miRNAs at the C. elegans neuromuscular junction: potential SMA modifiers

 描述(申请人提供)：脊髓性肌萎缩症(SMA)是一种以下运动神经元丧失为特征的神经系统疾病。这种退化是由导致运动神经元(SMN)蛋白存活水平下降的基因突变引起的。目前尚不清楚SMN减少是如何导致神经元死亡的，但最近的证据表明，miRNA的破坏可能起到了作用。MiRNAs是一种小的非编码RNA，被预测为调节大量mRNAs的蛋白质表达。在培养的小鼠运动神经元中，RNA解旋酶Gmin3与SMN和大量的miRNAs一起被下调。我推测，在SMN-1功能丧失(Lf)动物中，Gmin3相关的miRNAs调控不当，导致NMJ缺陷。SMN-1(Ok355)动物对敌百威有缺陷，泵减少和突触蛋白水平改变，提示神经肌肉接头(NMJ)缺陷。我的初步数据显示，线虫Gmin3直系同源基因Mel-46的缺失会导致类似的NMJ缺陷。利用组织特异性拯救分析和遗传上位性，我将证明MEL-46在影响NMJ信号转导的途径中位于SMN-1的下游。此外，我还鉴定了线虫Gmin3相关miRNAs的同源基因，这些miRNAs对于正常的NMJ突触传递是必要的，类似于SMN-1和MEL-46。我将使用在线生物信息学工具编制这些候选miRNAs的潜在mRNA靶点列表；将优先考虑具有已知突触功能的保守靶点。对于每个潜在的mRNA靶点，我将确认miRNA的调控，并调查SMN-1lf和MEL-46lf中miRNA的功能是否发生了变化。此外，我还设计了实验，证明SMN-1IF动物的miRNA功能丧失是由Mel-46减少引起的。使用SMA小鼠模型中的运动神经元，我将证明SMN、Gmin3和miRNA功能之间的关系在物种之间是保守的。这些实验将增进我们对SMN蛋白如何促进基本神经元功能的了解，并扩大我们对miRNA错误调控如何导致神经退化的理解。

 DESCRIPTION (provided by applicant): Spinal Muscular Atrophy (SMA) is a neurological disorder characterized by loss of lower motor neurons. This degeneration is caused by genetic mutations that lead to decreased levels of Survival of Motor Neuron (SMN) protein. It is currently unknown how SMN reduction results in neuronal death, however recent evidence suggests that miRNA disruption may play a role. miRNAs are small non-coding RNAs predicted to regulate protein expression of a vast number of mRNAs. The RNA helicase Gemin3 pulls down with both SMN and numerous miRNAs in cultured mouse motor neurons. I hypothesize that Gemin3-associated miRNAs are misregulated in smn-1 loss-of-function(lf) animals leading to NMJ defects. smn-1(ok355) animals are defective on aldicarb, have reduced pumping and altered levels of synaptic proteins, suggesting neuromuscular junction (NMJ) defects. My preliminary data shows that loss of the C. elegans Gemin3 ortholog, mel-46, results in similar NMJ defects. Using tissue-specific rescue analysis and genetic epistasis, I will show that mel-46 is downstream of smn-1 in a pathway influencing NMJ signaling. Additionally, I have identified C. elegans orthologs of Gemin3-associated miRNAs that are necessary for proper NMJ synaptic transmission similar to smn-1 and mel-46. I will compile lists of potential mRNA targets for these candidate miRNAs using online bioinformatics tools; assigning priority to conserved targets with known synaptic function. For each potential mRNA target, I will confirm miRNA regulation and investigate whether miRNA function is altered in smn-1 lf and mel-46 lf. Furthermore, I have designed experiments to show loss of miRNA function in smn-1 lf animals is caused by mel-46 reduction. Using motor neurons from a SMA mouse model, I will show that the relationship between SMN, Gemin3 and miRNA function is conserved across species. These experiments will advance our knowledge of how SMN protein contributes to essential neuronal functions and expand our understanding of how miRNA misregulation may contribute to neurodegeneration.