Abstract
Purpose: :
The intralysosomal accumulation of the non-degradable, autofluorescent macromolecule lipofuscin, impairs autophagy and may promote a decline in the function of glial cells of the lamina cribrosa which has been implicated in the pathogenesis of disease related biomechanical changes occurring at the optic nerve head (ONH) in glaucoma. This study aimed to compare levels of lipofuscin-like material in lamina cribrosa cells from normal (NLC) and glaucomatous (GLC) donor eyes and assess its effects on autophagy.
Methods: :
NLC and GLC cells were examined by transmission electron microscopy (TEM) and the number of peri-nuclear lysososomes per high powered field (x 20,000) recorded. The cells were stained with Sudan Black B, and peri-nuclear lipophilic body number assessed. Cellular autofluorescence was quantified by flow cytometry (emission at 563-607nm). Real-Time PCR (RT-PCR) measured the cell content of Cathepsin D and Autophagy protein 5 (ATG5) mRNA in NLC and GLC. Cellular protein levels of the former were analysed at Western blot.
Results: :
The number of peri-nuclear lysosomes was increased in GLC (11.1 +/- 3.8 v 4.2 +/- 3.7, p = 0.002) at TEM. The quantity of Sudan Black B stained peri-nuclear lipophillic bodies was also increased in GLC ( 22.10 +/- 3.57 v 13.77 +/- 5.66, p = 0.07). An increase in whole cell autofluorescence was observed in GLC ( 83062 +/- 45.1 v 41.01 +/- 3.9, p = 0.2). Cathepsin D mRNA and protein content did not differ significantly between the two cell groups. There were significantly higher levels of ATG5 mRNA in GLC samples compared to NLC.
Conclusions: :
We present evidence supportive of increased lipofuscin formation as being characteristic of lamina cribrosa cells derived from donors with glaucoma with important downstream effects on autophagy. Potential future anti-glaucoma strategies might therefore include attempts at stimulation of cellular degradation systems.
Keywords: ipofuscin • lamina cribrosa • glia