Microglial Neuroprotection and Neurotoxicity

描述(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，在脑部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的表达增加。在AD、PDAPP小鼠和实验性脑损伤模型中，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细胞增殖、细胞因子表达增加，以及其他炎症细胞的旁分泌激活。M-CSFR的过表达还通过FcGammaR依赖和非依赖机制导致小胶质细胞对Abeta的侵袭性吞噬。令人惊讶的是，小胶质细胞上M-CSFR的过表达可以保护神经元免受兴奋性毒性损伤，在使用生物生物学技术的切片培养中，以及在小胶质细胞-海马器型共培养系统中。此外，M-CSF保护器官培养中的神经元免受兴奋性毒性损伤。在拟议的实验中，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细胞表达将在NMDA处理后在生物转染模型和共培养系统中被量化。为了定位单个细胞类型的差异表达，将使用免疫组织化学和激光捕获显微切割从切片培养中获得均一组细胞，用于实时RT-PCR。为了确定神经保护因素，锤头状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。候选的神经保护因子也将通过将它们添加到器官型培养基中来测试从NMDA毒性中拯救神经元。M-CSFR激活的影响细胞增殖、细胞因子表达和吞噬作用的信号转导通路尚不清楚。因此，将进行实验以测试小胶质细胞Src激酶、PI3K和RAS介导的信号的激活，所有这些在其他类型的细胞的M-CSFR信号转导中都是重要的。这些研究将确定小胶质细胞M-CSFR的激活如何保护神经元，并确定M-CSFR激活小胶质细胞和诱导Aβ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导致促进Abeta清除和保护AD和其他大脑疾病中的神经元的新策略。小胶质细胞的负面作用一直受到人们的重视。虽然小胶质细胞的神经毒性无疑发生在许多疾病状态，但了解如何诱导小胶质细胞清除大脑中的异常蛋白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.