Abstract
Purpose :
The E50K Optineurin (OPTN) mutation is associated with normal tension glaucoma (NTG). Preliminary electron microscopy done by our group showed E50K mitochondria (Mt) to be fragmented, round, dilated and darker than controls. Such features are reported to be related to an immature type of metabolism, characterised by inner membrane proton leak, uncoupled phosphorylation, and increased glycolysis. The leak pore is believed to be composed of the c-subunit of ATP synthase. We hypothesised E50K Mt have a leak metabolism, in which ATP synthase operates in reverse, with the c-subunit acting as the pore, consuming ATP to restore the inner membrane potential
Methods :
We studied fibroblasts from 6 members of one family with NTG and the E50K-OPTN mutation and 6 age-matched healthy controls. To investigate ATP synthase reversal, we applied oligomycin 5µM (inhibitor of ATP synthase) in cells stained with TetraMethylRhodamine Methyl ester (TMRM) (Figure). Oxygen consumption rate (OCR) and glycolytic rate: measured using the XFe24 Analyzer. NADH/NAD+ and lactate levels: measured in fresh cells and media respectively. ATP levels: measured in the absence or presence of inhibitors of ATP synthase (Oligomycin) or glycolysis (2-deoxyglucose). Protein synthesis assessed by Western blot analysis of puromycin-labelled cells. ATP synthase beta and c-subunits quantified in mitochondrial lysates via western blotting
Results :
E50K cells have an accelerated cell growth rate and overall increased protein synthesis (P<0.05). Upon addition of Oligomycin, E50K Mt depolarised whereas controls hyperpolarised. E50K cells have increased OCR, glycolytic rate, NADH/NAD+ ratio, lactate levels and glycolytic ATP production (all P<0.05). E50K Mt exhibit an increase in ATP synthase c-subunit (P<0.05), while the beta subunit is relatively constant across cell lines
Conclusions :
E50K mutant Mt depolarise upon Oligomycin treatment suggesting the presence of a proton leak and ATP synthase reversal consuming rather, than producing, ATP (Figure). To sustain the ATP demand, glycolysis is upregulated. The data support that the leak channel is via the c-subunit of ATP synthase. Presence of a leak leads to uncoupled phosphorylation and consequently to an immature phenotype, as shown by increased cell growth rate and protein synthesis
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.