ABSTRACT
Elevated levels of SNCA have been implicated in the pathogenesis of Parkinson's disease (PD), while normal physiological levels of SNCA are needed to maintain neuronal function. We ought to develop new therapeutic strategies targeting the regulation of SNCA expression. DNA methylation at SNCA intron 1 regulates SNCA transcription, and PD brains showed differential methylation levels compared to controls. Thus, DNA methylation at SNCA intron 1 is an attractive target for fine-tuned downregulation of SNCA levels. Here we developed a system, comprising an all-in-one lentiviral vector, for targeted DNA methylation editing within intron 1. The system is based on CRISPR-deactivated Cas9 (dCas9) fused with the catalytic domain of DNA-methyltransferase 3A (DNMT3A). Applying the system to human induced pluripotent stem cell (hiPSC)-derived dopaminergic neurons from a PD patient with the SNCA triplication resulted in fine downregulation of SNCA mRNA and protein mediated by targeted DNA methylation at intron 1. Furthermore, the reduction in SNCA levels by the guide RNA (gRNA)-dCas9-DMNT3A system rescued disease-related cellular phenotype characteristics of the SNCA triplication hiPSC-derived dopaminergic neurons, e.g., mitochondrial ROS production and cellular viability. We established that DNA hypermethylation at SNCA intron 1 allows an effective and sufficient tight downregulation of SNCA expression levels, suggesting the potential of this target sequence combined with the CRISPR-dCas9 technology as a novel epigenetic-based therapeutic approach for PD.
Subject(s)
CRISPR-Cas Systems/genetics , DNA Methylation/genetics , Genetic Therapy , Parkinson Disease/genetics , alpha-Synuclein/genetics , Brain/metabolism , Brain/pathology , Cell Culture Techniques , Cell Differentiation/genetics , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methyltransferase 3A , Dopaminergic Neurons/metabolism , Dopaminergic Neurons/pathology , Gene Editing , Gene Expression Regulation/genetics , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/transplantation , Introns/genetics , Parkinson Disease/pathology , Parkinson Disease/therapy , RNA, Guide, Kinetoplastida/geneticsABSTRACT
INTRODUCTION: The α-synuclein (SNCA) gene has been implicated in the etiology of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). METHODS: A computational analysis of SNCA 3' untranslated region to identify potential microRNA (miRNA) binding sites and quantitative real-time polymerase chain reaction (PCR) to determine their expression in isogenic induced pluripotent stem cell-derived dopaminergic and cholinergic neurons as a model of PD and DLB, respectively, were performed. In addition, we performed a deep sequencing analysis of the SNCA 3' untranslated region of autopsy-confirmed cases of PD, DLB, and normal controls, followed by genetic association analysis of the identified variants. RESULTS: We identified four miRNA binding sites and observed a neuronal-type-specific expression profile for each miRNA in the different isogenic induced pluripotent stem cell-derived dopaminergic and cholinergic neurons. Furthermore, we found that the short structural variant rs777296100-polyT was moderately associated with DLB but not with PD. DISCUSSION: We suggest that the regulation of SNCA expression through miRNAs is neuronal-type-specific and possibly plays a part in the phenotypic heterogeneity of synucleinopathies. Furthermore, genetic variability in the SNCA gene may contribute to synucleinopathies in a pathology-specific manner.
