Sex-specific activation of cell death signalling pathways in cerebellar granule neurons exposed to oxygen glucose deprivation followed by reoxygenation

Abstract

Neuronal death pathways following hypoxia-ischaemia are sexually dimorphic, but the underlying mechanisms are unclear. We examined cell death mechanisms during OGD (oxygen-glucose deprivation) followed by Reox (reoxygenation) in segregated male (XY) and female (XX) mouse primary CGNs (cerebellar granule neurons) that are WT (wild-type) or Parp-1 [poly(ADP-ribose) polymerase 1] KO (knockout). Exposure of CGNs to OGD (1.5 h)/Reox (7 h) caused cell death in XY and XX neurons, but cell death during Reox was greater in XX neurons. ATP levels were significantly lower after OGD/Reox in WT-XX neurons than in XY neurons; this difference was eliminated in Parp-1 KO-XX neurons. AIF (apoptosis-inducing factor) was released from mitochondria and translocated to the nucleus by 1 h exclusively in WT-XY neurons. In contrast, there was a release of Cyt C (cytochrome C) from mitochondria in WT-XX and Parp-1 KO neurons of both sexes; delayed activation of caspase 3 was observed in the same three groups. Thus deletion of Parp-1 shunted cell death towards caspase 3-dependent apoptosis. Delayed activation of caspase 8 was also observed in all groups after OGD/Reox, but was much greater in XX neurons, and caspase 8 translocated to the nucleus in XX neurons only. Caspase 8 activation may contribute to increased XX neuronal death during Reox, via caspase 3 activation. Thus, OGD/Reox induces death of XY neurons via a PARP-1-AIF-dependent mechanism, but blockade of PARP-1-AIF pathway shifts neuronal death towards a caspase-dependent mechanism. In XX neurons, OGD/Reox caused prolonged depletion of ATP and delayed activation of caspase 8 and caspase 3, culminating in greater cell death during Reox.

Figure 1. Sex-related differences in OGD- and OGD/Reox-induced neuronal death

The cells were exposed to OGD for 1.5 h or to 1.5 h OGD followed by 7 h of Reox. (A) Morphological evidence of injury in WT- and Parp-1-KO-XX CGNs as compared with that in WT- and KO-XY neurons. A higher magnitude of cell injury was observed in both WT- and KO-XX CGNs. The Parp-1-KO-XY neurons exhibited lesser extent of injury than that in WT-XY CGNs. (B) Quantification of cell death by LDH release also revealed significantly higher percentage of cell death in female as compared with male CGNs after 7 h Reox following 1.5 h of OGD. Values are means±S.E.M. (n = 4), **P<0.01, WT-XX versus WT-XY; ***P<0.001, KO-XX versus KO-XY, and ⁺⁺⁺P<0.001 versus control. (C) Fluorometric TUNEL immunostaining (green) of CGNs exposed to OGD (1.5 h)/Reox (7 h) showed enhanced fluorescence of TUNEL (+) cells in XX cells of both WT and KO genotype as compared with WT-XY and KO-XY CGN cells. Quantification of TUNEL-(+) cells revealed significantly higher cell death in WT- and KO-XX than in WT-XY and KO-XY CGNs (means±S.E.M.; n = 4,*P<0.05 WT-XX versus WT-XY, **P<0.01 KO-XX versus KO-XY, ⁺⁺⁺P<0.001 versus control).

Figure 2. Effects of OGD (1.5 h) or OGD (1.5 h) followed by Reox (7 h) on neuronal viability and cellular ATP content in CGNs

(A) Neuronal survival and death was assessed using the fluorescent dyes calcein AM (green) and PI (red) incorporation following exposures. Control normoxia cells were viable as evident by intense calcein-specific green fluorescence (upper panel). Increased red fluorescence intensity of PI indicates enhanced cell death following OGD/Reox (lower panel). Quantification of calcein- and PI- (+) cells revealed a significantly higher percentage of cell death in WT-XX (73%) and KO-XX (74%) than in WT-XY (62%) and KO-XY (56%) respectively. Results are means±S.E.M. (n = 4, *P<0.05 WT-XX versus WT-XY and ***P<0.001 KO-XX versus KO-XY, ⁺⁺⁺P<0.001 versus control). (B) Exposure to OGD (1.5 h) or to OGD (1.5 h)/Reox (7 h) resulted in significant reduction in ATP content with much greater and prolonged reduction observed in WT-XX neurons than in WT- and KO-XY neurons. ATP concentration was assayed as described in the Materials and methods. Data were expressed as percentage of control (100%) and shown as means±S.E.M. for at least three separate experiments (***P<0.001, WT-XX versus WT-XY; ⁺⁺⁺P<0.001 versus control).

