Peroxisomal defects in microglial cells induce a disease-associated microglial signature

Abstract

Microglial cells ensure essential roles in brain homeostasis. In pathological condition, microglia adopt a common signature, called disease-associated microglial (DAM) signature, characterized by the loss of homeostatic genes and the induction of disease-associated genes. In X-linked adrenoleukodystrophy (X-ALD), the most common peroxisomal disease, microglial defect has been shown to precede myelin degradation and may actively contribute to the neurodegenerative process. We previously established BV-2 microglial cell models bearing mutations in peroxisomal genes that recapitulate some of the hallmarks of the peroxisomal β-oxidation defects such as very long-chain fatty acid (VLCFA) accumulation. In these cell lines, we used RNA-sequencing and identified large-scale reprogramming for genes involved in lipid metabolism, immune response, cell signaling, lysosome and autophagy, as well as a DAM-like signature. We highlighted cholesterol accumulation in plasma membranes and observed autophagy patterns in the cell mutants. We confirmed the upregulation or downregulation at the protein level for a few selected genes that mostly corroborated our observations and clearly demonstrated increased expression and secretion of DAM proteins in the BV-2 mutant cells. In conclusion, the peroxisomal defects in microglial cells not only impact on VLCFA metabolism but also force microglial cells to adopt a pathological phenotype likely representing a key contributor to the pathogenesis of peroxisomal disorders.

Immune response of BV-2 microglial cells is impacted by peroxisomal beta-oxidation

“…One notable finding from the transcriptomic analysis of the Abcd 1 −/− Abcd 2 −/− and Acox 1 −/− mutant BV-2 cell lines was that the term “Innate immune response” consistently ranked as the top hit across all mutant genotypes (Raas et al, 2023 ). Genes encoding cytokines and chemokines, defense receptors such as Toll-like receptors (TLRs), proteins involved in immune signaling, response to viruses or bacteria, and inflammasome figured among the DEGs.…”

“…Total RNA was extracted from three independent batches of BV-2 cells for each genotype (WT, Abcd1 −/− Abcd2 −/− , and Acox1 −/− ) using an RNeasy kit (Qiagen). RNA-seq library preparation, sequencing, and analysis were performed as previously described (Raas et al, 2023 ). Differential gene expression analyses were performed using R 3.3.2 and DESeq2 version 1.16.1 Bioconductor library (Love et al, 2014 ).…”

“…Furthermore, apart from the noted VLCFA-related findings and the crosstalks existing between peroxisomal ABC transporters and ACOX1, we also observed the accumulation of neutral lipids, alterations in membrane structure, the presence of lipid inclusions, and the occurrence of lipid droplets in the mutant cell lines. This was accompanied by a significant reprogramming of gene expression patterns associated with lipid metabolism that could be attributed in part to PPARβ activation, which is highly expressed in BV-2 microglial cells (Raas et al, 2023 ).…”

“…Thus, these cells serve as a useful tool and continue to be the most used cell line for studying microglial behavior and inflammatory response in vitro . Using the BV-2 cell models, we could demonstrate that a defect in peroxisomal fatty acid β-oxidation results in a reprograming of gene expression linked to lipid metabolism, lysosome, autophagy, and in the emergence of a disease-associated microglial-like (DAM-like) signature (Raas et al, 2023 ). Interestingly, some of the main hub genes of microglia ( Apoe, Tyrobp , and Trem2 ), which play pivotal roles in modulating microglial functions, were found to be significantly upregulated in the knockout genotypes.…”

Progress in leukodystrophies with zebrafish

“…Furthermore, microglia depletion is associated with excessive and ectopic myelin in this model organism (Hughes and Appel, 2020). Microglial dysfunction or loss of homeostasis has been characterized as a key pathological event in many leukodystrophies, including Krabbe disease, metachromatic leukodystrophy (MLD), CSF1R leukodystrophy, TREM2 leukodystrophy, and X-linked adrenoleukodystrophy (ALD) (Bergner et al, 2019;Ferrer, 2022;Raas et al, 2023). The recent identification of astrocyte-specific markers in zebrafish, such as Glast (Chen et al, 2020), should facilitate future work investigating the role played by astrocytes during myelination and the role of astrocytic dysfunction in leukodystrophies.…”