Proteomics of Apopotosis in GFP-Labeled Motor Neurons

Massachusetts General Hospital, Boston MA

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Abstract

This is a project to study Amyotrophic Lateral Sclerosis (ALS), an age-dependent neurodegenerative disease of spinal motor neurons. Mutations in the gene encoding for copper/zinc superoxide dismutase (SOD1) cause motor neuron degeneration in about 25% of familial ALS cases (FALS). Evidence indicates that the death process involves activation of apoptotic genes. The broad goal of this project is to elucidate the mechanism(s) governing mutant SODl-mediated apoptotic death. Our investigations in mouse neuroblastoma N2A cells and transgenic ALS mice show that multiple SOD1 mutants initiate a toxic cascade that entails sequential activation of caspase-1 (a slowly initiator of cell death) and caspase-3 (the final effector). The appearance of activated caspase-1 in the ALS mice months prior to motor neurons death indicates that caspase-1 plays a role as an early mediator of cell death probably by activating other apoptotic pathways. This suggests the hypothesis that at least in mutant SODl-related ALS, apoptosis is not a secondary phenomenon that contributes to the final demise of motor neurons, but rather a slow process that develops over time. There is a gradual response to the primary insult (the inherited molecular defect in the SOD1 protein) that over time enhances motor neurons susceptibility to either an exogenous stimulus (e.g. oxidative stress) or to age-related changes in apoptotic pathways. Consistent with this view, we have found that chronic activation of caspase-1 in the N2A cells does not provoke rapid cell death, but renders the cells more sensitive to an oxidative insult. At the end of the cascade, the close temporal relationship between caspase-3 activation and cell death argues that this executioner caspase directly contributes to motor neuron death. A detailed analysis of motor neuron-related apoptotic mechanisms may have a great importance in understanding the biology of motor neuron cell death. We now plan to dissect the individual steps in the cell death cascade over time in the ALS mice using high sensitive SELDI ProteinChip Technology in GFP-labeled primary motor neurons. To this end we propose to (1) Generate ALS mice in which the GFP fluorescent protein is specifically expressed in motor neurons, and (2) use the fluorescent-sorted motor neurons from these mice for SELDI analysis of apoptotic proteins.