Data Sheet 1_Deciphering motor dysfunction and microglial activation in mThy1-α-synuclein mice: a comprehensive study of behavioral, gene expression, and methylation changes.pdf

IntroductionGrowing recognition of microglia’s role in neurodegenerative disorders has accentuated the need to characterize microglia profiles and their influence on pathogenesis. To understand changes observed in the microglial profile during the progression of synucleinopathies, microglial gene expression and DNA methylation were examined in the mThy1-α-synuclein mouse model.
MethodsDisease progression was determined using behavioral tests evaluating locomotor deficits before DNA and RNA extraction at 7 and 10 months from isolated microglia for enzymatic methyl-sequencing and RNA-sequencing.
ResultsPathway analysis of these changes at 7 months indicates a pro-inflammatory profile and changes in terms related to synaptic maintenance. Expression and methylation at both 7 and 10 months included terms regarding mitochondrial and metabolic stress. While behavior symptoms progressed at 10 months, we see many previously activated pathways being inhibited in microglia at a later stage, with only 8 of 53 shared pathways predicted to be directionally concordant. Despite the difference in pathway directionality, 21 of the 22 genes that were differentially expressed and annotated to differentially methylated regions at both 7 and 10 months had conserved directionality changes.
DiscussionThese results highlight a critical period in disease progression, during which the microglia respond to α-synuclein, suggesting a transition in the role of microglia from the early to late stages of the disease.

引言
小胶质细胞（microglia）在神经退行性疾病中的作用日益得到学界认可，这使得精准表征小胶质细胞表型谱及其对疾病发病机制的影响成为亟待解决的重要课题。为阐明突触核蛋白病（synucleinopathies）进程中小胶质细胞表型的动态变化，本研究在mThy1-α-突触核蛋白小鼠模型中开展实验，检测了小胶质细胞的基因表达与DNA甲基化水平。

方法
本研究通过评估运动功能缺陷的行为实验确定疾病进展程度；于7月龄和10月龄时分离小胶质细胞，提取DNA与RNA，分别用于酶促甲基化测序（enzymatic methyl-sequencing）与RNA测序（RNA-sequencing）。

结果
对7月龄时的分子变化进行通路分析显示，小胶质细胞呈现促炎表型，且存在与突触维持相关的通路改变。7月龄与10月龄时的基因表达与甲基化分析均涉及线粒体与代谢应激相关的分子条目。尽管10月龄时动物的行为症状进一步加重，但后期小胶质细胞中多数此前激活的通路已被抑制；53个共有的富集通路中仅8个的调控方向保持一致。尽管通路调控方向存在显著差异，但在7月龄与10月龄时均出现差异表达且注释到差异甲基化区域（differentially methylated regions）的22个基因中，有21个的表达变化方向保持保守。

讨论
本研究结果揭示了疾病进程中的一个关键时期，此时小胶质细胞对α-突触核蛋白产生特异性应答，提示小胶质细胞在疾病早晚期的功能角色发生了显著转变。