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Background:Retinal ganglion cells (RGCs) are highly energy dependent due to their continuous action potential firing requirements and long unmyelinated axons hence highly susceptible to mitochondrial dysfunctions, observed in glaucoma. Dr. Das’s lab recently had identified Tank-binding kinase 1 (TBK1) inhibition by BX795 drug activates mitochondrial biogenesis and promotes RGC protection with glaucomatous Optineurin (OPTN-E50K) mutation. OPTN is a critical player for mitophagy. It is still not clear if activation of mito-biogenesis improved mitochondrial homeostasis which I investigated in this project.MethodsTo investigate mitochondrial homeostasis in human RGCs, I have used a robust well-characterized human stem cell differentiated RGC (hRGC) model with wild-type (WT) and E50K mutation background which Dr. Das’s lab routinely uses. To investigate mitochondrial homeostasis, I used JC1 live cell mitochondrial dye which fluoresces red when bound to healthy mitochondria and green when bound to damaged mitochondria. I used this assay on hRGCs treated with BX795 and mitochondrial stressor CCCP and measured red to green mitochondria ratio on confocal z-stacks using ImageJ.ResultsUnder basal level, we found hRGCsWT had a significantly increased healthy (red:green) mitochondria compared to hRGCsE50K. This suggests E50K mutation disrupts mitochondrial homeostasis. To gain mitochondrial homeostasis, it is possible that hRGCsE50K will produce more mitochondria over time than the WT. Indeed, we observed significant increase in healthy mitochondria for hRGCsE50K at 3h and 24h of DMSO and BX795 treatment, but not for hRGCsWT. We also observed under CCCP damage for 3h, hRGCsE50K had significantly higher amount of damaged mitochondria (p=0.051) while hRGCsWT maintained homeostasis.Conclusion and Potential ImpactMy study suggests glaucomatous OPTN-E50K mutation disrupts mitochondrial homeostasis and activation of mito-biogenesis by BX enriches healthy mitochondria leading to hRGCE50K protection. This study has high impact as further avenues for promoting mito-biogenesis could lead to glaucoma neuroprotection therapy.

Mitochondrial dysfunctions are widely afflicted in central nervous system (CNS) disorders with minimal understanding on how to improve mitochondrial homeostasis to promote neuroprotection. Here we used human stem cell differentiated retinal ganglion cells (hRGCs) of the CNS, which are highly sensitive towards mitochondrial dysfunctions due to their unique structure and function, to identify mechanisms for improving mitochondrial quality control (MQC). We found that hRGCs are efficient in maintaining mitochondrial homeostasis through rapid degradation and biogenesis of mitochondria under acute damage. Using a glaucomatous Optineurin mutant (E50K) stem cell lines, we saw that at basal level mutant hRGCs possess less mitochondrial mass and suffer mitochondrial swelling due to excess ATP production load. Activation of mitochondrial biogenesis through pharmacological inhibition of the Tank binding kinase 1 (TBK1) restored energy homeostasis, mitigated mitochondrial swelling with neuroprotection against acute mitochondrial damage for glaucomatousE50KhRGCs, revealing a novel neuroprotection mechanism.