Microglial Neuroprotection and Neurotoxicity

描述（由申请方提供）：巨噬细胞集落刺激因子（M-CSF）是一种在脑炎症反应中起重要作用的小胶质细胞激活剂。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表达导致增殖、细胞因子表达增加和其他炎性细胞的旁分泌激活。M-CSFR过表达还通过Fc γ R依赖性和非依赖性机制导致侵袭性小胶质细胞吞噬Abeta。令人惊讶的是，M-CSFR在小胶质细胞上的过表达在使用生物射弹转染的切片培养物中以及在小胶质细胞-海马器官型共培养系统中保护神经元免受兴奋性毒性损伤。此外，M-CSF保护器官型培养物中的神经元免受兴奋性毒性损伤。在所提出的实验中，在生物射弹转染模型和共培养系统中，将在NMDA处理后定量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。为了将差异表达定位于单个细胞类型，将使用免疫组织化学和激光捕获显微切割从切片培养物中获得均一的细胞群体用于实时RT-PCR。为了鉴定神经保护因子，锤头状核酶将用于阻断小胶质细胞表达，并且抗体将用于中和共培养物中的小胶质细胞因子。还将通过将候选神经保护因子添加到器官型培养基中来测试其对神经元免于NMDA毒性的拯救。由M-CSFR激活的影响增殖、细胞因子表达和吞噬作用的信号转导途径尚不清楚。因此，将进行实验以测试小胶质细胞Src激酶、PI3K和Ras介导的信号传导的活化，所有这些在其他细胞类型中的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Abeta吞噬作用的分子机制。了解M-CSFR及其配体在小胶质细胞中的功能可能会导致新的策略，以促进Abeta清除并保护AD和其他脑疾病中的神经元。小胶质细胞的负面作用一直受到重视。尽管小胶质细胞神经毒性无疑发生在许多疾病状态中，但重要的是要了解小胶质细胞如何被诱导以清除大脑中的异常蛋白质或提供神经保护。

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.