A key role for calpains in retinal ganglion cell death.
OBJECTIVE
The purpose of this study was to examine the importance of calpains in retinal ganglion cell (RGC) apoptosis and the protection afforded by calpain inhibitors against cell death.


METHODS
Two different models of RGC apoptosis were used, namely the RGC-5 cell line after either intracellular calcium influx or serum withdrawal and retinal explant culture involving optic nerve axotomy. Flow cytometry analysis with Annexin V/PI staining was used to identify RGC-5 cells undergoing apoptosis after treatment. TdT-mediated dUTP nick end labeling (TUNEL) was used to identify cells undergoing apoptosis in retinal explant sections under various conditions. Serial sectioning was used to isolate the cell population of the ganglion cell layer (GCL). Western blotting was used to demonstrate calpain cleavage and activity by detecting cleaved substrates.


RESULTS
In the RGC-5 cell line, the authors reported the activation of mu-calpain and m-calpain after serum starvation and calcium ionophore treatment, with concurrent cleavage of known calpain substrates. They found that the inhibition of calpains leads to the protection of cells from apoptosis. In the second model, after a serial sectioning method to isolate the cells of the ganglion cell layer (GCL) on a retinal explant paradigm, protein analysis indicated the activation of calpains after axotomy, with concomitant cleavage of calpain substrates. The authors found that inhibition of calpains significantly protected cells in the GCL from cell death.


CONCLUSIONS
These results suggest that calpains are crucial for apoptosis in RGCs after calcium influx, serum starvation, and optic nerve injury.
Cell Development and Disease Laboratory, Department of Biochemistry, Biosciences Institute, University College, Cork, Ireland.
Declan P
DP
McKernan
Marc B
MB
Guerin
Colm J
CJ
O'Brien
Thomas G
TG
Cotter
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D019771:Axotomy; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors / Q000502:physiology*; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D005434:Flow Cytometry; D005901:Glaucoma / Q000473:pathology; D020287:In Situ Nick-End Labeling; D051379:Mice; D008810:Mice, Inbred C57BL; D009900:Optic Nerve / Q000502:physiology; D009924:Organ Culture Techniques; D011419:Propidium / Q000378:metabolism; D012160:Retina / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*
D017304:Annexin A5; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; C071482:calpeptin; D011419:Propidium; D000001:Calcimycin; D002154:Calpain; D002118:Calcium; C058076:calpain inhibitor III