Identification of repurposable cytoprotective drugs in vanishing white matter disease patient-derived cells

Abstract

Background Vanishing white matter disease (VWMD) is a rare disease caused by mutations of the guanine exchange factor eIF2B. VWMD typically presents with juvenile onset, and there are few treatments for the disease. Recent progress in the field has established mitochondrial dysfunction and endoplasmic reticulum (ER) stress to be strongly implicated in observed glial cell pathology. Drug repurposing offers a rapid approach toward translation of therapeutics using already-licensed drugs. Objective The aim of this study was to use fibroblasts and induced pluripotent stem cell (iPSC)-derived astrocytes from patients bearing the EIF2B5R113H/A403V or EIF2B2G200V/E213G VWMD mutations to identify potential repurposable FDA-approved drugs based on in vitro assays. Methods Cell viability in the presence or absence of stress was assessed by resazurin reduction activity and assays for mitochondrial membrane potential and oxidative stress by tetramethylrhodamine and dichlorofluorescein-based assays, respectively. Integrated stress response markers, including eIF2α phosphorylation, GADD34 and CHOP were quantified by fluorescent western blot. Results Dysregulated GADD34 and CHOP were identified in patient fibroblasts and iPSC-derived astrocytes under induced stress conditions. A drug screen from a 2400 FDA-approved drug library with EIF2B5R113H/A403V VWMD patient fibroblasts identified 113 anti-inflammatory drugs as a major class of hits with cytoprotective effects. A panel of candidate drugs, including berberine, deflazacort, ursodiol, zileuton, guanabenz and Anavex 2–73, and preclinical ISRIB, increased cell survival of EIF2B5R113H/A403V or EIF2B2G200V/E213G VWMD astrocytes, and were further investigated for their effect on the integrated stress response and mitochondrial stress. Ursodiol demonstrated capacity to ameliorate oxidative stress and loss of mitochondrial membrane potential in VWMD patient iPSC-derived astrocytes in the presence or absence of stress conditions. Conclusion Patient-derived cells can be used to identify cellular phenotypes and for large-scale drug screening. Anti-inflammatory compounds, such as berberine, deflazacort, ursodiol and zileuton are potentially repurposable drug candidates for VWMD that should be further investigated for translation in vivo.

Revisiting the Resazurin-Based Sensing of Cellular Viability: Widening the Application Horizon

“…Usually, such studies are carried out by measuring dose-response curves at different concentrations of the compound of interest. Importantly, the resazurin assay enables studies on the cytoprotective [28], cytotoxic, and cytostatic effects of compounds [8], though we will only focus on the two latter categories. Figure 8 illustrates some examples of the variations and trends in this assay format that might prove useful for a deeper understanding of the obtained data.…”

An Optimized Direct Lysis Gene Expression Microplate Assay and Applications for Disease, Differentiation, and Pharmacological Cell-Based Studies

“…All experimental protocols were approved by the University of Wollongong Human Research Ethics Committee (HE17/522). The iPSCs were previously generated and characterized [ 6 ]. Fibroblasts were maintained in 5% CO 2 at 37 °C.…”

“…Human neural precursor cells (NPCs) were generated by dual SMAD inhibition with incubation of induced pluripotent stem-cell cultures with ALK inhibitors, 0.1 µM LDN193189 (HY-12071A-10MG, Focus Bioscience, Murarrie, Australia) and 10 µM SB431542 (HY-10431-10MG, Focus Bioscience, Murarrie, Australia) in neural medium (2% B27 supplement (17504001, Thermo Fisher Scientific, Waltham, MA, USA) and GlutaMAX in DMEM/F12) for 5 days, followed by replacement of small molecules with 20 ng/mL epidermal growth factor (EGF) (130-097-749, Miltentyi Biotec GmbH, Bergisch Gladbach, Germany) and 20 ng/mL basic fibroblast growth factor (FGF-2) (130-093-841, Miltentyi Biotec GmbH, Bergisch Gladbach, Germany) ( Figure S1 ) [ 6 ].…”

“…Neuronal and astrocyte cell cultures were analyzed during days 10–14 of differentiation ( Figure S1 ). The eIF2B/vanishing white-matter (VWM) disease astrocytes were differentiated and maintained by a previously described growth factor-directed differentiation method and analyzed during weeks 6–8 [ 6 ], generated from NPCs incubated in a ciliary neurotrophic factor (CNTF)-based medium for 4 weeks before incubation in DMEM/F12, 2% FBS, and Astrocyte Growth Supplement (1852, Sciencell, Carlsbad, CA, USA).…”

“…Inflammation stress response assays were conducted by incubation of astrocytes with poly(I:C) at 10 µg/mL or fibroblasts overnight with 0.1–1000 ng/µL poly(I:C). ER stress assays with VWM disease lines were performed as previously described with overnight incubation of 0.1 µM MG132 [ 6 ]. For neuronal maturation small-molecule assays, NPCs transduced with NGN2 were incubated with combinations of 5 µM DAPT, SU-5402, Y-27632, 10 ng/mL BDNF, or BDNF and glial-derived neurotrophic factor (GDNF).…”