Role of Stasimon Dysfunction in Spinal Muscular Atrophy

项目摘要 脊髓性肌萎缩症是一种以运动神经元缺失为特征的遗传性神经退行性疾病 和骨骼肌萎缩。SMA是婴儿期死亡的最常见遗传原因，但没有有效的治疗方法。 治疗目前可用。尽管已经确定运动神经元存活水平的降低 (SMN)SMN 1基因纯合突变引起的蛋白质引起SMA，SMA是运动神经元的分子基础。 神经元功能障碍是未知的。鉴定受SMN缺乏影响的分子， 有助于SMA病理学的研究不仅是阐明疾病机制， 开发有效的治疗方法。在以前的研究中，我们发现了一种新的，进化上保守的 跨膜蛋白-我们命名为Stasimon-其表达因SMN缺乏而减少， 导致果蝇和斑马鱼SMA模型中SMN依赖性运动神经元功能障碍。 由于这些动物模型不具有SMA患者特有的低稳定SMN水平，因此， 该项目将研究Stasimon功能降低是否有助于小鼠模型中的SMA病理学 与人类疾病更相似到目前为止，SMN靶基因与一个证明的作用， SMA小鼠的发病机制尚未描述。在目标1中，我们将分析SMN缺陷的影响 在SMA小鼠组织中的mRNA和蛋白质水平上的Stasimon表达，特别关注 脊髓激光捕获显微切割将用于分离选定的疾病相关神经元。 用于RNA分析。这些将包括腹侧的运动神经元（MN） 腰髓角和位于背根神经节（DRG）的本体感受神经元。变化 在Stasimon中的表达将在来自不同运动柱（外侧和内侧）的MN中进行研究， 不同的腰椎节段以时间依赖性方式受到SMN缺乏的不同影响。 免疫组织化学将用于确定在正常条件下Stasimon蛋白的体内分布。 条件和任何变化引起的SMN缺乏在相同的神经类型。在目标2中，我们将研究 增加Stasimon表达对SMA小鼠表型的影响，以评估其参与 SMA病理学。最近的研究表明，出生后通过全身性增加SMN水平， 注射从普遍存在的启动子表达SMN的腺相关病毒（AAV 9）拯救SMA， 严重小鼠模型中的表型。我们将使用这种先前验证的AAV 9介导的基因递送， SMA小鼠中Stasimon表达系统。然后将进行一系列全面的检测， 感觉运动回路形态和功能参数的监测Stasimon依赖性改善 SMA中严重受损的连接。总的来说，这些实验有可能 鉴定Stasimon作为导致小鼠SMA病理学SMN功能障碍的下游靶点 这种毁灭性的人类疾病的模型。

Project Summary Spinal muscular atrophy (SMA) is an inherited neurodegenerative disease characterized by motor neuron loss and skeletal muscle atrophy. SMA is the most common genetic cause of death in infancy, but no effective treatment is currently available. Although it is well established that reduced levels of the survival motor neuron (SMN) protein due to homozygous mutations in the SMN1 gene cause SMA, the molecular basis of motor neuron dysfunction are unknown. The identification of molecules that are affected by SMN deficiency and contribute to SMA pathology is critically needed not only for elucidation of disease mechanisms but also for development of effective therapies. In previous studies, we have identified a novel, evolutionarily conserved transmembrane protein-which we named Stasimon-whose expression is decreased by SMN deficiency and that contributes to SMN-dependent motor neuron dysfunction in Drosophila and zebrafish models of SMA. Since these animal models do not have the low steady levels of SMN characteristic of SMA patients, this project will investigate whether decreased Stasimon function contributes to SMA pathology in a mouse model that more closely resemble the human disease. To date, SMN target genes with a demonstrated role in the pathogenesis of SMA mice have not been described. In Aim 1, we will analyze the effects of SMN deficiency on Stasimon expression at the mRNA and protein levels in tissues of SMA mice, with a particular focus on the spinal cord. Laser capture microdissection will be employed to isolate selected, disease-relevant neuronal types from control and SMA mice for RNA analysis. These will include motor neurons (MNs) in the ventral horns of the lumbar spinal cord and proprioceptive neurons located in the dorsal root ganglia (DRG). Changes in Stasimon expression will be investigated in MNs from different motor columns (lateral and medial) as well as distinct lumbar segments that are differentially affected by SMN deficiency in a time-dependent manner. Immunohistochemistry will be used to define the in vivo distribution of Stasimon protein under normal conditions and any changes caused by SMN deficiency in the same neural types. In Aim 2, we will study the effect of increasing Stasimon expression on the phenotype of SMA mice in order to assess its involvement in SMA pathology. Recent studies demonstrated that increasing SMN levels postnatally through systemic injection of an adeno-associated virus (AAV9) expressing SMN from a ubiquitous promoter rescues the SMA phenotype in a severe mouse model. We will use this previously validated, AAV9-mediated gene delivery system for expression of Stasimon in SMA mice. A comprehensive set of assays will then be carried out to monitor Stasimon-dependent improvement in morphological and functional parameters of sensory-motor circuit connectivity that are severely compromised in SMA. Collectively, these experiments have the potential to identify Stasimon as a downstream target of SMN dysfunction that contributes to SMA pathology in a mouse model of this devastating human disease.