Analysis and Therapy of Age-Dependent Proteostasis Failure in Neurodegeneration

Central Arkansas Veterans Hlthcare Sys, North Little Rock AR

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Abstract

DESCRIPTION (provided by applicant): Most major neurodegenerative diseases are characterized by the formation of toxic protein aggregates which compromise a specific part of the central nervous system. Clinical diagnosis has been guided by the regions affected, but it is becoming increasingly evident that the basic processes underlying Alzheimer dementia are remarkably similar to those leading to Parkinson's and Huntington's diseases, and Amyotrophic Lateral Sclerosis. Although all cells are obliged to maintain proteostatic equilibrium, neurons are at particular risk due to high rates of oxidative metabolism. Specific genetic lesions (or possibly chemical or viral exposure, in sporadic cases) may determine the site of neuropathy as the weakest link based on the balance of pro- aggregation and countervailing factors. Factors favoring aggregation include high local abundance of vulnerable proteins and structural instability (determined by the native folded structure, exposed reactive groups, and triplet-array length, if present), oxidative damage, glycation, and protein-reactive metabolites or environmental toxicants that produce cross-linking or adduction. Protective factors include antioxidant defenses (SODs, GSTs, catalases, etc.), detoxification/clearance systems, levels of HSP/chaperones to refold transiently misfolded proteins (preventing their adherence to aggregates via exposed hydrophobic residues), protein repair of covalent molecular damage that would favor misfolding, and proteasomal degradation of ubiquitinylated proteins. Autophagy may be able to clear the large insoluble aggregates diagnostic of these neuropathologies, but it remains controversial whether those are indeed the most neurotoxic complexes, or are instead less-toxic, sequestered repositories formed from smaller, soluble aggregates which are more potent neurotoxins. This issue could be resolved through a careful, comparative analysis of soluble vs. insoluble aggregates, seeded by several of the diverse initiator proteins observed in neuropathies, defining proteins and post-translational modifications in C. elegans and human neuropathology samples. The current proposal has three intertwined and complementary aims: (1.) To define proteins that aggregate in C. elegans models of Alzheimer's, initiated or seeded by neuron-specific tau and Ass transgenes. Protein complexes from nematodes with age-dependent or induced aggregates will be pulled down with antibody to tau or Ass, and then separated into detergent-insoluble and soluble fractions. Proteins from each fraction will be resolved by gel electrophoresis, and peptides will be cleaved by trypsin and analyzed by LC-MS2 to define and quantify the proteins and their post-synthetic modifications. The most sensitive and reliable quantitation uses product-ion scans to seek diagnostic fragments from predicted peptides; in parallel, we will also conduct more general but less sensitive unbiased scans to identify and quantify the more abundant proteins, even if unforeseen. Results will be cross-checked and extended by unrestrictive searches to quantify postsynthetic modifications including phosphorylation, acetylation, oxidation, acylation, glycation, etc., and thus implicate mechanisms involved in their age-dependent occurrence. Dual-label procedures will allow robust relative quantitation, and the validation of proteins that aggregated in vivo rather than during handling. (2.) To seek similar aggregates in human samples. De-identified samples from affected and control brain sites from AD subjects will be studied along with comparable-site samples from normal controls, and their aggregates immunoprecipitated with antibodies to Ass and tau. Complexes will be fractionated and separated as for Aim 1, and aggregates subjected to LC-MS2 analyses as above, seeking protein markers or modifications including any were implicated in the C. elegans models. (3.) To assess which implicated proteins and modifications play functional roles in neurotoxicity, it will be made worse by RNA interference targeting protective genes, or relieved by therapeutic drugs or RNAi against pro-aggregative genes. Proteins or modifications in either soluble or insoluble aggregates, which vary concordantly with neurotoxicity, become functional candidates