Mechanisms of LRRK2 Mediated Neurotoxicity

描述(申请人提供)：帕金森病(PD)是第二种最常见的神经退行性疾病。预计到2030年，全球将有超过1000万人患有帕金森氏症，目前还没有已知的治疗方法可以减缓或阻止疾病的无情发展。富含亮氨酸重复蛋白2(LRRK2)基因的显性错义突变是帕金森病最常见的已知原因，在美国有多达4万到6万名致病性G2019S-LRRK2突变携带者。在德系或柏柏尔血统的个体中，G2019S-LRRK2突变的频率在PD患者中可能高达20%-30%。在我们的上一个项目期间，我们证明了G2019S-LRRK2的表达促进了由α-突触核蛋白过度表达引起的多巴胺能神经退行性变，并且LRRK2激酶抑制剂阻断了这些作用。然而，LRRK2与α-突触核蛋白神经毒性之间的联系机制尚不清楚。进一步剖析LRRK2在帕金森病中的作用可能会加速发现针对LRRK2和相关神经保护通路的新疗法。我们发现，在健康大脑中，LRRK2在许多与α-突触核蛋白作用更强的神经元中表达，但在含有α-突触核蛋白包涵体的疾病脑中，LRRK2也在髓系细胞中表达，这可以指导神经毒性细胞因子和活性氧物种的产生。在这一竞争性更新请求中，我们假设LRRK2的表达和激酶活性促进了髓系细胞向含有α-突触核蛋白包涵体的神经元募集，从而加剧了神经变性和α-突触核蛋白包涵体的扩散。利用在髓系细胞中诱导过表达G2019S-LRRK2的条件性转基因技术，我们将测试G2019S-LRRK2是否能通过促进髓系细胞募集来上调细胞因子反应和神经元α-突触核蛋白包涵体引起的氧化损伤。利用条件缺失技术，我们将测试髓系细胞中的LRRK2基因敲除是否会通过抑制髓系细胞的募集而下调神经毒性细胞因子和氧化损伤，以响应α-突触核蛋白的包涵体。我们将探索LRRK2蛋白如何通过作为Arp2/3控制肌动蛋白聚合的新型支架来控制髓系细胞的运动和趋化。我们预测，LRRK2与Arp2/3以一种依赖于激酶的方式相互作用，以促进移动髓系细胞前沿的肌动蛋白微刺，从而增强运动性。这些研究的完成将有助于进一步了解LRRK2如何导致帕金森病，LRRK2在髓系细胞中的表达如何促进神经退行性变，以及对髓系细胞趋化至关重要的新的肌动蛋白核复合体的定义。

DESCRIPTION (provided by applicant): Parkinson disease (PD) is the second most common neurodegenerative disorder. More than 10 million individuals worldwide are expected to have PD by the year 2030, and currently there are no known therapies that slow or halt the relentless progression of the disease. Dominant missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known cause of PD, with up to 40 to 60 thousand carriers of the pathogenic G2019S-LRRK2 mutation in the United States. In individuals of Ashkenazi or Berber descent, frequencies of the G2019S-LRRK2 mutation can be as high as 20-30% in PD patients. In our last project period, we demonstrated that G2019S- LRRK2 expression promotes dopaminergic neurodegeneration caused by α-synuclein over- expression and that LRRK2 kinase inhibitors blocked these effects. However, the mechanisms linking LRRK2 to α-synuclein neurotoxicity are not clear. Further dissection of LRRK2 action in PD may expedite the discovery of new therapies that target LRRK2 and related pathways for neuroprotection. We found that LRRK2 is expressed in many neurons tougher with α-synuclein in healthy brains, but in diseased brains that have α-synuclein inclusions, LRRK2 is also expressed in myeloid cells that can direct the production of neurotoxic cytokines and reactive-oxygen species. In this competitive renewal request, we hypothesize that LRRK2 expression and kinase activity promotes myeloid cell recruitment to neurons that have α-synuclein inclusions to exacerbate neurodegeneration and the spread of α-synuclein inclusions. Using conditional transgenics that inducibly over-express G2019S-LRRK2 in myeloid cells, we will test whether G2019S-LRRK2 can upregulate cytokine responses and oxidative damage caused by neuronal α-synuclein inclusions through promoting myeloid cell recruitment. Using conditional deletion technology, we will test whether LRRK2 knockout in myeloid cells will downregulate neurotoxic cytokines and oxidative damage in response to α-synuclein inclusions through inhibiting myeloid cell recruitment. We will explore how LRRK2 protein can control myeloid cell motility and chemotaxis by acting as a novel scaffold for Arp2/3 to control actin polymerization. We predict that LRRK2 interacts with Arp2/3, in a kinase-dependent manner, to facilitate actin microspikes at the leading front of mobile myeloid cells to enhance motility. Completion of these studies should allow for further insight into how LRRK2 can cause PD, how LRRK2 expression in myeloid cells might contribute to neurodegeneration, and definition of a novel actin nucleation complex important for myeloid cell chemotaxis.