The studies reported here address the role of neuropeptide processing in the brain’s response to ischemia. We first examined PC2, an enzyme that performs endoproteolytic processing of neuropeptide precursors in the secretory pathway, in ischemic cortices. Results of fluorescent in situ hybridization analyses of PC2 mRNA levels showed a transient upregulation after 100-min MCAO in rats (). This is not surprising, as it is known that the promoter region of the PC2 gene contains several elements that can potentially respond to ischemic stress (Jansen et al, 1997
; Yan et al, 2000
), and elevated PC2 mRNA levels in the brain have been reported for several pathophysiological conditions (Bhat et al, 1993
; Noel et al, 1998
; Oyarce et al, 1996
). Of particular interest is whether the PC2 protein in the brain could be processed properly to its active form after ischemia, when its expression levels were still high. The proteolytic activation of PC2, in addition to a dependence on 7B2—a chaperone-like protein (Westphal et al, 1999
; Zhu and Lindberg, 1995
)—is regulated by calcium concentrations in the secretory pathway (Guest et al, 1997
). Depletion of the ER calcium pool in ischemic cells has been well described (Hayashi and Abe, 2004
; Paschen and Doutheil, 1999
). Although there is still a lack of detailed information concerning changes in calcium concentrations in different compartments of the secretory pathway after brain ischemia, we suspect that the overall disruption of intraluminal homeostasis of the secretory pathway would not favor PC2 for either its processing from pro-PC2 to its matured form or its enzymatic activity, or both. Indeed, in ischemic brains, we found both a trend of accumulation of pro-PC2-sized protein and a decrease in PC2 activity in ischemic brains ( and ) at reperfusion time points at which total protein levels of PC2 remained high (). The observation of attenuated proteolytic processing of PC2 in ischemic NS20Y cells () provides a mechanistic explanation for changes of PC2 in ischemic brain tissues. The substantial loss of PC2 enzymatic activity, when its protein levels were still high, after brain ischemia () is also important to note. As discussed below, an ischemia-induced decrease in PC2 activity may have a significant impact on brain cell survival after ischemia.
Analyses of tissue levels of DYN-A(1–8), a cleavage product of pro-DYN by PC2 activity, revealed in decreased levels of this neuropeptide in ischemic cortices (). DYN-related peptides vary in their affinities to receptors and in their interactions with other proteins (Hauser et al, 2005
). In general, DYN-A-related neuropeptides preferably bind to the κ
-opioid receptor. Involvement of κ
-receptor in neuroprotection after focal ischemia has long been known (Chen et al, 2004
; Hall and Pazara, 1988
). DYN-A(1–8), in addition to the κ
-receptor, also binds to δ
- and μ
-receptors, and may have a higher affinity to the δ
-receptor (Bell and Traynor, 1998
; Schulz et al, 1984
), whose activation is anti-apoptotic and whose inactivation induces cell death in stressed cells (Cao et al, 2003
; Hayashi et al, 2002
; Ma et al, 2005
). Hence, our present finding that brain ischemia attenuated the production of DYN-A(1–8), especially at early reperfusion hours, may have a pathologic significance.
Our observation of reduced ischemic brain injury in animals receiving exogenous DYN-A(1–8) () is the first in describing a protective role of this neuropeptide under ischemic conditions. This result agrees well with a recent report by Charron et al (2008)
that the administration of κ
- or δ
-receptor agonist protects CA1 neurons from ischemic cell death in a global ischemia model in rats. Although it remains to be established whether the protective effect of DYN-A(1–8) is exerted through κ
- or δ
-receptor, or both, the results, nevertheless, show its potential role in preventing cell death after ischemia. The results also suggest an important role of PC2-mediated processing of DYN peptides in determining the brain’s response to ischemic stress.
mice are a mouse strain that expresses an inactive CPE protein because of a spontaneous mutation in the CPE gene ((Fricker et al, 1996
; Naggert et al, 1995
). Similar to our previous observation on Cpefat/fat
mice (Zhou et al, 2004
), PC2-null mice showed exacerbated ischemic brain injury after 30-min MCAO (). Although PC2-null and Cpefat/fat
mice share certain abnormalities in neuropeptide and peptide hormone processing, they differ in their neuroendocrine conditions. For example, adult male Cpefat/fat
mice will become severely obese and hyperglycemic at about 12 weeks of age (Leiter et al, 1999
), whereas PC2-null mice, in general, maintain a normal body weight and become hypoglycemic under fasting conditions (Furuta et al, 1997
). The exacerbating effect of hyperglycemia on ischemic brain injury is well established, whereas the effects of hypoglycemia have been less studied and it may be beneficial. A similar outcome after brain ischemia in these two different processing-deficient strains suggests that the increased vulnerability to brain ischemia in them is likely a consequence of defective neuropeptide processing, rather than that of chronic metabolic disorders. It would be interesting to see in future studies whether the exacerbated ischemic brain injury in PC2-null mice can be prevented by administrating DYN-A(1–8) to the animals.
It remains to be determined whether brain ischemia may also attenuate the processing of other PC2 substrate neuropeptides, and the consequences of it. It should be noted that functional roles of many opioid-like neuropeptides can be paradoxical in respect to pro- or anti-cell death after brain ischemia. Their possible excitotoxic effects through glutamate receptors, especially under pathophysiological conditions, as well as possible toxic effects of their precursor forms whose function are still poorly understood, are all important issues that must be studied in details in future work.
In summary, this study has shown a high responsiveness of PC2-mediated neuropeptide processing system to brain ischemia, adverse changes of this system under ischemic conditions, and the importance of sustaining the normal function of this system in protecting the brain from ischemic injury.