Impact of Presenilin 2 Mutation and Deficiency on Microglial Function

描述(申请人提供)：家族性阿尔茨海默病(AD)的研究发现了三个重要的AD基因，淀粉样前体蛋白(APP)，早老素1(PS1)和早老素2(PS2)，其中突变导致常染色体显性几乎完全外显的AD。最近的文献和我们的初步研究表明，早老素和β-分泌酶复合体在关键的细胞通路中发挥作用，包括对炎症刺激的反应。小鼠的条件性PS1/2抑制导致大脑皮层炎症基因上调和小胶质细胞数量增加。PS2缺陷小鼠会发展成成人肺纤维化，这种情况被认为是人类慢性炎症的结果。对多个基因组数据集的系统分析表明，PS2在免疫途径中过度表达，并与关键的先天免疫信号分子共同调节。我们已经证明，在人类AD脑组织的小胶质细胞和培养的受炎性刺激激活的小胶质细胞中，PS2蛋白增加。我们发现，炎症刺激也减少了β-分泌酶介导的APP裂解，而β-分泌酶抑制剂则增加了小胶质细胞对炎性细胞因子的释放。利用稳定表达针对PS2的短发夹状RNA(ShRNA)的小胶质细胞，我们发现PS2表达缺陷的小胶质细胞表现出夸大的炎症反应。我们建议通过研究PS2缺陷的小胶质细胞或表达家族性AD(FAD)PS2突变的小胶质细胞的表型来研究PS2在神经退行性疾病中的作用机制。我们假设PS2下调了一种失控的炎症反应，导致了神经毒性环境。我们将通过慢病毒基因转移将PS2Fad突变基因导入原代小胶质细胞，比较其细胞因子释放、神经毒性、吞噬行为和γ-分泌酶活性。通过比较表达PS2 FAD突变的小胶质细胞、表达PS2 shRNA的慢病毒载体或表达PS2 shRNA的慢病毒诱导的PS2缺失，目的是评估小胶质细胞中PS2 FAD突变的功能效应的丧失或获得。公共卫生相关性：在解决我们的假设时，我们希望对AD生物学的总体理解做出重大贡献，目的是揭示PS相关途径中的特定靶点，用于治疗干预，以及关于目前正在临床试验中评估的治疗的潜在重要信息。

DESCRIPTION (provided by applicant): Studies of familial Alzheimer's disease (AD) have revealed three important AD genes, Amyloid Precursor Protein (APP), Presenilin 1 (PS1) and Presenilin 2 (PS2), in which mutations lead to autosomal dominant AD with nearly complete penetrance. Recent literature and our preliminary studies suggest that the presenilins and the ?-secretase complex play a role in critical cellular pathways including the response to inflammatory stimuli. Conditional PS1/2 repression in mice leads to upregulation of inflammatory genes in the cortex and increased numbers of microglia. PS2 deficient mice develop adult pulmonary fibrosis, a condition presumed to be a result of chronic inflammation in the human. Systems analysis of multiple genomic datasets reveals PS2 is overrepresented in immune pathways and is co-regulated with critical innate immunity signaling molecules. We have shown that PS2 protein is increased in microglia from human AD brain tissue and in cultured microglia activated by inflammatory stimuli. We show that inflammatory stimuli also decrease ?-secretase mediated APP cleavage while ?-secretase inhibitors augment release of inflammatory cytokines by microglia. Using microglia stably expressing short hairpin RNA (shRNA) targeted against PS2 we have found that microglia deficient in PS2 expression manifest an exaggerated inflammatory response. We propose to study the mechanism by which PS2 contributes to neurodegenerative disease by characterizing the phenotype of microglia deficient in PS2 or expressing Familial AD (FAD) PS2 mutations in vitro. We hypothesize that PS2 down regulates an uncontrolled inflammatory response, contributing to a neurotoxic environment. We will compare cytokine release, neurotoxicity, phagocytosis behavior and y-secretase activity of cultured primary microglia following introduction of PS2 FAD mutation gene constructs using lentiviral gene transfer. Microglia expressing PS2 FAD mutations will be evaluated in comparison to microglia expressing control lentiviral vectors or lentivirus expressing PS2 shRNA to induce PS2 deficiency with the goal of assessing loss or gain of function effects of PS2 FAD mutation in microglia. PUBLIC HEALTH RELEVANCE: In addressing our hypothesis, we hope to significantly contribute to the general understanding of AD biology with the goal of revealing specific targets in PS related pathways for therapeutic intervention and potentially important information regarding therapies currently being evaluated in clinical trials.