Role of Microglial LRRK2 in Inflammation

描述（由申请人提供）：在大多数高加索人群中，LRRK2错义突变占晚发性PD病例的1%至5%。LRRK2在特发性PD中的作用尚不清楚，尽管更好地了解LRRK2突变背后的信号传导和生理学可能有助于阐明PD的致病机制。一些证据暗示神经炎症与帕金森病的发病机制有关。虽然LRRK2的作用尚不清楚，但一些证据表明LRRK2参与了先天免疫反应。最近的数据表明，LRRK2的表达和激酶活性对于tlr4诱导的小胶质细胞炎症反应是必要的。炎症标志物以及raav2 -突触核蛋白过表达引起的黑质多巴胺能神经元的选择性死亡是PD的一个缩影。我们假设LRRK2可能会改变α -突触核蛋白驱动的神经炎症，其中G2019S LRRK2会夸大促炎反应，而LRRK2抑制则会下调促炎反应。这些假设将在两个方面进行测试：第一个目的是确定α -突触核蛋白信号是否通过初级小胶质细胞中的LRRK2引发促炎反应。利用转基因小鼠、基因敲除小鼠和对照小鼠获得的原代小胶质细胞，研究细胞因子/趋化因子/生长因子对α -突触核蛋白和其他促炎刺激的反应。此外，原代的抗原处理/递呈能力

DESCRIPTION (provided by applicant): Missense mutations in LRRK2 account for between 1 and 5% of late onset PD cases in most Caucasian populations. The role of LRRK2 in idiopathic PD remains unclear, although better understanding of the signaling and physiology behind mutations in LRRK2 may help clarify pathogenic mechanisms in PD. Several lines of evidence implicate neuroinflammation in the pathogenesis of PD. While the role of LRRK2 remains unclear, several lines of evidence point to LRRK2 being involved in the innate immune response. Recent data show that LRRK2 expression and kinase activity are necessary for a full TLR4-induced pro- inflammatory response in microglia. Inflammatory markers as well as selective death of dopaminergic neurons in the substantia nigra caused by rAAV2-alpha-synuclein over-expression recapitulate aspects of PD. We hypothesize that LRRK2 may modify alpha-synuclein driven neuroinflammation, where G2019S LRRK2 exaggerates pro-inflammatory responses and LRRK2 inhibition down-regulates pro-inflammatory responses. These hypotheses will be tested in two aims: The first aim will determine whether alpha-synuclein signals through LRRK2 in primary microglia to elicit pro-inflammatory responses. Using primary microglia derived from transgenic, knockout, and control mice, the cytokine/chemokine/growth-factor response to alpha-synuclein and other pro-inflammatory stimuli will be examined. Additionally, the antigen processing/presentation capabilities of primary microglia harboring LRRK2 mutations will be examined. The second aim will determine whether LRRK2 activity and expression is necessary for rAAV2-alpha-synuclein induced inflammation and neurodegeneration, and whether G2019S-LRRK2 exacerbates neurodegeneration in the substantia nigra. Using the same animals as in aim 1, the second aim will utilize quantitative endpoints including unbiased stereological estimations of dopaminergic neurons in the substantia nigra, microglial activation, peripheral immune cell invasion, and microglia presenting antigen and inducing T-cell proliferation. Additionally, microglial polarization and activation wil be analyzed with flow cytometry. Overall, we expect to observe that G2019S LRRK2 exacerbates neurodegeneration, microglial activation, pro-inflammatory cytokine release, antigen processing/presentation, and polarization of microglia to an M1 phenotype while the knockout shows the opposite effect. The proposed training plan is sponsored by Dr. Andrew West and co-sponsored by Dr. David Standaert. Included in the training plan are experiences to help the PI gain a wide range of molecular and cellular neuroimmunology techniques, presentation of data in a written and oral format, responsibly conduct in research, and development into an independent researcher capable of carrying out high impact translation research. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) is a chronic and progressive neurodegenerative disorder affecting between 1 and 5% of the population over 65 with an estimated annual economic impact of $35.5 billion in the US. Current therapeutic approaches treat only the symptoms of PD and offer neither a cure nor aid in slowing the progression of disease. The proposed research plan aims to uncover the molecular basis of PD and move closer to developing the most efficacious therapeutic strategies.