GSE191034
Mitochondrial respiratory chain dysfunction alters ER sterol sensing and mevalonate pathway activity
Mitochondrial dysfunction induces a strong adaptive retrograde signaling response, however many of the down-stream effectors remain to be discovered. Here, we studied the shared transcriptional responses to three different mitochondrial respiratory chain inhibitors in human primary skin fibroblasts using QuantSeq 3'RNA-sequencing. We found that mevalonate pathway genes were concurrently downregulated irrespective of the respiratory chain complex affected. Targeted metabolomics demonstrated that impaired mitochondrial respiration at any of the three affected complexes also had functional consequences on the mevalonate pathway, reducing cholesterol precursor metabolites. A deeper study of complex I inhibition showed a reduced activity of ER-bound sterol sensing enzymes through impaired processing of the transcription factor SREBP2 and accelerated degradation of the ER cholesterol sensors SQLE and HMGCR. These adaptations of mevalonate pathway activity neither affected total intracellular cholesterol levels nor the cellular free (non-esterified) cholesterol pool.  Measurement of intracellular cholesterol using the fluorescent cholesterol binding dye filipin revealed that complex I inhibition elevated cholesterol on intracellular compartments. Our study shows that mitochondrial respiratory chain dysfunction elevates intracellular free cholesterol levels and therefore attenuates the expression of mevalonate pathway enzymes, which lowers endogenous cholesterol biosynthesis, disrupting the metabolic output of the mevalonate pathway. Intracellular disturbances in cholesterol homeostasis may alter systemic cholesterol management in diseases associated with declining mitochondrial function. Overall design: 48 samples, 6 replicates, 4 conditions, 2 timepoints. Control (0.1% DMSO), 100 nM rotenone, 500 nM antimycin A, 1 µM Oligomycin A. Cells treated for 1 or 5 days.