Abstract
Purpose :
Glaucoma is the leading cause of irreversible blindness worldwide. The role of mitochondrial dysfunction leading to retinal ganglion cell (RGC) death has been implicated in the pathogenesis of primary open-angle glaucoma. In addition to supporting the high bioenergetic needs of RGCs, mitochondria also play a central role in oxidative stress, calcium dysregulation, and apoptosis pathways. Restoring mitochondrial function by transplantation is a recent development and has been a target of therapeutic intervention.
Methods :
Mitochondria were isolated from hSkMCs by magnetically binding to the TOM22 surface protein. Western blot and PCR analysis with gene-specific primers for mitochondrial DNA confirmed the purity of the isolated mitochondria. The integrity of the purified mitochondria was confirmed by transmission electron microscopy. We incubated human induced pluripotent stem cell-derived RGCs (hiPSC-RGCs) with exogenous mitochondria from metabolically active human skeletal muscle cells (hSkMCs). SkMCs were transduced with lentiviral mito-GFP to label donor mitochondria for identification. To test the functional impact of mitochondrial transfer, we treated hiPSC-RGCs with rotenone to induce oxidative stress. The effects of exogenous mitochondria in hiPSC-RGCs were evaluated for oxygen consumption by Oroboros, mitochondrial membrane potential, reactive oxygen species (ROS), and ATP production.
Results :
Treatment of hiPSC-RGCs with 730 nM of rotenone induced significant mitochondrial depletion by 12 hours of exposure and thereby decreased ATP production while increasing ROS and superoxide production. hiPSC-RGCs incubated with isolated mitochondria accept them by 1 hour, and the uptake increases with mitochondrial dosage, incubation time, and presence of PEP-1 peptide. ATP production and oxygen consumption by hiPSC-RGCs under oxidative stress improved following mitochondrial transfer. Furthermore, intravitreally injected exogenous mitochondria into C57BL/6 mouse eye were detected in the ganglion cell layer of the murine retina, and its expression co-localized with BRN3, TUJ1, and TOM22 markers.
Conclusions :
Through our studies, we hope to demonstrate that mitochondrial transplantation is a viable, novel therapy to rescue RGC function and can be used as a mitigating treatment for glaucoma and other optic neuropathies before the occurrence of permanent vision loss.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.