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

1.Nurr1决定帕金森病模型小鼠中脑多巴胺能神经元的优先变性 中脑DA神经元的功能特性和存活依赖于核受体相关1(Nurr1)的构成活性，也称为核受体亚家族4，A组，成员2(NR4A2)(Perlmann和Wallen-Mackenzie，2004)。Nurr1调控酪氨酸羟化酶(TH)、多巴胺转运体(DAT)、受体酪氨酸激酶Ret和其他对中脑DA神经元的功能和维持至关重要的多巴胺能基因的表达。从生殖细胞中基因消融Nurr1会导致缺乏成熟的中脑DA神经元和过早死亡(Zetterstrom等人，1997)，而在中脑DA神经元的末端分化后缺失Nurr1会导致中脑DA神经元标记蛋白的类似PD的进行性丢失，最终导致中脑DA神经元的死亡(Kadkhodaei等人，2009)。Nurr1的表达在转录和翻译后水平上都受到动态调节。核因子B(核因子B)和cAMP反应元件结合蛋白(CREB)通路被认为可以调节Nurr1mRNA的转录(McEworth等人，2002年)。与此同时，已发现多种翻译后修饰，如磷酸化、泛素化(Jo等人，2009)、SUMO化(Gallegulos等人，2004)，可能还有乙酰化(Kang等人，2010)，在各种细胞事件中调节Nurr1蛋白的稳定性和功能。更有趣的是，在死后帕金森病患者的大脑中，中脑DA神经元中Nurr1的表达显著减少，这些神经元中也含有α-syn阳性包涵体(Chu等人，2006年)。然而，在帕金森病大脑中的观察并没有说明Nurr1水平的降低是否只是一种终末期的结果，或者是一种更动态的依赖于SYN的致病机制，直接与疾病有关。 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，我们通过选择性地驱动PD相关的人α-SYN A53T错义突变在中脑DA神经元中的表达，建立了一个新的α-SYN转基因小鼠系。突变的小鼠出现了严重的运动障碍，以及健壮和进行性的中脑DA神经元退化，概括了帕金森病的关键临床和神经病理特征。除神经元丢失外，突变小鼠中脑DA神经元的内质网/高尔基体网络和自噬/溶酶体通路的结构/功能以及递质多巴胺的释放也受到显著影响。更重要的是，在中脑DA神经元中，α-syn的过度表达显著抑制了Nurr1mRNA的转录，并诱导了Nurr1蛋白的蛋白酶体依赖性降解。综上所述，我们的发现证明了Nurr1抑制在阿尔法-SYN介导的帕金森病中脑DA神经元优先易损性中的意义。作为一项原则证明，阻断蛋白酶体依赖的Nurr1降解挽救了α-SYN诱导的中脑DA神经元的丢失。 *文稿正在为上述数据做准备 2.帕金森病相关A53Tα-突触核蛋白的星形细胞表达导致小鼠神经变性。 帕金森氏病(PD)是最常见的运动障碍。虽然突触核蛋白的神经元沉积是帕金森病和路易小体痴呆的病理标志，但星形胶质细胞中也存在突触核蛋白阳性的蛋白聚集体。然而，星形细胞聚积-突触核蛋白的病理后果尚不清楚。在这里，我们展示了帕金森病相关的A53T突变体&突触核蛋白，当选择性地在星形胶质细胞中表达时，会在小鼠中诱导快速进展的瘫痪。在症状前和有症状的小鼠大脑中发现了越来越多的突触核蛋白聚集体的积累，并与反应性星形胶质细胞增生的扩大有关。星形胶质细胞的正常功能受到损害，表现为脑微出血和星形胶质细胞谷氨酸转运体下调，这也导致炎症反应增加和小胶质细胞激活。有趣的是，小胶质细胞的激活主要见于中脑、脑干和脊髓，其中多巴胺能神经元和运动神经元显著丢失。与小胶质细胞的激活相一致，环氧合酶1(COX-1)在有症状的小鼠脑中的表达水平显著上调，在经A53T-突触核蛋白过度表达的星形胶质细胞条件培养液处理后的小胶质细胞中也显著上调。因此，抑制COX-1活性延长了突变小鼠的存活时间，这表明由反应性星形胶质细胞引发的过度炎症反应可能有助于神经元的退化。我们的研究结果表明，星形胶质细胞突触核蛋白在启动神经元的非细胞自主杀伤过程中起着关键作用，这表明反应性星形胶质细胞和小胶质细胞的活性是帕金森病和其他神经退行性疾病的潜在治疗靶点。

1. Nurr1 determines the preferential degeneration of midbrain dopaminergic neurons in a Parkinsons disease mouse model The functional properties and survival of midbrain DA neurons rely on the constitutive activities of nuclear receptor related 1 (Nurr1), also known as nuclear receptor subfamily 4, group A, member 2 (NR4A2) (Perlmann and Wallen-Mackenzie, 2004). Nurr1 controls the expression of tyrosine hydroxylase (TH), dopamine transporter (DAT), receptor tyrosine kinase Ret, and other dopaminergic genes critical for the function and maintenance of midbrain DA neurons. Genetic ablation of Nurr1 from the germ cells results in a lack of mature midbrain DA neurons and premature lethality (Zetterstrom et al., 1997), while deletion of Nurr1 after the terminal differentiation of midbrain DA neurons leads to a PD-like progressive loss of midbrain DA neuron marker proteins and eventually the death of midbrain DA neurons (Kadkhodaei et al., 2009). The expression of Nurr1 is dynamically regulated at both the transcriptional and post-translational levels. Nuclear factor-κB (NF-κB) and cAMP response element-binding protein (CREB) pathways have been suggested to regulate the transcription of Nurr1 mRNA (McEvoy et al., 2002). Meanwhile, multiple post-translational modifications such as phosphorylation, ubiquitination (Jo et al., 2009), sumoylation (Galleguillos et al., 2004), and perhaps acetylation (Kang et al., 2010) have been found to modulate the stability and function of Nurr1 protein in various cellular events. More interestingly, in postmortem PD brains the expression of Nurr1 is significantly decreased in the midbrain DA neurons that also contain