In this study, we demonstrate that HSV-mediated overexpression of viral caspase inhibitor p35 significantly protects against ischemic neuronal loss after stroke. In addition, we found that p35 not only inhibits caspase 3 activity, but also blocks cytosolic cytochrome c release and nuclear AIF translocation. In addition, crmA showed a trend towards attenuating ischemic neuronal loss and blocking the above apoptotic signals, as compared with the control vector.
We had previously shown that the delivery of various genes by the HSV system reduced ischemic neuronal loss after stroke. These genes include the glucose transporter Glut-1 that enhances ATP generation (Lawrence et al., 1995
), Heat shock protein 70 that repairs misfolded proteins (Yenari et al., 1998
), calbindin 28 that buffers an increase in intracellular calcium (Yenari et al., 2001
) and several anti-oxidant genes that scavenge free radicals (Hoehn et al., 2003
, Gu et al., 2004
). However, caspase-dependent apoptosis is the dominant cell death mechanism in the ischemic penumbra (Wei et al., 2004
). Therefore, it is intriguing to study whether the ischemic penumbra can be targeted by gene therapy, and whether gene therapy with caspase inhibitors blocks ischemic damage. We have previously shown that the HSV based vectors start to express a few hours after delivery (Fink et al., 1997
, Yenari et al., 1998
). In the current study, we successfully delivered vectors into the ischemic penumbra or the ischemic margin, and found that gene delivery of p35 reduces ischemic neuronal loss after stroke. In contrast, crmA overexpression showed only a trend towards protection. As we have discussed, these differential effects of p35 and crmA may lie in their different ability to inhibit caspase activity. Cowpox viral crmA physiologically inhibits only caspases 1 and 8 (Zhou et al., 1997
), while baculoviral p35 protein acts as a pan-caspase inhibitor, including inhibition of caspases 1 and 8 as well as 3 and others (Bump et al., 1995
). A straightforward implication is that p35's superior neuroprotective capacity following ischemia is directly due to its role as a pan-caspase inhibitor, inhibiting caspases involved in both the intrinsic and extrinsic apoptotic pathways, while crmA can only modulate caspases involved in the extrinsic pathway. Indeed, consistent with the protective effects, p35, but not crmA, significantly blocks caspase-3 activity after stroke. Plausibly related to this, prior studies have shown them to have differing effects, depending on the nature of the insult model. We have previously demonstrated that both p35 and crmA inhibit neuronal death caused by domoic acid and heat shock in primary mixed neuronal culture (Roy et al., 2001
), but do not attenuate sodium cyanide-induced hypoxic damage. Interestingly, caspase activity was absent after domoic acid insult (Roy et al., 2001
), suggesting the protection is not executed through blocking this classical apoptotic pathway. Instead, such caspase inhibitors reduce cell damage by attenuating ATP depletion and mitochondrial potential decreases. In addition, their neuroprotective effects differ in vivo from in vitro. While p35 reduces CA3 damage after kainic acid exposure, crmA does not. However, p35 does not attenuate damage in the dentate gyrus caused by the electron transport uncoupler, 3-acetyl pyridine (3-AP), and crmA treatment worsens such injury (Roy et al., 2002
). Such differentially protective effects of p35 and crmA in vivo seem consistent with the current findings of the differing effects of the two. Taken together, these results suggest that the protective effects of p35 and crmA depend on damage models or types of insults.
In addition to inhibiting caspase 3 activity, p35 blocks cytochrome c release and AIF translocation as well. Although caspase activation is cytochrome c-dependent in mitochondrial-mediated apoptosis, it has been demonstrated that caspase activity can form a positive feedback loop onto mitochondria and cause more cytochrome c release, thereby amplifying the apoptotic pathway (Chen et al., 2000
). A recent study suggests that deficit in caspase-3 and −7 preserves mitochondrial membrane potential and blocks AIF nuclear translocation (Lakhani et al., 2006
). Consistent with this, we have demonstrated that blocking caspase activity can inhibit delayed cytochrome c release in a forebrain ischemia model (Zhao et al., 2005b
). In our current study, although we found that the caspase inhibitor blocks cytochrome c release and AIF nuclear translocation, whether it achieves these results by inhibiting the feed back effect of caspase on cytochrome c release or by blocking cross talk between AIF and caspase pathways needs further study.
In the ischemic cortex, the number of positive cells immunostained with active caspase-3, cytochrome c and AIF appears less in non-infected cells in brains treated with P35 vectors than in those treated with other vectors. This raises the possibility that overexpression of p35 may not only protect infected neurons but, through indirect mechanisms, protect neighboring neurons as well; we also observe evidence for such “good neighbor” effects in primary cultures (upublished data). Whether such effect exists in vivo needs further study.
There are some limitations to this study. HSV based gene transfer can only infect individual neurons. It does not reduce overall infarct size. Therefore, it may not improve the overall neurological outcome. In the future, gene delivery in multiple sites in the ischemic cortex after focal ischemia should be performed to determine if it improves neurological function. In addition, viral vectors were injected before stroke, which is not entirely clinically relevant, since most strokes are not predictable; a future study should address whether vectors delivered post-stroke reduce neuronal loss. Moreover, it might be more appropriate in the future to deliver such genes into the hippocampal region after global ischemia to target delayed selective neuronal death and address its efficacy for preservation of brain function.
In conclusion, our study demonstrated that the viral caspase inhibitor p35 is significantly neuroprotective, while crmA only shows a trend toward neuroprotection, in the ischemic penumbra in a permanent MCAO rat model. P35 expression may block neuronal death by inhibiting both caspase and AIF dependent apopotic pathways.