Investigation of the role of SMN in Neuronal Apoptosis

描述(申请人提供)：运动神经元病脊髓性肌萎缩症(SMA)患者的SMN1(存活运动神经元)基因发生突变。SMN蛋白广泛表达，其神经元特异性调节细胞存活的机制尚不清楚。我们之前已经开发了Sindbis病毒载体系统，既可以将相关基因直接传递到神经元，也可以作为细胞死亡刺激。利用这个模型，我们首次证明了SMN在组织培养和体内保护神经元(但不保护各种其他类型的细胞)。脊髓运动神经元也受到Sindbis病毒载体SMN的保护，导致瘫痪减轻，而SMN-D7则增加了运动神经元的死亡和瘫痪。全长SMN的过度表达可保护培养的运动神经元免受谷氨酸的影响，这表明SMN介导的保护作用可能位于兴奋性毒性刺激的下游。我们现在建议继续定义SMN调节神经元死亡的分子通路。在目标1中，我们将研究通过表达SMN或SMN衍生物改变培养的脊髓运动神经元存活的细胞内机制。将评估细胞形态、半胱氨酸天冬氨酸氨基转移酶和钙蛋白酶的激活、钙内流、活性氧产生、细胞色素C定位和DNA片段化。随后的实验将使用药理学和遗传学方法来调节SMN-D7诱导的杀伤和SMN诱导的保护的关键成分。在目标2中，我们将把这些发现扩展到SMA的活体模型。我们将确定遗传或药物机制是否可以调节来自SMN转基因小鼠的分离和器官型神经元培养中发生的神经元死亡。最终，利用从这些实验中收集的信息，基因交叉和阻断策略(即抗氧化剂、谷氨酸受体阻滞剂或凋亡抑制剂)将在SMA小鼠中被利用，以确定这些细胞死亡途径在SMA中的重要性和潜在的治疗相关性。

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.