Ceramide-induced Mitochondrial Damage in Diabetic Retinopathy

Abstract

Purpose : Increase in acid sphingomyelinase (ASM) activity and ceramide production were shown to promote diabetes-induced pro-inflammatory and pro-apoptotic changes in the retina and retinal pigment epithelial (RPE) cells. Mitochondria were recently shown to contain sphingolipids, as well as enzymes of sphingolipid pathway, including ASM. This study addressed the role of diabetes-induced increase in ASM and ceramide in mitochondrial damage in the retina and RPE cells.

Methods : Retinal mitochondria from control, STZ-induced diabetic and ASM-/- rodent models were isolated by differential centrifugation. Sphingolipids were analyzed by electrospray ionization high resolution mass spectrometry. Human (H)RPE cells were isolated from control and diabetic donors. Mitochondrial morphology and ceramide localization were measured by fluorescence microscopy. mRNA was measured by qPCR. Respiration was measured using a novel custom designed microrespirometer.

Results : Relative mitochondrial ceramide (mCer) abundance increased in short term (7 wks) diabetic rat retina (38 ± 4%) compared to control (26.5 ± 6.5%, P<0.05, n=3). Mitochondrial sphingomyelin (mSM) decreased in diabetic retina (62 ± 1%) compared to control (72.5 ± 7%, P<0.05, n=3). Long term (36 wks) diabetic (n=4) rat retina showed significant increases in short chain (C16:0, C18:0) mCer compared to control (n=3, P<0.05). Wildtype and ASM-/- mouse retinas were used to determine if mCer changes are due to ASM. ASM-/- retina showed increased mSM (85.0 ± 6.7%) compared to wildtype (77.0 ± 2.8%) and decreased mCer (11.5 ± 3.6%) compared to wildtype (17.6 ± 3.6%, P<0.05, n=3). We determined if similar changes occur in RPE layer, a mitochondrial rich cell population missing from retina preparations. HRPE isolated from diabetic donor retinas showed increased IL1ß, IL6 and ASM expression and a 2- fold increase in Cer compared to control (P<0.05, n=3). Average mitochondrial length decreased in diabetic HRPE (1.2 ± 0.57 μm) compared to control (3.4 ± 0.78 μm, P<0.05, n=3). Whole cell respiratory control ratio was decreased greater than 2-fold in diabetic HRPE compared to control.

Conclusions : Diabetes-induced elevation of ASM expression and activity leads to increased ceramide abundance in mitochondria isolated from diabetic retina and RPE cells. This increase is associated with upregulation of inflammatory markers and mitochondrial dysfunction in diabetic retina and RPE cells.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.