Abstract
Accumulation of α-synuclein is a main underlying pathological feature of Parkinson’s disease and α-synucleinopathies, for which lowering expression of the α-synuclein gene (SNCA) is a potential therapeutic avenue. Using a cell-based luciferase reporter of SNCA expression we performed a quantitative high-throughput screen of 155,885 compounds and identified A-443654, an inhibitor of the multiple functional kinase AKT, as a potent inhibitor of SNCA. HEK-293 cells with CAG repeat expanded ATXN2 (ATXN2-Q58 cells) have increased levels of α-synuclein. We found that A-443654 normalized levels of both SNCA mRNA and α-synuclein monomers and oligomers in ATXN2-Q58 cells. A-443654 also normalized levels of α-synuclein in fibroblasts and iPSC-derived dopaminergic neurons from a patient carrying a triplication of the SNCA gene. Analysis of autophagy and endoplasmic reticulum stress markers showed that A-443654 successfully prevented α-synuclein toxicity and restored cell function in ATXN2-Q58 cells, normalizing the levels of mTOR, LC3-II, p62, STAU1, BiP, and CHOP. A-443654 also decreased the expression of DCLK1, an inhibitor of α-synuclein lysosomal degradation. Our study identifies A-443654 and AKT inhibition as a potential strategy for reducing SNCA expression and treating Parkinson’s disease pathology.
Highlights
Synucleinopathies suggests overexpression and toxic gain of function as a causative factor of disease, supported by increased neuronal cell death being associated with altered SNCA expression in patients with PD and in model systems [7,8,9,10,11,12,13]
Cotransfection of CRISPRi plasmids in HEK293-SNCA-luc cells resulted in 45% reduction of luciferase expression from the SNCA locus compared with untransfected cells, whereas transfection of CRISPRi plasmids in HEK-293CMV-luc control cells did not inhibit luciferase expression (Fig. S1)
We screened a total of 155,885 compounds, with 3 to 11 doses per compound depending on how the libraries were originally formatted, and measured the effects on SNCA-luc expression [21]
Summary
Synucleinopathies suggests overexpression and toxic gain of function as a causative factor of disease, supported by increased neuronal cell death being associated with altered SNCA expression in patients with PD and in model systems [7,8,9,10,11,12,13]. The screening assay was generated using genome editing to insert the luciferase gene downstream and in frame of the SNCA gene in HEK293 cells [19].
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