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

Abstract

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.

Fig. 1

Involvement of MMPs in apoptotic cell death in rat brain at 48-h post-ischemic reperfusion. (a) Representative DAB-TUNEL staining showing increased TUNEL-positive cells in saline vehicle-treated compared to BB-1101-treated cortex (CTX), caudate (CDAT), and piriform cortex (PFC). Scale bar = 50 μm. (b) Apoptotic body-like staining in the cytoplasm of apoptotic neurons in vehicle-treated infarct PFC (arrows). (c) Representative TUNEL-fluorescence and DAPI-nuclear staining showing increased DNA fragmentation in ischemic infarct cortex without BB-1101 treatment. Scale bar = 20 μm. (d) TUNEL-positive cell stereology in whole ischemic hemispheres. A significant increase in TUNEL-positive cells (×10³/mm³) was observed in ischemic hemispheres both with and without BB-1101 treatment compared to sham animals (*), p < 0.01. Sham group n = 3, vehicle group n = 5, BB-1101 group n = 4. Significantly less TUNEL-positive cells were detected in the BB-1101-treated ischemic hemispheres (#) compared to vehicle, p < 0.05. Inset diagram, stereological analysis of the entire ischemic (right) hemisphere was done over a distance of 3.0, 1.5 mm rostral and caudal to the bregma. Images in panel (a) were obtained from the infarct areas indicated (red, cortex; blue, caudate; green, piriform cortex). (e) Representative confocal images of double-staining for NeuN and TUNEL show that most TUNEL-positive cells are neurons in ischemic infarct cortex at 48-h reperfusion. Scale bar = 20 μm.

Fig. 2

Induction of gelatinase activity and nuclear localization of MMP-2 and -9 in cortical neurons after OGD. (a) In situ zymography (ISZ) was performed on living neurons after 0, 3-, 6-, 12-, and 24-h reoxygenation as described in Experimental Procedures. ISZ-positive cells are stained green in column 1, nuclei are stained blue with DAPI in column 2, and neurons are identified with a neuron-specific marker (anti-MAP2 polyclonal antibody) in column 3. Superimposition of ISZ, DAPI, and MAP2 shows gelatinase activity in neurons. Scale bar = 100 μm. (b) The stages of gelatinase induction associated with apoptosis are shown. Stage I, early apoptosis with nuclear condensation, strong nuclear gelatinase activity, and retraction of cellular processes. Stage II, mid-stage apoptosis with further condensation of the nucleus, collapse of the cytoskeleton, and loss of MAP2 staining. Stage III, late-stage apoptosis with clear nuclear fragmentation and intense uniform gelatinase activity throughout the cell. (c) Using immunocytochemistry, MMP-2 was detected in nuclei in both control and OGD-treated cells and increased in the nucleus 24 h after OGD. MMP-9 could not be detected in the nucleus of control cells by immunocytochemistry but was detectable in neuronal nuclei after OGD.

Fig. 3

Gelatinase activity after OGD is inhibited by MMP-2/9 inhibitor II. The percentage of gelatinase-positive neurons was measured 24 h after OGD. (a) Both control and OGD experiments were performed with and without treatment with MMP-2/9 inhibitor II. Scale bar = 100 μm. The percentage of cells with gelatinase activity for each condition was determined from 4 experiments and results are shown graphically in (b) A statistically significant (p < 0.01) difference in the percent of ISZ-positive cells between control and OGD-treated cells (*) and between OGD and OGD plus inhibitor was observed (#).

