MicroRNA Regulation of Phospholipid Homeostasis in Alzheimer's Disease Pathogenesis

项目总结 载脂蛋白E4是散发性阿尔茨海默病的最强的遗传危险因素，具有抗体依赖和抗体非依赖性 疾病发病机制。然而，阿尔茨海默病中APOE4致病的分子机制 还没有完全阐明。在上一个供资期间(2017年7月1日至今)，我们取得了重大进展 目的了解微小RNA(MiRNA)对APOE4诱导的AD患者脑磷脂失调的调节作用。 我们发现了针对APOE4相关认知缺陷的miR-195的一种新的调节机制 AD患者存在溶酶体缺陷。值得注意的是，我们确定miR-195是参与APOE- 使用人ROSMAP和小鼠微阵列数据调节脑磷酸肌醇二磷酸(PIP2)途径。 在APOE4+的人和小鼠的大脑中，miR-195的水平明显较低，在人的诱导性脑中 多能干细胞(IPSC)来源的神经元和星形胶质细胞，与APOE4对应的细胞相比。过了- 表达miR-195可降低其顶靶突触素1(Synj1)的表达水平，从而降解大脑中的PIP2 酵素。上调miR-195改善APOE4+小鼠的认知障碍和AD病理 APOE4+IPSC脑细胞溶酶体缺陷。此外，我们的初步结果支持miR-195的作用 作为一种抗炎的miRNA，调节小胶质细胞的功能。我们的单细胞(Sc)-RNA序列。E4FAD分析 具有miR-195过表达的小鼠大脑提示miR-195改变了小胶质细胞亚单位的分子特征. 集群。ApoE4+小胶质细胞在基线时miR-195水平较低，synj1表达较高，表现为 与APOE3+小胶质细胞相比，吞噬功能受损和溶酶体缺陷。-下调对 Synj1或过表达miR-195可以挽救这些表型。除了synj1，炎症基因pdcd4 和Smad7也是miR-195的预测目标。MiR-195在小胶质细胞中的过表达抑制作用 内毒素诱导的Smad7和pdcd4表达增加，减弱内毒素诱导的 促进炎症细胞因子的释放，增强抗炎反应。此外，外显子派生 APOE4/4星形胶质细胞(ADE)的miR-195含量低于APOE3/3星形胶质细胞(ADE)，并且过表达 APOE4/4星形胶质细胞中的miR-195可增加ADE中miR-195的水平，从而减轻内毒素诱导的PRO-195 炎性细胞因子释放。因此，我们推测miR-195可能具有抗炎作用。 通过下调小胶质细胞synj1调节溶酶体功能，靶向小胶质细胞炎症 外体miR-195对神经炎症和tau扩散的调节、基因表达和反应。 我们建议表征在AD发病过程中miR-195对小胶质细胞功能的调节。 续签申请。我们将：1)确定miR-195对小胶质细胞功能和APOE调节的影响 铜酮(CPZ)诱导的男性和女性EFAD患者体内AD的神经炎症反应(目标1) 小鼠模型(人类ApoE4在5xFAD背景下敲入)；2)通过以下方法表征分子机制 利用小胶质细胞培养和3-D共培养，哪个miR-195调节AD相关神经炎症(AIM 2) EFAD和APOE KI小鼠以及synj1/-、APOE-/-和TREM2-/-小鼠的脑细胞系统 (具有miR-195级别的操作)；3)使用scRNA执行高分辨率多尺度网络建模- 来自小鼠脑的SEQ数据集(目标1)以及来自小胶质细胞和三维共培养的RNA-SEQ和miR-SEQ数据集 系统(目标2)以鉴定由miR-195驱动的小胶质细胞特异性分子特征；以及4)以验证所识别的 人死后脑标本中miR-195驱动的小胶质细胞特征及其与 疾病进展过程中与AD相关的神经炎症的发展(目标2)。这样做的目的是 应用目的在于阐明miR-195保护猪瘟病毒的新途径和分子特征 载脂蛋白E4诱导的小胶质细胞功能障碍在AD发病机制中的作用 一种更个性化的靶向治疗阿尔茨海默病相关神经炎的方法。

PROJECT SUMMARY APOE4 is the strongest genetic risk factor for sporadic AD with Ab-dependent and Ab-independent effects on disease pathogenesis. However, the molecular mechanisms underlying the pathogenic nature of APOE4 in AD are not fully elucidated. In previous funding period (07/01/2017-present), we have made significant progress toward understanding micro-RNA (miRNA) regulation of APOE4-induced brain phospholipid dysregulation in AD. We have uncovered a novel regulatory mechanism of miR-195 targeted at APOE4-associated cognitive deficits and lysosomal defects in AD. Notably, we identified miR-195 as a top miRNA candidate involved in the APOE- regulated brain phosphoinositol biphosphate (PIP2) pathway using human ROSMAP and mouse microarray data. Levels of miR-195 are significantly lower in APOE4+ human and mouse brains, and in human inducible pluripotent stem cells (iPSC)-derived neurons and astrocytes when compared to APOE4- counterparts. Over- expressing miR-195 reduces expression levels of its top target synaptojanin 1 (synj1), the brain PIP2 degrading enzyme. Elevating miR-195 ameliorates cognitive deficits and AD pathology in APOE4+ mice and rescues lysosomal defects in APOE4+ iPSC brain cells. Furthermore, our preliminary results support the