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错义突变占迟发性帕金森病病例的1%至5%。LRRK2在特发性帕金森病中的作用尚不清楚，尽管更好地了解LRRK2突变背后的信号和生理可能有助于阐明帕金森病的发病机制。有几条证据表明神经炎与帕金森病的发病机制有关。虽然LRRK2的作用尚不清楚，但有几条证据表明LRRK2参与了先天免疫反应。最近的数据表明，LRRK2的表达和激酶活性是TLR4诱导的小胶质细胞全面促炎反应所必需的。炎症标记物以及由rAAV2-α-突触核蛋白过度表达引起的黑质多巴胺能神经元的选择性死亡概括了帕金森病的一些方面。我们假设LRRK2可能改变α-突触核蛋白驱动的神经炎症，其中G2019S LRRK2夸大促炎反应，而抑制LRRK2下调促炎反应。这些假说将在两个目标中得到验证：第一个目标是确定初级小胶质细胞中的α-突触核蛋白信号是否通过LRRK2引发促炎反应。使用来自转基因小鼠、基因敲除小鼠和对照小鼠的原代小胶质细胞，将检测细胞因子/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，初级患者的抗原处理/提呈能力 我们将检查携带LRRK2突变的小胶质细胞。第二个目的将确定LRRK2的活性和表达是否是rAAV2-α-突触核蛋白诱导的炎症和神经变性所必需的，以及G2019S-LRRK2是否加剧了黑质的神经变性。使用与目标1相同的动物，第二个目标将利用定量终点，包括对黑质多巴胺能神经元、小胶质细胞激活、外周免疫细胞入侵和小胶质细胞呈递抗原和诱导T细胞增殖的无偏见体视学评估。此外，还将用流式细胞术分析小胶质细胞的极化和激活情况。总体而言，我们希望观察到G2019S LRRK2加剧了神经变性、小胶质细胞激活、促炎细胞因子释放、抗原处理/递呈以及小胶质细胞极化为M1表型，而基因敲除则显示出相反的效果。拟议的培训计划由安德鲁·韦斯特博士赞助，大卫·斯坦德尔特博士协办。培训计划包括帮助PI获得广泛的分子和细胞神经免疫学技术、以书面和口头形式呈现数据、负责任地进行研究以及发展成为能够进行高影响力翻译研究的独立研究人员的经验。 公共卫生相关性：帕金森氏病(PD)是一种慢性进行性神经退行性疾病，影响65岁以上人口的1%至5%，在美国估计每年造成355亿美元的经济影响。目前的治疗方法只治疗帕金森病的症状，既不能治愈疾病，也不能帮助减缓疾病的发展。拟议的研究计划旨在揭示帕金森病的分子基础，并更接近于开发最有效的治疗策略。

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.