Function and Pathogenic Mechanism of alpha-synuclein in Parkinson's Disease

帕金森病（PD）的病理特征在于中脑多巴胺能（mDA）神经元的优先损失和称为路易体（LB）和路易神经突（LN）的α-突触核蛋白（α-syn）阳性胞质内包涵体的存在（Schapira，1997;Spillantini et al.，1997年）。虽然α-syn的错义突变和倍增突变都引起早发性常染色体显性家族性PD，但α-syn基因座也与更常见的散发性PD相关（Polymeropoulos et al. 1997;Singleton等人，2003;Simon-Sanchez等人，2009;Satake等人，2009年）。总之，这些遗传和病理学研究清楚地指出了α-syn在PD发病机制中的重要作用。 已经进行了广泛的研究以了解α-syn诱导的细胞损失的潜在致病机制。已经表明，野生型和PD相关突变体α-syn的过表达导致多种细胞毒性，包括蛋白酶体和溶酶体活性的损害（Cuervo等人，2004;Stefanis等人，2001;Tanaka等人，2001;Chen等人，2006），ER-高尔基体运输的破坏（库珀等人，2006;Gosavi等人，2002;Lin等人，2009）、线粒体功能的扰动（Hsu等人，2000;Martin等人，2006;Song等人，2004;中村等人，2011）和突触传递的抑制（Nemani等人，2010年）。然而，这些结果中的大多数是从细胞系和非mDA神经元获得的。这仍然是为了确定这些致病途径是否是病理生理相关的mDA神经元的变性。 虽然黑质DA神经元的损失是PD的主要运动综合征的基础，但PD研究的进展尤其受到缺乏携带PD相关基因突变并发展mDA神经元进行性变性的有效小鼠遗传模型的阻碍（Hisahara和Shimohama，2010）。先前已经产生了许多PD相关突变体α-syn转基因小鼠品系;然而，它们中很少表现出mDA神经元的稳健和进行性变性（Kahle et al.，2001;货车der等人，2000;Matsuoka等人，2001;Lee等人，2002;Lin等人，2009;Chesselet，2008;Harvey等人，2008;里奇菲尔德等人，2002;Gispert等人，2003;Wakamatsu等人，2008;Thiruchelvam等人，2004年）。值得注意的是，在这些突变小鼠的mDA神经元中仅观察到很少或低水平的转基因α-syn表达，其中转基因α-syn通常在泛神经元启动子或大鼠酪氨酸羟化酶（TH）启动子的转录控制下。 为了研究体内α-syn依赖性多巴胺能功能障碍的发病机制，我们通过使用二元四环素依赖性诱导基因表达系统驱动PD相关A53 T α-syn在mDA神经元中的表达来产生新的α-syn转基因小鼠品系。突变小鼠出现严重的运动障碍以及稳健和进行性mDA神经变性，从而可以提供有价值的小鼠遗传模型来研究α-syn如何诱导mDA神经元的变性。朝着这个方向，我们系统地研究了α-syn介导的突变小鼠的mDA神经元的亚细胞异常。此外，我们确定了核受体相关蛋白1（Nurr 1），一个主转录因子的发展和维护的mDA神经元，作为一个关键的下游分子靶点的α-syn诱导的优先变性的mDA神经元。 先前已经提出了Nurr 1功能障碍在PD中的作用（Le等人，2003;Chu等人，2002;Baptista等人，2003年）。我们扩展了这些早期研究，并证明野生型和A53 T α-syn的过度表达促进了Nurr 1的蛋白酶体依赖性降解。我们进一步证明，抑制蛋白酶体介导的Nurr 1降解可改善alpha-syn诱导的mDA神经元丢失。这些数据表明，抑制Nurr 1蛋白表达的α-syn是一个关键的分子决定因素的优先功能障碍和损失的mDA神经元在PD。与此观点一致，与A53 T条件性转基因小鼠相比，mDA神经元中Nurr 1的条件性缺失导致非常相似的直立障碍和mDA神经变性（Kadkhodaei等人，2009年）。已经发现多种翻译后修饰调节Nurr 1蛋白的稳定性和功能，包括磷酸化、泛素化（Jo et al.，2009）、sumoylation（Galleguillos等人，2004）和乙酰化（Kang等人，2010年）。alpha-syn可能通过多种分子级联调节Nurr 1蛋白的降解。此外，还需要确定其他PD相关基因是否也影响Nurr 1蛋白在PD中的表达和稳定性。 总之，本研究描述了一种新的alpha-syn A53 T转基因小鼠品系，其显示出mDA神经元的稳健和进行性变性。alpha-syn对mDA神经元Nurr 1蛋白稳定性的动态调节不仅有助于阐明mDA神经元在PD中优先易感性的分子机制，而且可能为PD的治疗提供新的治疗靶点。

Parkinsons disease (PD) is pathologically characterized by a preferential loss of midbrain dopaminergic (mDA) neurons and the presence of alpha-synuclein (alpha-syn)-positive intracytoplasmic inclusions named Lewy bodies (LBs) and Lewy neurites (LNs) (Schapira, 1997;Spillantini et al., 1997). While both missense and multiplication mutations of alpha-syn cause early-onset autosomal dominant familial form of PD, the alpha-syn gene locus also associates with the more common sporadic PD (Polymeropoulos et al., 1997;Singleton et al., 2003;Simon-Sanchez et al., 2009;Satake et al., 2009). Together, these genetic and pathological studies clearly point out an important role of alpha-syn in the pathogenesis of PD. Extensive studies have been performed to understand the underlying pathogenic mechanisms of alpha-syn-induced cell loss. It has been shown that over-expression of both wild-type and PD-related mutant alpha-syn leads to a variety of cytotoxicity, including the impairment of proteasome and lysosome activities (Cuervo et al., 2004;Stefanis et al., 2001;Tanaka et al., 2001;Chen et al., 2006), the disruption of ER-Golgi transport (Cooper et al., 2006;Gosavi et al., 2002;Lin et al., 2009), the perturbation of the mitochondrial function (Hsu et al., 2000;Martin et al., 2006;Song et al., 2004;Nakamura et al., 2011), and the inhibition of synaptic transmission (Nemani et al., 2010). However, most of these results were obtained from cell lines and non-mDA neurons. It remains to determine whether these pathogenic pathways are pathophysiologically relevant to the degeneration of mDA