Role of Microglial LRRK2 in Inflammation

小胶质细胞 LRRK2 在炎症中的作用

• 批准号: 8554785
• 负责人: Mark S Moehle
• 金额: $ 3.21万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-28 至 2015-09-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8554785
• 关键词: Accounting; Affect; Animals; Antigen Presentation Pathway; Antigens; Area; Basal Ganglia; Caucasians; Caucasoid Race; Cell Lineage; Cells; Cessation of life; Chronic; Clinical; Cognitive; Critical Pathways; Crohn' s disease; Data; Disease Progression; Dopamine; Flow Cytometry; Gene Mutation; Genes; Genetic; Growth Factor; Human; Immune; Immune response; Individual; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Inflammatory Response; Knock-out; Leprosy; Lesion; Link; Mediating; Microglia; Missense Mutation; Modeling; Molecular; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Oral; PARK8 gene; Parkinson Disease; Pathogenesis; Pathway interactions; Peripheral; Phenotype; Phosphotransferases; Physiological; Physiology; Population; Prevalence; Protein Kinase; Proteins; Publishing; Research; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Transduction; Signal Transduction Pathway; Staging; Stimulus; Substantia nigra structure; Symptoms; T-Cell Proliferation; T-Lymphocyte; TLR4 gene; Techniques; Testing; Therapeutic; Training; Transgenic Animals; Transgenic Organisms; Translational Research; Virus; Writing; alpha synuclein; base; chemokine; cytokine; dopaminergic neuron; economic impact; experience; genome wide association study; in vivo Model; inflammatory marker; knockout animal; leucine-rich repeat kinase 2; macrophage; neuroimmunology; neuroinflammation; neurotoxicity; pars compacta; research and development; response; Accounting; Affect; Animals; Antigen Presentation Pathway; Antigens; Area; Basal Ganglia; Caucasians; Caucasoid Race; Cell Lineage; Cells; Cessation of life; Chronic; Clinical; Cognitive; Critical Pathways; Crohn' s disease; Data; Disease Progression; Dopamine; Flow Cytometry; Gene Mutation; Genes; Genetic; Growth Factor; Human; Immune; Immune response; Individual; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Inflammatory Response; Knock-out; Leprosy; Lesion; Link; Mediating; Microglia; Missense Mutation; Modeling; Molecular; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Oral; PARK8 gene; Parkinson Disease; Pathogenesis; Pathway interactions; Peripheral; Phenotype; Phosphotransferases; Physiological; Physiology; Population; Prevalence; Protein Kinase; Proteins; Publishing; Research; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Transduction; Signal Transduction Pathway; Staging; Stimulus; Substantia nigra structure; Symptoms; T-Cell Proliferation; T-Lymphocyte; TLR4 gene; Techniques; Testing; Therapeutic; Training; Transgenic Animals; Transgenic Organisms; Translational Research; Virus; Writing; alpha synuclein; base; chemokine; cytokine; dopaminergic neuron; economic impact; experience; genome wide association study; in vivo Model; inflammatory marker; knockout animal; leucine-rich repeat kinase 2; macrophage; neuroimmunology; neuroinflammation; neurotoxicity; pars compacta; research and development; response

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.

描述（由申请人提供）：在大多数高加索人群中，LRRK2中的错义突变占晚期PD病例的1-5％。 LRRK2在特发性PD中的作用尚不清楚，尽管对LRRK2突变背后的信号传导和生理学的了解可能有助于阐明PD中的致病机制。几条证据暗示了PD发病机理中的神经炎症。尽管LRRK2的作用尚不清楚，但几条证据表明LRRK2参与了先天免疫反应。最近的数据表明，LRRK2表达和激酶活性对于完全TLR4诱导的小胶质细胞中的炎性反应是必需的。由Raav2-α-alpha-synuclein引起的黑质中多巴胺能神经元的炎症标志物以及多巴胺能神经元的选择性死亡，过表达了PD的方面。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夸大了促炎反应，而LRRRK2抑制下调促炎反应。这些假设将以两个目的进行检验：第一个目标将确定α-突触核蛋白信号是否通过原代小胶质细胞中的LRRK2信号是否引起促炎反应。使用源自转基因，基因敲除和对照小鼠的原发性小胶质细胞，将检查细胞因子/趋化因子/生长因子对α-核蛋白和其他促炎性刺激的反应。此外，主要的抗原处理/表现能力 将检查具有LRRK2突变的小胶质细胞。第二个目的将确定LRRK2活性和表达对于RaAV2-α-α-突触核蛋白诱导的炎症和神经变性是否是必需的，以及G2019S-LRK2是否加剧了黑质Nigra中的神经变性。使用与AIM 1相同的动物，第二个目标将利用定量终点，包括对底底植物中多巴胺能神经元的公正立体估计，小胶质细胞激活，外周免疫细胞浸润以及呈现抗原的小胶质细胞和诱导T细胞增殖的小胶质细胞。另外，将通过流式细胞仪分析小胶质极化和激活。总体而言，我们希望观察到G2019S LRK2加剧了神经变性，小胶质细胞激活，促炎性细胞因子释放，抗原加工/表现以及将小胶质细胞对M1表型的极化，而敲除效果相反。拟议的培训计划由Andrew West博士赞助，并由David Standaert博士共同赞助。培训计划中包括的经验是帮助PI获得广泛的分子和细胞神经免疫性技术，以书面和口头形式呈现数据，在研究中进行负责任的进行，并发展成为能够进行高影响力翻译研究的独立研究人员。