Intranuclear matrix metalloproteinases promote DNA damage and apoptosis induced by oxygen-glucose deprivation in neurons

Abstract

Degradation of the extracellular matrix by elevated matrix metalloproteinase (MMP) activity following ischemia/reperfusion is implicated in blood-brain barrier disruption and neuronal death. In contrast to their characterized extracellular roles, we previously reported that elevated intranuclear MMP-2 and -9 (gelatinase) activity degrades nuclear DNA repair proteins and promotes accumulation of oxidative DNA damage in neurons in rat brain at 3-h reperfusion after ischemic stroke. Here, we report that treatment with a broad-spectrum MMP inhibitor significantly reduced neuronal apoptosis in rat ischemic hemispheres at 48-h reperfusion after a 90-min middle cerebral artery occlusion (MCAO). Since extracellular gelatinases in brain tissue are known to be neurotoxic during acute stroke, the contribution of intranuclear MMP-2 and -9 activities in neurons to neuronal apoptosis has been unclear. To confirm and extend our in vivo observations, oxygen-glucose deprivation (OGD), an in vitro model of ischemia/reperfusion, was employed. Primary cortical neurons were subjected to 2-h OGD with reoxygenation. Increased intranuclear gelatinase activity was detected immediately after reoxygenation onset and was maximal at 24h, while extracellular gelatinase levels remained unchanged. We detected elevated levels of both MMP-2 and -9 in neuronal nuclear extracts and gelatinase activity in neurons co-localized primarily with MMP-2. We found a marked decrease in PARP1, XRCC1, and OGG1, and decreased PARP1 activity. Pretreatment of neurons with selective MMP-2/9 inhibitor II significantly decreased gelatinase activity and downregulation of DNA repair enzymes, decreased accumulation of oxidative DNA damage, and promoted neuronal survival after OGD. Our results confirm the nuclear localization of gelatinases and their nuclear substrates observed in an animal stroke model, further supporting a novel role for intranuclear gelatinase activity in an intrinsic apoptotic pathway in neurons during acute stroke injury.

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.