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.
INTRODUCTION
The alpha-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’UTR to identify potential microRNA binding-sites, and quantitative real-time-PCR to determine their expression in isogenic iPSC-derived dopaminergic and cholinergic neurons as a model of PD and DLB, respectively. Additionally, deep sequencing analysis of SNCA-3’UTR of autopsy confirmed cases of PD, DLB, and normal 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 iPSC-derived neurons, i.e. dopaminergic and cholinergic. 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.
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