Age-dependent susceptibility of the retinal ganglion cell layer to cell death.
OBJECTIVE
The purpose of this study was to determine the susceptibility of the retinal ganglion cell layer (GCL) to apoptosis after optic nerve transection and excitotoxic stimulus and to investigate the regulation of apoptosis in the GCL during development. The authors also sought to determine the role played by caspases in cell death and their expression during development.


METHODS
TdT-mediated dUTP nick end labeling (TUNEL) was used to identify cells undergoing apoptosis during mouse retinal development from postnatal day (P)3 to P5 and in retinal explant sections under various conditions. The expression of active caspases was determined by immunohistochemistry (IHC) using an antibody that detects the cleaved large subunit. IHC was also used to detect the expression levels of procaspase-3, procaspase-9, and Apaf-1 in P6 and P60 whole eye sections. Retinal ganglion cells at ages P6 and P60 were purified by immunopanning, total RNA was extracted, and mRNA levels of the above proteins were determined by semiquantitative PCR.


RESULTS
After optic nerve transection, a significant number of TUNEL-positive cells were seen 24 hours after lesion in P6 retinas. This death was caspase dependent, as shown by IHC and caspase inhibition with zVAD-fmk. In contrast, adult GCL was resistant to apoptosis under these conditions. Similarly, after excitotoxic stimulus, the GCL of the P6 retinas underwent apoptosis at 6 hours and was caspase dependent, whereas adult GCL was resistant. Developmental apoptosis in the GCL between P2 and P6 was shown to involve caspase-3 and caspase-9. Significant downregulation of Apaf-1 and caspase-3 was detected in the P60 GCL at both the mRNA and the protein levels.


CONCLUSIONS
Adult GCL is more resistant to apoptosis than neonatal GCL after ON transection and excitotoxic stimulus. The expression of caspase-3 and Apaf-1 is significantly reduced in adult GCL. The authors suggest that age-dependent susceptibility to apoptosis may be caused by this reduced expression.
McKernan
Declan P
DP
Cell Development and Disease Laboratory, Department of Biochemistry, Biosciences Institute, University College Cork, Cork, Ireland.
Caplis
Ciara
C
Donovan
Maryanne
M
O'brien
Colm J
CJ
Cotter
Thomas G
TG
D000375:Aging / Q000502:physiology*; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000502:physiology*; D053477:Apoptotic Protease-Activating Factor 1; D019771:Axotomy; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D007124:Immunoenzyme Techniques; D020287:In Situ Nick-End Labeling; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009900:Optic Nerve / Q000502:physiology; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000254:growth & development; D012165:Retinal Ganglion Cells / Q000201:enzymology / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
C506593:Apaf1 protein, mouse; D053477:Apoptotic Protease-Activating Factor 1; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D011506:Proteins; D012333:RNA, Messenger; D020169:Caspases