REST-Activated Program of Gene Expression in Ischemia

 描述(申请人提供)：REST，也被称为NRSF，是一种基因沉默因子，在多能干细胞和神经前体细胞中广泛表达，在这些细胞中它是活跃的 抑制大量对突触可塑性和结构重塑重要的编码和非编码基因，包括microRNAs。REST和microRNAs之间的相互作用是一个新的新兴话题1-4。越来越多的证据表明，microRNAs在K+通道的表达调控中起着关键作用。我们的研究发现，神经元损伤触发了成熟海马神经元REST的诱导，以及对神经元功能至关重要的靶基因miR-132和Kv7.1表达的依赖于REST的沉默/减弱，这表明REST参与了缺血性中风11的发病机制。我们的发现是，miR-132在海马神经元的过表达提供了强有力的保护，证明了miR-132的丢失与神经元死亡之间的因果关系，并强调了我们工作的临床相关性11。总体目标是研究我们最近在神经元中发现的两个有效的REST靶点miR132和KCNQ1/Kv7.1在与全球缺血相关的神经退化和认知障碍中的潜在作用。推动这项研究的中心假设是，全球缺血上调了成年神经元的休眠状态，而休眠状态则促进了一部分“转录反应”基因网络的沉默，这些基因网络维持了神经元死亡与存活之间的平衡。我们试图在以下特定目标中检验这一假说：1)检测候选REST靶点miR-132在缺血诱导的神经元死亡中的作用。实验将1)验证miR-132表现出静息依赖的表观遗传学重塑和对缺血损伤的基因表达的改变；2)确定REST是否与缺血诱导的CA1神经元miR-132表达和/或功能的改变有关，包括对REST的抑制；3)确定miR-132是否与缺血诱导的神经元死亡有关；4)通过miR下拉实验确定促进神经元死亡的新的miR-132靶点；5)检测缺血预适应预防缺血诱导的静息激活、抑制miR-132、挽救神经元死亡和认知缺陷的能力。2)确定新的静息靶基因KV7/KCNQ家族电压门控K+通道是否与缺血诱导的神经元死亡有关。实验将：1)记录在控制和缺血条件下的候选REST靶点KCNQ1通道的存在；2)通过芯片验证缺血对Kv7.1-3启动子上REST结合改变和表观遗传修饰的影响；3)通过qPCR验证Kv7.1-3基因表达的变化，通过蛋白质和电生理学验证Kv7.1-3的功能；4)检测REST的遗传操作模拟或防止缺血诱导的Kv7.1-3表达和/或功能变化的能力，包括REST CKO小鼠；5)检测Kv7.1基因操作影响神经元死亡和认知缺陷的能力；6)检测在神经元和可能的休息靶点表达的新的microRNAs，如133和375，调节KCNQ1在受损神经元中表达和功能的能力。

 DESCRIPTION (provided by applicant): REST, also known as NRSF, is a gene silencing factor that is widely expressed in pluripotent stem cells and neural progenitors, where it actively represses a large array of coding and non-coding genes important to synaptic plasticity and structural remodeling including microRNAs. The interplay between REST and microRNAs is a new, emerging topic1-4. Emerging evidence indicates that microRNAs play a key role in regulation of K+ channel expression. Our findings that neuronal insults trigger induction of REST in mature hippocampal neurons and REST- dependent silencing/attenuation of miR-132 and Kv7.1 expression, target genes essential for neuronal function, implicate REST in the pathogenesis of ischemic stroke11. Our finding that overexpression of miR-132 in hippocampal neurons affords robust protection documents a causal relation between loss of miR-132 and neuronal death and underscores the clinical relevance of our work11. The overall objective is to examine a potential role for two validated REST targets miR132 and KCNQ1/Kv7.1, which we recently discovered in neurons, in the neurodegeneration and cognitive impairment associated with global ischemia. The central hypothesis driving the research is that global ischemia upregulates REST in adult neurons and that REST promotes silencing of a subset of "transcriptionally-responsive" and gene networks that maintain the balance between neuronal death and survival. We seek to test this hypothesis in the following Specific Aims: 1) Examine a role for miR-132, a candidate REST target, in ischemia-induced neuronal death. Experiments will 1) verify that miR-132 exhibits REST-dependent epigenetic remodeling and altered gene expression in response to ischemic insults; 2) Determine whether REST is causally related to ischemia-induced alterations in miR-132 expression and/or function in insulted CA1 neurons by overexpression or inhibition of REST, including a floxed REST KO mouse; 3) Determine whether miR-132 is causally related to ischemia-induced neuronal death; 4) Identify novel miR-132 targets that promote neuronal death by miR pull-down assay; 5) Examine the ability of ischemic preconditioning to prevent ischemia-induced activation of REST, suppression of miR-132 and rescue of neuronal death and cognitive deficits 2) Determine whether the Kv7/KCNQ family of voltage-gated K+ channels, novel REST target genes, are causally related to ischemia-induced neuronal death. Experiments will: 1) Document the presence of functional KCNQ1 channels, a candidate REST target, in under control and ischemic conditions; 2) Validate the impact of ischemia on altered REST binding and epigenetic modifications at Kv7.1-3 promoters by ChIP; 3) Validate alterations in gene expression of Kv7.1-3 by qPCR, protein by Westerns and function by electrophysiology; 4) Examine the examine ability of genetic manipulation of REST to mimic or prevent ischemia-induced alterations in Kv7.1-3 expression and/or function by genetic manipulation, including a REST cKO mouse; 5) Examine the ability of genetic manipulation of Kv7.1 to influence neuronal death and cognitive deficits; and 6) Examine the ability of novel microRNAs, such as 133 and 375, expressed in neurons and putative targets of REST, to regulate KCNQ1 expression and function in insulted neurons.