Increased intranuclear matrix metalloproteinase activity in neurons interferes with oxidative DNA repair in focal cerebral ischemia.

Increased matrix metalloproteinase (MMP) activity is implicated in proteolysis of extracellular matrix in ischemic stroke. We recently observed intranuclear MMP activity in ischemic brain neurons at early reperfusion, suggesting a possible role in nuclear matrix proteolysis. Nuclear proteins, poly-ADP-ribose polymerase-1 (PARP-1) and X-ray cross-complementary factor 1 (XRCC1), as well as DNA repair enzymes, are important in DNA fragmentation and cell apoptosis. We hypothesized that intranuclear MMP activity facilitates oxidative injury in neurons during early ischemic insult by cleaving PARP-1 and XRCC1, interfering with DNA repair. We induced a 90-min middle cerebral artery occlusion in rats. Increase activity of MMP-2 and -9, detected in the ischemic neuronal nuclei at 3 h, was associated with DNA fragmentation at 24 and 48 h reperfusion. The intranuclear MMPs cleaved PARP-1. Treatment of the rats with a broad-spectrum MMP inhibitor, BB1101, significantly attenuated ischemia-induced PARP-1 cleavage, increasing its activity. Degradation of XRCC1 caused by ischemic insult in rat brain was also significantly attenuated by BB1101. We found elevation of oxidized DNA, apurinic/apyrimidinic sites, and 8-hydroxy-2'-deoxyguanosine, in ischemic brain cells at 3 h reperfusion. BB1101 markedly attenuated the early increase of oxidized DNA. Using tissue from stroke patients, we found increased intranuclear MMP expression. Our data suggest that intranuclear MMP activity cleaves PARP-1 and XRCC1, interfering with oxidative DNA repair. This novel role for MMPs could contribute to neuronal apoptosis in ischemic injuries.

Intranuclear localization of apoptosis-inducing factor and endonuclease G involves in peroxynitrite-induced apoptosis of spiral ganglion neurons.

OBJECTIVES: The present study was designed to determine whether or not the caspase-independent apoptotic pathway participated in the cellular death of spiral ganglion neurons (SGNs) after exposure to peroxynitrite (ONOO(-)), with particular attention given to the intranuclear translocation of mitochondrial apoptosis-inducing factor (AIF) and endonuclease G (Endo G) in this process. METHODS: The rat SGNs were isolated and primary cultured in vitro and were exposed to ONOO(-) with pre-treatment of pan-caspase inhibitor. Morphological changes of SGNs were observed by acridine orange cytochemistry staining, and apoptosis was examined by flow cytometry. The translocation of mitochondrial AIF and Endo G was detected by immunocytochemistry and Western blot. The protein expressions of Bcl-2 family in SGNs exposed to ONOO(-) were determined by Western blot. RESULTS: Treatment of SGNs with ONOO(-) resulted in the occurrence of caspase-independent apoptosis as evidenced by acridine orange staining and flow cytometry analysis. The immunocytochemical analysis showed that AIF and Endo G labeling were marked in neuronal nuclei, while the Western blot demonstrated the intranuclear localization of AIF and Endo G in SGNs treated with ONOO(-). Western blot analysis demonstrated that ONOO(-) increased the Bax expression while reducing Bcl-2 expression, which was not prevented by pre-treatment with caspase inhibitor. CONCLUSION: These data indicate that ONOO(-) can trigger caspase-independent apoptosis in SGNs associated with mitochondrial AIF and Endo G intranuclear localization.