Poster presented at ISSCR 2022 by Vukasin Jovanovic. The poster is entitled: Proteomics Analysis of Alexander Disease Patient iPSC-derived Astrocytes Identifies New Pathophysiological Features and Druggable Targets.

Authors (underlined, presenting): Vukasin M. Jovanovic, John Braisted, Chaitali Sen, Carlos A. Tristan, Pinar Ormanoglu, Ilyas Singeç

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Abstract

Alexander disease (AxD) is a rare fatal leukodystrophy caused by mutations in the GFAP gene resulting in protein aggregates (Rosenthal fibers, RF) and other abnormalities. We recently demonstrated that astrocytes derived from an AxD-patient recapitulate hallmarks of disease including RF formation and activation of stress and autophagy pathways. Here, we performed two-dimensional gel electrophoresis of membrane bound fraction of proteins isolated from healthy and diseased iPSC- astrocytes under basal conditions and after 24 h treatment with inflammatory cytokines. This comparative approach enabled detection of differentially expressed proteins as potential targets for therapeutic development. The identity of a total of 92 proteins was confirmed by MALDI/TOF mass spectrometry. Top upregulated hits in AxD-astrocytes included diverse categories such as protein chaperones (CALR, CALU), cytoskeleton (VIM, ACTG, TPM1/3), cell adhesion molecules (VCAM1, ITA2), oxidative stress (SODM), and innate immunity (C1QBP). Among the top downregulated hits were proteins and enzymes involved in metabolic pathways such as glycolysis (ENOA, GAPDH), pyruvate cycle (MAOM), antioxidant activity (PRDX2) and NADH redox balance (AATM). Exposure to inflammatory cytokines had a particularly strong effect on glycolytic enzymes and decreased the expression of GAPDH and ENOA in AxD- astrocytes. These findings were functionally validated by real-time metabolic analysis using a standardized glycolysis stress test (Searhorse XF Analyzer). Guided by these hits, we then queried the NIH Pharos database (https://pharos.nih.gov) to find potential disease-modifying drugs. Indeed, 18 out of 92 proteins could be linked to disease-relevant FDA-approved small molecule drugs and are currently tested for drug repurposing. Furthermore, we profiled the secretome of AxD-astrocytes under basal and “reactive” conditions and found that specific cytokines involved in innate immunity were not produced as compared to healthy astrocytes. To develop assays amenable for quantitative high-throughput screening, we generated reporter cell lines to monitor GFAP expression levels and phagocytic activity and systematic screening experiments are currently underway.