Analytical subcellular fractionation of microglial BV-2 cells with peroxisomal beta-oxidation defect

Peroxisomes have gained increasing attention and are now considered vital players in normal physiological functions. To gain further insight into how peroxisomal defects influence cellular functions, we developed BV-2 microglial models featuring CRISPR/Cas9 gene-edited mutations in peroxisomal Acox1 or Abcd1 and Abcd2 genes. The Acox1^-/- BV-2 cell line we generated lacks acyl-CoA oxidase 1, the key enzyme that initiates peroxisomal β-oxidation. In contrast, the double mutant Abcd1/d2^-/- BV-2 cell line carries mutations in the genes encoding the membranous ABC transporters ABCD1 and ABCD2, which are responsible for transporting fatty acyl-thioesters inside peroxisome. Here, for the first time, we used analytical fractionation to compare these three genotypes. Through flow cytometry, we observed an increase in cell granularity in these mutant cells, which could be associated with alterations in peroxisome distribution and mitochondrial dynamics. Additionally, the analysis of organelle markers in microglial cells, employing differential centrifugation, exhibited an enrichment of peroxisomes particularly in both L and P fractions of these BV-2 cell line models. The use of an isopycnic Nycodenz density gradient showed that peroxisomes sedimented with a median density of 1.18 g/ml. Notably, our results revealed no significant differences in the distribution profiles of organelles when comparing microglial BV-2 Wt cells with deficient Acox1^‒/‒ or Abcd1/d2^-/‒ BV-2 cells, which lack peroxisomal fatty acid beta-oxidation. Our study is the first to report on the fractionation of brain-derived microglial cells, laying valuable groundwork for future proteomic and/or metabolomic analyses of peroxisome fractions.