DJ-1 and Parkinson's Disease

背景和具体目标： DJ-1基因的一个大外显子缺失和一个点突变（L166 P）最初在两个不同的常染色体早发性PD家族中报道。DJ-1相关PD似乎通过功能丧失机制表现出来，因为首次报道的突变包含导致蛋白质和mRNA缺失的大外显子缺失，或导致蛋白质快速降解的L166 P突变。为了更好地了解DJ-1及其在PD中的作用，我们开发了DJ-1基因敲除小鼠。因此，我们对该项目的具体目标是： 目的1：建立DJ-1基因敲除小鼠模型并鉴定其生物学特性; 目的2：研究DJ-1的功能。 目的3：探讨DJ-1基因与其他PD相关基因突变的相互作用。 将在这些DJ-1敲除小鼠中检查运动行为、多巴胺能传递和神经病理学。同时，我们还将通过免疫细胞化学方法检测DJ-1在培养的中脑神经元中的亚细胞定位，并通过免疫沉淀法鉴定DJ-1相关蛋白，从而研究DJ-1蛋白的功能。 进展报告和未来方向： 为了研究DJ-1在体内的生理功能，我们产生了DJ-1敲除（DJ-1 -/-）小鼠。年轻（< 1岁）的DJ-1 -/-小鼠活动减退，有轻度步态异常。然而，与野生型同窝仔相比，年龄较大的DJ-1 -/-;表现出体重和握力下降以及更严重的步态不规则。细胞外多巴胺的基础水平、诱发的多巴胺释放和多巴胺受体D2敏感性在DJ-1/小鼠的纹状体中表现正常，这与DJ-1 -/-;和多巴胺能系统特异性行为范例中的对照之间的类似结果一致。对脊髓、神经和肌肉组织的检查未能发现任何与观察到的运动缺陷一致的病理变化。综上所述，我们的研究结果表明，DJ-1的损失导致渐进的行为变化，而没有显着改变黑质纹状体多巴胺能和脊髓运动系统。 虽然DJ-1剂量的丧失不会导致小鼠中的DA神经元变性，但缺乏DJ-1的小鼠在纹状体中表现出多巴胺能信号传导的缺陷。由于海马含有相对高水平的DJ-1，PD患者往往认知功能受损，我们评估了DJ-1缺陷对海马CA 1区突触可塑性的影响。在DJ-1 -/-小鼠中，LTP轻微受损，LTD被消除，而DJ-1+/-小鼠在突触可塑性方面没有表现出改变。多巴胺受体D2/3激动剂quinpirole拯救了DJ-1 -/-小鼠的LTD，表明多巴胺能信号转导受损在海马LTD缺陷中的作用。 作为我们先前在DJ-1 KO小鼠上的工作的延伸，我们有兴趣研究DJ-1在其他PD相关基因突变的发病机制中的作用。我们最近成功地开发了几种新型的转基因小鼠，它们在中脑DA神经元中选择性地表达PD相关的α-突触核蛋白A53 T和LRRK 2 G2019 S突变。α-突触核蛋白A53 T转基因小鼠以年龄依赖性方式发展PD样临床表型。研究DJ-1的存在是否会影响突变体α-突触核蛋白介导的运动行为和神经病理学异常的进展将是有趣的。我们将使α-突触核蛋白转基因小鼠与DJ-1 KO小鼠杂交以在DJ-1无效背景下产生α-突触核蛋白转基因小鼠的群组。我们期望在没有DJ-1的情况下观察到α-突触核蛋白介导的神经病理学的加速。然后，我们将研究DJ-1如何通过体内小鼠建模和体外神经生物学方法的组合来调节α-突触核蛋白诱导的细胞毒性的分子机制。

Background and Specific Aims: A large exonic deletion and a point mutation (L166P) in the DJ-1 gene were initially reported in two different families with autosomal early-onset PD. DJ-1-associated PD appears to manifest via a loss of function mechanism, because the first reported mutations contained either large exonic deletions resulting in an absence of protein and mRNA, or a L166P mutation that resulted in rapid protein degradation. To better understand DJ-1 and its role in PD, we developed a DJ-1 gene knockout mouse. Thus, our specific aims for this project are: Aim 1: To generate and characterize DJ-1 knockout mice; Aim 2: To study the function of DJ-1. Aim 3: To investigate the genetic interactions between DJ-1 and other PD-related genetic mutations. Motor behavior, dopaminergic trans-mission, and neuropathology will be examined in these DJ-1 knockout mice. Meanwhile, we will study the function of DJ-1 protein by determining the subcellular localization of DJ-1 via immunocytochemistry of cultured midbrain neurons and identifying DJ-1-associated proteins via immunoprecipitation. Progress Report and Future Directions: To investigate the physiological functions of DJ-1 in vivo, we generated DJ-1 knockout (DJ-1 -/-) mice. Younger (< 1 year) DJ-1 -/- mice were hypoactive and had mild gait abnormalities. Older DJ-1 -/-;, however, showed decreased body weight and grip strength and more severe gait irregularities compared to wild-type littermates. The basal level of extracellular dopamine, evoked dopamine release and dopamine receptor D2 sensitivity appeared normal in the striatum of DJ-1&#8722;/&#8722; mice, which was consistent with similar results between DJ-1 -/-; and controls in behavioral paradigms specific for the dopaminergic system. An examination of spinal cord, nerve and muscle tissues failed to identify any pathological changes that were consistent with the noted motor deficits. Taken together, our findings suggest that loss of DJ-1 leads to progressive behavioral changes without significant alterations in nigrostriatal dopaminergic and spinal motor systems. Although the loss of DJ-1 dose not lead to DA neuron degeneration in mice, mice lacking DJ-1 exhibit a deficit in dopaminergic signaling in the striatum. Because the hippocampus contains relatively high levels of DJ-1, and PD patients are often cognitively impaired, we evaluated the effects of DJ-1 deficiency on the plasticity of hippocampal CA1 synapses. LTP was slightly impaired and LTD was abolished in DJ-1 -/- mice, whereas DJ-1+/- mice exhibited no alterations in synaptic plasticity. The dopamine receptor D2/3 agonist quinpirole rescued LTD in DJ-1 -/- mice, suggesting a role for impaired dopaminergic signaling in the hippocampal LTD deficit. As an extension of our previous work on DJ-1 KO mice, we are interested in studying the role of DJ-1 in the pathogenesis of other PD-related genetic mutations. We have recently successfully developed several novel lines of transgenic mice that selectively express PD-related alpha-synuclein A53T and LRRK2 G2019S mutations in the midbrain DA neurons. The alpha-synuclein A53T transgenic mice developed PD-like clinical phenotypes at an age dependent manner. It would be interesting to investigate whether the presence of DJ-1 will affect the progression of mutant alpha-synucelin-mediated motor behavioral and neuropathological abnormalities. We will crossbreed the alpha-synuclein transgenic mice with DJ-1 KO mice to generate a cohort of alpha-synuclein transgenic mice in the DJ-1 null background. We expect to observe an acceleration of alpha-synuclein-mediated neuropathology in the absence of DJ-1. We will then investigate the molecular mechanism of how DJ-1 modulates the cytotoxicity induced by alpha-synuclein by a combination of in vivo mouse modeling and in vitro neurobiology approaches.