REST-Activated Program of Gene Expression in Ischemia

描述（由申请人提供）：心脏骤停期间出现的短暂前脑或全脑缺血每年影响20万美国人，在许多情况下导致海马神经元延迟死亡和严重的认知缺陷。迄今为止，与全脑缺血相关的神经变性和认知缺陷的治疗是一个未满足的需求。REST（阻遏物元件1沉默转录因子）是一种基因沉默转录因子，在胚胎发生过程中广泛表达，并在多能干细胞向神经元的转变中发挥战略作用。在神经元分化的晚期阶段，REST下调对神经表型的获得至关重要。在多能细胞和神经祖细胞中调节REST丰度的基本机制是通过SCF（Skp 1-Cul 1-F-box蛋白）/？TrCP依赖的、基于泛素的蛋白酶体降解。我们发现局部缺血抑制了？- TrCP在成年神经元中的作用，以及蛋白酶体抑制剂即使在没有损伤的情况下也能激活REST。我们的靶向ChIP芯片分析表明，REST抑制了缺血后神经元中靶基因的离散子集。总体目标是了解神经元损伤如何激活REST，并研究两个新发现的REST靶点Kv 7和TRPV 1在与全脑缺血相关的神经变性中的潜在作用。推动这项研究的中心假设是，全脑缺血通过泛素蛋白酶体降解来上调成年神经元中的REST，并且REST促进了“转录响应”靶基因的子集的沉默，这驱动了神经元死亡。具体目标是：目标1。通过药理学和遗传学方法（包括floxed REST敲除小鼠）确定缺血调节CA 1神经元REST的分子机制。1)检查缺血对泛素E3连接酶的影响？TrCP、蛋白酶体活性和REST稳定性; 2）检查蛋白酶体降解抑制剂增加REST丰度、增强REST稳定性、沉默REST靶点和在无损伤情况下诱导神经元死亡的能力; 3）检查？- TrCP敲低和显性阴性（dn）？- TrCP增强REST丰度和稳定性，沉默REST靶点并诱导神经元死亡;以及4）检查？TrCP和Cul 1过表达抑制REST上调和REST靶点沉默，促进缺血后CA 1神经元存活。目标二。确定通过靶向ChIP芯片分析鉴定的新型REST靶点是否在功能上发生改变，并与神经元死亡存在因果关系，并确定实现REST沉默特异性的机制。1)验证鉴定为阳性命中的候选基因表现出表观遗传失调（技术验证）; 2）记录表观遗传失调导致基因表达、蛋白质丰度和突触功能的改变3）证明新的REST靶标Kv 7和TRPV 1与通过遗传操作的神经元死亡有因果关系;和4）鉴定在受损伤的CA 1神经元中实现REST沉默的特异性的机制。这些转化研究将加速开发新的治疗策略，以改善这一严重的人类发病率和死亡率的原因。

DESCRIPTION (provided by applicant): Transient forebrain or global ischemia arising during cardiac arrest affects 200,000 Americans each year and in many cases results in delayed death of hippocampal neurons and severe cognitive deficits. To date, treatment of the neurodegeneration and cognitive deficits associated with global ischemia is an unmet need. REST (repressor element 1 silencing transcription factor) is a gene silencing transcription factor that is widely expressed during embryogenesis and plays a strategic role in the transition from pluripotent stem cells to neurons. During the late stages of neuronal differentiation, REST downregulation is critical to acquisition of the neural phenotype. A fundamental mechanism by which REST abundance is regulated in pluripotent cells and neural progenitors is via SCF (Skp1-Cul1-F-box protein)/?-TrCP-dependent, ubiquitin-based proteasomal degradation. We found that ischemia suppresses ?-TrCP in adult neurons, and that proteasomal inhibitors activate REST even in the absence of insult. Our targeted ChIP-on-chip analysis indicates that REST suppresses a discrete subset of target genes in postischemic neurons. The overall objectives are to understand how neuronal insults activate REST and examine a potential role for two newly identified REST targets, Kv7 and TRPV1, in the neurodegeneration associated with global ischemia. The central hypothesis driving the research is that global ischemia acts via ubiquitin proteasomal degradation to upregulate REST in adult neurons and that REST promotes silencing of a subset of "transcriptionally-responsive" target genes, which drives neuronal death. Specific Aims are: Aim 1. Identify the molecular mechanisms by which ischemia regulates REST in CA1 neurons by pharmacological and genetic approaches including a floxed REST knockout mouse. 1) Examine impact of ischemia on the ubiquitin E3 ligase ?-TrCP, proteasome activity and REST stability; 2) Examine ability of inhibitors of proteasomal degradation to increase REST abundance, enhance REST stability, silence REST targets and induce neuronal death in the absence of insult; 3) Examine ability of ?-TrCP knockdown and dominant-negative (dn)?-TrCP to enhance REST abundance and stability, silence REST targets and induce neuronal death; and 4) Examine ability of ?-TrCP and Cul1 overexpression to suppress REST upregulation and silencing of REST targets and promote survival of postischemic CA1 neurons. Aim 2. Determine whether novel REST targets identified by a targeted ChIP-on-chip analysis are altered functionally and causally related to neuronal death and identify mechanisms by which specificity of REST silencing is achieved. 1) Verify that candidate genes identified as positive hits exhibit epigenetic dysregulation (technical validation); 2) Document that epigenetic dysregulation results in alterations in gene expression, protein abundance and synaptic function (biological validation); 3) Document that novel REST targets Kv7 and TRPV1 are causally related to neuronal death by genetic manipulation; and 4) identify mechanisms by which specificity of REST silencing is achieved in insulted CA1 neurons. These translational studies will accelerate development of novel therapeutic strategies to ameliorate this serious cause of human morbidity and mortality.