Using BAC-transgenic and Proteomic Approach to Study LRRK2 Biology and Pathology

描述(由申请人提供)：帕金森氏病(PD)的发病机制尚不清楚。LRRK2(Park8)的突变最近被认为与最常见的家族性形式(常染色体显性遗传)以及一些散发性帕金森氏病(PD)有关。LRRK2蛋白含有多个保守结构域，包括一个激酶和一个GTPase结构域。最近对LRRK2的表征表明，与PD相关的LRRK2突变导致激酶活性增强，这与神经元培养中的神经毒性有关。我们的长期目标是阐明LRRK2在中枢神经系统中的结构和功能，明确突变型LRRK2在帕金森病中的致病途径，为最终了解帕金森病的发病机制提供信息。有证据表明，家族性PD基因突变引起的病理仅限于大脑，这突显了大脑特定背景在PD的发生和发展中的重要性。在此之前，我们结合了BAC(细菌人工染色体)介导的转基因小鼠、蛋白质组学和生化研究的集成系统，在脑的背景下研究了LRRK2的细胞功能。我们已经从转基因脑中纯化了LRRK2蛋白，并发现与其他组织或细胞培养的LRRK2蛋白相比，脑中的LRRK2蛋白具有较强的激酶和GTPase活性。因此，我们假设LRRK2的激酶/GTPase活性受大脑中的辅助因素(例如蛋白质和脂类)特异性地调节。我们将使用从大脑中纯化的LRRK2来专门测试这一假设(目标1)。为了进一步了解LRRK2酶活性的调节，我们鉴定了LRRK2在小鼠脑中表达的磷酸化位点。我们还开发了一种针对LRRK2特异性磷酸化的抗体，以辅助磷酸化LRRK2的功能分析。由于LRRK2激酶的自磷酸化水平与神经毒性相关，我们建议研究LRRK2中已发现的磷酸化和自磷酸化之间的关系，并评估已发现的LRRK2磷酸化在LRRK2介导的发病机制中的功能意义。此外，我们将测试LRRK2的特定磷酸化调节LRRK2激酶/GTPase活性并潜在地修改LRRK2介导的脑内病理过程的假设(目标2)。最后，尽管最近有证据表明LRRK2的激酶活性和GTP结合增加与神经毒性有关，但这些研究主要是在细胞培养中进行的。在这项应用中，我们将使用我们的BAC介导的LRRK2转基因小鼠来验证这些体外研究，并使用动物模型进一步验证LRRK2激酶在体内帕金森病发病机制中“过度活跃”的假说(目标3)。通过将生物化学、细胞生物学和新的小鼠转基因方法相结合，我们的研究有望为LRRK2蛋白的生物学和病理学提供机械性的见解，LRRK2蛋白被认为是治疗帕金森病的一个有前途的药物靶点。公共卫生相关性：帕金森病(PD)是一种主要的人类神经退行性疾病，但其发病机制尚不清楚。这项应用将研究PD相关基因的中枢功能，并确定由该基因在中枢神经系统中的PD突变所介导的致病途径。它有望为最终了解帕金森病的病因和验证治疗帕金森病的药物靶点提供信息。

DESCRIPTION (provided by applicant): The disease mechanism underlying Parkinson's disease (PD) is poorly understood. Mutations in LRRK2 (PARK8) have recently been linked to the most common familial forms (autosomal dominant) as well as some sporadic forms of Parkinson's disease (PD). LRRK2 protein contains multiple conserved domains including a kinase and a GTPase domain. Recent characterization of LRRK2 suggests that PD-associated mutations of LRRK2 cause enhanced kinase activity, which is linked to the neurotoxicity in neuron cultures. Our long-term goal is to elucidate the structure/function of LRRK2 in the CNS, to define the mutant LRRK2-mediated pathogenic pathways in PD, and to provide information for ultimate understanding of the pathogenesis of PD. The evidence that the pathology caused by familial PD gene mutations is restricted to the brain underscores the importance of brain-specific context in the onset and development of PD. Previously we have used an integrated system combining BAC (bacterial artificial chromosome)-mediated transgenic mice, proteomics and biochemical study to investigate the cellular function of LRRK2 in the context of brain. We have purified LRRK2 protein from the transgenic brain and found that the brain LRRK2 is associated with robust kinase and GTPase activity as compared to that from other tissues or cell cultures. Thus, we hypothesize that the kinase/GTPase activities of LRRK2 are specifically regulated by co-factors (e.g., proteins and lipids) in the brain. We will specifically test this hypothesis using purified LRRK2 from the brain (Aim 1). To further understand the regulation of LRRK2 enzymatic activity, we have identified phosphorylation sites in LRRK2 expressed in mouse brain. We have also developed an antibody against specific phosphorylation of LRRK2 to assist in the functional analysis of phospho- LRRK2. Since the levels of autophosphorylation of LRRK2 kinase are correlated with neurotoxicity, we propose to investigate the relationship between the identified phosphorylation and autophosphorylation in LRRK2, and to assess the functional significance of the identified phosphorylation of LRRK2 in LRRK2-mediated pathogenesis. In addition, we will test the hypothesis that specific phosphorylation of LRRK2 regulates LRRK2 kinase/GTPase activity and potentially modifies LRRK2-mediated pathological process in the brain (Aim 2). Finally, despite the recent evidence linking increased kinase activity and GTP binding of LRRK2 to neurotoxicity, the studies were performed mostly in cell cultures. In this application, we will use our BAC-mediated LRRK2 transgenic mice to validate these in vitro studies and further test the hypothesis of "hyperactivity" of LRRK2 kinase in the pathogenesis of PD in vivo using animal models (Aim 3). By integrating biochemistry, cell biology and novel mouse transgenic approaches, our study is expected to provide mechanistic insight into the biology and pathology of LRRK2 protein, which is considered a promising drug target for the treatment of PD. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) is a major human neurodegenerative disease, but the pathogenic mechanism is not clear. This application will investigate the central function of a PD-related gene and define the pathogenic pathway mediated by PD mutations of the gene in the central nervous system. It is expected to provide information for ultimate understanding of the etiology of PD and validation of drug targets for treatment of PD.