In this project we will define a role for copper-zinc superoxide dismutase (SOD1) in the regulation of cell survival and death. While the wild-type (WT) SOD1 is a pro-survival protein, amyotrophic lateral sclerosis (ALS)-linked SOD1 mutants are toxic both in vitro and in vivo. We recently found that both WT and mutant SOD1 interact with the anti-apoptotic protein Bcl-2. However, the nature of the mutant SOD1 binding with Bcl-2 differs from WT SOD1. Contrary to WT SOD1, mutant SOD1 specifically localizes to spinal cord mitochondria where it forms SDS-resistant high molecular weight aggregates that bind and entrap Bcl-2. (Pasinelli et al, 2004, Neuron 43: 19-30). These studies suggest a potentially novel function for WT SOD1 in regulating cell survival and death, and a novel, toxic gain-of-function for mutant SOD1. Thus, while WT SOD1 may protect against cell death through its interaction with Bcl-2, mutant SOD1 may become toxic by aberrantly binding to Bcl-2 and converting Bcl-2 into a toxic or non-functional protein. In support of this hypothesis, we now have preliminary data indicating that both WT and mutant SOD1 might require Bcl-2 to exert their anti-and-pro apoptotic function respectively. With the present proposal we intend to characterize the anti and pro-death function of WT and mutant SOD1 and their respective interactions with Bcl-2. The ultimate goal is to understand the mechanism(s) of mutant SOD1-mediated toxicity and to define a potential role for the mitochondrial mutant SOD1/Bcl-2 complex in ALS pathogenesis.
The specific aims are: 1) (A) To determine whether WT SOD1 pro-survival activity depends on its binding to Bcl-2, and (B) to determine whether mutant SOD 1-mediated toxicity depends on the aberrant interaction with Bcl-2. 2) (A) To identify the region(s) in SOD1 essential for the binding with Bcl-2, and (B) to study the difference in binding strength between WT SOD1 and Bcl-2 and mutant SOD1 and Bcl-2. 3) (A) To determine whether Bcl-2 undergoes conformational modifications upon binding with mutant SOD1 and.(B) to test the potential benefit of Bcl-2 and SOD1 like-peptides that abolish binding between Bcl-2 and mutant SOD1 on our cell culture model of mutant SOD1-linked ALS. 4) To determine whether Bcl-2 mediates mutant SOD1 mitochondrial translocation. 5) To study the correlation between mutant SOD1/Bcl-2-containing aggregates and ALS using transgenic ALS mice and patients. ? ?
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