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

描述（由申请人提供）：帕金森病（PD）的发病机制尚不清楚。LRRK 2（PARK 8）突变最近已与最常见的家族形式（常染色体显性遗传）以及一些散发形式的帕金森病（PD）有关。LRRK 2蛋白含有多个保守结构域，包括激酶和GT3结构域。最近对LRRK 2的表征表明，LRRK 2的PD相关突变导致激酶活性增强，这与神经元培养物中的神经毒性有关。我们的长期目标是阐明LRRK 2在中枢神经系统中的结构/功能，以确定突变LRRK 2介导的PD致病途径，并为最终理解PD的发病机制提供信息。由家族性PD基因突变引起的病理仅限于大脑的证据强调了大脑特异性背景在PD发病和发展中的重要性。以前，我们已经使用了一个整合的系统相结合的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，以研究LRRK 2在脑的背景下的细胞功能。我们已经从转基因脑中纯化了LRRK 2蛋白，并发现与来自其他组织或细胞培养物的LRRK 2蛋白相比，脑LRRK 2与强激酶和GT3活性相关。因此，我们假设LRRK 2的激酶/GTP酶活性是由辅因子（例如，蛋白质和脂质）。我们将使用从大脑中纯化的LRRK 2专门测试这一假设（目的1）。为了进一步了解LRRK 2酶活性的调节，我们鉴定了小鼠脑中表达的LRRK 2的磷酸化位点。我们还开发了针对LRRK 2的特异性磷酸化的抗体，以辅助磷酸化LRRK 2的功能分析。由于LRRK 2激酶的自磷酸化水平与神经毒性相关，因此我们建议研究LRRK 2中鉴定的磷酸化和自磷酸化之间的关系，并评估鉴定的LRRK 2磷酸化在LRRK 2介导的发病机制中的功能意义。此外，我们将检验LRRK 2的特异性磷酸化调节LRRK 2激酶/GT3活性并潜在地改变脑中LRRK 2介导的病理过程的假设（目的2）。最后，尽管最近有证据表明LRRK 2的激酶活性增加和GTP结合与神经毒性有关，但这些研究主要在细胞培养中进行。在本申请中，我们将使用我们的BAC介导的LRRK 2转基因小鼠来验证这些体外研究，并使用动物模型进一步测试LRRK 2激酶在体内PD发病机制中“过度活跃”的假设（目的3）。通过整合生物化学、细胞生物学和新型小鼠转基因方法，我们的研究有望为LRRK 2蛋白的生物学和病理学提供机理性见解，LRRK 2蛋白被认为是治疗PD的有前途的药物靶点。 公共卫生相关性：帕金森病（PD）是一种重要的人类神经退行性疾病，但其发病机制尚不清楚。本申请将研究PD相关基因的中枢功能，并确定中枢神经系统中由PD基因突变介导的致病途径。为进一步了解PD的病因学和确定治疗PD的药物靶点提供了信息。

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.