Figure 3. Mitochondrial release of AIF and nuclear translocation in WT-XY and WT-XX CGNs following OGD exposure

(A) Primary CGNs were immunostained with AIF (red), mitochondrial marker ATP synthase (green); DAPI stained nucleus of cells (blue) and analysed by confocal microscopy. Under normal conditions, AIF and ATP synthase are co-localized in mitochondria (yellow). Exposure at different time periods of OGD showed mitochondrial release of AIF at 30 min in WT-XY cells as AIF-specific immunofluorescence was localized in the perinuclear region of cytoplasm (red) and nuclear localization at 1 h OGD (purple), but remained localized in the mitochondria of WT-XX neurons under identical conditions. No AIF or ATP synthase-specific immunofluorescence was observed in cells incubated with non-immune IgG (negative controls). (B) AIF translocation was also assessed by subcellular fractionations and Western-blot analyses. Quantification of AIF-specific protein band showed significant decrease in AIF protein levels in the mitochondrial fraction from WT-XY CGNs with concomitant increase in the nuclear fraction of WT-XY CGNs in comparison with WT-XX cells (C). Values represent means±S.E.M. (n = 4) (⁺P<0.05, ⁺⁺⁺P<0.001 versus control normoxia cells; *P<0.05, **P<0.01 WT-XY versus WT-XX). Parp-1-KO-XY and -XX CGNs retained AIF within the mitochondria under similar exposure. Prohibitin and histone were used as a control for purity of the mitochondrial and nuclear fraction respectively, which also served as loading controls. Representative blots are shown.

Figure 4. Mitochondrial release of Cyt C in XY and XX CGNs following OGD exposure

(A) Primary CGNs were immunostained with Cyt C (red), mitochondrial marker VDAC (green); DAPI-stained nucleus of cells (blue). Confocal microscopy of double immunofluorescence staining with Cyt C and VDAC and merged images (yellow) detected Cyt C predominantly localized within the mitochondrial compartment of control CGN cells. OGD exposure resulted in Cyt C release from mitochondria into the cytoplasmic region (red) within 30 min OGD onset and more pronounced release at 1 h in WT-XX cells in comparison with WT-XY neurons. No Cyt C- or VDAC-specific immunofluorescence was observed in cells incubated with non-immune IgG (negative controls). (B) Subcellular fractionation and quantification of the Cyt C-specific protein band showed OGD-dependent decrease in Cyt C protein levels in the mitochondrial fraction with simultaneous increase in the cytoplasmic fraction of WT-XX and KO-XX CGNs (C). Unlike AIF, Cyt C release was also observed in Parp-1-KO-XY cells than in WT-XY neurons. Values represent means±S.E.M. (n = 4; *P<0.05 WT-XX versus WT-XY. ⁺⁺⁺P<0.001, ⁺P<0.05 versus controls). Prohibitin and actin were used as a control for purity of the mitochondrial and cytosolic fraction respectively, which also serve as loading controls. Representative blots are shown.

Figure 5. Sex-specific activation of caspase 3 in CGNs exposed to OGD (1.5 h) or OGD (1.5 h) followed by Reox (7 h)

(A) Caspase 3-like activity assay from total cellular extract showed no change in caspase 3 activity after 1.5 h OGD, but caspase 3 activity was significantly increased in WT-XX CGNs following Reox (7 h) in comparison with WT-XY cells. Interestingly, an increase in caspase 3-like activity was also seen in Parp-1-KO-XX and KO-XY CGN cells. Results are means±S.E.M. (n = 3; ***P<0.001 WT-XX versus WT-XY, **P<0.01 KO-XY and KO-XX versus WT-XY CGNs; ⁺P<0.05, ⁺⁺P<0.001 versus normoxic control). (B) Western immunoblot using cleaved caspase 3-specific antibody detected increased cleaved caspase 3 protein band in WT-XX CGNs and in CGNs from Parp-1-KO genotype of either sex. Values are normalized to procaspase-3 and expressed as a percentage of control (100%) (means±S.E.M., n = 3; ***P<0.001, **P<0.01 and *P<0.05 in comparison with WT-XY; ⁺⁺P<0.001 in comparison with normoxic control). Representative blots are shown. (C) Caspase 3-specific immunostaining and quantification of caspase 3 (+) cells at 7 h Reox following 1.5 h of OGD showed similar increase in cleaved caspase 3 (+) cells, which paralleled the increased caspase 3-like activity (A). Cleaved caspase 3 (+) cells at 1.5 h of OGD were not significantly different from normoxic control cells (results not shown). Values are means±S.E.M. (n = 3, *P<0.05 WT-XX versus WT-XY; ⁺⁺P<0.001 versus normoxic control cells).

Figure 6. Sex-specific activation of caspase 8 in CGNs exposed to OGD (1.5 h) or OGD (1.5 h) followed by Reox (7 h)

(A) Results showed increased caspase 8-specific peptide cleavage activity following 7 h of Reox in WT- and KO-XX cells only, but not in WT- and KO-XY CGNs. Results are means±S.E.M., n = 3 (**P<0.01, *P<0.05 in comparison with both WT- and KO-XY neurons respectively; ⁺P<0.05, ⁺⁺P<0.001 versus control). (B) Cellular extracts were subjected to fractionation after the indicated time of exposure, and the nuclear fraction was analysed by Western-blot analysis for the full-length (57 kDa) and the cleaved form (45 kDa). Quantification of cleaved active fragments of caspase 8 (normalized to 57 kDa bands) showed significant increase of cleaved caspase 8 at 7 h Reox in WT- and Parp-1-KO-XX cells only. Results are expressed as a percentage of control (100%) (means±S.E.M., n = 3, ***P<0.001 in comparison with both WT- and KO-XY neurons, ⁺⁺P<0.001 in comparison with control). Representative blots are shown.