Epigenomic Characterization of Alzheimer's Disease Neurons from iPSCs

描述（由申请人提供）：阿尔茨海默病（AD）是最常见的与痴呆相关的神经退行性疾病，虽然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中，CK-p25小鼠，我们发现染色质重塑的改变，通过抑制组蛋白去乙酰化酶（HDAC）活性，改善了突触和认知障碍。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HDAC 2，HDAC 2通过与突触可塑性和记忆形成相关基因的调控元件结合在认知中起着关键作用。我们的初步数据表明，HDAC 2表达诱导神经毒性刺激，包括A？42和H2 O2处理。重要的是，在死后人类AD大脑和AD小鼠模型中，HDAC 2显示出显著的上调，这伴随着HDAC 2与记忆基因的关联增加以及这些基因表达的急剧减少。我们建议研究HDAC 2介导的抑制人类诱导多能干细胞（iPSC）衍生的神经元中的可塑性基因的机制，这些神经元来自对照个体以及家族性和散发性AD患者。我们将检查HDAC 2 mRNA和蛋白在这些iPSC衍生的神经元中的表达，并将对APOE基因型的每个散发性AD系进行PCR测序。然后，我们将这些神经元暴露于神经毒性刺激，并将通过用HDAC 2抗体进行染色质免疫沉淀，然后进行Illumina第二代测序，来检查对照，家族性和散发性AD神经元中的表观遗传学改变。我们还将通过进行RNA测序来检查这些iPSC衍生的神经元的转录组的变化，有和没有神经毒性刺激。拟议的研究将测试的假设，即来自家族性或散发性AD患者的神经元比来自对照组的神经元更容易受到神经毒性损伤，并且这种提高的敏感性导致HDAC 2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HDAC 2敲低以及用几种已知和新的HDAC抑制剂化合物治疗对HDAC 2介导的对照、家族性和散发性AD神经元中的学习和记忆基因表达改变的影响，有和没有神经毒性刺激。本申请中概述的实验将测试表观遗传改变作为AD相关认知下降的新机制的作用。 公共卫生相关性：我们以前已经表明，I类组蛋白脱乙酰基酶，HDAC 2，响应神经毒性结合的启动子，并抑制表达，基因的关键正常的学习和记忆。目前的提案将使用来自患者诱导多能干细胞的人类神经元来测试以下假设：来自阿尔茨海默病患者的神经元更容易通过HDAC 2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.