role of miR-195 as an anti-inflammatory miRNA in regulating microglial function. Our single cell (sc)-RNA seq. analysis of E4FAD mouse brains with miR-195 over-expression suggests that miR-195 alters molecular signatures of microglia sub- clusters. APOE4+ microglia with lower miR-195 levels and higher synj1 expression at baseline, manifests with impaired phagocytic activities and lysosomal defects when compared to APOE3+ microglia. Down-regulation of synj1 or over-expression of miR-195 can rescue these phenotypes. Beside synj1, inflammatory genes pdcd4 and smad7 are predicted targets of miR-195 as well. Over-expression of miR-195 in microglia inhibits lipopolysaccharide (LPS)-induced increases in smad7 and pdcd4 expression, attenuates LPS-induced proinflammatory cytokine release and augments anti-inflammatory responses. In addition, exosomes derived from APOE4/4 astrocytes (ADEs) contain less miR-195 than those in APOE3/3 ADEs, and over-expression of miR-195 in APOE4/4 astrocytes increases miR-195 levels in ADEs which can attenuate LPS-induced pro- inflammatory cytokine release. Therefore, we hypothesize that miR-195 may exhibit anti-inflammatory effects through down-regulation of microglial synj1 to regulate lysosomal function, direct target at microglial inflammatory gene expression and responses, and modulation of neuro-inflammation and tau spread by exosomal miR-195. We propose to characterize the regulation of microglial function by miR-195 during AD pathogenesis in this renewal application. We will: 1) determine the impact of miR-195 on microglia function and APOE-regulated neuro-inflammation in AD in vivo (Aim 1) using cuprizone (CPZ)-induced inflammation in male and female EFAD mouse models (human ApoE4 knock-in at 5xFAD background); 2) to characterize the molecular mechanisms by which miR-195 regulates AD-associated neuro-inflammation (Aim 2) using microglial culture and 3-D co-culture system of mouse brain cells from EFAD and APOE KI mice, as well as from synj1-/-, APOE-/- and TREM2-/- mice (with manipulations of miR-195 levels); 3) to perform high resolution multiscale network modeling using scRNA- seq dataset from mouse brains (Aim 1) and RNA-seq and miR-seq datasets from microglia and 3-D co-culture system (Aim 2) to identify microglia-specific molecular signatures driven by miR-195; and 4) to validate identified microglial signature driven by miR-195 in postmortem human brain samples and investigate their correlation with the development of AD-associated neuro-inflammation during disease progression (Aim 2). The goals of this application aim to elucidate novel pathways and molecular signatures driven by miR-195 protective against APOE4-induced microglial dysfunction in AD pathogenesis, which will facilitate identification and development of a more personalized targeted therapeutic approach to AD-associated neuro-inflammation.