Pharmacological restoration of impaired autophagy in retinal ganglion cells prevents abnormal mitochondrial accumulation and glaucomatous neurodegeneration

Abstract Background Progressive loss of retinal ganglion cells (RGCs) and degeneration of optic nerve (ON) axons are the key pathological hallmarks of glaucoma, the leading cause of irreversible blindness. Elevated intraocular pressure (IOP), primarily due to dysfunction of the trabecular meshwork (TM), remains the most significant and only known modifiable risk factor. However, vision loss persists in some patients despite effective IOP control, highlighting the critical need to elucidate the mechanisms driving glaucomatous neurodegeneration. Emerging evidence links mitochondrial dysfunction to glaucomatous neurodegeneration, yet the precise mechanisms remain poorly defined. Here, we investigate whether defective autophagy/mitophagy, which removes damaged mitochondria, contributes to mitochondrial accumulation, oxidative stress, and neurodegeneration in glaucoma. We further explore the therapeutic potential of enhancing autophagy to improve mitochondrial turnover, mitigate RGC loss, and preserve visual function. Methods Glucocorticoid (GC)-induced and myocilin (MYOC)-associated glaucoma mouse models were used to assess the expression of mitochondrial markers (TOM20/COX IV), oxidative DNA damage (8-OHdG), and mitophagy/autophagy-related proteins (p62, LC3, Phospho-ubiquitin (Ser65), and LAMP1) in retinal tissues. Transmission electron microscopy (TEM) was employed to analyze mitochondrial accumulation in glaucomatous ON. Mitophagy flux was assessed at early and late stages of neurodegeneration using mitophagy reporter Mt-Keima mice. The effect of RGC-specific autophagy deficiency on mitochondrial accumulation and neurodegeneration was further investigated using Atg5 flox/flox mice, in which Atg5 deletion was induced by AAV2-Cre delivery. Additionally, the therapeutic effect of enhancing autophagy with Torin 2 to restore mitochondrial turnover and prevent glaucomatous neurodegeneration was evaluated in both GC-induced and myocilin-associated glaucoma models, as well as in ex vivo human retinal explants. Results Chronic IOP elevation led to increased mitochondrial accumulation, oxidative DNA damage, and impaired mitophagy/autophagy in glaucomatous retina. TEM analysis further confirmed the accumulation of structurally abnormal mitochondria in glaucomatous ON. In Mt-Keima mice, chronic IOP elevation significantly reduced mitophagy flux prior to RGC loss, indicating that mitophagy impairment precedes neurodegeneration. RGC-specific Atg5 deletion induced the accumulation of damaged mitochondria, leading to neurodegeneration in Atg5 flox/flox mice. Notably, pharmacological restoration of impaired autophagy with Torin 2 prevented mitochondrial accumulation and preserved the structural and functional integrity of RGCs and their axons in glaucoma mouse models and ex vivo human retinal explant cultures. Conclusion Our study indicates impaired autophagy contributes to damaged mitochondrial accumulation and oxidative stress, leading to glaucomatous neurodegeneration. Enhancing autophagy in RGCs represents a promising therapeutic strategy to prevent glaucomatous neurodegeneration. Graphical Abstract