INVESTIGATION OF THE ROLE OF SMN IN NEURONAL APOPTOSIS

本研究的目的是探讨运动神经元存活蛋白（SMN）在神经元凋亡中的作用。 我们建立了一个辛德毕斯病毒模型系统，以研究运动神经元存活蛋白（SMN）在体内神经元凋亡中的作用。 辛德毕斯病毒是一种嗜神经病毒，可引起以神经元凋亡为特征的幼龄小鼠严重感染。 在该模型系统中，病毒既作为细胞死亡刺激物，又作为在CNS神经元中特异性表达SMN的载体。 初步研究揭示了第一个证据表明，SMN调节神经元的易感性，在体内程序性细胞死亡，我们提出，这种功能的基础上看到的脊髓性肌萎缩症（SMA）患者的神经元死亡。 SMN蛋白不仅保护神经元免于凋亡性死亡，而且在SMA患者中发现的天然突变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，在经历细胞凋亡的神经元中，SMN被胱天蛋白酶-6切割，产生类似于SMA患者中发现的截短SMN蛋白的截短蛋白，并且这种截短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调节细胞凋亡的机制。 我们将采用辛德毕斯病毒载体模型系统进行体内基因递送和诱导神经元凋亡。 将研究对促凋亡和抗凋亡功能至关重要的SMN结构域，并确定凋亡期间产生的SMN切割产物的身份。 将检查胱天蛋白酶-6在体内SMN切割中的作用以及这种切割的抑制是否调节SMN保护神经元免于凋亡的能力。 调查将研究潜在的机制，SMN的能力，以调节神经元凋亡，包括是否SMN衍生物调节细胞凋亡过程中所选的前mRNA转录的差异剪接。 最后，将采用转基因小鼠的方法，以进一步的机制探索SMN的能力，改变神经元的生存，并确定SMN在神经发育和介导细胞对凋亡刺激的反应中的作用。

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.