A study of ZCCHC17 regulation of synaptic genes in Alzheimers disease

描述(由申请者提供)：越来越多的证据表明，阿尔茨海默病(AD)最初是一种突触障碍。研究表明，毒性水平的β-淀粉样蛋白在萎缩和细胞死亡发生之前很久就与突触功能障碍和记忆丧失有关，这一观点得到了支持。受到这种观察的启发，我使用生物信息学工具来寻找阿尔茨海默病突触功能障碍的原因。我已经确定了一种名为ZCCHC17的转录因子，它在AD的脑组织中被降低，我的假设是，这种转录因子的低水平与AD的突触功能障碍有关。我的假设得到了我的初步数据的支持(见下文)。根据其结构和核定位，ZCCHC17有望调控基因表达。然而，它并没有得到很好的研究，以前也没有被认为与AD有关。进一步研究ZCCHC17‘S在AD突触功能障碍中的作用，可能为挽救AD突触可塑性和记忆提供一种治疗策略。我通过计算和湿法实验室技术的结合得出了我的假设。我开始使用新的统计技术，从激光解剖的人类AD和对照大脑的神经元中筛选出RNA表达谱。我的目标是找出转录因子：1)被预测调节大量突触靶点，2)被预测在AD中功能受损。我在这项工作中最有力的候选者是ZCCHC17。我的研究表明：1)ZCCHC17蛋白在AD脑组织匀浆中的表达水平降低；2)ZCCHC17主要定位于神经元核，在AD患者中表达降低。尽管令人信服，但这些数据仍然只是相互关联的。由于ZCCHC17尚未得到很好的研究，因此本研究的目标是产生支持我的假设的基础数据，即ZCCHC17对突触功能很重要，并且在AD中受损。这些数据将证明在未来的项目中对ZCCHC17进行更深入的研究是合理的。在目标1中，我将检验ZCCHC17是一种调节突触基因的转录因子的假设。我将首先使用Chip-seq来完成这项工作，它将允许我确定ZCCHC17是否与DNA结合。如果ZCCHC17与DNA调控区结合，则支持ZCCHC17是调控基因表达的转录因子的假说。我将在几个不同的组织中进行CHIP-SEQ，以加深我对ZCCHC17的S在神经元功能(人组织、SY5Y细胞、小鼠海马神经元和成年小鼠脑)中作用的理解。我还将对最后三个组织中的ZCCHC17进行siRNA敲除。如果ZCCHC17的siRNA下调导致ZCCHC17‘S预测的突触靶基因表达减少，那么这支持了ZCCHC17调控这些突触基因表达的假说。在目标2中，我将检验β-淀粉样蛋白水平升高导致ZCCHC17蛋白水平降低的假设。我有支持这一假设的初步数据，我将在SY5Y细胞、小鼠海马神经元培养物和成年小鼠皮质中直接测试这一假设。如果这一预测是正确的，那么这支持了β-淀粉样蛋白损害ZCCHC17功能的假设。

DESCRIPTION (provided by applicant): Accumulating evidence suggests that Alzheimer's Disease (AD) begins as a synaptic disorder. This view is supported by work showing that toxic levels of β-amyloid protein are associated with synaptic dysfunction and memory loss long before atrophy and cell death occur. Inspired by this observation, I have used bioinformatics tools to search for causes of synaptic dysfunction in AD. I have identified a transcription factor called ZCCHC17 that is reduced in AD brain tissue, and my hypothesis is that low levels of this transcription factor contribute to synaptic dysfunction in AD. My hypothesis is supported by my preliminary data (see below). Based on its structure and nuclear localization, ZCCHC17 is predicted to regulate gene expression. However, it is not well studied, and has not previously been linked to AD. Further study of ZCCHC17's role in synaptic dysfunction in AD may provide a therapeutic strategy to rescue synaptic plasticity and memory in this disease. I arrived at my hypothesis using a combination of computational and wet-lab techniques. I began by using novel statistical techniques to screen RNA expression profiles from laser-dissected neurons taken from human AD and control brains. My goal was to identify transcription factors that: 1) Are predicted to regulate a large number of synaptic targets, and 2) Are predicted to have impaired function in AD. My strongest candidate from this work is ZCCHC17. I have now shown that 1) ZCCHC17 protein levels are decreased in AD brain homogenate, and 2) ZCCHC17 is primarily located in neuronal nuclei and is decreased in AD. Although compelling, this data is still only correlational. Since ZCCHC17 is not well studied, the goal of this proposal is to generate foundational data that supports my hypothesis that ZCCHC17 is important for synaptic function and is impaired in AD. This data will justify a more in-depth study of ZCCHC17 in a future project. In Aim 1, I will test the hypothesis that ZCCHC17 is a transcription factor that regulates synaptic genes. I will do this first using ChIP-seq, which will allow me to determine if ZCCHC17 binds to DNA. If ZCCHC17 binds to DNA regulatory regions, then this supports the hypothesis that ZCCHC17 is a transcription factor that regulates gene expression. I will perform ChIP-seq in several different tissues in order to increase my understanding of ZCCHC17's role in neuronal function (human tissue, SY5Y cells, mouse hippocampal neurons, and adult mouse brain). I will also perform siRNA knock-down of ZCCHC17 in the last three tissues. If siRNA knock- down of ZCCHC17 causes reduced expression of ZCCHC17's predicted synaptic targets, then this supports the hypothesis that ZCCHC17 regulates the expression of these synaptic genes. In Aim 2, I will test the hypothesis that elevated β-amyloid levels lead to lower ZCCHC17 protein levels. I have preliminary data supporting this hypothesis, and I will directly test this hypothesis in SY5Y cells, mouse hippocampal neuronal cultures, and in adult mouse cortex. If this prediction is true, then this supports the hypothesis that β-amyloid impairs ZCCHC17 function.