X-linked adrenoleukodystrophy patient fibroblast-iPSC-derived astrocytes reveal phenotype-specific metabolic, inflammatory, and microRNA alterations

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. corrected P -values, with adjusted-p < 0.05 and mean fold change >2 as the threshold for statistical significance. Boxplots were drawn of the miRNA abundance (log10) within the three groups, for the set of commonly altered miRNA. and G-banding pattern with no evidence of structural or numerical changes. This suggests that reprogramming and differentiation did not alter or induce chromosomal abnormalities in AMN and cALD astrocytes compared Our revealed a higher DRP-1 expression in cALD than in AMN suggesting higher In line with this, we observed a significant decrease in mtDNA/nuclear DNA ratio in AMN and most decrease in cALD astrocytes suggesting a decrease in mitochondrial content compared to that in CTL astrocytes. Accumulation of dysfunctional mitochondria may result in ROS production. Although, we did not detect increased ROS, cALD astrocytes showed higher expression of superoxide catalyzing NADPH oxidases and overexpression of NDUFA6 in OXPHOS immunoblotting. We observed reduced SDHB (complex II) protein in both AMN and cALD astrocytes compared to CTL astrocytes in OXPHOS immunoblotting implying an impaired electron transport. These data suggest that activated STAT3 may play a role in inflammation in cALD astrocytes and alters mitochondrial-OCR. effect on and astrogliosis via interaction with insulin-like growth factor-1 gene which is crucial for cell growth and proliferation. 46 Additionally, we document upregulated miR-409-3p in AMN and cALD astrocytes compared to CTL astrocytes. The IPA network illustrates that miR-409-3p targets IL-17A and is regulated by IL-17A creating a feedback loop. IL-17A also regulates vascular endothelial growth factor (VEGF) and is in turn regulated by mTOR pathway. Reports exist where miR-409-3p contributes to IL-17-induced inflammatory cytokine production in astrocytes via STAT3 signaling pathway in experimental autoimmune encephalomyelitis (EAE) mice. 21 Interestingly, we recently documented decreased miR-409-3p expression in plasma of AMN patients with increasing disease severity. 47 Similarly, our differential gene IPA network shows that miR-145-3p is associated with HAND1, ATRX, and RAP2C genes. Altered ATRX and RAP2C have been reported to be associated with occurrence of gliomas. 48, 49 However, further studies are needed to explore association of miR-145-3p and these genes in neuroinflammation in AMN and cALD astrocytes. increased proinflammatory cytokines IFN-γ, IL-23α, TNF-α, Nos2, IL-17A, and IL-22. Elevated NOX expression and reduced SDHB and UQCRQ activity in addition to Stat3 activation is detrimental to mitochondria. Substantial increased TLR activity, mitochondrial dysfunction, and simultaneous hyperglycolytic state are potential contributors to neuroinflammation in cALD astrocytes. Our findings support our belief that AMN astrocytes show anti-inflammatory (A2) phenotype whereas cALD astrocytes demonstrate highly reactive and proinflammatory (A1) phenotype. Abbreviations : AMN: