Role of Microglial LRRK2 in Inflammation

描述（由申请方提供）：在大多数高加索人群中，LRRK 2错义突变占迟发性PD病例的1 - 5%。LRRK 2在特发性PD中的作用仍不清楚，尽管更好地理解LRRK 2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。一些证据表明，神经炎症在PD的发病机制。虽然LRRK 2的作用仍不清楚，但几条证据表明LRRK 2参与先天免疫应答。最近的数据显示，LRRK 2表达和激酶活性对于小胶质细胞中完全的TLR 4诱导的促炎反应是必需的。炎症标志物以及由rAAV 2-α-突触核蛋白过表达引起的黑质中多巴胺能神经元的选择性死亡概括了PD的各个方面。我们假设LRRK 2可以修饰α-突触核蛋白驱动的神经炎症，其中G2019 S LRRK 2夸大促炎反应，LRRK 2抑制下调促炎反应。这些假设将在两个目标中进行测试：第一个目标将确定α-突触核蛋白是否通过原发性小胶质细胞中的LRRK 2发出信号以引发促炎反应。使用来自转基因、基因敲除和对照小鼠的原代小胶质细胞，将检查细胞因子/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，原代细胞的抗原加工/呈递能力 将检查携带LRRK 2突变的小胶质细胞。第二个目的将确定LRRK 2活性和表达是否是rAAV 2-α-突触核蛋白诱导的炎症和神经变性所必需的，以及G2019 S-LRRK 2是否加剧黑质中的神经变性。使用与目标1相同的动物，第二个目标将利用定量终点，包括黑质中多巴胺能神经元的无偏体视学估计、小胶质细胞活化、外周免疫细胞侵袭和小胶质细胞呈递抗原并诱导T细胞增殖。此外，将用流式细胞术分析小胶质细胞极化和活化。总的来说，我们期望观察到G2019 S LRRK 2加剧神经变性、小胶质细胞活化、促炎细胞因子释放、抗原加工/呈递和小胶质细胞极化为M1表型，而敲除显示相反的效果。拟议的培训计划由Andrew West博士赞助，大卫斯坦达特博士共同赞助。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，以书面和口头形式展示数据，负责任地进行研究，并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）是一种慢性和进行性神经退行性疾病，影响1%至5%的65岁以上人口，在美国估计每年的经济影响为355亿美元。目前的治疗方法仅治疗PD的症状，既不能治愈也不能帮助减缓疾病的进展。拟议的研究计划旨在揭示PD的分子基础，并更接近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.