In this study, we demonstrate that caspase-1– and caspase-2–like
proteases play an important role in photoreceptor apoptosis during
retinal degeneration in the RCS rat. Activation of caspase-1–like
protease contributes to photoreceptor apoptosis in the RCS rat.
Caspase-1 was first identified as the mammalian homologue of the
ced-3 gene, initially identified in
Caenorhabditis
elegans. Caspase-1 is known to promote apoptosis during
development of the nematode, and, currently, 10 Ced-3–related cysteine
proteases, termed caspase, have been cloned.
3 4 These
family members participate in one of two distinct signaling pathways:
activation of proinflammatory cytokines and activation of apoptotic
cell death.
4 All the known caspase family proteases are
synthesized as inactive proenzymes, which require cleavage to liberate
one large and one small subunit to form the active
enzyme.
4 Activation of one caspase can lead to cleavage
and activation of another molecule of the same caspase, another
caspase, or both, leading to an amplified apoptotic
cascade.
4 Indeed, in the Fas-mediated apoptosis of
lymphoid cells, activation of caspase-3 via specific cleavage of the
proenzyme by caspase-1 has been demonstrated.
4 A number of
experimental studies suggest that activation of the caspase family
plays a critical role in the execution of apoptotic
events.
3 7 8 9 Caspase-1 was implicated in death caused by
superoxide dismutase downregulation in PC12 cells but not in withdrawal
of trophic factor support.
9 Conversely, the antisense
constructed to downregulate caspase-2 in PC12 cells inhibited cell
death by withdrawal of trophic support but not oxidative
stress.
7 The activation of distinct caspases in the same
cells thus can promote apoptosis initiated by the various
stimuli.
7
In the present study, immunohistochemical analysis showed that
caspase-1 and caspase-2 were colocalized in the same cell. Furthermore,
on immunoblot analysis and measurement of enzymatic activities
(Figs. 3 4) , the active forms of caspase-1– and caspase-2–like proteases
were shown to be upregulated in P28 RCS rat retinas. These results
suggest that caspase-1 and caspase-2 are correlated with the
photoreceptor apoptotic process in RCS rats. However, it remains to be
determined how these two caspases are activated in photoreceptor
degeneration. To estimate the potential role of caspase-1–like
protease in photoreceptor apoptosis more directly, we examined in vivo
effects of a specific inhibitor of caspase-1 on the number of apoptotic
photoreceptors. When administrated into the vitreous, Ac-YVAD-CHO
reduced the number of apoptotic cells
(Fig. 5) . This suggests that
caspase-1–like protease induced photoreceptor apoptosis in the RCS rat
and further suggests that an inhibitor of such caspases could inhibit
apoptosis in other photoreceptor degenerative diseases. In fact,
inhibitors of caspase-1 and caspase-3 can inhibit neuronal apoptosis in
brain injury induced by ischemia and by trauma.
14
Compared with the number of TUNEL-positive cells, immunopositive cells
were fewer in this study. It is difficult to give satisfactory
explanation to this apparent discrepancy. However, there are four
possible explanations or speculations for this. First, low level
expression of apoptosis-related proteins may not be detectable by the
immunohistochemical methods used in this study. Second, photoreceptor
apoptosis proceeds very slowly in RCS rats. TUNEL-positive cells
accounted for as many as one third of the total cell number in the ONL
at P28; if these TUNEL-positive cells were digested rapidly by
surrounding cells, all the photoreceptors would disappear within
several days. However, because photoreceptors survive until
P60,
2 dying cells may accumulate and remain over a long
period in the ONL. Thus, only a few cell
s that have recently
entered the apoptotic process and that overexpress the caspase-like
proteases may be present in any individual tissue sections. A third
possibility is that numerous other mechanisms may be involved in the
apoptotic process in RCS rats, and we may be aware of only a small
portion of this apoptotic process. For example, caspases other than
caspase-1 and caspase-2 may play a more important role in the apoptotic
process of the RCS rat retina. Finally, it is likely that the
persistence of apoptotic nuclei for long periods reflects a failure of
phagocytosis.
To our knowledge, this is the first report of a correlation between
photoreceptor apoptosis and the caspase family. Although this study
suggests an important role for caspase-1 and caspase-2 in the process
of photoreceptor apoptosis in the RCS rat retina, further studies are
necessary to clarify the molecular mechanism underlying retinal
degeneration in these animals.