Analysis of apoptotic and survival mediators in the early post-natal and mature retina.
Apoptosis, a cellular process critical to retinal neurogenesis, has been implicated in several neurodegenerative disorders. As the retina matures the suppression of apoptosis occurs and the emphasis shifts towards survival. To identify the cellular changes that bring about this critical shift in the balance, we performed an expression analysis of pro- and anti-apoptotic mediators in the immature, post-natal day 6 (P6) and the post-mitotic adult P60 mouse retina. Laser capture microdissection (LCM) of the P6 and the P60 retina, followed by reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to elucidate changes in the mRNA expression of Apaf-1, caspase-3 and caspase-9 in the individual retinal layers in the young and mature tissue. RT-PCR and Western blotting of whole P6 and P60 retinal preparations was carried out to determine changes in other caspases and key survival mediators at the mRNA and protein level, respectively. Our results demonstrate that each neuronal cell layer in the adult retina down-regulates the gene expression of Apaf-1 and caspase-3, and to a lesser extent, caspase-9. The protein expression levels of other executioner and initiator caspases are also reduced in the adult tissue. Interestingly, XIAP, a potent caspase inhibitor, increases in expression in the adult retina. Additionally, we demonstrate age-dependent increased expression and activation status of the components of the MAPK transduction cascade. Conversely, we observe decreased PI3-K and AKT expression and decreased activity of AKT (pAKT) in the adult retina. Furthermore, results from RNAi experiments demonstrate an additional mechanism of PI3-K regulation in photoreceptor cells. Our findings suggest that a survival strategy adopted by the post-mitotic retina involves a down-regulation of key pro-apoptotic factors concomitant with changes in expression and activation status of certain pro-survival mediators.
O'Driscoll
Carolyn
C
Cell Development and Disease Laboratory, Biochemistry Department, Bioscience Research Institute, University College Cork, Cork, Ireland. carolyn.omahony@ucc.ie
Donovan
Maryanne
M
Cotter
Thomas G
TG
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053477:Apoptotic Protease-Activating Factor 1 / Q000096:biosynthesis / Q000235:genetics; D015153:Blotting, Western / Q000379:methods; D053148:Caspase 3 / Q000096:biosynthesis / Q000235:genetics; D053453:Caspase 9 / Q000096:biosynthesis / Q000235:genetics; D002470:Cell Survival / Q000502:physiology; D058534:Class I Phosphatidylinositol 3-Kinases; D015536:Down-Regulation; D005136:Eye Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D042282:Microdissection / Q000379:methods; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D012160:Retina / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
C506593:Apaf1 protein, mouse; D053477:Apoptotic Protease-Activating Factor 1; D005136:Eye Proteins; D012333:RNA, Messenger; D019869:Phosphatidylinositol 3-Kinases; C509622:1-phosphatidylinositol 3-kinase p110 subunit, mouse; D058534:Class I Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D020929:Mitogen-Activated Protein Kinase Kinases; C505409:Casp3 protein, mouse; C506434:Casp9 protein, mouse; D053148:Caspase 3; D053453:Caspase 9