alpha-syn-positive inclusions (Chu et al., 2006). However, the observations in PD brains do not address whether the decreased Nurr1 levels are just an end-stage consequence or a more dynamic α-syn-dependent pathogenic mechanism directly involved in the disease. To investigate the pathogenic mechanism of alpha-syn-dependent dopaminergic dysfunction in vivo, we generated a new line of alpha-syn transgenic mice by selectively driving the expression of PD-related human alpha-syn A53T missense mutation in the midbrain DA neurons. The mutant mice developed profound movement disorders, as well as robust and progressive midbrain DA neuron degeneration, recapitulating the key clinical and neuropathological features of PD. In addition to the neuronal loss, the structure/function of ER/Golgi networks and autophagosome/lysosome pathways, and the release of transmitter dopamine were also significantly affected in the midbrain DA neurons of mutant mice. More importantly, over-expression of alpha-syn substantially abrogated the transcription of Nurr1 mRNA and evoked the proteasome-dependent degradation of Nurr1 protein in midbrain DA neurons. Together, our findings demonstrate the significance of Nurr1 inhibition in alpha-syn-mediated preferential vulnerability of midbrain DA neurons in PD. As a proof-of-principle, the blockage of proteasome-dependent degradation of Nurr1 rescued the alpha-syn-induced loss of midbrain DA neurons. * Manuscript is in preparation for the above data. 2. Astrocytic expression of Parkinson's disease-related A53T alpha-synuclein causes neurodegeneration in mice. Parkinson's disease (PD) is the most common movement disorder. While neuronal deposition of α-synuclein serves as a pathological hallmark of PD and Dementia with Lewy Bodies, α-synuclein-positive protein aggregates are also present in astrocytes. The pathological consequence of astrocytic accumulation of α-synuclein, however, is unclear. Here we show that PD-related A53T mutant α-synuclein, when selectively expressed in astrocytes, induced rapidly progressed paralysis in mice. Increasing accumulation of α-synuclein aggregates was found in presymptomatic and symptomatic mouse brains and correlated with the expansion of reactive astrogliosis. The normal function of astrocytes was compromised as evidenced by cerebral microhemorrhage and down-regulation of astrocytic glutamate transporters, which also led to increased inflammatory responses and microglial activation. Interestingly, the activation of microglia was mainly detected in the midbrain, brainstem and spinal cord, where a significant loss of dopaminergic and motor neurons was observed. Consistent with the activation of microglia, the expression level of cyclooxygenase 1 (COX-1) was significantly up-regulated in the brain of symptomatic mice and in cultured microglia treated with conditioned medium derived from astrocytes over-expressing A53T α-synuclein. Consequently, the suppression of COX-1 activities extended the survival of mutant mice, suggesting that excess inflammatory responses elicited by reactive astrocytes may contribute to the degeneration of neurons. Our findings demonstrate a critical involvement of astrocytic α-synuclein in initiating the non-cell autonomous killing of neurons, suggesting the viability of reactive astrocytes and microglia as potential therapeutic targets for PD and other neurodegenerative diseases.