MicroRNA-195调节长期大脑低灌注诱发海马神经退行性变的分子机制研究

Chronic brain hypoperfusion (CBH ) could induce increase of Aβ, hyperphosphorylation of Tau, loss of neuron and synapses of hippocampus, and lead to the impairment of learning and study. Our previous study reported that CBH resulted downregulation of miR-195, which contributed to the increase of Aβ by negative regulation of APP and BACE1 expression. However, whether miR-195 could participate in CBH induced hyperphosphorylation of Tau, loss of neuron and synapses of hippocampus is unknown. In this study, we would like to elucidate the effect of miR-195 on CBH induced neurodegeneration by integrated application of multi-techniques including internet database, lentivirus and miR-masking and raleted molecular biology techniques. Study contains: 1. To assess the level of p-Tau, number of neurons and synapses, the level of miR-195 and it's targeted proteins in the hippocampus of 2VO rat, APP/PS1 mice and deceased patients with VaD; 2. To clarify the regulation effect of miR-195 on it's targets; 3. To evaluate the effect of upregulation of miR-195 by hippocampal stereotaxic injection of miR-195 oligonucleotide fragment carried by lentivirus vector (lenti-pre- miR-195) on the level of p-Tau, number of neurons and synapses, as well as related proteins. The present study would like to further uncover the effect and mechanism of miR-195 on CBH induced dementia, therefore provide the basic evidence for clinical drug development

长期大脑低灌注（CBH）使海马Aβ增多、 Tau过度磷酸化及神经元和突触丢失，导致学习记忆功能下降。我们前期研究表明：CBH时miR-195下降，负性调控APP和BACE1的表达，使Aβ生成增多。但miR-195是否参与p-Tau及神经元和突触丢失的过程尚不清楚。本研究联合使用网络数据库、慢病毒转染、miR-masking及分子生物学技术探讨miR-195在CBH引起p-Tau、神经元和突触丢失中的作用及机制。研究内容：1.评价CBH大鼠、APP/PS1转基因小鼠及血管性痴呆患者海马p-Tau、神经元及突触密度、miR-195及目的蛋白的表达；2.检测miR-195对目的蛋白的调控作用;3.评价慢病毒局部注射对CBH大鼠和APP/PS1小鼠海马p-Tau，神经元和突触密度及相关蛋白的作用。本研究在前期研究基础上结合临床标本阐明miR-195在CBH诱发痴呆中的作用，为临床诊断和用药提供依据

长期脑低灌注被认为是老年痴呆的临床前阶段，但是其分子机制不清。本研究联合使用网络数据库、电镜，慢病毒转染，miR-masking及相关分子生物学技术探讨在实验性CBH大鼠痴呆模型中miR-195在CBH引起的神经退行性变化中的作用及其机制。研究内容：1.评价2VO大鼠海马组织Tau 蛋白的磷酸化，神经元的丢失以及突触密度的变化及miR-195和相应目的蛋白的表达；2.通过细胞转染技术检测miR-195对目的蛋白的调控作用;3.评价慢病毒载体局部海马注射对2VO大鼠海马p-Tau，神经元丢失以及突触密度的影响以及相关蛋白的作用。研究结果表明：CBH可以明显诱发大鼠学习记忆功能下降，其机制与长期大脑缺氧促进核转录因子NF-κB的活化抑制miR-195的表达有关。miR-195转录后上调APP，BACE1蛋白表达促进Aβ沉积，并进一步激活CDK5/p35通路，促进Tau蛋白Thr231及Ser262位点的过度磷酸化，促进LCMT-1和PME-1表达抑制PP2A活性促进，激活N-APP/DR6/caspase信号通路引起的神经元树突退化和神经元死亡。在这一过程中miR-195也直接转录后上调CDK5/p35表达，PME-1以及DR6的表达为Tau蛋白磷酸化，促进PP2A失活和促进神经元树突退化和神经元死亡提供底物蛋白。外源性给予miR-195能够延缓CBH引起的上述Aβ级联反应蛋白的表达，并预防CBH引起的大鼠认知功能障碍。本研究为CBH诱发痴呆的治疗提供了一个有价值的治疗靶点。