Microglial Neuroprotection and Neurotoxicity

小胶质细胞的神经保护和神经毒性

• 批准号: 6729930
• 负责人: GREER M MURPHY
• 金额: $ 36.06万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6729930
• 关键词: Alzheimer' s disease; amyloid proteins; biological signal transduction; colony stimulating factor; enzyme linked immunosorbent assay; gene expression; immunocytochemistry; laboratory rat; laser capture microdissection; microglia; mixed tissue /cell culture; neurons; neuroprotectants; neurotoxicology; nitric oxide; polymerase chain reaction; transfection

DESCRIPTION (provided by applicant): Macrophage colony stimulating factor (M-CSF) is a microglial activator that is important in the cerebral inflammatory response. M-CSF expression is increased in AD and in the PDAPP transgenic mouse model for AD. The M-CSF receptor (M-CSFR) is increased on microglia in AD, in the PDAPP mouse, and in experimental models for brain injury. Overexpression of M-CSFR on microglia results in proliferation, increased expression of cytokines, and paracrine activation of other inflammatory cells. M-CSFR overexpression also results in aggressive microglial phagocytosis of Abeta by both FcgammaR-dependent and - independent mechanisms. Surprisingly, overexpression of M-CSFR on microglia protects neurons from excitotoxic injury in slice cultures transfected using biolistics, and in a microglial-hippocampal organotypic co-culture system. Also, M-CSF protects neurons in organotypic cultures from excitotoxic injury. In the proposed experiments, M-CSFR-induced microglial expression of potentially protective neurotrophins, cytokines, chemokines, and nitric oxide will be quantified after NMDA treatment in the biolistic transfection model and in the co-culture system. To localize differential expression to individual cell types, immunohistochemistry and laser capture microdissection will be used to obtain homogeneous populations of cells from slice cultures for real-time RT-PCR. To identify neuroprotective factors, hammerhead ribozymes will be used to block microglial expression and antibodies will be used to neutralize microglial factors in the co-culture. Candidate neuroprotective factors will also be tested for rescue of neurons from NMDA toxicity by adding them to organotypic culture medium. The signal transduction pathways activated by the M-CSFR that affect proliferation, cytokine expression, and phagocytosis are unknown. Hence, experiments will be performed to test activation of microglial Src kinases, PI3K, and Ras-mediated signaling, all of which are important in M-CSFR signal transduction in other cell types. These studies will define how activation of microglial M-CSFR protects neurons, and identify the molecular mechanisms by which the M-CSFR activates microglia and induces Abeta phagocytosis. Understanding the functions of M-CSFR and its ligand in microglia may lead to new strategies to promote Abeta clearance and to protect neurons in AD and other brain disorders. Much emphasis has been placed on the negative role of microglia. Although microglial neurotoxicity undoubtedly occurs in many disease states, it is important to understand how microglia might be induced to clear abnormal proteins from the brain or provide neuroprotection.

描述（由申请人提供）：巨噬细胞刺激因子（M-CSF）是一种小胶质激活因子，在脑炎症反应中很重要。在AD和AD的PDAPP转基因小鼠模型中，M-CSF表达增加。在AD，PDAPP小鼠中的小胶质细胞和脑损伤的实验模型中，M-CSF受体（M-CSFR）增加。 M-CSFR对小胶质细胞的过表达导致其他炎症细胞的增殖，细胞因子的表达增加以及旁分泌激活。 M-CSFR的过表达还会导致通过FCGAMMAR依赖性和 - 独立机制对Abeta的侵袭性小胶质细胞增多症。出乎意料的是，M-CSFR对小胶质细胞的过表达可保护神经元在使用生物学转染和小胶质细胞器官型共培养系统中转染的切片培养物中的兴奋性毒性损伤。此外，M-CSF可保护器官培养基中的神经元免受兴奋性毒性损伤。在提出的实验中，M-CSFR诱导的小胶质细胞表达在NMDA处理后，将在生物学转染模型和共培养系统中对NMDA处理后进行定量的潜在保护性神经营养蛋白，细胞因子，趋化因子和一氧化氮。为了将差异表达定位到单个细胞类型中，免疫组织化学和激光捕获显微解剖将用于从Slice培养物中获取用于实时RT-PCR的细胞的均匀种群。为了鉴定神经保护因素，将使用锤头核酶来阻断小胶质细胞表达，抗体将用于中和共培养中的小胶质细胞因子。候选神经保护因素还将通过将其添加到器官培养基中，以从NMDA毒性中拯救神经元。由M-CSFR激活的信号转导途径影响增殖，细胞因子表达和吞噬作用是未知的。因此，将进行实验以测试小胶质细胞SRC激酶，PI3K和RAS介导的信号传导的激活，所有这些信号在其他细胞类型中都很重要。这些研究将定义小胶质细胞M-CSFR的激活如何保护神经元，并确定M-CSFR激活小胶质细胞并诱导Abeta吞噬作用的分子机制。了解M-CSFR及其在小胶质细胞中的配体的功能可能会导致新的策略来促进ABETA清除并保护AD和其他脑部疾病中的神经元。非常重视小胶质细胞的负面作用。尽管小胶质细胞神经毒性无疑是在许多疾病状态发生的，但重要的是要了解如何诱导小胶质细胞清除大脑的异常蛋白质或提供神经保护作用。