The survival of motor neuron protein in axonal mRNA localization

描述（由申请人提供）：脊髓性肌萎缩症（SMA）是婴儿死亡的主要遗传原因，也是全球第二常见的常染色体隐性遗传病。SMA的特征在于运动神经元的逐渐丧失，导致肌肉神经支配失败，从而导致进行性肌无力和最终因呼吸窘迫而死亡。SMA是由编码运动神经元存活蛋白（SMN）的SMN 1基因缺失或突变引起的。SMN及其相关的复合蛋白，功能是组装剪接体小核核糖核蛋白（snRNP）复合物。与snRNP组装中的作用一致，在SMA动物模型中观察到剪接缺陷，但是它们本身不太可能解释SMA患者中脊髓运动神经元的选择性脆弱性。了解SMN在这些脊髓运动神经元中的作用将有助于深入了解病理生理学的机制，并确定开发新SMA疗法的新靶点。SMN定位于脊髓运动神经元的轴突和树突。在轴突中，SMN与指导轴突mRNA转运的多种mRNA结合蛋白共定位和共转运。我们实验室使用严重SMA小鼠模型的初步数据表明，在这些培养的脊髓运动神经元中存在轴突mRNA定位缺陷，对mRNA的稳态水平没有影响。考虑到mRNA转运在促进轴突功能所需的局部mRNA翻译中的重要性，我们预测这种mRNA定位缺陷有助于SMA的疾病进展。我们假设SMN通过mRNA和相关蛋白（mRNP）颗粒的组装和/或转运介导轴突生长和维持所需的mRNA的轴突定位。我们将首先定义SMN在mRNA定位中的作用。在具体目标一中，使用来自野生型和SMA小鼠模型的培养的脊髓运动神经元;我们将使用原位方法来测试mRNP颗粒的组装和转运。在具体的目标二，我们将扩大SMN缺陷的运动神经元中已知的错误定位的轴突mRNA的列表，使用干细胞衍生的运动神经元结合区室化培养和RNA测序方法。RNA-seq方法将为我们提供轴突中具有改变的稳态水平的所有转录物的全基因组视图。通过这项工作，我们将进一步追求我们的长期目标，了解mRNA定位作为轴突生长和稳态调节剂的重要性，以及mRNA定位缺陷如何促进SMA的病理生理学。通过鉴定新的错误定位的mRNA转录本和SMN在mRNA定位中的作用，本研究将为SMA疗法的开发确定新的靶点。

DESCRIPTION (provided by applicant): Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality and the second most common autosomal recessive genetic disorder worldwide. SMA is characterized by gradual loss of motor neurons leading to failed innervation of muscles which results in progressive muscle weakness and eventual death due to respiratory distress. SMA results from either a deletion or mutation in the SMN1 gene, encoding the survival of motor neuron protein (SMN). SMN and its associated complex proteins, function to assemble spliceosomal small nuclear ribonucleoprotein (snRNP) complexes. Consistent with a role in snRNP assembly, splicing defects are seen in SMA animal models, however they alone unlikely explain the selective vulnerability of spinal motor neurons in SMA patients. Understanding the role for SMN in these spinal motor neurons will provide insight into the mechanisms underlying the pathophysiology and identify new targets for development of new SMA therapies. SMN localizes to axons and dendrites in spinal motor neurons. In the axon, SMN co- localizes and co-transports with multiple mRNA binding proteins that direct axonal mRNA transport. Preliminary data from our lab using a severe SMA mouse model demonstrate an axonal mRNA localization defect in these cultured spinal motor neurons, without effects on the steady state levels of mRNAs. Given the importance of mRNA transport in facilitating localized mRNA translation needed for axonal function, we predict that this defect in mRNA localization contributes to disease progression in SMA. We hypothesize that SMN mediates axonal localization of mRNAs required for growth and maintenance of the axon, through the assembly and/or transport of mRNA and associated protein (mRNP) granules. We will first define the role for SMN in mRNA localization. In specific aim one, using cultured spinal motor neurons derived from both wildtype and an SMA mouse model; we will use in situ approaches to test the assembly and transport of mRNP granules. In specific aim two we will expand the list of known mislocalized axonal mRNAs in SMN-deficient motor neurons, using stem cell-derived motor neurons combined with compartmentalized cultures and an RNA-sequencing approach. The RNA-seq method will give us a genome wide view of all transcripts with an altered steady-state level in the axon. Through this work we will further pursue our long-term goal of understanding the importance of mRNA localization as a regulator of axon growth and homeostasis and how defects in mRNA localization contribute to the pathophysiology of SMA. Through identification of novel mislocalized mRNA transcripts and a role for SMN in mRNA localization, this research will identify novel targets for the development of SMA therapies.