Role of Stasimon Dysfunction in Spinal Muscular Atrophy

描述（由申请人提供）：脊髓性肌萎缩症（SMA）是一种遗传性神经退行性疾病，其特征为运动神经元缺失和骨骼肌萎缩。SMA是婴儿期死亡的最常见遗传原因，但目前尚无有效的治疗方法。虽然已经确定SMN 1基因纯合突变导致运动神经元存活（SMN）蛋白水平降低会导致SMA，但运动神经元功能障碍的分子基础尚不清楚。鉴定受SMN缺陷影响并导致SMA病理学的分子不仅是阐明疾病机制的关键，也是开发有效疗法的关键。在以前的研究中，我们已经确定了一种新的，进化上保守的跨膜蛋白，我们命名为Stasimon，其表达减少SMN缺陷，并有助于SMN依赖的运动神经元功能障碍的果蝇和斑马鱼模型的SMA。由于这些动物模型不具有SMA患者特有的低稳定SMN水平，因此本项目将研究Stasimon功能降低是否有助于更接近人类疾病的小鼠模型中的SMA病理学。迄今为止，尚未描述在SMA小鼠发病机制中发挥作用的SMN靶基因。在目的1中，我们将分析SMN缺乏对SMA小鼠组织中Stasimon表达的影响，特别是脊髓。将采用激光捕获显微切割从对照和SMA小鼠中分离选定的疾病相关神经元类型用于RNA分析。这些将包括腰脊髓腹角中的运动神经元（MN）和位于背根神经节（DRG）中的本体感受神经元。将在来自不同运动柱（外侧和内侧）以及不同腰椎节段的MN中研究Stasimon表达的变化，这些节段以时间依赖性方式受到SMN缺乏的差异影响。免疫组织化学将用于确定正常条件下Stasimon蛋白的体内分布以及相同神经类型中SMN缺乏引起的任何变化。在目的2中，我们将研究增加Stasimon表达对SMA小鼠表型的影响，以评估其在SMA病理学中的参与。最近的研究表明，通过全身注射从普遍存在的启动子表达SMN的腺相关病毒（AAV 9），出生后增加SMN水平，挽救了严重小鼠模型中的SMA表型。我们将使用这种先前验证的AAV 9介导的基因递送系统在SMA小鼠中表达Stasimon。然后将进行一组全面的试验，以监测SMA中严重受损的感觉-运动回路连接的形态和功能参数的Stasimon依赖性改善。总的来说，这些实验有可能将Stasimon鉴定为SMN功能障碍的下游靶标，该SMN功能障碍有助于这种毁灭性人类疾病的小鼠模型中的SMA病理学。 公共卫生相关性：SMA是一种无法治愈的运动神经元疾病，是婴儿死亡的主要遗传原因。我们将确定一个新的基因，我们已经确定其在疾病过程中的潜在参与是否有一个直接的作用，在神经肌肉病理学的小鼠模型的SMA。如果成功，这些研究将揭示一种重要的分子，用于阐明SMA的分子机制，以及开发这种遗传性疾病新治疗策略的候选靶点。

DESCRIPTION (provided by applicant): 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 is 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. PUBLIC HEALTH RELEVANCE: SMA is an incurable motor neuron disease and the leading genetic cause of death in infancy. We will establish whether a novel gene we have identified for its potential involvement in the disease process has a direct role in the neuromuscular pathology of a mouse model of SMA. If successful, these studies will reveal an important molecule for elucidation of the molecular mechanisms of SMA as well as a candidate target for development of new therapeutic strategies for this genetic disorder.