Fig. 4

Measurement of MMP-2 and MMP-9 levels in neuronal nuclei after OGD. The levels of MMP-2 and -9 in the nuclei of neurons were evaluated by gelatin zymography 24 h after OGD or mock treatment with or without MMP-2/9 inhibitor II. In agreement with immunocytochemistry (Fig. 2c), MMP-9 was undetectable in the nucleus by gelatin zymography in control cells and was elevated following OGD. MMP-2 was observed in the nucleus in control cells and was elevated after OGD. (a) Gelatin zymogram showing MMP-2 and -9 levels (photographically negative). M, HT1080 culture medium standard for MMP-2 and -9. Lane 1, control, lane 2, control plus inhibitor, lane 3, OGD, lane 4, OGD plus inhibitor. Molecular weights indicate MMP-9 glycosylated and pro forms, 94 and 88 kDa, respectively, and MMP-2 pro, intermediate, and activation forms, 68, 64, and 62 kDa, respectively. (b) Control Western blot showing fractionation of nuclear and cytoplasmic proteins. Five micrograms neuronal nuclear (N) and cytoplasmic (C) extract were immunoblotted with antibody to HDAC1, nuclear protein histone deacetylase 1, left panel, or cytoplasmic protein glyceraldehyde 3-phosphate dehydrogenase, GAPDH, right panel. (c–e) Graphical representations of relative MMP-9 and -2 levels in nuclear extracts shown as the mean and standard deviation of 4 independent experiments. Units are arbitrary. (c) Total MMP-9 level. (d and e) Levels of pro and intermediate and activation forms of MMP-2, respectively. Asterisks indicate statistically significant differences between control and OGD, while pound signs indicate significant differences between OGD and OGD plus inhibitor, p < 0.01.

Fig. 5

Decreased levels of DNA repair proteins and measurement of oxidative DNA damage and PARP1 activity in neuronal nuclei after OGD. (a) The levels of DNA repair proteins PARP1, XRCC1, OGG1, and APE1 in nuclear extracts of neurons exposed to control (lane 1), control plus inhibitor (lane 2), OGD (lane 3), or OGD plus inhibitor (lane 4) conditions were measured by Western blot. (b) Graphical representation of normalized relative levels of DNA repair proteins. Units are arbitrary. For PARP1 and XRCC1, protein levels were significantly lower in OGD-treated cells compared to controls (*), and significantly higher in OGD plus inhibitor compared to OGD (#), p < 0.01. OGG1 significantly decreased after OGD (*) and no significant changes in APE1 were observed. (c) Level of PAR present in the nucleus of neurons in pg PAR per μg nuclear extract. OGD caused a statistically significant decrease in nuclear PAR level compared to control cells (*), p < 0.01. In the presence of MMP-2/9 inhibitor II, a significant difference in nuclear PAR levels between OGD and OGD plus inhibitor was observed (#), p < 0.01. (d) After OGD and 24-h reoxygenation, 8-oxo-dG in genomic DNA (pg per μg DNA) was measured as described in Experimental Procedures. 8-oxo-dG was significantly elevated in OGD-treated cells compared to controls (*), p < 0.01, and significantly lower in OGD plus inhibitor compared to OGD (#), p < 0.05. Results are means and standard deviations from 4 independent experiments.

Fig. 6

Apoptosis and co-localization of apoptosis and ISZ in neurons. Apoptosis in neurons was measured by TUNEL assay after OGD and 24-h reoxygenation as described in Experimental Procedures. (a) Apoptotic cells are labeled green and superimposed with blue DAPI-stained nuclei. Scale bar = 100 μm. (b) Co-localization of TUNEL and ISZ. Apoptotic cells (red) are superimposed with ISZ (green). When superimposed, cells positive for both TUNEL and ISZ appear yellow (indicated by down arrows) while healthy cells (blue) are stained with DAPI alone (indicated by up arrows). (c) Percent apoptotic cells for each condition as determined by counting cells from 4 independent experiments. Results are shown as means with standard deviation. A statistically significant difference in apoptosis was observed between control and OGD (*) and OGD plus inhibitor and OGD (#), p < 0.01.

Fig. 7

Measurement of gelatinase levels in neuronal culture medium after OGD. Gelatinase levels in culture medium after OGD or mock treatment with or without MMP-2/9 inhibitor II were measured by gelatin zymography. (a) Gelatin zymogram showing MMP-2 and -9 levels (photographically negative). M, HT1080 culture medium standard for MMP-2 and -9. Lane 1, control, lane 2, control plus inhibitor, lane 3, OGD, lane 4, OGD plus inhibitor. (b and c) Graphical representations of relative MMP-9 and -2 levels in culture medium shown as the mean and standard deviation of 4 independent experiments. Units are arbitrary.