The COPA vesicle protein and pathogenesis of spinal muscular atrophy

描述(申请人提供)：我们发现SMN蛋白与α-COP结合，α-COP是七聚体COPI囊泡的最大成分。本项目的研究假设是，SMA的发病机制是由于这种货物运输复合体无法维持运动神经元的功能完整性所致。我们探讨了SMN蛋白与α-COP的相互作用在轴突发育和维持中的作用。重要的是，我们观察到，在SMN耗尽的NSC34细胞中，α-COP的过度表达可以恢复轴突的发育。新的数据显示，低水平的SMN改变了内质网-高尔基体运输的功能，这表明在这一蛋白质加工途径上存在以前未被认识到的影响。我们最近报道，α-COP复合体整合了分化的NSC-34细胞总转录组中的~800个特异RNA。其中很高比例的mRNA3‘端非翻译区含有一个G-四链基序，该基序在轴突定位中起着重要作用。我们的目标是识别依赖SMN与COPI复合体相关的mRNAs，从而能够研究α-COP和SMN在这些RNA进入轴突的运输中的作用，并表征它们对神经元发育的要求。为了检验SMN与α-COP相互作用的生物生理学意义，以及随后的机制研究，我们产生了新的转基因小鼠，α-COP蛋白水平降低，并预测这将导致运动单位功能障碍。我们还创造了一种转基因菌株，可以过度表达标记的人α-COP。这些小鼠将与低水平SMN的SMA模型小鼠杂交，以检验以下假设：增加α-COP和COPI囊泡促进SMN依赖的货物运送到轴突，恢复运动技能和延长寿命。这些实验小鼠模型将成为研究神经退行性变机制以及蛋白质和特定RNA向轴突和轴突内运输的重要资源。药物诱导COPI通路可能是治疗SMA的一种新手段。

DESCRIPTION (provided by applicant): We discovered that the SMN protein binds to alpha-COP, the largest constituent of the heptameric COPI vesicle. The hypothesis to be investigated in this project is that pathogenesis of SMA results from inability of this cargo transport complex to sustain the functional integrity of motor neurons. We explore the role of the SMN protein's interaction with alpha-COP in neurite development and maintenance. Importantly, we observed that over-expression of alpha-COP restores neurite development in SMN depleted NSC34 cells. New data reveal that low levels of SMN alter the functionality of endoplasmic reticulum-Golgi trafficking, suggesting a previously unrecognized effect on this protein processing pathway. We recently reported that the alpha-COP complex incorporates ~800 specific RNAs from the total transcriptome of differentiated NSC-34 cells. A high fraction of these mRNAs contain in their 3' untranslated regions a G-quadruplex motif, which has been assigned a role in neurite localization. Our goal is to identify the mRNAs that depend on SMN for association with the COPI complex, enabling studies of the roles of alpha-COP and SMN in the trafficking of these RNAs into the axon and characterization of their requirement for neuronal development. To examine the biological physiologic significance of the interaction of SMN with alpha-COP and subsequent mechanistic studies, we generated novel transgenic mice with reduced levels of alpha-COP protein, with the prediction this will result in motor unit dysfunction. We also have created a transgenic strain that over-expresses tagged human alpha- COP. These mice will be crossed with SMA model mice with low levels of SMN to test the hypothesis that increased levels of alpha-COP and COPI vesicles promote SMN dependent cargo delivery to the axon and restore motor skills and increase lifespan. These experimental mouse models will be important resources to study the mechanism of neurodegeneration and the transport of proteins and specific RNAs to and within the axon. Pharmacologic induction of the COPI pathway may represent a novel means to treat SMA.