neurons. While the loss of nigral DA neurons underlie the main motor syndrome of PD, the progress of PD research has been especially hindered by a lack of effective mouse genetic model that carries PD-related genetic mutations and develops progressive degeneration of mDA neurons (Hisahara and Shimohama, 2010). Many lines of PD-related mutant alpha-syn transgenic mice have been generated previously; however, few of them exhibited robust and progressive degeneration of mDA neurons (Kahle et al., 2001;van der et al., 2000;Matsuoka et al., 2001;Lee et al., 2002;Lin et al., 2009;Chesselet, 2008;Harvey et al., 2008;Richfield et al., 2002;Gispert et al., 2003;Wakamatsu et al., 2008;Thiruchelvam et al., 2004). Noticeably, only a scarce or low levels of transgenic alpha-syn expression were observed in the mDA neurons of these mutant mice, in which the transgenic alpha-syn is often under the transcriptional control of pan neuronal promoters or a rat tyrosine hydroxylase (TH) promoter. To investigate the pathogenic mechanism of alpha-syn-dependent dopaminergic dysfunction in vivo, we generated a new line of alpha-syn transgenic mice by driving the expression of PD-related A53T alpha-syn in the mDA neurons using a binary tetracycline-dependent inducible gene expression system. The mutant mice developed profound movement disorders as well as robust and progressive mDA neurodegeneration, which thereby may provide a valuable mouse genetic model to investigate how alpha-syn induces the degeneration of mDA neurons. Towards to this direction, we systematically examined the alpha-syn-mediated subcellular abnormalities in the mDA neurons of mutant mice. Moreover, we identified nuclear receptor related 1 protein (Nurr1), a master transcription factor for the development and maintenance of mDA neurons, as a key downstream molecular target for the alpha-syn-induced preferential degeneration of mDA neurons. A contribution of Nurr1 dysfunction in PD has been proposed previously (Le et al., 2003;Chu et al., 2002;Baptista et al., 2003). We extended these early studies and demonstrated that over-expression of both wild-type and A53T alpha-syn promoted a proteasome-dependent degradation of Nurr1. We further demonstrated that inhibition of proteasome-mediated degradation of Nurr1 ameliorated alpha-syn-induced loss of mDA neurons. These data suggest that the suppression of Nurr1 protein expression by alpha-syn is a key molecular determinant for the preferential dysfunction and loss of mDA neurons in PD. In line with this notion, a conditional deletion of Nurr1 in the mDA neurons results in a very similar rearing impairments and mDA neurodegeneration compared to the A53T conditional transgenic mice (Kadkhodaei et al., 2009). Multiple post-translational modifications have been found to modulate the stability and function of Nurr1 protein, including phosphorylation, ubiquitination (Jo et al., 2009), sumoylation (Galleguillos et al., 2004), and acetylation (Kang et al., 2010). alpha-syn may regulate the degradation of Nurr1 protein through various molecular cascades. In addition, it remains to determine whether other PD-related genes also affect the expression and stability of Nurr1 protein in PD. In summary, this study describes a new line of alpha-syn A53T transgenic mice that display robust and progressive degeneration of mDA neurons. The dynamic regulation of Nurr1 protein stability by alpha-syn in the mDA neurons may not only help to address the molecular mechanism of the preferential susceptibility of mDA neurons in PD, but may also provide new therapeutic targets for the treatment of the disease.