Mutant alpha-synuclein alters GATA1-dependent transcriptional regulation of the lysosomal ATPase ATP6V0A1 with downstream impact on autophagy
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Poster session at: 5th World Parkinson Congress ; Kyoto ; 04/06/2019 - 07/06/2019 ; Mutations and multiplications of the SNCA (aSyn) gene cause familial Parkinson´s disease (PD). Here we investigate the functional role of ATP6V0A1 (V-type proton ATPase 116kDa subunit isoform 1) identified as a genetic risk factor in a PD-GWAS with a single-nucleotide polymorphism (SNP) associated to PD (rs9897702) in its promoter region. An ancestral or PD-associated reporter gene assay (ATP6V0A1 promoter in front of the Gaussia luciferase) was transfected into SK-N-SH cell lines stably overexpressing WT and mutant aSyn and the effect of the SNP on the transcriptional regulation was measured. Interestingly, A53T-aSyn overexpressing cells showed increased activity of the ancestral promoter compared to the PD-associated one. Moreover, both promoter variants showed a higher activity when compared to control cells, respectively. In addition, we observed an enhanced ATP6V0A1 mRNA expression in A53T-aSyn overexpressing cells, compared to SK-N-SH. In silico analysis of the promoter region revealed a binding site for the GATA1 transcription factor on the same promoter sequence containing the PD-associated rs9897702 variant which reduced the predicted binding to GATA1. Consistently, EMSA (electrophoretic mobility shift assay) experiments showed a decreased GATA1 binding to the PD-associated promoter sequence, compared to the ancestral one. Western blot analysis for LC3B showed a decrease in the autophagic flux in cell lines with the overexpression of aSyn (WT and mutant). To dissect the functional consequences of endogenous rs9897702, we obtained human PBMCs (Peripheral blood mononuclear cells) from three carriers of the homozygous wildtype allele (G/G) and three carriers of the homozygous allele, which is associated to PD (A/A). The PBMCs were then immortalized with Eppstein-Barr virus (lymphoblasts). No difference could be observed in the expression levels of ATP6V0A1 mRNA between the genotypes, when measured using digital droplet PCR (ddPCR). mRNA levels are being investigated upon transient overexpression of aSyn in the lymphoblasts. We have shown that the ATP6V0A1 SNP has a functional role in modulating gene transcription. These results further suggest that ATP6V0A1 transcription is critically modulated by aSyn via a GATA1-dependent mechanism. Importantly, autophagy appears as a cellular process affected by aSyn dysregulation, indicating the importance of this pathway in PD.