Synthesis of sphingosine is essential for oxidative stress-induced apoptosis of photoreceptors.
OBJECTIVE
Oxidative stress is involved in inducing apoptosis of photoreceptors in many retinal neurodegenerative diseases. It has been shown that oxidative stress increases in photoreceptors the synthesis of ceramide, a sphingolipid precursor that then activates apoptosis. In several cell types, ceramide is converted by ceramidases to sphingosine (Sph), another apoptosis mediator; hence, this study was undertaken to determine whether Sph participates in triggering photoreceptor apoptosis.


METHODS
Rat retina neurons were incubated with [(3)H]palmitic acid and treated with the oxidant paraquat (PQ) to evaluate Sph synthesis. Sph was added to cultures with or without docosahexaenoic acid (DHA), the major retina polyunsaturated fatty acid and a photoreceptor survival factor, to evaluate apoptosis. Synthesis of Sph and sphingosine-1-phosphate (S1P), a prosurvival signal, were inhibited with alkaline ceramidase or sphingosine kinase inhibitors, respectively, before adding PQ, C(2)-ceramide, or Sph. Apoptosis, mitochondrial membrane polarization, cytochrome c localization, and reactive oxygen species (ROS) production were determined.


RESULTS
PQ increased [(3)H]Sph synthesis in photoreceptors and blocking this synthesis by inhibiting alkaline ceramidase decreased PQ-induced apoptosis. Addition of Sph induced photoreceptor apoptosis, increased ROS production, and promoted cytochrome c release from mitochondria. Although DHA prevented this apoptosis, inhibiting Sph conversion to S1P blocked DHA protection.


CONCLUSIONS
These results suggest that oxidative stress enhances formation of ceramide and its subsequent breakdown to Sph; ceramide and/or Sph would then trigger photoreceptor apoptosis. Preventing Sph synthesis or promoting its phosphorylation to S1P rescued photoreceptors, suggesting that Sph is a mediator of their apoptosis and modulation of Sph metabolism may be crucial for promoting photoreceptor survival.
Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB), Universidad Nacional del Sur (UNS)-CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas), Buenos Aires, Argentina.
Carolina E
CE
Abrahan
Gisela E
GE
Miranda
Daniela L
DL
Agnolazza
Luis E
LE
Politi
Nora P
NP
Rotstein
D000818:Animals; D017209:Apoptosis; D002478:Cells, Cultured; D045304:Cytochromes c / Q000378:metabolism; D004281:Docosahexaenoic Acids / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D008246:Lysophospholipids / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial; D008856:Microscopy, Fluorescence; D016877:Oxidants / Q000633:toxicity; D018384:Oxidative Stress / Q000187:drug effects*; D019308:Palmitic Acid / Q000494:pharmacology; D010269:Paraquat / Q000633:toxicity; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism / Q000473:pathology*; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D000099157:Sphingosine Kinase
D045304:Cytochromes c; D004281:Docosahexaenoic Acids; D004791:Enzyme Inhibitors; D008246:Lysophospholipids; D016877:Oxidants; D019308:Palmitic Acid; D010269:Paraquat; D017853:Phosphotransferases (Alcohol Group Acceptor); D017382:Reactive Oxygen Species; D013110:Sphingosine; D000099157:Sphingosine Kinase; C064769:N-acetylsphingosine; C060506:sphingosine 1-phosphate