Identification of alpha-synuclein and ataxin-3 expression modifying compounds

Abstract

Neurodegenerative diseases (NDDs) are affecting millions of people worldwide. A common feature of all NDDs is the neuronal demise in the brain leading to the clinically observed decline of motor- and cognitive capabilities of patients. Despite enormous scientific efforts, no therapies to halt NDDs have yet proven to be effective. This work focussed on the identification of transcriptional modulators for endogenous regulation of the Parkinson Disease (PD)- and Spinocerebellar Ataxia 3 (SCA3) causing genes alpha-synuclein (<em>SNCA</em>) and ataxin-3 (<em>ATXN3</em>) by using a novel luciferase (LUC) reporter cell line-based compound screening approach. The human neuroblastoma screening cell lines were genetically modified by using the CRISPR/Cas9- based gene editing to insert a <em>GFP-T2A-LUC</em> cassette at the respective genomic locus which resulted in full-length <em>SNCA-GFP-T2A-LUC</em> and <em>ATXN3-Exon4-GFP-T2A-LUC</em> fusions. These cell lines were used for the high throughput screening (HTS) of libraries containing 1,649 (alpha-synuclein) and 2,640 (ataxin-3) bioactive- and U.S. Food and Drug Administration (FDA) approved drugs. While no inhibitors were identified in both HTS, activators of endogenous alpha-synuclein and ataxin-3 expression were found. Three non-related compounds increasing alpha-synuclein expression were revealed. A potential mechanism explaining the induction of alpha-synuclein expression levels became evident for Emodin which was shown to have histone deacetylase (HDAC) inhibitory effects. Treatment of SH-SY5Y wild type cells led to increased levels of histone marks for H3K4 trimethylation and H3/H4 acetylation which might facilitate binding of specific transcription factors (TF) to the <em>SNCA</em> promotor. The HTS for modulators of ataxin-3 expression revealed four statins increasing LUC signal in the screening cell line. Simvastatin was used for further validation in SK-N-SH wild type cells. Statins are potent inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). Inhibition leads to cholesterol deprivation and to the activation of sterol regulatory element binding protein (SREBP) TFs. The observed increase of ataxin-3 expression was likely mediated by Simvastatin induced binding of SREBP1 TF to the <em>ATXN3</em> promotor. Finally, Simvastatin treatment increased endogenous ATXN3 protein levels in a SCA3 patient-derived cell line supporting potential clinical implications. These findings suggest that ataxin-3 is a direct target of SREBP1 and indicate a putative role of ATXN3 in cholesterol homeostasis.