Cumulative mtDNA damage and mutations contribute to the progressive loss of RGCs in a rat model of glaucoma

Abstract

Glaucoma is a chronic neurodegenerative disease characterized by the progressive loss of retinal ganglion cells (RGCs). Mitochondrial DNA (mtDNA) alterations have been documented as a key component of many neurodegenerative disorders. However, whether mtDNA alterations contribute to the progressive loss of RGCs and the mechanism whereby this phenomenon could occur are poorly understood. We investigated mtDNA alterations in RGCs using a rat model of chronic intraocular hypertension and explored the mechanisms underlying progressive RGC loss. We demonstrate that the mtDNA damage and mutations triggered by intraocular pressure (IOP) elevation are initiating, crucial events in a cascade leading to progressive RGC loss. Damage to and mutation of mtDNA, mitochondrial dysfunction, reduced levels of mtDNA repair/replication enzymes, and elevated reactive oxygen species form a positive feedback loop that produces irreversible mtDNA damage and mutation and contributes to progressive RGC loss, which occurs even after a return to normal IOP. Furthermore, we demonstrate that mtDNA damage and mutations increase the vulnerability of RGCs to elevated IOP and glutamate levels, which are among the most common glaucoma insults. This study suggests that therapeutic approaches that

High Pressure-Induced mtDNA Alterations in Retinal Ganglion Cells and Subsequent Apoptosis

“…The isolation protocol for purified RGCs followed our previously described two-step immunopanning-magnetic separation method (Wu et al, 2015; Gao et al, 2016). Briefly, 2-day-old Sprague-Dawley rats were sacrificed to obtain the retinas.…”

“…The cultured RGCs were harvested and mitochondria were isolated using Mitochondria Isolation Kit (Pierce Biotechnology, Rockford, IL, USA) as previously reported (Wu et al, 2015). The isolated mitochondrial pellet was resuspended in 100 μL of mannitol (210 mM), sucrose (70 mM), HEPES (5 mM), EGTA (1 mM) and fatty-acid-free bovine serum albumin (BSA, 0.5% (w/v) Sigma-Aldrich, St. Louis, MO, USA) solution.…”

“…RGC protein extractions and Western blot analysis were performed as previously reported (Wu et al, 2015). Briefly, cultured RGCs were lysed and total proteins were extracted on ice by use of cell lysis buffer (Cell Signaling Technology, Boston, MA, USA) and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA).…”

“…Moreover, many studies have indicated that mitochondrial abnormalities occur in primary open-angle glaucoma patients, including increasing mitochondrial DNA (mtDNA) pathogenic mutations and content as well as decreasing mitochondrial respiratory activity (Abu-Amero et al, 2011; Nucci et al, 2013; Sundaresan et al, 2015). Our previous studies have shown that progressively increased mtDNA damage and mutations contributed to aggressive RGC death in rat model of experimental glaucoma, and the prevention of mtDNA alterations improved RGC survival after IOP elevation (Wu et al, 2015). However, the underlying mechanisms triggering mtDNA mutations and damage in glaucoma remain unknown because of the complexity of the in vivo microenvironment of the retina after IOP elevation.…”

Neurodegenerative Eye Disorders

“…Damage to the mtDNA can cause an energy crisis in the RGCs, making them susceptible to glaucomatous injury. 16,17 It has already been established that mitochondrial abnormalities leading to RGC apoptosis and axonal injury at the optic nerve head are the major underlying pathology in LHON. 18,19 The role of mitochondrial abnormalities in optic disorders like Kjer optic neuropathy (also known as ADOA), diabetic retinopathy, agerelated macular degeneration (AMD), chronic progressive external ophthalmoplegia (CPEO), and glaucoma is well documented.…”