Autophagy Impairment in Retinal Ganglion Cells Following Hypoglycemia in Mice.
Diabetic retinopathy (DR), caused by hypo- and hyperglycemia, is a leading cause of blindness. Hypoglycemia induces endoplasmic reticulum stress and retinal cell death in mice, and low glucose conditions induce macroautophagy/autophagy defects in 661W photoreceptor cells and retinal explants. To further understand the effect of hypoglycemia on the mouse retina, we studied autophagy in vivo. Retinal explants cultured under low glucose conditions exhibited autophagy activation; a similar pattern was observed in the presence of autophagy modulators. Intraocular injection of rapamycin and 5 h hypoglycemia had similar effects, specifically in the ganglion cell layer, as shown by green fluorescence protein (GFP) colocalization with different ganglion cell markers. We then isolated retinal ganglion cells (RGCs) from two mouse lines and cultured them at different glucose concentrations. We also assessed autophagosome/lysosome fusion in C57BL/6 RGCs infected with the RFP-GFP-LC3 lentivirus and cultured under different conditions. p62 protein expression and GFP fluorescence increased when RGCs were cultured with 1 or 25 mM glucose in the presence of rapamycin, whereas no sign of autophagy was detected with 5 or 25 mM glucose alone. The fusion process was defective at 1 and 25 mM glucose in the presence of chloroquine, but normal with 5 and 25 mM glucose alone. This study showed that 5 h hypoglycemia induces autophagosome formation in mouse RGCs; however, a defect in the fusion process inhibits the protective effect of autophagy. Therefore, the modulation of both autophagic and apoptotic pathways might be important to avoid complications associated with DR. Overall design: RNA-Seq analyses on six samples after laser capture microdissection of ganglion cell layer from 3 GFP-LC3 mice underwent a 5 h hypoglycemia and 3 controls mice that were maintain euglycemic during 5 h.