Investigation of the role of SMN in Neuronal Apoptosis

描述（由申请人提供）：患有运动神经元疾病脊髓性肌萎缩症（SMA）的患者具有SMN1（存活运动神经元）基因突变。SMN蛋白普遍表达，其神经元特异性调节细胞存活的机制尚不清楚。我们之前已经开发出Sindbis病毒载体系统，既可以将相关基因直接传递给神经元，也可以作为细胞死亡刺激。使用该模型，我们首次证明了SMN在组织培养和体内保护神经元（而不是各种其他细胞类型）。通过Sindbis病毒载体传递SMN也可保护脊髓运动神经元，从而减少瘫痪，而SMN- d7则可增强运动神经元的死亡和瘫痪。培养的运动神经元通过过表达全长SMN来保护谷氨酸，这表明SMN介导的保护可能是在兴奋毒性刺激的下游。我们现在建议继续确定SMN调节神经元死亡的分子途径。在目的1中，我们将研究过度表达SMN或SMN衍生物的培养脊髓运动神经元存活改变的细胞内机制。将评估细胞形态、半胱天蛋白酶和钙蛋白酶激活、钙内流、活性氧产生、细胞色素C定位和DNA片段。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的体内模型。我们将确定遗传或药理学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到的信息，基因杂交和阻断策略（即抗氧化剂，谷氨酸受体阻断或凋亡抑制剂）将在SMA小鼠中使用，以确定SMA中这些细胞死亡途径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.