INVESTIGATION OF THE ROLE OF SMN IN NEURONAL APOPTOSIS

SMN 在神经元凋亡中的作用研究

• 批准号: 6024691
• 负责人: DOUGLAS A KERR
• 金额: $ 10.08万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6024691
• 关键词: RNA splicing; Sindbis virus; apoptosis; cysteine endopeptidases; disease /disorder model; enzyme activity; genetically modified animals; laboratory mouse; laboratory rat; motor neurons; neural growth associated protein; neuroregulation; neurotropic virus; precursor mRNA; protein structure function; site directed mutagenesis; superoxide dismutase; transfection /expression vector

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.

该提案的目的是研究运动神经元存活（SMN）蛋白在神经元凋亡中的作用。 我们建立了辛德比斯病毒模型系统来研究运动神经元存活（SMN）蛋白在体内神经元凋亡中的作用。 辛德比斯病毒是一种嗜神经病毒，可引起幼鼠严重感染，其特征是神经元凋亡。 在这个模型系统中，病毒既充当细胞死亡刺激物，又充当特异在 CNS 神经元中表达 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敏感性的第一个证据，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 蛋白不仅可以保护神经元免于细胞凋亡，而且在 SMA 患者中发现的自然发生的突变 SMN 蛋白也会加速神经元死亡，导致小鼠死亡率增加。进一步的数据表明，在一个模型中，SMN 在经历凋亡的神经元中被 Caspase-6 裂解，产生一种截短的蛋白质，类似于 SMA 患者中发现的截短的 SMN 蛋白质，并且这种截短的蛋白质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。 我们将采用辛德比斯病毒载体模型系统进行体内基因传递和诱导神经元凋亡。 我们将研究对促凋亡和抗凋亡功能都至关重要的 SMN 结构域，并确定细胞凋亡过程中产生的 SMN 裂解产物的身份。 将检查 Caspase-6 在 SMN 体内裂解中的作用，以及抑制这种裂解是否会调节 SMN 保护神经元免于凋亡的能力。 研究将检查 SMN 调节神经元凋亡能力的潜在机制，包括 SMN 衍生物是否在凋亡过程中调节选定的前 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对凋亡刺激的反应中的作用。