iPSC-derived astrocytes to model phenotype-specific differential neuroinflammatory and metabolic responses in X-linked adrenoleukodystrophy

Abstract

Objective: The role of astrocytes largely remains to be explored in X-linked adrenoleukodystrophy (X-ALD). Due to limited study models and inaccessibility to patient-tissue samples, we differentiated astrocytes from patient fibroblast-derived induced pluripotent stem cell (iPSCs) which provided a unique system to investigate molecular and etiopathogenetic mechanisms, identify targets, and develop therapeutic agents for X-ALD. Methods: We reprogrammed fibroblasts from adrenomyeloneuropathy (AMN) and cerebral adrenoleukodystrophy (cALD) patients carrying ABCD1 with pathogenic variants and a control patient to generate iPSCs. We differentiated iPSCs into astrocytes and performed transmission electron microscopy, gene expression, immunoblotting, enzyme-linked immunosorbent assay, miRNA-Seq, and lipidomics to characterize phenotypic and molecular features of patient-derived astrocytes. Results: These differentiated astrocytes exhibit diseased phenotypes and replicate biochemical and molecular changes found in patients. We confirmed the deletion of ABCD1 gene-encoded ALD protein and identified ABCD1 variant-driven very long chain fatty acid deposition in AMN and cALD astrocytes. Especially, cALD astrocytes showed increased glycolysis, increased signal transducer and transcription activator (STAT)3 activation, higher miR-9 expression in miRNA-Seq analysis, and reduced expression of anti-inflammatory cytokines such as arginase-1 and mannose receptor C-type-1. Consequently, Toll-like receptor-signaling via myeloid differentiation primary response gene 88 (MyD88) and nuclear factor-kappa B (NF-κB; p52 and p65) induced STAT3 and an altered miR-9 expression is a potential contributor to inflammatory milieu in cALD while interleukin-6 -induced anti-inflammatory cytokine production and increased chemotactic CCL-2 (MCP-1) production in AMN potentially favors microglial recruitment protecting its further progression. Interpretation: We demonstrate for the first time that patient iPSC-derived astrocytes mimic and recapitulate neuroinflammatory and biochemical defects of X-ALD and provide an in vitro cellular system to study X-ALD.

Generating human AMN and cALD iPSC-derived astrocytes with potential for modeling X-linked adrenoleukodystrophy phenotypes

“…In the present study, we generated and characterized astrocytes obtained from iPSC lines reprogrammed from skin fibroblasts of additional two male patients each of CTL, AMN and cALD phenotype. In line with our recent report [17], ABCD1 mutation did not affect the reprogramming ability of AMN and cALD skin fibroblasts and the iPSC-lines were successfully differentiated into astrocytes. Immunocytochemical expression analysis of differentiated astrocytes indicated that they were positive for mature astrocyte markers including GFAP, EAAT, S100β and ALD1H1 consistent with previous studies [17,22].…”

“…However, they are unable to account for the differential response seen in AMN and cALD phenotype. We recently documented differential accumulation of VLCFA in patient iPSC-derived astrocytes from AMN and cALD phenotypes [17]. In line with these, VLCFA levels were also differentially increased in AMN and cALD patient iPSC-derived astrocytes in the present study with higher accumulation in cALD patient iPSC-derived astrocytes.…”

“…Human iPSCderived cell models of brain cells provide an opportunity to investigate early events in disease development and also to test novel therapeutic strategies. This study documents iPSC-derived astrocytes generated for apparently healthy controls (CTL), AMN and cALD phenotypes of X-ALD and adds biological replicates to our recent report [17]. Non-integrating mRNA-based reprogramming used in the present work is advantageous to generate clinical grade stem cells as there is no genome integration of the reprogramming factors.…”

“…Total RNA was extracted with the miRNeasy kit (Qiagen) and 1µ RNA was used for cDNA synthesis. RT-qPCR were conducted using CFX96 Real-Time PCR Detection System (BioRad) using IQ SYBR Green Supermix (BioRad), as described previously [17]. Gene expression was normalized to 60S ribosomal L27 gene and samples were run in triplicate.…”

“…CTL, AMN and cALD astrocytes were homogenized in radioimmunoprecipitation (RIPA) buffer with protease inhibitor cocktail (Thermo Fisher Scientific). 60 μg of total protein was electrophoresed as described previously [17]. Antibodies used are listed in the Appendix 2.…”