Investigating Argonaute phosphorylation and miR-134 as novel therapeutic targets for Alzheimer's disease

在阿尔茨海默病（AD）的临床前期，突触传递和树突棘丢失的显着缺陷发展。这一过程巩固了在疾病后期出现的电路功能障碍和认知能力下降。microRNA（miRNAs）是小的非编码RNA，作为RNA诱导沉默复合物（RISC）的一部分，通过Argonaute（Ago）参与RNA介导的突触蛋白沉默。已知miRNA参与突触和树突形态的调节，并且特定的miRNA已经涉及AD的发病机制。然而，RISC蛋白机器如何受到AD病理学的影响还不太清楚。来自该实验室的初步数据表明，在AD的hAPP-J20小鼠模型中，S387处的Ago磷酸化和Ago与RISC支架蛋白GW 182的结合增加。这表明miRNA活性可能是AD相关病理学的结果，并导致脊柱丢失和突触功能障碍。假设：阻断淀粉样蛋白病模型中S387处的Ago磷酸化将通过重新设置miRNA activity.Methods减少在这些模型中观察到的突触缺陷和脊柱收缩：在过表达APP的原代神经元培养物中，将内源性Argonaute替换为磷酸无效突变体，并使用携带关键突触蛋白的3 'UTR种子序列的荧光素酶报告构建体进行双荧光素酶测定来定量miRNA活性。在淀粉样蛋白病的小鼠模型中，通过使用立体定向注射到海马的CA 1区中，将使用以下三种挽救策略：a）用磷酸无效突变体慢病毒替换内源Argonaute，B）关键突触蛋白3的慢病毒过表达结合和抑制关键突触miRNA活性的拮抗剂。共聚焦显微镜以评估突触功能和树突形态。

During the pre-clinical period of Alzheimer's Disease (AD) significant deficits in synaptic transmission and dendritic spine loss develop. This process underpins the circuit dysfunction and cognitive decline seen in later stages of the disease. MicroRNAs (miRNAs) are small non-coding RNAs involved in RNA-mediated silencing of synaptic proteins via Argonaute (Ago) as part of the RNA-induced Silencing Complex (RISC). miRNAs are known to be involved in regulation of synapses and dendrite morphology and specific miRNAs have been implicated in the pathogenesis of AD. However, how RISC protein machinery is affected by AD pathology is less well understood. Preliminary data from this lab suggests that phosphorylation of Ago at S387 and binding of Ago to RISC scaffold protein GW182 is increased in the hAPP-J20 mouse model of AD. This suggests that miRNA activity may be dysregulated as a consquence of AD-related pathology and contributing to spine loss and synaptic dysfunction. Hypothesis: Blocking Ago phosphorylation at S387 in models of amyloidopathy will reduce synaptic deficits and spine shrinkage observed in these models via resetting of miRNA activity.Methods: Endogenous Argonaute will be replaced with phospho-null mutant in primary neuronal cultures overexpressing APP and miRNA activity will be quantified using dual luciferase assays using luciferase reporter constructs carrying the 3'UTR seed sequence of key synaptic proteins. In a mouse model of amyloidopathy the following three rescue strategies will be utilised by use of stereotactic injection into the CA1 region of the hippocampus: a) lentiviral replacement of endogenous Argonaute with a phospho-null mutant, b) lentiviral overexpression of key synaptic protein 3) Antagomirs to bind and repress activity of key synaptic miRNAs.Slices will be prepared from these mice for electrophysiological recording and spine analysis by confocal microscopy to assess synaptic function and dendrite morphology.