特异性5hmC丢失对阿尔茨海默病神经元退行性病变的影响及意义

The aging of brain has been associated with abnormal alterations in multiple epigenetic systems including aberrant DNA methylation patterns. DNA 5-Hydroxymethylcytosin (5hmC), a new epigenetic hallmark which was discovered recently is becoming an interesting topic. The exact function of this base remains being poorly elucidated, but it is thought to involve gene expression or prompt active DNA demethylation. The levels of 5hmC in genomic DNA vary significantly depending on the cell type. The highest levels are found in neuronal cells of the central nervous system, implying its importance in gene regulation of central nervous system. While alterations of histone modification and DNA methylation have been well studied in Alzheimer's disease (AD), the extent of ten eleven Ten-eleven translocation enzymes TET-mediated conversion from DNA 5-methylcytosine (5mC) to 5hmC and their involvement in the pathology of the main neurodegenerative disorders is still mystery. The core hypothesis of this application is that loss of 5-hydroxymethylcytosine plays a critical and possibly vital supporting role in the death of neurons during late-onset Alzheimer's disease (AD). We present preliminary data showing that a significant loss of 5hmC was exclusively found in specific brain regions in postmortem samples from patients with confirmed diagnosis of Alzheimer's disease (AD) in comparison to age and sex matched healthy control subjects. These results indicate that the involvement of 5hmC in the pathology of neurodegenerative disorders and points out common and particular signatures that might aid in differential diagnosis as biomarkers or in the discovery of novel therapeutic targets. We will examine the level of 5mC and 5hmC as well as TET family proteins in human AD postmortem brain samples and assess whether loss of 5hmC is an hallmark related to classic neuropathological features found in AD. We will focus on exploring whether loss of 5hmC is a direct factor of neurodegeneration in human AD frontal and temporal cortex as well as hippocampus samples. We have developed preliminary data that demonstrate that a significant loss of 5hmC is selectively found in human AD as well as 3xTg AD mouse frontal cortex and hippocampus. We will quantify these findings by deep sequencing analyses of 5mC MeDIP and 5hmC hMeDIP in human patient samples. We will focus on the mechanism of loss of 5hmC in driving the selective degeneration of hippocampal and cortical neurons in AD by using 3xTg AD animal model. We have developed a new preliminary data that demonstrate that Tet-mediated the conversion from 5mC to 5hmC in primary neurons was downregulated by treatment of beta-amyloid (Aβ). We will examine whether the manipulation of TETs have directly effects on learning and memory loss in AD mouse modelby live mouse hippocampus lentiviral micro-injection as well as mouse hippocampal slice cultures with viral infection or CRISPR/Cas9 gene-editting.

5-羟甲基化胞嘧（5hmC）是一个新近发现的表观遗传标记，由于其在神经细胞内所呈现的高水平，提示它对神经元功能可能有重要作用。我们初步的研究结果证实在AD病人的大脑皮层及海马等区域的特定神经元5hmC含量发生明显地降低；同时，在AD 小鼠模型的大脑皮层及海马神经细胞也发现明显的选择性5hmC 减少。另外，我们也发现给予可溶性淀粉样蛋白小肽（Aβ）可直接诱导海马及皮层神经细胞发生显著的5hmC 降低。我们计划首先确认Aβ是否影响TETs 酶活性以及机制。另外，利用CRISPR/Cas9技术结合在体神经核团微量注射病毒靶基因或者通过离体特定脑片培养对TETs酶蛋白活性进行调控来观察5hmC 水平变化对AD神经退行性病变的影响。本课题的研究目的是确定特异性DNA 5-羟甲基胞嘧啶（5hmC）的变化在AD神经细胞退行性病变过程的作用及机制，为研发新的早期诊断及治疗手段提供一个全新理论与实验依据。

大量的研究已经揭示在AD等神经退行性疾病进行性病变过程中会发生各种各样的细胞和分子变化损伤，这其中就包括表观遗传系统的遗传改变。通常这些改变都是病理性的，并且可能对病变过程起到重要的影响作用。大量研究表明， 在AD病变过程中，DNA 甲基化/去甲基化修饰会发生异常改变，但是DNA去甲基化的重要中间产物5hmC如何变化，以及其介导的表观调控失调与AD病变关系如何？目前都不清楚。该研究结果表明5hmC丢失是一个新的AD神经元退行性病变过程中的重要生物标志物；利用甲基化DNA免疫沉淀纯化方法和DNA深度测序来分析和确定了5hmC改变对神经细胞和突触小体退行性病变的调控机理；利用在体微量注射病毒人源性TET3基因到AD小鼠海马，能够显著改善AD小鼠海马神经元的退行性病变进程、以及学习记忆功能。该研究证据提示5hmC 改变是AD神经退行性病变的一个新的重要的表观遗传学生物标记，同时也与AD神经元退行性病变密切相关。

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