Measurement of Systemic Mitochondrial Function in Advanced Primary Open-Angle Glaucoma and Leber Hereditary Optic Neuropathy

Abstract

Primary Open Angle Glaucoma (POAG) is a common neurodegenerative disease characterized by the selective and gradual loss of retinal ganglion cells (RGCs). Aging and increased intraocular pressure (IOP) are glaucoma risk factors; nevertheless patients deteriorate at all levels of IOP, implying other causative factors. Recent evidence presents mitochondrial oxidative phosphorylation (OXPHOS) complex-I impairments in POAG. Leber Hereditary Optic Neuropathy (LHON) patients suffer specific and rapid loss of RGCs, predominantly in young adult males, due to complex-I mutations in the mitochondrial genome. This study directly compares the degree of OXPHOS impairment in POAG and LHON patients, testing the hypothesis that the milder clinical disease in POAG is due to a milder complex-I impairment. To assess overall mitochondrial capacity, cells can be forced to produce ATP primarily from mitochondrial OXPHOS by switching the media carbon source to galactose. Under these conditions POAG lymphoblasts grew 1.47 times slower than controls, whilst LHON lymphoblasts demonstrated a greater degree of growth impairment (2.35 times slower). Complex-I enzyme specific activity was reduced by 18% in POAG lymphoblasts and by 29% in LHON lymphoblasts. We also assessed complex-I ATP synthesis, which was 19% decreased in POAG patients and 17% decreased in LHON patients. This study demonstrates both POAG and LHON lymphoblasts have impaired complex-I, and in the majority of aspects the functional defects in POAG were milder than LHON, which could reflect the milder disease development of POAG. This new evidence places POAG in the spectrum of mitochondrial optic neuropathies and raises the possibility for new therapeutic targets aimed at improving mitochondrial function.

Fig 1. Impaired population doubling time in POAG and LHON lymphoblasts when forced to rely on OXPHOS.

Control, POAG and LHON lymphoblasts were grown either in glucose media or galactose media, which forces cells to rely on OXPHOS for proliferation and survival. Proliferation curves were used to calculate population doubling time. A) POAG and B) LHON lymphoblasts had significantly longer population doubling time in galactose media when compared to controls. Data is median (IQR), Mann-Whitney test, n = 20 cont1, n = 15 POAG, n = 6 cont2 and n = 6 LHON, * = p< 0.05, ** = p< 0.01. Glucose media (RPMI-1640 media containing 2mg/ml glucose, 15% heat-inactivated serum, 2.05mM l-glutamine); Galactose media (glucose-free RPMI-1640 containing 5mM galactose, 4.5mM sodium pyruvate, 15% dialysed heat inactivated FBS, 2.05mM l-glutamine).

Fig 2. Impaired OXPHOS complex-I in POAG and LHON lymphoblasts.

There was a significant decrease in complex-I specific activity in A) POAG and C) LHON lymphoblasts when specific activities were ratioed to that of the Kreb’s cycle enzyme citrate synthase. There was no change in the specific activity of Complex-IV for B) POAG or D) LHON lymphoblasts when ratioed to citrate synthase. Data is mean ± SD, students t-test, n = 20 cont1, n = 15 POAG, n = 6 cont2 and n = 6 LHON, * = p< 0.05, ** = p< 0.01.

Fig 3. Impaired ATP synthesis in POAG and LHON lymphoblasts.

Digitonin-permeabilised lymphoblasts from POAG, LHON and control patients were used to measure maximal ATP synthesis by mitochondrial respiration. There was a significant decrease in complex-I (glutamate + malate) driven ATP synthesis in both A) POAG and C) LHON lymphoblasts. There was a significant decrease in complex-II (succinate + rotenone) driven ATP synthesis in B) POAG lymphoblasts, but not in D) LHON lymphoblasts. Data is median (IQR), Mann-Whitney test, n = 20 cont1, n = 15 POAG, n = 6 cont2 and n = 6 LHON, * = p< 0.05.

Fig 4. Decreased NAD⁺/NADH redox balance in LHON lymphoblasts.

Lymphoblasts were grown in regular RPMI media for 48 hours prior to lymphoblasts being harvested to measure NAD⁺ and NADH levels. There was no significant difference in A) NAD⁺ levels between any groups; however there was a significant increase in B) NADH levels in LHON patients leading to C) a significant decrease in the NAD⁺/NADH ratio in LHON lymphoblasts. There was no difference between any of these measured parameters in POAG lymphoblasts, and cellular protein content was similar between groups (data not shown). For NAD⁺ and NADH data is mean ± SD, students t-test, and for NAD⁺/NADH data is median (IQR), Mann-Whitney test, n = 20 cont1, n = 15 POAG, n = 6 cont2 and n = 6 LHON, * = p< 0.05, ** = p< 0.01.

Fig 5. No changes in cellular lactate production in POAG or LHON lymphoblasts.

Lymphoblasts were incubated in high glucose media (RPMI supplemented with glucose increased to 4mg/ml from the standard RPMI level of 2, uridine at 50 μg/ml, pyruvate at 1mM + 15% heat-inactivated FCS) for 48 hours prior to lymphoblast media supernatant being harvested for lactate measurement. There was no significant difference in extracellular lactate levels (reflective of cellular lactate production) between POAG lymphoblasts and controls, or between LHON lymphoblasts and controls. Data is median (IQR), n = 20 cont1, n = 15 POAG, n = 6 cont2 and n = 6 LHON.