Mechanism Of Pathologic Neuronal Apoptosis Several converging lines of inquiry suggest that delayed neuronal loss associated with diseases such as stroke or spinal chord trauma occurs by a process known as apoptosis. Enhanced understanding of the common and distinct mechanism by which apoptosis is triggered may thus reveal novel therapeutic approaches to limit the neurologic disability associated with these diseases. We previously demonstrated that oxidative death in cortical neurons can be abrogated by the antioxidant, N-acetylcysteine (NAC, 100 muM), a drug commonly used in humans to treat acetaminophen toxicity. To determine whether NAC is broadly applicable as an anti-apoptotic agent, we examined the effects of this antioxidant in another paradigm of neuronal apoptosis: infection with Sindbis Virus (SV). SV is a neurotropic virus which can be utilized to model encephalitis due to related human neurotropic viruses. We found that NAC prevented SV-induced apoptosis, but that much higher concentrations of the drug were required (30mM). In preliminary studies, we correlated the protective effects NAC (in two cultured cell lines) with its ability to inhibit SV-induced nuclear translocation of the redox-sensitive transcription factor, NF-kappa B (NF- kappaB). Inhibition of NF-kappaB directly using molecular approaches prevented SV-induced death in one or two cell lines examined. These studies leave unanswered two important questions: 1) What is NF-kappaB's role in SV induced apoptosis in neurons? and 2.) What is the mechanism of action of NAC in abrogating SV-induced NF-kappaB activation and death? In specific Aim 1 we will investigate whether SV-induced activation of NF- kappaB is required for apoptosis in primary neuronal cultures. We will utilize whether SV-induced activation of NF-kappaB is required for apoptosis in primary neuronal cultures. We will utilize phosphorothioate double stranded oligonucleotides containing NF-kappaB binding sites as decoys to inhibit binding of NF-kappaB to authentic DNA sites. Additionally, we will utilize SV as a vector not only to initiate apoptosis, but also to deliver a protein inhibitor of NF-kappaB activation.
In Specific Aim 2, we will investigate whether SV-induced NF- kappaB activation or apoptosis involves the generation of an oxidant second messenger. The multidisciplinary training emphasized in this proposal will allow great flexibility in approaching the critical issues at hand, and offer excellent preparation for a career integrating clinical investigation and molecular neuroscience.
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