Comprehensive miRNome-Wide Profiling in a Neuronal Cell Model of Synucleinopathy Implies Involvement of Cell Cycle Genes.

Elisabeth Findeiss,Thomas W Rösler,Matthias Höllerhage,Günter U Höglinger,Niko-Petteri Nykänen,Wolfgang Wurst,Michael Kohl,Tasnim Chakroun,Yimin Shen,Sigrid C Schwarz,Jörg Tost,Valentin Evsyukov

doi:10.3389/fcell.2021.561086

Abstract

Growing evidence suggests that epigenetic mechanisms like microRNA-mediated transcriptional regulation contribute to the pathogenesis of parkinsonism. In order to study the influence of microRNAs (miRNAs), we analyzed the miRNome 2 days prior to major cell death in α-synuclein-overexpressing Lund human mesencephalic neurons, a well-established cell model of Parkinson’s disease (PD), by next-generation sequencing. The expression levels of 23 miRNAs were significantly altered in α-synuclein-overexpressing cells, 11 were down- and 12 upregulated (P < 0.01; non-adjusted). The in silico analysis of known target genes of these miRNAs was complemented by the inclusion of a transcriptome dataset (BeadChip) of the same cellular system, revealing the G0/G1 cell cycle transition to be markedly enriched. Out of 124 KEGG-annotated cell cycle genes, 15 were present in the miRNA target gene dataset and six G0/G1 cell cycle genes were found to be significantly altered upon α-synuclein overexpression, with five genes up- (CCND1, CCND2, and CDK4 at P < 0.01; E2F3, MYC at P < 0.05) and one gene downregulated (CDKN1C at P < 0.001). Additionally, several of these altered genes are targeted by miRNAs hsa-miR-34a-5p and hsa-miR-34c-5p, which also modulate α-synuclein expression levels. Functional intervention by siRNA-mediated knockdown of the cell cycle gene cyclin D1 (CCND1) confirmed that silencing of cell cycle initiation is able to substantially reduce α-synuclein-mediated cytotoxicity. The present findings suggest that α-synuclein accumulation induces microRNA-mediated aberrant cell cycle activation in post-mitotic dopaminergic neurons. Thus, the mitotic cell cycle pathway at the level of miRNAs might offer interesting novel therapeutic targets for PD.

Highlights

In recent years, epigenetic regulation by microRNAs has been linked to the pathogenesis of Parkinson’s disease (PD) (Goodall et al, 2013; Singh and Sen, 2017; Goh et al, 2019; Ravanidis et al, 2020)
A miRNome-wide screen was performed in Lund human mesencephalic (LUHMES) neurons overexpressing either αSyn or green fluorescent protein (GFP) 4 days post transduction
We found that hsa-miR-34c-5p expression significantly increased between DIV5 (2.7 relative quantity (RQ) ± 0.03 standard error of the mean (SEM); P = 0.0164) and DIV7 (4.84 RQ ± 0.06 SEM; P < 0.001) and decreased on DIV8 (3.8 RQ ± 0.9 SEM; P < 0.001) in Adenoviral vectors (AV) SNCA samples compared to AV GFP control

Summary

Introduction

Epigenetic regulation by microRNAs (miRNAs) has been linked to the pathogenesis of Parkinson’s disease (PD) (Goodall et al, 2013; Singh and Sen, 2017; Goh et al, 2019; Ravanidis et al, 2020). We chose a robust PD model with adenoviral overexpression of human wild-type αSyn in post-mitotic Lund human mesencephalic (LUHMES) neurons (Lotharius et al, 2005; Höllerhage et al, 2014) Epigenetic mechanisms such as miRNA-mediated regulation of gene expression have been suggested to be involved in the etiology of PD (Goh et al, 2019). The study of in vitro and in vivo PD models revealed a link between several miRNAs and PD pathology, such as miR-7 and miR-153 (Je and Kim, 2017; Titze-de-Almeida and Titzede-Almeida, 2018) Both have been shown to regulate SNCA mRNA and the αSyn protein levels in mouse models of PD (Junn et al, 2009; Doxakis, 2010). It is currently unknown whether certain miRNAs are involved in biological compensation processes at an early stage of αSyn upregulation and could be used as novel drug targets to attenuate synucleinopathies

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