Microglial Neuroprotection and Neurotoxicity

描述（由申请人提供）：巨噬细胞集落刺激因子（M-CSF）是一种小胶质细胞激活剂，在脑炎症反应中起重要作用。在AD和PDAPP转基因AD小鼠模型中，M-CSF表达增加。在AD、PDAPP小鼠和脑损伤实验模型中，M-CSF受体（M-CSFR）在小胶质细胞上增加。M-CSFR在小胶质细胞上的过度表达导致增殖、细胞因子表达增加和其他炎症细胞的旁分泌激活。M-CSFR过表达也会通过fcgammar依赖性和非依赖性机制导致Abeta的侵袭性小胶质吞噬。令人惊讶的是，在生物学转染的切片培养和小胶质-海马器官型共培养系统中，M-CSFR在小胶质细胞上的过表达可以保护神经元免受兴奋性毒性损伤。此外，M-CSF保护器官型培养的神经元免受兴奋性毒性损伤。在我们提出的实验中，将在生物转染模型和共培养系统中对NMDA处理后，m - csfr诱导的潜在保护性神经营养因子、细胞因子、趋化因子和一氧化氮的小胶质表达进行量化。为了将差异表达定位到单个细胞类型，免疫组织化学和激光捕获显微解剖将用于从切片培养中获得均匀的细胞群，用于实时RT-PCR。为了确定神经保护因子，锤头核酶将被用来阻断小胶质细胞的表达，抗体将被用来中和共培养中的小胶质细胞因子。候选神经保护因子也将通过将其添加到器官型培养基中来测试其对NMDA毒性神经元的拯救作用。M-CSFR激活的影响增殖、细胞因子表达和吞噬的信号转导途径尚不清楚。因此，将进行实验来测试小胶质Src激酶、PI3K和ras介导的信号传导的激活，所有这些在其他细胞类型的M-CSFR信号转导中都很重要。这些研究将明确小胶质细胞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吞噬的分子机制。了解M-CSFR及其配体在小胶质细胞中的功能可能会为促进Abeta清除和保护AD和其他脑部疾病中的神经元提供新的策略。许多人强调小胶质细胞的负面作用。虽然小胶质细胞神经毒性无疑发生在许多疾病状态中，但了解小胶质细胞如何被诱导清除大脑中的异常蛋白质或提供神经保护是很重要的。

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.