Abstract
Purpose :
Mitochondrial dysfunction is implicated in optic neuropathies such as normal-tension glaucoma (NTG) and a missense mutation in the mitophagy gene Optineurin (OPTNE50K), has a strong association (17% prevalence) with NTG. To date, mechanism to improve mitochondrial homeostasis in human RGC neurons for developing RGC neuroprotection is unclear. Here, using human stem cell differentiated RGC neurons and cutting-edge cell biology approaches, we set out to identify RGC protection mechanism by improving mitochondrial homeostasis
Methods :
Small molecule-based stem cell differentiation and immunopurification (MACS) was done to obtain pure, well characterized RGCs (originally developed in Don Zack lab). CRISPR mutated human embryonic stem cells with OPTNE50K mutation was used for glaucoma modeling. Mitochondrial damage was inflicted by potent stressor, CCCP. Literature evidence suggests Tank-binding kinase1 (TBK1) inhibits mitochondrial biogenesis and we tested if TBK1 inhibition by a potent inhibitor BX795 could promote mitochondrial biogenesis. Mitochondrial mass was measured by immunofluorescence (Tom20), western blot (Tom70) and flow cytometry. Activation of mitobiogenesis was measured by qPCR and western blot of biogenesis genes (AMPK, PGC1a). Statistical comparison between two independent data sets was done using student’s t-test.
Results :
Mitochondrial homeostasis or Mitochondrial quality control (MQC) process is maintained by biogenesis of healthy mitochondria, fission/fusion, and degradation of damaged mitochondria (mitophagy). Our data shows hRGCs promote mitochondrial biogenesis under mitochondrial stress. Aggregates of OPTNE50K are dissolved by TBK1 inhibitor, BX795, (p<0.05, 24hrs; <0.005, 48hrs) treatment. Furthermore, TBK1 inhibition also enhanced mitochondrial mass (<0.05, WT and <0.01, E50K hRGCs) by activating biogenesis pathway and improved cell viability (p<0.01, WT and <0.05, E50K hRGCs) with reduction in caspase activity (p <0.01, WT).
Conclusions :
Protein aggregates are often found in neurodegenerative conditions and increased mitochondrial biogenesis is often seen as a stress response. TBK1 inhibition leads to dissolution of OPTNE50K aggregates, with increased mitochondrial biogenesis resulting in neuroprotection of both WT and E50K hRGCs. Our study, for the first time, shows RGC neuroprotection mechanism by improving MQC, which is applicable for multiple optic neuropathies.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.