SIRT1 Limits Microglial Toxicity in Alzheimer's Disease

描述(由申请人提供)：长期以来，小胶质细胞的激活被认为与阿尔茨海默病(AD)的发病机制有关。除了对神经元和突触功能造成直接毒性作用外，淀粉样β蛋白和/或tau的积聚还可以刺激小胶质细胞的激活和炎性细胞因子的表达，从而导致进一步的神经元损伤。阻断小胶质细胞激活过程中的毒性途径，可有效防止神经退行性变。然而，调节小胶质细胞环的分子机制仍然难以捉摸。我们之前在原代皮质培养中的研究表明，小胶质细胞中的NF-κB激活在小胶质细胞介导的Aü毒性中起着关键作用。组蛋白脱乙酰基酶的sirtuin家族成员sirtuin家族成员sirtuin抑制NF-κB，可保护经A？处理的原代培养细胞免受小胶质细胞的毒性。阿尔茨海默病患者脑内SIRT1水平显著降低。培养的小胶质细胞中SIRT1的表达明显减少。基于这些发现，我们假设SIRT1的减少是导致AD小胶质细胞毒性的关键事件，并且小胶质细胞SIRT1通过抑制NF-κB的激活来限制A？介导的神经元缺陷。为了检验这一假设，我们提出了三个具体目标。在目标1中，我们将灭活表达人淀粉样前体蛋白(HAPP)的小鼠小胶质细胞中的SIRT1，并系统地研究小胶质细胞SIRT1失活如何影响炎症反应和A？相关神经元/行为缺陷。在目标2中，为了确定小胶质细胞SIRT1是否通过抑制NF-κB的激活而发挥神经保护作用，我们将确定HAPP小鼠小胶质细胞中规范的NF-κB信号的结构性激活是否以类似于SIRT1缺失的方式加剧了这种缺陷。在互补性实验中，我们将确定抑制NF-κB信号是否会通过在脑内注入有效的NF-κB抑制剂或在小胶质细胞中注射抑制NF-κB的病毒载体来改善A？相关神经元缺陷。在目标3中，为了确定小胶质细胞sirt1抑制NF-κB的机制，我们将系统地检测sirt1是否通过去乙酰化RELA和/或通过减少髓系细胞中RELA的磷酸化来抑制NF-κB。利用染色质免疫沉淀(CHIP)分析，我们将确定SIRT1诱导的对NF-κB激活的抑制是否涉及髓系细胞中组蛋白(H3K56Ac)的去乙酰化。拟议研究的完成将为调节神经退行性变中小胶质细胞环的分子机制提供新的见解。这些研究也将为我们开发SIRT1增强策略作为AD新的治疗方法的长期目标奠定基础。

DESCRIPTION (provided by applicant): Microglial activation has long been proposed to contribute to the pathogenesis of Alzheimer's disease (AD). Besides causing direct toxic effects on neuronal and synaptic functions, accumulation of amyloid beta (Aß) and/or tau stimulates microglial activation and expression of inflammatory cytokines, which can induce further neuronal damage. Blocking the toxic pathway in microglial activation could effectively protect against neurodegeneration. However, the molecular mechanisms modulating the microglial loop remain elusive. Our previous studies in primary cortical cultures suggest that NF-κB activation in microglia plays a critical role in microglial-mediated Aß toxicity. Inhibition of NF-κB by SIRT1, a member of the sirtuin family of histone deacetylases, protected against microglia toxicity in Aß-treated primary cultures. In AD brains, SIRT1 levels were markedly reduced. SIRT1 expression in cultured microglia was significantly diminished by Aß treatment. Based on these findings, we hypothesize that SIRT1 reduction is a key event leading to microglial toxicity in AD and that microglial SIRT1 limits Aß-mediated neuronal deficits by suppressing NF-κB activation. To test this hypothesis, we propose three Specific Aims. In Aim 1, we will inactivate SIRT1 in microglia of mice expressing human amyloid precursor protein (hAPP) and systematically examine how microglial SIRT1 inactivation affects inflammatory responses and Aß-related neuronal/behavioral deficits. In Aim 2, to determine if microglial SIRT1 exerts neuroprotection by suppressing NF-κB activation, we will determine if constitutive activation of canonical NF-κB signaling in microglia of hAPP mice exacerbates the deficits in a manner similar to SIRT1 deletion. In complementary experiments, we will determine if inhibiting NF-κB signaling will ameliorate Aß-associated neuronal deficits by infusing a potent NF-κB inhibitor in the brain or injection of a viral vector that inhibits NF-κB in microglia. In Aim 3, to determine the mechanism by which microglial SIRT1 inhibits NF-κB, we will systematically examine if SIRT1 inhibits NF-κB by deacetylating RelA and/or by reducing RelA phosphorylation in myeloid cells. Using chromatin immunoprecipitation (ChIP) analyses, we will then determine if SIRT1- induced suppression of NF-κB activation involves deacetylation of histones (H3K56Ac) in myeloid cells. Completion of the proposed studies will provide new insight into the molecular mechanisms modulating the microglial loop in neurodegeneration. These studies will also lay the foundation for our long-term goal of developing SIRT1-enhancing strategies as a new therapeutic approach for AD.