Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS) and prion protein diseases all feature misfolded proteins and their aggregates, which appear to play a role in disease pathogenesis. However, the mechanism(s) and pathways by which misfolded proteins couple to the cell death program is poorly understood. We have recently found that misfolded proteins, which trigger endoplasmic reticulum stress (ER stress), induce a novel intrinsic apoptotic pathway that is independent of Apaf-1 and mitochondria (Rao et al., 2001; Rao et al., 2002a; Rao et al., 2002b). In order to define the molecular requirements of this pathway, we have developed a cell-free system of ER stress-induced apoptosis. In this system, microsomes isolated from cells lacking ER stress fail to activate cytosolic extracts, whereas microsomes isolated from cells undergoing ER stress activate caspases-3 and -9 in cytosolic extracts. Using a set of complementary approaches including protein purification procedures, 2D MALDI-TOF/nano-ESMS (two-dimensional matrix-assisted laser desorption ionization-time of flight/nanoelectrospray mass spectrometry), immunodepletion of candidate proteins, and an RNA interference (RNAi) approach, we have identified the initial candidate biochemical mediators of this novel apoptotic pathway, and here we propose to continue to identify the components of this ER stress-induced apoptotic pathway. Because an understanding of the relationship between the accumulation of misfolded proteins, cellular stress response, and cell death programs should facilitate the development of new therapeutic strategies for neurodegenerative disorders that feature misfolded proteins, we propose to integrate the findings from our cell work with an animal model of ALS, and address the following questions: (1) What are the proteins that mediate ER stress-induced cell death? (2) Within the set of relevant proteins that are differentially expressed, what are the crucial proteins for cell death induction? (3) Is there evidence of activation of the ER stress-induced apoptotic pathway in transgenic mice expressing mutant (vs. wild type) CuZnSOD? (4) Does recombinant mutant CuZnSOD protein induce normal organelles to initiate a specific cell death pathway? We believe that the results of the proposed experiments may offer insight into the pathogenesis of neurodegenerative disorders that feature misfolded proteins, and should enhance the usefulness of our system for development of therapeutics.
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