This proposal uses in vitro and in vivo approaches to explore neurodegenerative and neuroprotective mechanisms that are active in motor neuron diseases (MNDs). There are two main disease conditions that are targeted: familial amyotrophic lateral sclerosis (FALS) associated with mutations in CU-Zn superoxide dismutase type 1 (SOD1)(FALS) (1-3) and the wasted (wst/wst) mouse which is associated with a deletion in a regulatory region of the PCNA (proliferating cell nuclear antigen) gene (4). One of the hypothesis in this project is the there is a similar pathway with respect to neuronal death in both of these disease processes. A major goal of this project is to clarify the mechanisms of cell death, and to identify agents and therapeutic approaches that are important in rescue with respect to MND-specifically SOD1 linked FALS and the wasted mouse. In the first specific aim, we will investigate the """"""""toxicity"""""""" of mutant SOD1s and identify agents that rescue neurons from mutant SOD1- induced cell death. We will express wild type and mutant SOD1 genes in primary neurons in culture and in the central nervous system (CNS) using recombinant replication-defective adenoviruses (AdVs) as vectors, and begun to determine the effect of this expression on free radical accumulation, neuronal function and viability, and aggregation of SOD1. We will also express wild type and mutant SOD1 genes in yeast to clarify whether a mutation of change in the enzyme leads to aggregation and insolubility. We will test varied agents for their ability to rescue in vitro cultured cells from mutant SOD1-induced apoptosis, and then test whether these agents will affect disease in the FALS transgenic mouse (in the third specific aim). In the second specific aim we will investigate the reasons for neuronal death in the wasted mice. We will study neurons from the wasted mouse to test whether these cells have a decreased level of PCNA message, undergo spontaneous apoptosis (in vitro and in vivo), or are more susceptible to stress conditions such as oxidative stress. A better understanding of the pathogenesis of MN death in FALS and the wasted mouse may clarify the mechanism(s) of cell death in sporadic ALS and other neurodegenerative processes and also potentially lead to the identification of effective treatments for MNDs.
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