Epigenomic Characterization of Alzheimer's Disease Neurons from iPSCs

描述(申请人提供)：阿尔茨海默病(AD)是与痴呆症相关的最常见的神经退行性疾病，尽管其他主要死亡原因在过去十年中有所下降，但AD的发病率却惊人地上升。在AD样神经变性的小鼠模型CK-p25小鼠中，我们表明，通过抑制组蛋白脱乙酰酶(HDAC)活性，染色质重塑的改变可以改善突触和认知障碍。此外，我们发现，认知功能的增强特别需要抑制I类组蛋白脱乙酰酶HDAC2，它通过与突触可塑性和记忆形成相关基因的调控元件在认知中发挥关键作用。我们的初步数据显示，神经毒性刺激可以诱导HDAC2的表达，包括A？42和H_2O_2处理。重要的是，在死后的人类AD脑和AD小鼠模型中，HDAC2显著上调，伴随着HDAC2与记忆基因的关联增加，以及这些基因的表达急剧减少。我们建议研究HDAC2在人类诱导的多能干细胞(IPSC)来源的神经元中抑制可塑性基因的机制，这些神经元来自对照组以及家族性和散发性AD患者。我们将检测HDAC2mRNA和蛋白在这些IPSC来源的神经元中的表达，并将对每个零星的AD系进行APOE基因的聚合酶链式反应测序。然后我们将这些神经元暴露在神经毒性刺激下，并将通过进行抗HDAC2抗体的染色质免疫沉淀和Illumina第二代测序来检查对照、家族性和散发性AD神经元的表观遗传学变化。我们还将通过进行RNA测序来检查这些IPSC来源的神经元在有和没有神经毒性刺激的情况下转录组的变化。这些拟议的研究将检验一种假设，即家族性或散发性AD患者的神经元比对照组更容易受到神经毒性侮辱，并且这种高度的敏感性导致HDAC2上调对神经可塑性基因的抑制增加。重要的是，我们还将研究shRNA介导的HDAC2基因敲除以及几种已知和新的HDAC抑制剂化合物对HDAC2介导的对照组、家族性和散发性AD神经元在神经毒性刺激下学习和记忆基因表达变化的影响。本申请中概述的实验将测试表观遗传改变作为AD相关认知下降的一种新机制的作用。 公共卫生相关性：我们之前已经证明，I类组蛋白脱乙酰酶HDAC2通过与启动子结合并抑制对正常学习和记忆至关重要的基因的表达来响应神经毒性。目前的提议将使用来自患者诱导的多能干细胞的人类神经元来测试这一假设，即阿尔茨海默病患者的神经元更容易通过HDAC2进行神经毒性诱导的染色质重塑。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common neurodegenerative disorder associated with dementia and, while other major causes of death have declined in the past ten years, the incidence of AD has risen alarmingly. In a mouse model of AD-like neurodegeneration, the CK-p25 mouse, we showed that alterations in chromatin remodeling, via the inhibition of histone deacetylase (HDAC) activity, ameliorated synaptic and cognitive impairments. Moreover, we found that the enhancement of cognitive function specifically required inhibition of the class I histone deacetylase, HDAC2, which plays a critical role in cognition by binding to the regulatory elements of genes implicated in synaptic plasticity and memory formation. Our preliminary data show that HDAC2 expression is induced upon neurotoxic stimulation, including A?42 and H2O2 treatments. Importantly, in postmortem human AD brains and in AD mouse models, HDAC2 shows a marked upregulation, which is accompanied by increased association of HDAC2 with memory genes and the drastic reduction in the expression of these genes. We propose to examine the mechanism underlying the HDAC2-mediated inhibition of plasticity genes in human induced pluripotent stem cell (iPSC)-derived neurons derived from control individuals as well as both familial and sporadic AD patients. We will examine the expression of HDAC2 mRNA and protein in these iPSC-derived neurons, and will PCR-sequence each sporadic AD line for the APOE genotype. We will then expose these neurons to neurotoxic stimuli, and will examine the epigenetic alterations in the control, familial, and sporadic AD neurons by conducting chromatin immunoprecipitation with antibodies against HDAC2, followed by Illumina second-generation sequencing. We will also examine changes in the transcriptome of these iPSC-derived neurons, with and without neurotoxic stimuli, by conducting RNA sequencing. The proposed studies will test the hypothesis that neurons derived from familial or sporadic AD patients are more susceptible to neurotoxic insults than those from controls, and that this heightened sensitivity results in an increased suppression of neural plasticity genes by HDAC2 upregulation. Importantly, we will also examine the effects of shRNA-mediated HDAC2 knockdown, as well as treatment with several HDAC inhibitor compounds, both known and novel, upon the HDAC2-mediated alterations in learning and memory gene expression in control, familial, and sporadic AD neurons with and without neurotoxic stimulation. The experiments outlined in this application will test the role of epigenetic alteration as a novel mechanism underlying AD-associated cognitive decline. PUBLIC HEALTH RELEVANCE: We have previously shown that class I histone deacetylase, HDAC2, responds to neurotoxicity by binding to the promoters, and suppressing the expression, of genes critical to normal learning and memory. The current proposal will use human neurons derived from patient induced pluripotent stem cells to test the hypothesis that neurons from Alzheimer's disease patients are more susceptible to neurotoxicity-induced chromatin remodeling via HDAC2.