Autophagy and Mitochondrial Dysfunction in Tenon Fibroblasts from Exfoliation Glaucoma Patients

Abstract

PurposeTo test the hypothesis that autophagy dysfunction is involved in exfoliation syndrome (XFS), a systemic disorder of extracellular elastic matrices that causes a distinct form of human glaucoma.MethodsFibroblasts derived from tenon tissue discards (TFs) from filtration surgery to relieve intraocular pressure in XFS patients were compared against age-matched TFs derived from surgery in primary open-angle glaucoma (POAG) patients or from strabismus surgery. Differential interference contrast light, and electron microscopy were used to examine structural cell features. Immunocytochemistry was used to visualize LOXL1 and Fibulin-5, lysosomes, endosomes, Golgi, and microtubules. Light scatter, Cyto-IDTM and JC1 flow cytometry were used to measure relative cell size, autophagic flux rate and mitochondrial membrane potential (MMPT), respectively. Enhanced autophagy was induced by serum withdrawal.ResultsIn culture, XFS-TFs were 1.38-fold larger (by light scatter ratio, p = 0.05), proliferated 42% slower (p = 0.026), and were morphologically distinct in 2D and 3D culture compared to their POAG counterparts. In extended 3D cultures, XFS-TFs accumulated 8–10 times more Fibulin-5 than the POAG-TFs, and upon serum withdrawal, there were marked deficiencies in relocation of endosomes and lysosomes to the perinuclear area. Correspondingly, the XFS-TFs displayed significant accumulation of the autophagasome marker LC3 II (3.9 fold increase compared to POAG levels, p = 0.0001) and autophagic flux rate as measured by Cyto-ID dye was 53% lower in XFS-TFs than in POAG-TFs (p = 0.01), indicating reduced clearance of autophagasomes. Finally the percent of cells with diminished MMPT was 3–8 times larger in the XFS-TFs than in POAG-TFs (p = 0.02).ConclusionsOur results provide for the first time a link between XFS pathology to autophagy dysfunction, a major contributor to multiple age related diseases systemically throughout the body, in the brain and in the retina. A diminished capacity for degradation of denatured protein and aging cellular organelles may underpin the development of extracellular protein aggregates in XFS.

Genetic association study of exfoliation syndrome identifies a protective rare variant at LOXL1 and five new susceptibility loci

“…For example, the marked downregulation of POMP (Figure 3), a ubiquitously expressed proteasome maturation protein in XFS tissues suggests a reduction of ubiquitin conjugating enzymes in XFS tissues 65 . Abnormalities in the closely related autophagy pathway have also been implicated in XFS 66 , a process that also involves ubiquitinproteasome signaling 67,68 . TMEM136 expression was predominantly localized to vascular endothelia.…”

Recent advances in risk factors associated with ocular exfoliation syndrome

“…An in vitro study using fibroblast cells excised from Tenon's capsule of XFS and POAG patients during filtration surgery and strabismus surgery showed that, compared to POAG counterparts, Tenon's cells from XFS patients had different phenotypic signs of reduced clearance of autophagosomes due to autophagy dysfunction (Want et al. ). In a following in vitro study, it was shown that those functional and structural differences were statistically significant between POAG and XFS‐derived Tenon capsule fibroblasts (Wolosin et al.…”

“…Non‐functioning mitochondria have been observed in XFS in vitro models, which could reduce the efficiency of respiration, leading to reduced ATP with increased reactive oxygen species production (Want et al. ). During ageing, some ATG s are downregulated and autophagy function declines in humans, which is believed to be linked with age‐related disease (Leidal et al.…”

“…Given the association between its dysfunction and different diseases, autophagy has been considered as potential therapeutic target (Want et al. ; Galluzzi et al. ; Leidal et al.…”

“…It is now well established that mutual interplay between mitochondria and lysosomes is crucial in maintaining cell homeostasis, and impaired autophagy has been linked with many different diseases (Audano et al 2018). Non-functioning mitochondria have been observed in XFS in vitro models, which could reduce the efficiency of respiration, leading to reduced ATP with increased reactive oxygen species production (Want et al 2016). During ageing, some ATGs are downregulated and autophagy function declines in humans, which is believed to be linked with age-related disease (Leidal et al 2018).…”