Impaired mitochondrial homeostasis of optic nerve cells in glaucoma

Currently, little is known about how mitochondrial homeostasis can be improved to promote neuroprotection of optic nerve cells. Yet mitochondrial dysfunction is common in central nervous system (CNS) diseases. A research group at Indiana University School of Medicine has been studying mitochondrial dysfunction in detail in optic nerve stem cell-differentiated human retinal ganglion cells (hRGCs). In their recently published study, the researchers came a decisive step closer to unravelling the mysteries surrounding mitochondrial dysfunctions.¹

Mutated hRGCs have fewer mitochondria

In their research project, they identified mechanisms to improve mitochondrial quality control (MQC). They showed that hRGCs were able to efficiently maintain mitochondrial homeostasis of acute damage. This was possible through rapid degradation and subsequent biogenesis of mitochondria. Using a glaucomatous optineurin-mutant (E50K) stem cell line, the research group brought to light an important element of the glaucomatous pathomechanism: The mutant glaucoma-mimicking hRGCs have a lower mitochondrial mass in the ground state. Due to the resulting excessive ATP production load, there is a swelling of the existing mitochondria.¹

Pharmacological inhibition of tank-binding kinase 1 offers new opportunities for glaucoma therapy

In further experiments, the research group could restore energy homeostasis within the overloaded mutant hRGCs. This was possible through pharmacological inhibition of Tank-binding kinase 1 (TBK1). By inhibiting this kinase, mitochondrial biogenesis could be activated. The result was a decrease in mitochondrial swelling and concomitant neuroprotection against acute mitochondrial damage in glaucomatous E50K hRGCs. This result was groundbreaking in that it revealed a completely new neuroprotection mechanism.¹

Acute damage to hRGCs by carbonyl cyanide m-chlorophenyl hydrazone

In a next step, the research group induced mitochondrial damage within the hRGCs by carbonyl cyanide m-chlorophenylhydrazone (CCCP). It was known from the literature that as little as 10 μM of CCCP can effectively alter the mitochondrial morphology of cultured cells within a few seconds. By triggering acute mitochondrial CCCP-induced damage, the research group was able to investigate whether and in what way hRGCs can restore mitochondrial homeostasis under stress. Using immunofluorescence, the researchers were able to determine the total mitochondrial mass of the cells before and after damage using optical criteria. For this purpose, they stained the mitochondria-specific protein Tom20.

This allowed direct measurement of mitochondrial DNA copy number. The research group found that total mitochondrial mass fluctuated around the dimethyl sulfoxide (DMSO) control state over the course of CCCP treatment in both wild-type (WT) and E50K hRGCs. They concluded that there must be strict MQC mechanisms within hRGCs to ensure stable total mitochondrial mass even in the face of acute damage.¹

Measurement of mitochondrial total mass: New versus degraded mitochondria

The total mass of mitochondria, which could previously be determined by immunofluorescence, did not provide information about the proportion of newly synthesised mitochondria. To distinguish the newly synthesised mitochondrial population from mitochondria that had already been degraded, the research group used the mitochondria-specific dye for living cells: MitoTracker Deep Red (MTDR). They then carried out flow cytometric measurements. In this way, the researchers were able to directly measure mitochondrial degradation and biogenesis and distinguish between them when determining mitochondrial mass.¹

Results that first seemed controversial, finally make sense

The research group was able to visualise the damaged and the healthy mitochondria by using the dye JC1. Healthy mitochondria appear red and damaged ones green. Initially, the researchers were able to see an increase in red JC1 intensity and a decrease in green JC1 mitochondria in both WT and E50K-hRGCs after CCCP damage. Actually, they had expected an increase in green (damaged) JC1 mitochondria with a simultaneous decrease in red (healthy) mitochondria. One reason for this could be an efficient removal of the damaged mitochondria and compensation through biogenesis by the hRGCs.¹

In the baseline condition, the wild-type hRGCs and the mutant E50K hRGCs showed a similar ratio of healthy to damaged mitochondria. However, it was striking that after the recovery phase (CCCP washout after CCCP exposure), the glaucoma-mimicked E50K hRGCs had a lower mass of healthy mitochondria - compared to the mitochondrial mass in the control condition.¹

Dysfunctional mitochondrial homeostasis after acute damage in glaucomatous E50K hRGCs

The key difference between wild-type and E50K hRGCs was as follows: after the recovery phase following CCCP damage and washout, WT hRGCs had a similar mitochondrial distribution as in the control (DMSO) state. In contrast, E50K hRGCs had a lower mass of healthy mitochondria compared to the control condition. This suggests that glaucomatous E50K hRGCs have difficulty restoring mitochondrial homeostasis after acute injury.¹

Surma M. et al. (2023). Enhanced mitochondrial biogenesis promotes neuroprotection in human pluripotent stem cell derived retinal ganglion cells. Commun Biol. 2023 Feb 24;6(1):218.
Zhang, N., Wang, J., Li, Y. et al. Prevalence of primary open angle glaucoma in the last 20 years: a meta-analysis and systematic review. Sci Rep 11, 13762 (2021).
(Only in German) Kremmer S. et al. (2000). Das kardiovaskuläre Risikoprofil bei der Progression der Glaukomerkrankung. Dtsch Arztebl 2000; 97(34-35): A-2241 / B-1909 / C-1793.