INVESTIGATION OF THE ROLE OF SMN IN NEURONAL APOPTOSIS

本研究旨在探讨运动神经元存活蛋白（SMN）在神经元凋亡中的作用。我们建立了一个Sindbis病毒模型系统来研究运动神经元存活蛋白（SMN）在体内神经元凋亡中的作用。Sindbis病毒是一种嗜神经病毒，可引起以神经元凋亡为特征的幼鼠严重感染。在这个模型系统中，病毒既作为细胞死亡刺激，又作为在中枢神经系统神经元中特异性表达SMN的载体。初步研究揭示了SMN在体内调节神经元对程序性细胞死亡的易感性的第一个证据，我们提出这种功能是脊髓性肌萎缩症（SMA）患者神经元死亡的基础。SMN蛋白不仅保护神经元免于凋亡死亡，而且在SMA患者中发现的自然发生的突变SMN蛋白加速神经元死亡，导致小鼠死亡率增加。进一步的数据表明，在一个模型中，发生凋亡的神经元中的SMN被Caspase-6切割，产生一个截断的蛋白，类似于SMA患者中发现的截断的SMN蛋白，并且该截断的蛋白可能具有促死亡功能。我们现在建议进一步分析以确定SMN调控细胞凋亡的机制。我们将采用Sindbis病毒载体模型系统进行体内基因传递和诱导神经元凋亡。对促凋亡和抗凋亡功能至关重要的SMN结构域将被研究，细胞凋亡过程中产生的SMN切割产物的身份将被确定。Caspase-6在体内SMN切割中的作用以及抑制这种切割是否会调节SMN保护神经元免于凋亡的能力将被研究。研究将研究SMN调节神经元凋亡能力的潜在机制，包括SMN衍生物是否在细胞凋亡过程中调节选择的前mrna转录物的差异剪接。最后，将采用转基因小鼠方法进一步探索SMN改变神经元存活能力的机制，并确定SMN在神经发育和介导细胞凋亡刺激反应中的作用。

The goal of this proposal is to investigate the role of the Survival of Motor Neuron (SMN) protein in neuronal apoptosis. We have generated a Sindbis virus model system to investigate the role of Survival of Motor Neuron (SMN) protein in neuronal apoptosis in vivo. Sindbis virus is a neuronotropic virus that causes a severe infection in young mice characterized by neuronal apoptosis. In this model system, the virus serves both as a cell death stimulus and as a vector expressing SMN specifically in CNS neurons. Preliminary studies reveal the first evidence that SMN modulates the susceptibility of neurons to programmed cell death in vivo and we propose that this function underlies the neuronal death seen in patients with Spinal Muscular Atrophy (SMA). Not only does SMN protein protect neurons from apoptotic death, but naturally occurring mutant SMN proteins found in SMA patients accelerate neuronal death, causing increased mortality in mice. Further data suggest a model in which SMN is cleaved by Caspase-6 in neurons undergoing apoptosis, creating a truncated protein that resembles a truncated SMN protein found in a patient with SMA, and that this truncated protein may have pro-death function. We now propose to further this analysis to determine the mechanisms that underlie the regulation of apoptosis by SMN. We will employ the Sindbis virus vector model system for in vivo gene delivery and induction of neuronal apoptosis. Domains of SMN critical for both pro- and anti-apoptotic functions will be investigated, and the identity of the SMN cleavage product created during apoptosis will be determined. The role of Caspase-6 in cleavage of SMN in vivo and whether suppression of this cleavage modulates the ability of SMN to protect neurons from apoptosis will be examined. Investigations will examine potential mechanisms that underlie the ability of SMN to modulate neuronal apoptosis, including whether SMN derivatives modulate differential splicing of selected pre-mRNA transcripts during apoptosis. Finally, a transgenic mouse approach will be employed to further the mechanistic exploration of SMN's ability to alter neuronal survival, and to determine the role of SMN in neural development and in mediating cellular responses to apoptotic stimuli.