. Although NPC1b mutants manifest no gross morphological defects or visible neuronal dysfunction, a small percentage of larvae show a double mouth-hook and double vertical plate phenotype similar to that of several Drosophila mutants with defects in the ecdysone steroid hormone synthesis pathway. To investigate whether loss of NPC1b function results in ecdysone deficiency, NPC1b mutants were fed ecdysone exogenously in an attempt to rescue larval lethality. Upon feeding ecdysone, the percentage of NPC1b mutants that survive until at least the second-to-third instar larval transition or beyond was found to increase dramatically. We are currently testing the hypothesis that NPC1b function is required for cholesterol trafficking in the larval ring gland, the primary steroidenergic tissue in Drosophila. We are also constructing more severe alleles of the NPC1a gene and generating NPC1a NPC1b double mutants to further investigate the function of NPC1a and the possibility of functional redundancy in these genes. Finally, we are attempting to generate a Drosophila cell culture model of NPCD using RNAi to attenuate NPC1a and NPC1b function in established Drosophila cell lines. Cholesterol accumulation can be easily visualized in cell culture using the cholesterol-binding drug, filipin. This cell culture model will be used to pursue specific mechanistic hypotheses using combinatorial RNAi, as well as high throughput screens for modifiers using a large collection of RNAi’s corresponding to all genes in the Drosophila genome (constructed in Norbert Perrimon’s lab). These cell culture modifier screens will be combined with modifier screens in flies to identify factors involved in intracellular trafficking of cholesterol and possibly neurodegeneration.
Our studies on NPC1 function have been supported by the Jim Lambright Niemann-Pick Foundation and the Ara Parseghian Medical Research Foundation .
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Parkinson's Disease (PD) (PD) is a prevalent neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra and the accumulation of proteinacous intraneuronal inclusions known as Lewy Bodies. While the molecular mechanisms responsible for neurodegeneration in PD remain poorly understood, previous work implicates aberrant proteolytic degradation and oxidative damage arising from mitochondrial dysfunction or byproducts of dopamine metabolism. Recently, mutations of the parkin gene, which encodes a ubiquitin-protein ligase, were found to underlie a familial form of PD known as autosomal recessive juvenile PD (AR-JP). To explore the specific biochemical pathways affected by parkin mutations we have created a Drosophila model of AR-JP through mutational inactivation of a Drosophila parkin ortholog. Drosophila parkin null mutants exhibit reduced lifespan, locomotor defects and male sterility. The locomotor defects derive from apoptotic cell death of muscle subsets, whereas the male-sterile phenotype derives from a spermatid individualization defect at a late stage of spermatogenesis. Parkin mutants also manifest dopaminergic neuron structural abnormalities. Mitochondrial pathology is the earliest manifestation of muscle degeneration and a prominent characteristic of individualizing spermatids in parkin mutants. These results suggest that the tissue-specific phenotypes observed in Drosophila parkin mutants result from mitochondrial dysfunction and raise the possibility that similar mitochondrial impairment triggers the selective cell loss seen in AR-JP. We are currently using genomic, proteomic, and classical genetic methods to investigate the molecular mechanism by which loss of parkin function results in muscle and germline pathology. |
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In addition to our work on parkin, we have also recently begun genetic studies of the DJ-1 and alpha-synuclein genes. Loss-of-function mutations of the DJ-1 gene, which encodes a protein of unknown function, were recently shown to result in an autosomal recessive form of PD. We have recently generated null alleles of a pair of closely related Drosophila DJ-1 counterparts and are currently investigating the phenotypes of these mutants. Missense alleles or increased gene dosage of alpha-synuclein, which encodes a component of Lewy body inclusions, result in an autosomal dominant form of PD. The finding that alpha-synuclein is a component of Lewy body inclusions suggests that this protein may also be an important causative factor in sporadic cases of PD. Although Drosophila does not appear to have a counterpart to the alpha-synuclein gene, a model of PD was recently created by ectopically expressing human alpha-synuclein in Drosophila. We are testing the involvement of candidate genes in alpha-synuclein mediated pathogenesis in an effort to investigate the mechanism of alpha-synuclein toxicity and to possibly identify additional genetic factors involved in PD.
Our studies on parkin, DJ-1 and alpha-synuclein are supported by the National Institutes of Health and the Michael J. Fox Foundation.
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Spinocerebellar Ataxia Type 2 (SCA2) is an autosomal dominant neurodegenerative disorder characterized by ataxia and selective neuronal cell loss caused by the expansion of a translated CAG repeat encoding a polyglutamine tract in ataxin-2, the SCA2 gene product. The normal cellular function of ataxin-2 and the mechanism by which polyglutamine expansion of ataxin-2 causes neurodegeneration remain unknown. We are using a mutational approach to investigate the normal function of a Drosophila SCA2 ortholog (Datx2). The long-term goal of our studies is to explore the hypothesis that SCA2 pathology results from polyglutamine-mediated alteration of ataxin-2 function. We recently showed that altering the dosage of Datx2 results in locomotor defects, tissue degeneration and lethality. Examination of the tissues affected by altered Datx2 gene dosage revealed defects in actin filament organization and the appearance of polymerized actin aggregates. Datx2 is a cytoplasmic polypeptide, but fails to bind directly to monomeric actin or actin filaments, suggesting that its role in actin filament formation is indirect. Given that loss of cytoskeletal-dependent dendritic structure defines an early event in SCA2 pathogenesis, our findings suggest the possibility that dysregulation of actin cytoskeletal structure resulting from altered ataxin-2 activity is responsible for neurodegeneration in SCA2.
We are now interested in exploring the mechanism by which Datx2 regulates actin filament formation. In comparing the sequences of ataxin-2 family members from a variety of organisms, we discovered several evolutionarily conserved domains that suggest that this family of proteins assembles with RNA. Specifically, these domains include SM motifs and a poly(A)-binding protein interaction domain. To investigate the hypothesis that Datx2 assembles with RNA we recently carried out sucrose density gradient experiments and find that Datx2 sediments with RNA and polysomes. Further, this sedimentation profile is sensitive to RNAse treatment and translation inhibitors. We obtained similar results in studies of human ataxin-2. We are now investigating a role for Datx2 in translation using a variety of in vitro and in vivo approaches, including RNAi in Drosophila cell lines. We are also investigating the Datx2 domains and trans acting factors responsible for Datx2 recruitment to RNA, and are pursuing identification of the transcripts subject to Datx2 regulation using microarray analysis. These studies will provide a foundation for directly investigating the effect of polyglutamine expansion on ataxin-2 activity.
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