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

帕金森病(PD)的病理特征是中脑多巴胺(MDA)神经元的优先丢失和胞浆内α-突触核蛋白(α-SYN)阳性包涵体的存在，称为路易小体(LBS)和路易神经节(LNS)(Schapira，1997；Spillantini等人，1997)。虽然α-syn的错义和倍增突变都会导致早发性常染色体显性家族性帕金森病，但α-syn基因座也与更常见的散发性帕金森病有关(Polymeropoulos等人，1997；Singleton等人，2003；Simon-Sanchez等人，2009；Satake等人，2009)。综上所述，这些遗传学和病理学研究清楚地指出了α-SYN在帕金森病发病机制中的重要作用。 人们已经进行了广泛的研究，以了解α-突触蛋白诱导的细胞丢失的潜在致病机制。已经证明，野生型和PD相关突变体α-syn的过度表达会导致多种细胞毒性，包括蛋白酶体和溶酶体活性的损害(Cuervo等人，2004；Stefan is等人，2001；Tanaka等人，2001；Chen等人，2006)，ER-Golgi转运的中断(Cooper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；Nakamura等人，2011年)和突触传递的抑制(Nemani等人，2010年)。然而，这些结果大多来自细胞系和非丙二醛神经元。这些致病途径是否与丙二醛神经元的退变有关还有待进一步研究。 虽然黑质DA神经元的丢失是PD的主要运动综合征的基础，但PD的研究进展尤其受到缺乏有效的小鼠遗传模型的阻碍，该模型携带PD相关的基因突变并导致MDA神经元的进行性退化(Hisahara和Shimohama，2010)。之前已经产生了许多与PD相关的突变型α-syn转基因小鼠；然而，其中很少有表现出丙二醛神经元的稳健和进行性退化(Kahle等人，2001；van der等人，2000；Matsuoka等人，2001；Lee等人，2002；Lin等人，2009；Chesslet，2008；Harvey等人，2008；Richfield等人，2002；Gispert等人，2003；Wakamatsu等人，2008；Thiruchelvam等人，2004)。值得注意的是，在这些突变小鼠的丙二醛神经元中只观察到很少或很低水平的转基因α-syn的表达，其中转基因的α-syn通常受PAN神经元启动子或大鼠酪氨酸羟化酶(TH)启动子的转录控制。 为了探讨α-SYN依赖的多巴胺能功能障碍在体内的发病机制，我们利用二进制四环素依赖的诱导基因表达系统，通过驱动PD相关的A53Tα-SYN在MDA神经元中的表达，产生了一种新的α-SYN转基因小鼠系。突变的小鼠表现出严重的运动障碍和健壮的进行性丙二醛神经元变性，这可能为研究α-syn如何诱导丙二醛神经元变性提供了一个有价值的小鼠遗传学模型。朝着这个方向，我们系统地研究了α-syn介导的突变小鼠丙二醛神经元的亚细胞异常。此外，我们发现核受体相关1蛋白(Nurr1)是丙二醛神经元发育和维持的主要转录因子，是α-突触蛋白诱导的丙二醛神经元优先变性的关键下游分子靶点。 先前已提出Nurr1功能障碍在帕金森病中的作用(Le等人，2003；Chu等人，2002；Baptista等人，2003)。我们扩展了这些早期研究，证明野生型和A53Tα-syn的过度表达促进了Nurr1蛋白酶体依赖的降解。我们进一步证明，抑制蛋白酶体介导的Nurr1降解可以改善α-SYN诱导的丙二醛神经元的损失。这些数据表明，α-SYN抑制Nurr1蛋白的表达是PD中MDA神经元优先功能障碍和丢失的关键分子决定因素。与这一概念一致，与A53T条件转基因小鼠相比，有条件地缺失MDA神经元中的Nurr1会导致非常相似的养育障碍和MDA神经变性(Kadkhodaei等人，2009年)。已发现多种翻译后修饰调节Nurr1蛋白的稳定性和功能，包括磷酸化、泛素化(Jo等人，2009)、SUMO化(Gallegulos等人，2004)和乙酰化(Kang等人，2010)。Alpha-syn可能通过多种分子级联途径调节Nurr1蛋白的降解。此外，其他PD相关基因是否也影响Nurr1蛋白在PD中的表达和稳定性还有待进一步研究。 综上所述，这项研究描述了一种新的α-syn A53T转基因小鼠，它们表现出健壮的和进行性的丙二醛神经元退化。α-SYN对MDA神经元中Nurr1蛋白稳定性的动态调节，不仅有助于阐明PD时MDA神经元优先易感的分子机制，而且可能为该病的治疗提供新的靶点。

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.