Patients with Spinal Muscular Atrophy (SMA) have mutations in the SMN1 (survival motor neuron) gene leading to the loss of spinal cord motor neurons. SMA is one of the two most common genetic causes of mortality in children. The motor neuron deficiency in SMA disease is apparently due to inappropriate cell death rather than a defect in neurogenesis. However, the molecular mechanisms by which SMN mutations lead to the premature death of this neuron subset are not known. We have developed the Sindbis virus vector system for assessing the functions of apoptosis regulators in cultured neurons and in the central nervous system (CNS) of mice. Sindbis virus is potently neuronotropic, specifically targeting neurons of the brain and spinal cord including spinal cord motor neurons. Using this model we have shown that SMN protects primary neurons (but not a variety of cell lines) from apoptotic cell death induced by Sindbis virus. SMN also protects mice from hind limb paralysis and death. However, patient mutants of SMN not only fail to protect, but rather accelerate apoptosis of neurons in culture and in mice leading to increased paralysis and early mortality. In addition, transient expression of these mutant SMN proteins in differentiated neuron-like cells also triggers apoptotic cell death in the absence of a virus infection. We will now explore the molecular mechanisms by which SMN modulates the neuronal cell death pathway. In the first aim, we seek to understand how SMNA7, commonly found in SMA patients, kills neurons. We will determine if SMN d7-induced neuronal death requires caspases or other components of the cell death machinery. The causality of assigned pathways will be explored using caspase inhibitors, transgenic and knockout mouse lines. SMN appears to be cleaved during neuronal injury. In the second aim, the role of this cleavage event in cell death, the proteases involved and the function of stable SMN cleavage fragments will be pursued. In addition, a panel of new antibodies that can distinguish cleaved and wild type forms of SMN will be developed to explore the role of proteolysis in SMN function. Finally, we will pursue the mechanisms by which full length SMN enhances neuronal survival with specific emphasis on spinal cord motor neurons.
