MicroRNA Regulation of Phospholipid Homeostasis in Alzheimer's Disease Pathogenesis

项目摘要 APOE 4是散发性AD最强的遗传风险因子，对AD具有Ab依赖性和Ab非依赖性影响。 发病机理然而，在AD中APOE 4致病本质的分子机制， 还没有完全阐明。在上一个资助期（2017年1月7日至今），我们取得了重大进展 旨在了解AD中APOE 4诱导的脑磷脂失调的micro-RNA（miRNA）调节。 我们发现了一种新的miR-195调控机制，靶向APOE 4相关的认知缺陷 和溶酶体缺陷。值得注意的是，我们将miR-195鉴定为参与APOE-1的最佳miRNA候选者。 调节的脑磷酸肌醇二磷酸（PIP 2）途径，使用人ROSMAP和小鼠微阵列数据。 miR-195的水平在APOE 4+人和小鼠脑中显著较低，在人诱导型脑中显著较低。 多能干细胞（iPSC）衍生的神经元和星形胶质细胞相比，APOE 4-对应物。过- 表达miR-195会降低其最高靶点synaptojanin 1（synj 1）的表达水平， 酵素升高miR-195可改善APOE 4+小鼠的认知缺陷和AD病理学， APOE 4 + iPSC脑细胞中的溶酶体缺陷。此外，我们的初步结果支持miR-195的作用， 作为一种抗炎性的miRNA来调节小胶质细胞的功能。我们的单细胞（sc）-RNA测序。E4 FAD分析 miR-195过表达的小鼠大脑表明，miR-195改变了小胶质细胞亚群的分子特征。 集群基线时miR-195水平较低且synj 1表达较高的APOE 4+小胶质细胞， 与APOE 3+小胶质细胞相比，吞噬活性受损和溶酶体缺陷。下调 synj 1或过表达miR-195可以挽救这些表型。除了synj 1，炎症基因pdcd 4 和smad 7也是miR-195的预测靶点。miR-195在小胶质细胞中的过表达抑制了 脂多糖（LPS）诱导的smad 7和pdcd 4表达增加，减弱LPS诱导的 促炎细胞因子释放并增强抗炎反应。此外，外泌体来源于 APOE 4/4星形胶质细胞（ADE）中的miR-195含量低于APOE 3/3 ADE， APOE 4/4星形胶质细胞中的miR-195增加ADE中的miR-195水平，这可以减弱LPS诱导的促凋亡作用。 炎症细胞因子释放。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synj 1来调节溶酶体功能，直接靶向小胶质细胞炎症 基因表达和应答，以及外泌体miR-195对神经炎症和tau扩散的调节。 我们建议在本研究中描述AD发病过程中miR-195对小胶质细胞功能的调节。 续期申请。我们将：1）确定miR-195对小胶质细胞功能和APOE调节的影响。 在雄性和雌性EFAD中使用铜腙（CPZ）诱导的炎症的体内AD神经炎症（目的1） 小鼠模型（在5xFAD背景下的人ApoE 4敲入）; 2）通过以下方法表征分子机制： 使用小胶质细胞培养和3-D共培养， 来自EFAD和APOE KI小鼠以及来自synj 1-/-、APOE-/-和TREM 2-/-小鼠的小鼠脑细胞系统 (with miR-195水平的操作）; 3）使用scRNA-195进行高分辨率多尺度网络建模。 来自小鼠脑的seq数据集（Aim 1）和来自小胶质细胞和3-D共培养物的RNA-seq和miR-seq数据集 系统（目的2）鉴定由miR-195驱动的小胶质细胞特异性分子特征;和4）验证鉴定的 在死后人脑样品中由miR-195驱动的小胶质细胞特征，并研究它们与 疾病进展期间AD相关神经炎症的发展（目的2）。这个的目标 本申请旨在阐明由miR-195驱动的新途径和分子特征， APOE 4诱导的AD发病机制中的小胶质细胞功能障碍，这将有助于识别和开发 一种针对AD相关神经炎症的更个性化的靶向治疗方法。

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.