Caspaselike proteases activated in apoptotic photoreceptors of Royal College of Surgeons rats.
OBJECTIVE
To study the role of caspase-like proteases, especially roles of more extensively characterized caspase-1 and caspase-2, in apoptotic photoreceptor cell degeneration in Royal College of Surgeons (RCS) rats.


METHODS
Both RCS and Sprague-Dawley rats were used. Cryosections of the retinas at various postnatal times were immunostained with antibodies against caspase-1 (interleukin-1beta-converting enzyme, ICE) and caspase-2 (Nedd2/Ich-1). Double staining with TdT-dUTP terminal nick-end labeling (TUNEL), propidium iodide, and the antibodies was also performed. To evaluate the time course of protein expression, western blot analysis was carried out. The temporal profile of caspase-like protease activity was studied using a fluorogenic tetrapeptide substrate, acetyl-tyrosyl-valyl-alanyl-aspartic acid alpha (4-methyl-coumaryl-7-amide) (Ac-YVAD-MCA). Intravitreal injection of a caspase-1 inhibitor, acetyl-tyrosyl-valyl-alanyl-aspartic-aldehyde (Ac-YVAD-CHO), at postnatal days 21 (P21) and P26 was performed to see if this caused a decrease in apoptotic cell number at P28.


RESULTS
TUNEL-positive photoreceptors of RCS rats stained strongly with antibodies against caspase-1 and caspase-2. Double staining studies revealed that caspase-1 and caspase-2 were coexpressed in apoptotic cells. Western blot analysis showed that active forms of caspase-1-like and caspase-2-like proteases were upregulated at P28, concurrent with the peak in TUNEL-positive cells. The enzymatic activity of caspase-1-like protease was elevated in RCS rat retinas at P28, and the inhibitor of caspase-1 transiently reduced the number of the apoptotic photoreceptors.


CONCLUSIONS
Activation of caspase-like proteases plays an important role in photoreceptor apoptosis of RCS rats.
Department of Ophtalmology, Shinshu University School of Medicine, Matsumoto, Japan.
N
N
Katai
T
T
Kikuchi
H
H
Shibuki
S
S
Kuroiwa
J
J
Arai
T
T
Kurokawa
N
N
Yoshimura
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D020170:Caspase 1 / Q000378:metabolism*; D053143:Caspase 2; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D003374:Coumarins; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D020287:In Situ Nick-End Labeling; D007202:Indicators and Reagents; D009842:Oligopeptides / Q000494:pharmacology; D020419:Photoreceptor Cells, Vertebrate / Q000201:enzymology* / Q000473:pathology; D051381:Rats; D011922:Rats, Mutant Strains; D017207:Rats, Sprague-Dawley; D012162:Retinal Degeneration / Q000201:enzymology* / Q000473:pathology
D061945:Caspase Inhibitors; D003374:Coumarins; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D009842:Oligopeptides; C096022:acetyl-tyrosyl-valyl-alanyl-aspartyl-7-amino-4-methylcoumarinamide; C096429:L 709049; D053143:Caspase 2; D020169:Caspases; D020170:Caspase 1