Epigenetic dysregulation of enhancers in neurons is associated with Alzheimer’s disease pathology and cognitive symptoms (RNA-Seq). Epigenetic dysregulation of enhancers in neurons is associated with Alzheimer’s disease pathology and cognitive symptoms (RNA-Seq)

Epigenetic control of enhancers alters neuron functions and may be involved in Alzheimer’s disease (AD). Here, we identify enhancers in neurons contributing to AD by comprehensive fine-mapping of DNA methylation at enhancers, genome-wide. We examine 1.2 million CpG and CpH sites in enhancers in prefrontal cortex neurons of individuals with no/mild, moderate, and severe AD pathology (n=101). We identify 1,224 differentially methylated enhancer regions; most of which are hypomethylated at CpH sites in AD neurons. CpH methylation losses occur in normal aging neurons, but are accelerated in AD. Integration of epigenetic and transcriptomic data demonstrates a pro-apoptotic reactivation of the cell cycle in post-mitotic AD neurons. Furthermore, AD neurons have a large cluster of significantly hypomethylated enhancers in the DSCAML1 gene that targets BACE1. Hypomethylation of these enhancers in AD is associated with an upregulation of BACE1 transcripts and an increase in amyloid plaques, neurofibrillary tangles, and cognitive decline. Overall design: In order to identify enhancers involved in AD, we comprehensively mapped DNA methylation at enhancers, genome-wide, in neurons isolated from the prefrontal cortex of 106 individuals with no/mild, moderate and severe AD pathology (Braak stage: I-II n=38, III-IV n=32, and V-VI n=36). We first isolated neuronal nuclei using an established antibody (NeuN+) and flow cytometry-based approach. We then fine-mapped DNA methylation at enhancer elements using a targeted bisulfite sequencing strategy, known as bisulfite padlock probe sequencing.

增强子的表观遗传调控可改变神经元功能，且可能参与阿尔茨海默病（Alzheimer’s disease, AD）的发病过程。本研究通过全基因组范围内对增强子区域DNA甲基化的精细化定位，筛选出参与AD发病的神经元增强子。我们对101例（n=101）具有无/轻度、中度及重度AD病理改变的个体的前额叶皮层神经元增强子区域内的120万个CpG及CpH位点进行了检测，共鉴定出1224个差异甲基化增强子区域，其中多数在AD神经元的CpH位点呈现低甲基化状态。正常衰老神经元中即可出现CpH甲基化丢失，但该过程在AD患者神经元中被显著加速。表观组与转录组整合分析显示，有丝分裂后AD神经元出现了促凋亡的细胞周期重激活现象。此外，AD神经元中DSCAML1基因内存在大量显著低甲基化的增强子，该基因可靶向调控BACE1。AD中这些增强子的低甲基化状态与BACE1转录本表达上调、淀粉样斑块形成、神经原纤维缠结及认知功能下降均存在显著关联。

实验设计：为筛选参与AD发病的增强子，我们对106例具有无/轻度、中度及重度AD病理改变的个体的前额叶皮层分离神经元进行了全基因组范围内的增强子DNA甲基化精细化定位。其中，布拉克分期（Braak stage）I-II期38例、III-IV期32例、V-VI期36例。我们首先采用已验证的NeuN+抗体及基于流式细胞术的方法分离神经元细胞核，随后采用靶向亚硫酸氢盐测序策略——即亚硫酸氢盐锁式探针测序（bisulfite padlock probe sequencing）——对增强子区域的DNA甲基化进行精细化定位。