Searching for the molecular pathways involved in ceramide-induced death of retina photoreceptors.

PRADO SPALM F.H.; VERA M; DIBO M; POLITI LE; ROTSTEIN N.P.

Buenos Aires

Congreso; II Reunión Conjunta de Sociedades de Biociencias; 2017

SAIC-SAIB-SAA-SAFE-SAB-SAFIS-SAH-SAI-SAP

SEARCHING FOR THE MOLECULAR PATHWAYS INVOLVED IN CERAMIDE-INDUCED DEATH OF RETINA PHOTORECEPTORSProgrammed cell death of photoreceptors (PhR) is a hallmark of neurodegenerative diseases of the retina, irrespective of their cause. Identifying the mediators and molecular mechanisms involved is essential for designing new treatments. Ceramide (Cer), a simple sphingolipid, signals cell death in many cell types. Our laboratory demonstrated that it induces PhR death in culture and Cer was then shown to participate in different retina pathologies. We now investigated the molecular mechanisms involved in this death.Rat retina neuronal cultures were treated at day 3 with or without 10 µM C2-Ceramide (Cer) for 6 hours. Cer decreased neuronal viability to 50%, as determined by MTT assays. Cer selectively increased PhR with pyknotic nuclei to 50 %, compared to 5 % in controls, without augmenting their TUNEL labeling, whereas amacrine neurons were not affected. Cer decreased PhR mitochondrial functionality and promoted AIF translocation from mitochondria to nuclei. Noteworthy, no cleaved-caspase 3 increase was observed; pretreatment with Z-VAD-FMK, a caspase pan inhibitor, failed to prevent PhR death, suggesting this death was caspase-independent. Cer did not increase LDH release and a necroptosis inhibitor did not protect PhR, implying programmed necrosis was not involved. Autophagy was also ruled out, as the autophagy inhibitor 3-MA did not prevent Cer-induced death. Next, we investigated the involvement of cathepsins and calpains. Pretreatment with ALLN, a calpain and cathepsin inhibitor, significantly increased viability from 50% in Cer-treated cultures to over 80%, decreasing the percentage of pyknotic PhR. Of note, inhibiting both cathepsins (Pepstatin A, CA-074) and lysosomal activity (Bafilomycin A1) did not block PhR death. As a whole, our results suggest that Cer promotes programmed cell death in PhR in a caspase-independent manner, activating calpain proteases, and inducing mitochondrial permeabilization followed by translocation of AIF and nuclear pyknosis.