Investigation of the role of SMN in Neuronal Apoptosis

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DESCRIPTION (provided by applicant): Patients with the motor neuron disease Spinal Muscular Atrophy (SMA) have mutations in the SMN1 (survival motor neuron) gene. The SMN protein is ubiquitously expressed and the mechanism for its neuron-specific modulation of cellular survival is not clear. We have previously developed the Sindbis virus vector system both to deliver relevant genes directly to neurons and to serve as a cell-death stimulus. Using this model, we demonstrated for the first time that SMN protects neurons (but not a variety of other cell types) in tissue culture and in vivo. Spinal motor neurons are also protected by delivery of SMN via the Sindbis virus vector, resulting in diminished paralysis, while SMN-D7 enhanced motor neuron death and paralysis. Cultured motor neurons are protected from glutamate by over expression of full-length SMN indicating that SMN-mediated protection may be downstream of excitotoxic stimuli. We now propose to continue defining the molecular pathways involved in the modulation of neuronal death by SMN. In Aim 1, we will examine the intracellular mechanisms underlying the altered survival of cultured spinal motor neurons over expressing SMN or SMN derivatives. Cellular morphology, caspase and calpain activation, calcium influx, reactive oxygen species production, cytochrome C localization and DNA fragmentation will be assessed. Subsequent experiments will then use pharmacologic and genetic approaches to modulate critical components of SMN-D7-induced killing and SMN-induced protection. In Aim 2, we will extend these findings to an in vivo model of SMA. We will determine if genetic or pharmacologic mechanisms can modulate the neuronal death that occurs in dissociated and organotypic neuron cultures derived from SMN transgenic mice. Ultimately, using information gleaned from these experiments, genetic crosses and blocking strategies (i.e. anti-oxidants, glutamate receptor blockade or apoptotic inhibitors) will be utilized in SMA mice to determine the importance and potential therapeutic relevance of such cell death pathways in SMA.