REST-Activated Program of Gene Expression in Ischemia

描述（由申请人提供）：在心脏骤停期间引起的短暂性前脑或全身缺血每年影响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，治疗与全脑缺血相关的神经变性和认知缺陷是一个未满足的需求。REST （repressor element 1 silencing transcription factor）是一种在胚胎发生过程中广泛表达的基因沉默转录因子，在多能干细胞向神经元的转变中起着战略性作用。在神经元分化的后期，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，REST丰度调节的一个基本机制是通过SCF （Skp1-Cul1-F-box蛋白）/？- trcp依赖性，泛素基础的蛋白酶体降解。我们发现缺血抑制？-TrCP在成年神经元中，并且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ChIP-on-chip分析表明，REST抑制后图式神经元中目标基因的一个离散子集。总体目标是了解神经元损伤如何激活REST，并检查两个新发现的REST靶点Kv7和TRPV1在与全局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，全身缺血通过泛素蛋白酶体降解来上调成人神经元中的REST，而REST促进了“转录反应”靶基因子集的沉默，从而导致神经元死亡。具体目标是：目标1。通过药理学和遗传学方法确定缺血调节CA1神经元REST的分子机制，包括floxed REST敲除小鼠。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)检测蛋白酶体降解抑制剂在无损伤情况下增加REST丰度、增强REST稳定性、沉默REST靶点和诱导神经元死亡的能力；3)考查能力？-TrCP敲低和显性阴性（dn）？-TrCP增强REST丰度和稳定性，沉默REST靶点，诱导神经元死亡；4)考查？-TrCP和Cul1过表达抑制REST上调和REST靶点沉默，促进缺血后CA1神经元的存活。目标2。确定通过靶向ChIP-on-chip分析确定的新的REST靶点是否在功能上发生改变，是否与神经元死亡有因果关系，并确定REST沉默特异性实现的机制。1)验证被鉴定为正命中的候选基因表现出表观遗传失调（技术验证）；2)证明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（生物学验证）；3)通过基因操作证明新的REST靶点Kv7和TRPV1与神经元死亡有因果关系；4)确定在受损伤的CA1神经元中实现REST特异性沉默的机制。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和死亡原因。

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. PUBLIC HEALTH RELEVANCE: Global ischemia arising as a consequence of cardiac arrest affects ~200,000 Americans per year and in many cases results in neurological deficits. To date, treatment of the neurodegeneration and cognitive deficits associated with global ischemia is an unmet need. These translational studies will accelerate the development of novel therapeutic strategies to ameliorate this serious cause of human morbidity and mortality. Findings from these studies have broad implications not only for ischemic stroke, but also for other neurodegenerative disorders including spinal cord injury, ALS, Huntington's disease, Parkinson's disease and Alzheimer's disease.