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.
Highlights
Exfoliation syndrome (XFS) is an age-related systemic disorder characterized by the accumulation of amorphous protein aggregates within the extracellular matrix of multiple tissues and organs [1] such as blood vessels, skin, and the gallbladder, kidneys, lungs and heart [2,3,4]
XFS-TFs were 1.38-fold larger, proliferated 42% slower (p = 0.026), and were morphologically distinct in 2D and 3D culture compared to their primary open-angle glaucoma (POAG) counterparts
The XFS-TFs displayed significant accumulation of the autophagasome marker light chain 3 (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
Summary
Exfoliation syndrome (XFS) is an age-related systemic disorder characterized by the accumulation of amorphous protein aggregates within the extracellular matrix of multiple tissues and organs [1] such as blood vessels, skin, and the gallbladder, kidneys, lungs and heart [2,3,4]. Carried along with the aqueous humor flow (the fluid circulation systems that provide nutrients to the internal avascular organs of the eye) the pigment granules and/or extracellular aggregates deposit at the trabecular meshwork, the sieving structure of the eye’s outflow facility. These deposits block fluid outflow, because aqueous humor production is not sensitive to the increased resistance, and a sustained elevated intraocular pressure ensues. XFS is the most common identifiable cause of open-angle glaucoma worldwide [10]
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