Replication of the mitochondrial genome is essential to sustain cellular energy conversion, and defects in this process cause severe, progressive mitochondrial diseases that have long lacked curative treatment. This is now beginning to change: mechanistic insight into the replication machinery has started to translate into therapy, with the first disease-modifying treatment recently approved and others entering clinical evaluation. Here we review the molecular mechanisms of mitochondrial DNA replication, with emphasis on replication initiation, deletion formation, and replication completion, and discuss how insights into these processes are opening new therapeutic possibilities for treating mitochondrial disease.
ClC-7 is a lysosomal 2Cl−/1H+ antiporter essential for normal lysosomal function. PIKfyve-generated PI(3,5)P2 negatively regulates ClC-7 activity, and gain-of-function mutations (e.g., Y715C) that abolish this regulation cause severe genetic disorders. At the cellular level, these variants induce extensive endolysosomal vacuolization accompanied by impaired autophagic flux; however, the precise molecular mechanism driving vacuole enlargement remained elusive. Here, we report that prominent vacuole formation in cells expressing ClC-7 gain-of-function variants is closely associated with a marked intracellular ammonium accumulation. Furthermore, vacuole enlargement was attenuated by an ammonium scavenger and accelerated by exogenous glutamine or ammonium chloride, while pharmacological glutaminase inhibition suppressed both vacuole size and ammonium accumulation. Pharmacological inhibition of PIKfyve likewise induced vacuolization and ammonium accumulation, both of which were suppressed by ClC-7 knockout. Using a newly modified lipid-probe, we found that unlike PIKfyve inhibition, PI(3,5)P2 is maintained on ClC-7 gain-of-function vacuoles. The formation of vacuoles induced by the overexpression of SNX10, which downregulates PI(3,5)P2 synthesis, or by expression of a gain-of-function ClC-6 mutant was also accompanied by a marked accumulation of cellular ammonium. Additionally, cells overexpressing ClC-3 or ClC-5 exhibited vacuolization associated with cellular ammonium accumulation under the control of the accessory protein TMEM9. The impairment of autophagic flux triggered by either ClC-7 gain-of-function expression or PIKfyve inhibition was restored by glutaminase inhibition.Collectively, these findings demonstrate that glutamine-derived ammonium drives endolysosomal vacuolization and autophagic dysfunction downstream of the PIKfyve–ClC-7 axis, identifying glutaminase inhibition as a potential therapeutic strategy for associated genetic disorders.
Renal tubular epithelial–interstitial fibroblast communication maintains kidney homeostasis but promotes disease when dysregulated. Tubular epithelial cells undergo large physiological osmotic fluctuations; however, it remains unclear whether epithelial hyperosmotic stress sends signals to neighboring fibroblasts. Herein, we examined how epithelial hyperosmotic stress regulates fibroblast autophagy and transcription factor EB (TFEB) activity. We used a Transwell co-culture system with rat proximal tubular epithelial cells (NRK-52E) and renal interstitial fibroblasts (NRK-49F), which allowed evaluation of the paracrine effect on fibroblasts mediated by tubular epithelial cells. Hyperosmotic stress in epithelial cells induced the robust nuclear translocation of TFEB in fibroblasts, increased the expression of lysosomal and autophagy-related genes, and enhanced autophagic flux without altering fibroblast viability or proliferation. Although fibroblasts internalized epithelial-derived extracellular vesicles, they did not activate TFEB, suggesting an extracellular vesicles-independent mechanism. RNA sequencing revealed that hyperosmotic stress markedly alters the expression of epithelial solute carrier genes, particularly those encoding amino acid transporters. Analysis of amino acid metabolism revealed corresponding changes in conditioned media, including an increase in extracellular glutamate. Glutamate alone induced TFEB nuclear translocation and autophagy activation in fibroblasts. Both epithelial hyperosmotic co-culture and glutamate treatment activated AMP-activated protein kinase and inhibited mTORC1 signaling, thereby enabling TFEB activation. These results identify extracellular glutamate as a metabolite-based paracrine signal that couples epithelial osmotic stress with TFEB-dependent autophagy in interstitial fibroblasts.
