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性前脑或全脑缺血每年影响20万美国人，在许多情况下会导致海马神经元延迟死亡和严重的认知缺陷。到目前为止，治疗与全球缺血相关的神经变性和认知缺陷是一个尚未得到满足的需求。REST(Repressor Element 1 Silving转录因子)是一种基因沉默转录因子，在胚胎发育过程中广泛表达，在多能干细胞向神经元转化过程中发挥重要作用。在神经元分化的后期，静息下调是获得神经表型的关键。调节多能细胞和神经前体细胞静止丰度的一个基本机制是通过SCF(Skp1-Cul1-F-box Protein)/？-TrCP依赖的、基于泛素的蛋白酶体降解。我们发现，缺血抑制了成年神经元中的？-TrCP，而蛋白酶体抑制剂即使在没有受到伤害的情况下也能激活REST。我们的靶向芯片上分析表明，REST抑制缺血后神经元中的一个离散的靶基因子集。总体目标是了解神经元损伤是如何激活REST的，并研究两个新发现的REST靶点KV7和TRPV1在与全球缺血相关的神经退行性变中的潜在作用。推动这项研究的中心假说是，全球缺血通过泛素蛋白酶体降解作用于成年神经元上调REST，而REST促进转录反应的靶基因子集沉默，从而导致神经元死亡。具体目的是：目的1.通过药理学和遗传学方法，包括一种REST基因敲除小鼠，鉴定缺血调节CA1神经元静息的分子机制。1)检测缺血对泛素E3连接酶？-TrCP、蛋白酶体活性和静息稳定性的影响；2)检测蛋白酶体降解抑制剂在无损伤的情况下增加静息丰度、增强静息稳定性、沉默静息靶点和诱导神经元死亡的能力；3)检测？-TrCP敲除和显性阴性(DN)？-TrCP增强静息丰度和稳定性、静息靶点沉默和诱导神经元死亡的能力；以及4)检测？-TrCP和Cul1过表达抑制静息靶点的静息上调和沉默以及促进缺血后CA1神经元存活的能力。目的2.确定靶向芯片分析确定的新的REST靶点是否在功能和因果关系上与神经元死亡相关，并确定实现REST沉默特异性的机制。1)证实被确认为阳性HITS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(生物验证)；3)记录新的REST靶标KV7和TRPV1通过基因操作与神经元死亡相关；以及4)确定在受干扰的CA1神经元中实现静息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，以改善这一人类发病率和死亡率的严重原因。 公共卫生相关性：心脏骤停导致的全球缺血每年影响约20万美国人，在许多情况下会导致神经功能障碍。到目前为止，治疗与全球缺血相关的神经变性和认知缺陷是一个尚未得到满足的需求。这些转化研究将加速开发新的治疗策略，以改善这一人类发病率和死亡率的严重原因。这些研究的结果不仅对缺血性中风有广泛的影响，而且对其他神经退行性疾病也有广泛的影响，包括脊髓损伤、肌萎缩侧索硬化症、亨廷顿病、帕金森病和阿尔茨海默病。

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.