The P.I. proposes to use the nematode C. elegans as a model system to study the pathophysiology underlying Alzheimer's disease. In his preliminary results he has expressed a fragment of human amyloid precursor protein (APP), called unc-54/b1-42, in the somatic musculature of the worm. Extracellular deposits of APP are most likely one of the underlying causes of Alzheimer's disease in humans, although it is unclear how they cause the widespread neuronal death typical of this disease. Expression of the APP fragment b1-42 leads to intracellular deposits in the musculature, although no extracellular secretion could be noted. However, vacuoles in the CNS developed which in other mutations are sign of cell death. The proposal entails the following four sets of experiments: 1. Analysis of the phenotype of the existing APP expressing lines in greater detail: TEM and specific markers will be used to establish in which cells of the CNS vacuoles appear; suspected are glia cells surrounding the neurons. Other experiments address the pathophysiological connection between b-peptide expression and vacuole formation. Intracellular Ca levels will be monitored by injection of the fluorochrome fura-2. b1-42 expressing animals will be reared in different ionic conditions to test the influence of these ions on vacuole formation. Sodium-channel blockers will be applied to test whether sodium channels are involved in vacuole formation. A number of mutations known to suppress vacuole formation during regular neuronal cell death will be tested as to their ability to also suppress vacuoles in b-peptide expressing animals. 2. Generation of novel lines in which other b1-42 variants and different regulatory elements will be used to achieve expression in other tissues. In particular, promoters of the mec-7, unc-4 and unc-119 genes will be used which should drive expression in subsets of neurons. To achieve secretion of the b1-42, several experiments will be carried out. It will be first established whether the too small size of the peptide is responsible, by fusing the peptide with green fluorescent protein (GFP) and thereby increasing its size. Alternatively, the signal sequence used in the unc-54/b1-42 construct may be faulty; signal sequences which successfully have lead to secretion of another peptide, transthyretin, will be used. Finally, a C. elegans gene, bli-4, encoding a pro-protease routed to the secretory pathway will be fused with b-peptide to ensure its secretion. Several new constructs in which selected residues of the b peptide are changed in vitro will be transformed into worms to test for increase or decrease of the phenotype caused. The P.I. is guided by vertebrate cell culture studies in which the corresponding residue changes were shown to modify the toxicity of the APP deposits in a certain way. 3. Coexpression of other human proteins suspected to modify the deposition of APP together with b1-42 peptide. Preliminary results had shown that co-expression of b1-42 and wild-type transthyretin reduces the number of amyloid-like deposits in transgenic worms. The interaction between the two proteins suggested by this result will be further tested by coexpressing a mutant form of transthyretin which in an in vitro assay does not interact with b-peptide. In another experiment, the gene encoding apolipoprotein E which according to clinical and in vitro data enhances the formation of amyloid, will be co-expressed with b1-42 to test whether the interaction can be replicated in the worm system. 4. Genetic screen for C. elegans genes modifying the phenotype caused by b1-42 expression is proposed. Three screens are proposed: A. b1-42 expressing animals will be treated with EMS; the F2 will be stained with the histological stain Congo Red to visualize whether increase or decrease in the amount of deposits has occurred. Animals of interest will be recovered and their progeny retested for altered deposits. Animals with altered deposits will be tested by ELISA whether the level of expression of b1-42 has not changed, since only mutations in genes which affect the ability of deposit formation are of interest. B. Screen for suppressors of the paralysis phenotype: it was observed that b-peptide expressing animals suffer from paralysis, possibly caused by intramuscular amyloid deposits. An EMS mutagenesis to obtain mutations in genes which affect the paralysis phenotype is proposed. C. Screen for suppressors of the vacuole phenotype. Since the vacuoles in b1-42 expressing animals are only infrequently observed, possibly due to the fragmentation of the degenerating cell, the ced-2 mutant which allows dead cells to persist will be introduced into the unc-54/b1-42 background.
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