Up-regulation of Glis2 involves in neuronal apoptosis after intracerebral hemorrhage in adult rats

Abstract

The novel Krüppel-like zinc finger protein Gli-similar 2 (Glis2), one member of the transcription factors, is involved in controlling the flow of genetic information and the modulation of diverse cellular activities. Accumulating evidence has demonstrated its important roles in adult development and several diseases. However, information regarding the regulation and possible function of Glis2 in the central nervous system is still limited. In this study, we explored the roles of Glis2 during the pathophysiological process of intracerebral hemorrhage (ICH). An ICH rat model was established and assessed by behavioral tests. Expression of Glis2 was significantly up-regulated in brain areas surrounding the hematoma following ICH. Immunofluorescence showed that Glis2 was strikingly increased in neurons, but not astrocytes or microglia. Up-regulation of Glis2 was found to be accompanied by the increased expression of active caspase-3 and Bax and decreased expression of Bcl-2 in vivo and vitro studies. Moreover, knocking down Glis2 by RNA-interference in PC12 cells reduced active caspase-3 and Bax expression while increased Bcl-2. Collectively, we speculated that Glis2 might exert pro-apoptotic function in neurons following ICH.

Fig. 1

Assessment and scores of behavioral tests at different time points on rats suffering from ICH. Forelimb placing (a) and corner turn testing scores (b) showed that the ICH group exhibited remarkable deficits compared with the sham-operated group over the first 5 days (*p < 0.05, significantly different from the sham-operated group), with no significant difference at baseline or 5 days later

Fig. 2

Expression profiles of Glis2 around the hematoma at each time point after ICH by Western blot analysis. Glis2 was relatively low in the sham-controlled group, but increased gradually after ICH, peaked at day 2 and reduced thereafter (a). The bar graph  indicated the relative density of Glis2 versus GAPDH at each time point (b). The data are mean ± SEM. (*p < 0.05, significantly distinct from the sham group)

Fig. 3

Representative microphotographs for Glis2 immunohistochemistry in the rat brain surrounding the hematoma. Low level of Glis2 was detected in the sham-operated group (a, b). At 2 days after ICH, the contralateral group showed no significant difference in Glis2 expression (c, d), while the Glis2-positive cells in the ipsilateral group were greatly increased (e, f). g Quantitative analysis of Glis2-positive cells in the rat brain in the three groups. (*p < 0.05, significantly distinct from the sham-operated and contralateral groups) Scale bar left column 100 μm; right column 50 μm

Fig. 4

Double immunofluorescence staining for Glis2 with different phenotype-specific markers in rat brain around hematoma. Horizontal sections were labeled with Glis2 (red, b, f, j) and different cell markers, including neuron marker (green, a, NeuN), astrocyte marker (green, e, GFAP), and microglia marker (green, i, CD11b). The yellow color in the merged images represented co-localization of Glis2 with different phenotype-specific markers (c, d, g, h, k, l). Neurons marked by NeuN specifically contain Glis2 (c, d). No positive signal was found in the negative control (m, n). o Quantitative analysis of NeuN-positive cells expressing Glis2 (%) in the sham group and 2 days after ICH. (*p < 0.05, significant difference of ICH group compared with the sham) Scale bar: 50 μm (a–n)

Fig. 5

Correlations of Glis2 with cell apoptosis following ICH. The expression of active caspase-3 and Bax increased, peaked at day 2 following ICH (a, c). The expression of Bcl-2 decreased after ICH and reached valley at 2 days (c). The bar graphs indicated the relative density of active caspase-3, Bax, and Bcl-2 versus GAPDH at each time point (b, d). Data are presented as mean ± SEM. (*^,# p < 0.05, significantly distinct from the sham group). Immunofluorescent staining showed co-staining of NeuN (green) and Glis2 (green) with active caspase-3 (red) in rat brain around hematoma (e). Scale bar 25 μm (e)

Fig. 6

Modulation of Glis2 on cell apoptosis in vitro. PC12 cells were incubated with hemin at 100 μmol/L for different times. Glis2, active caspase-3, and Bax were up-regulated, peaked at 12 h, while Bcl-2 had the opposite regulation (a). The bar graph indicated the relative density of Glis2, active caspase-3, Bax, and Bcl-2 versus GAPDH at each time point (b). The data are mean ± SEM (^{*,^,#,&} p < 0.05, significantly different from the control group). The knockdown of Glis2 induced decreasing levels of active caspase-3 and Bax and had down-regulation of Bcl-2 expression (c). The bar graph indicated the relative density of Glis2, active caspase-3, Bax, and Bcl-2 versus GAPDH (d). Data are presented as mean ± SEM. (^{*,^,#,&} p < 0.05, significantly different from the control-siRNA-treated group)

Fig. 7

Immunofluorescent staining showed the relationship of Glis2 and PC12 cell apoptosis. Compared with the control group, active caspase-3 and nuclei condensation were observed in PC12 cells at 12 h after the stimulation of hemin, but when Glis2 siRNA was used, these changes were reduced. The yellow color in the merged images represented co-localization of Glis2 (green) with active caspase-3 (red). Scale bars 50 μm