Molecular mechanisms underlying Primary CoQ10 deficiency: A nexus between cholesterol metabolism, CoQ10 biosynthesis, and mitochondrial genome maintenance

Abstract

itochondria are the "powerhouses" present in all cells producing energy in the form of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS). An essential element in OXPHOS is Coenzyme Q10 (CoQ10). Primary CoQ10 deficiencies are a group of rare diseases caused by biallelic recessive mutations in one of the COQ genes required in the CoQ10 biosynthesis pathway. The associated clinical manifestations are very heterogeneous and mainly affect the central and peripheral nervous systems, kidneys, skeletal muscle, and heart.

Mitochondria possess their own DNA, the mitochondrial DNA (mtDNA), organised into structures called nucleoids and confined in the mitochondrial matrix.

We observed that the primary CoQ10 deficiency due to the lack of either COQ4 or COQ6 induces a faster recovery rate of mtDNA after an induced depletion. Since cholesterol and CoQ10 share part of the synthesis pathway, we hypothesised that blocking CoQ10 production could lead to the accumulation of isoprenoid precursors within mitochondrial or extramitochondrial membranes. This accumulation might stimulate cholesterol synthesis, increasing the number of cholesterol-rich domains in mitochondrial membranes. Since replicative nucleoids preferentially anchor to cholesterol-enriched membrane regions1, a rise in mitochondrial cholesterol could enhance the replication activity of mtDNA. Alternatively, it is also possible that CoQ10 deficiency activates mitochondrial quality control mechanisms, leading to a more dynamic mitochondrial turnover that could favour mitophagy and the formation of new mitochondria, along with replication of their genome. These mechanisms may contribute to the accelerated mtDNA recovery observed in CoQ10-deficient models.

To study the relationship between cholesterol, CoQ10, and mtDNA, we used cellular models with either alterations in cholesterol metabolism or CoQ10 primary deficiency.

We first examined fibroblasts from patients with Niemann-Pick type C (NPC) and Smith-Lemli-Opitz syndrome (SLOS), diseases in which cholesterol trafficking and synthesis are profoundly impaired. In NPC cells, cholesterol accumulation was evident, and although CoQ10 levels were only slightly reduced, respiration was dramatically impaired. Interestingly, these cells had more than twice as many mtDNA copies as controls. SLOS fibroblasts, on the other hand, showed a clear lack of cholesterol. In particular, SLOS1 showed a significant deficit of CoQ10, COQ proteins and also a severe defect in mitochondrial respiration. Importantly, both SLOS models exhibited elevated mtDNA copy numbers.

To control SLOS patient-specific variability, we created the DHCR7-deficient cell line. Our findings indicate that cholesterol deficiency reduces the copy number of mtDNA and mitochondrial respiration, although the reduction in mitochondrial respiration is not as pronounced as that observed in SLOS1. Importantly, we found that cholesterol deficiency does not impact mtDNA recovery after induced depletion. Moreover, we observed a significant reduction in cholesterol content in COQ4 KO cells. These findings disprove our initial hypothesis that CoQ10 deficiency could redirect the flow of isoprenoids towards cholesterol biosynthesis to facilitate mtDNA replication. Although CoQ10 deficiency results from defects in downstream steps of the mevalonate pathway, our results suggest that it exerts regulatory effects upstream, influencing cholesterol biosynthesis. CoQ10 deficiency appears to downregulate this pathway, highlighting a broader metabolic crosstalk between CoQ10 and cholesterol metabolism.

Functional assays on COQ4 KO model indicated more intense mtDNA synthesis, an increase in mitochondrial membrane potential, and greater mitochondrial mass. Moreover, these changes were aligned with a cell cycle delay and a high number of cells blocked in the G1 phase, suggesting that the absence of CoQ10 affects not only energy production but also cell cycle progression, possibly due to its role in supporting mitochondrial pyrimidine synthesis.

Interestingly, uridine supplementation successfully normalised the cell cycle bypassing CoQ10-supported pyrimidine synthesis.

Finally, we explored the global transcriptional changes in gene expression to identify the possible factors responsible for the more efficient recovery of mtDNA in the COQ4 KO and DHCR7 KO (this experiment is going on at this moment) cellular models. The transcriptomic analysis of nucleoid-related genes revealed the upregulation of POLG, along with the downregulation of its co-factors TWNK and TFAM. Furthermore, we observed a significant reduction in the ATAD3B transcript, a gene involved in the stability of nucleoids. Interestingly, we observed reduced levels of transcripts involved in cholesterol synthesis (IDI1, TSPO), OXPHOS subunits, and mitoribosomal proteins.

Genes related to mitophagy (BCL2L13, PINK1) and mitochondrial biogenesis (HELZ2, CREBBP) were upregulated, indicating a cellular attempt to adapt to mitochondrial damage both through the removal of defective mitochondria and the regeneration of new ones. Notably, both transcriptomic and protein analyses indicated an increase in mitophagy.

The COQ4 KO model showed signs of ferroptosis vulnerability: transcripts that normally regulate iron homeostasis (CP, PCBP1) were reduced, while pro-ferroptotic markers like MAP1LC3 were increased. This was consistent with a redox imbalance exacerbated by the absence of CoQ10 that acts as antioxidant.

In the last part, we focused on the comparative transcriptomic analysis between the COQ4 KO model and the fibroblasts of nine patients with COQ4 gene mutations. Mutations in this gene causes primary CoQ10 deficiency with highly variable manifestations and, in many cases, with fatal outcomes. The analysis enabled the identification of commonly regulated genes that could be useful for a better understanding of pathophysiology. The genes THNSL2, TPD52L1, and VSTM2L were upregulated in most patients and in the COQ4 KO cells, representing potential diagnostic markers. Moreover, patients with more severe symptoms (P1, P2) shared specific transcriptomic characteristics with the KO model -such as the upregulation of ABAT, suggesting possible links to neurological phenotypes like epilepsy. Several key pathways emerged as recurrently altered: Wnt signalling (involved in embryonic development and adult tissues homeostasis), the presenilin pathway (implicated in Alzheimer’s), and inflammatory cascades.

Altogether, this work highlights how the biosynthesis of CoQ10 and cholesterol, the maintenance of the mitochondrial genome, and cellular stress responses are tightly coordinated, with subtle imbalances triggering extensive compensatory changes. Understanding these dynamics may open new perspectives for the diagnosis and treatment of mitochondrial diseases.