CREB5高甲基化参与帕金森病的神经节苷脂GM1治疗。

CREB5 hypermethylation involved in the ganglioside GM1 therapy of Parkinson's disease.

作者信息

Wang Rui, Tong Shanshan, Wang Mengdi, Zou Junjie, Wang Nan, Sun Fengjiao, Zhou Xiaosheng, Chen Jinbo, Wang Hongcai

机构信息

Department of Neurology, Binzhou Medical University Hospital, Binzhou, Shandong, China.

Department of Neurology, Penglai People's Hospital, Yantai, China.

出版信息

Front Aging Neurosci. 2023 May 31;15:1122647. doi: 10.3389/fnagi.2023.1122647. eCollection 2023.

INTRODUCTION

The treatment with monosialotetrahexosylganglioside (GM1) improves the symptoms of Parkinson's disease (PD). The alteration of DNA methylation in the blood was examined to investigate epigenetic modification by GM1 treatment.

METHODS

After a 28-day continuous intravenous infusion of GM1 (100mg), the motor and non-motor symptoms were evaluated by UPDRS III, Mini-mental state examination (MMSE) scores, FS-14, SCOPA-AUT, and PDQ-8. Moreover, blood samples were collected and PBMC was isolated. Genome-wide DNA methylation was performed by an 850K BeadChip. RNA levels and apoptosis were examined by RT-PCR and flow cytometry in rotenone-based cell models. The CREB5 plasmid was transfected by electroporation into SH-SY5Y cells. We also identified 235 methylation variable positions achieving genome-wide significance in 717558 differentially methylated positions (DMPs) ( = 0.0003) in comparison of pre-treatment with post-treatment measurements (statistical analysis paired-samples -test).

RESULTS

By searching the Gene Expression Omnibus (GEO) dataset and GWAS, 23 methylation variable positions were screened. Moreover, there are 7 hypomethylated methylation variable positions correlated with the scores of motor symptoms (UPDRS III scale). According to KEGG pathways enrichment analysis, the methylated genes CACNA1B (hypomethylated), CREB5 (hypermethylated), GNB4 (hypomethylated), and PPP2R5A (hypomethylated) were enriched in the dopaminergic synapse pathway. Pretreated with GM1 (80 μM) for 1 h, cell apoptosis and impaired neurite outgrowth were inhibited in rotenone-induced PD cell models. The RNA expression of CREB5 was increased in rotenone-treated SH-SY5Y cells. GM1 treatment decreased rotenone-induced CREB5 gene expression. The enhancement of CREB5 gene expression suppressed the protective role of GM1 in rotenone-induced cell apoptosis.

DISCUSSION

The application of GM1 improves the motor and non-motor symptoms of PD associated with the decreased CREB5 expression and the hypermethylation of CREB5.

CLINICAL TRIAL REGISTRATION

https://www.chictr.org.cn/showproj.html?proj=120582t, identifier ChiCTR2100042537.

引言

单唾液酸四己糖神经节苷脂（GM1）治疗可改善帕金森病（PD）症状。检测血液中DNA甲基化变化以研究GM1治疗引起的表观遗传修饰。

方法

连续28天静脉输注GM1（100mg）后，通过帕金森病统一评分量表第三部分（UPDRS III）、简易精神状态检查表（MMSE）评分、FS-14、自主神经症状评分量表（SCOPA-AUT）和帕金森病问卷-8（PDQ-8）评估运动和非运动症状。此外，采集血样并分离外周血单个核细胞（PBMC）。采用850K基因芯片进行全基因组DNA甲基化分析。在基于鱼藤酮的细胞模型中，通过逆转录-聚合酶链反应（RT-PCR）和流式细胞术检测RNA水平和细胞凋亡情况。通过电穿孔将CREB5质粒转染至人神经母细胞瘤细胞系（SH-SY5Y）。在比较治疗前和治疗后测量值时，我们还在717558个差异甲基化位点（DMPs）（P = 0.0003）中鉴定出235个达到全基因组显著性的甲基化可变位点（统计分析采用配对样本t检验）。

结果

通过检索基因表达综合数据库（GEO）数据集和全基因组关联研究（GWAS），筛选出23个甲基化可变位点。此外，有7个低甲基化的甲基化可变位点与运动症状评分（UPDRS III量表）相关。根据京都基因与基因组百科全书（KEGG）通路富集分析，甲基化基因钙通道亚基α1B（CACNA1B，低甲基化）、环磷腺苷效应元件结合蛋白5（CREB5，高甲基化）、鸟嘌呤核苷酸结合蛋白β亚基4（GNB4，低甲基化）和蛋白磷酸酶2A调节亚基Bα（PPP2R5A，低甲基化）在多巴胺能突触通路中富集。在鱼藤酮诱导的PD细胞模型中，用GM1（80μM）预处理1小时可抑制细胞凋亡和受损的神经突生长。在鱼藤酮处理的SH-SY5Y细胞中，CREB5的RNA表达增加。GM1治疗可降低鱼藤酮诱导的CREB5基因表达。CREB5基因表达的增强抑制了GM1对鱼藤酮诱导的细胞凋亡的保护作用。