IDH1 Associated with Neuronal Apoptosis in Adult Rats Brain Following Intracerebral Hemorrhage

Abstract

Isocitrate dehydrogenase 1 (IDH1), one member of the IDH family can convert isocitrate to α-ketoglutarate (α-KG) via oxidative decarboxylation. IDH1 and IDH2 mutations have been identified in multiple tumor types and the mutations confer neomorphic activity in the mutant protein, resulting in the conversion of α-KG to the oncometabolite, D-2-hydroxyglutarate (2-HG). The subsequent accumulation of 2-HG results in epigenetic dysregulation via inhibition of α-KG-dependent histone and DNA demethylase. And the glutamate levels are reduced in IDH mutant cells compared to wild-type. We have known that diffuse gliomas contain a high frequency of mutations in the IDH1 gene. However, the expression of IDH1 and its roles in Intracranial hemorrhage (ICH) remain largely unknown. We observed increased expression of IDH1 in neurons after intracerebral hemorrhage. Up-regulation of IDH1 was found to be accompanied by the increased expression of active caspase-3 and pro-apoptotic Bcl-2-associated X protein and decreased expression of anti-apoptotic protein B cell lymphoma-2 in vivo and vitro studies. So we hypothesized that IDH1 was involved in the regulation of neuronal apoptosis. The present research for the first time detected the expression and variation of IDH1 surrounding the hematoma, and all data proved the involvement of IDH1 in neuronal apoptosis following ICH.

Keywords: Active caspase-3; Apoptosis; ICH; IDH1; Neuron.

Fig. 1

Estimations and scores of behavioral tests on rats suffering from ICH. Behavioral tests were executed in rats after ICH or sham operation. 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

Western blot analysis protein level change of Bex1 after ICH. Western blot was performed to study the protein level of IDH1 surrounding the hematoma at various survival times. a Time courses of IDH1 expression after ICH, peaked at day 2, and declined thereafter. b Quantification graphs (relative optical density) of the intensity of staining of IDH1 to β-actin at each time point. β-actin was used to confirm equal amount of protein was run on gel. The data are mean ± SEM. (*P < 0.05, significantly different from the sham-operated group)

Fig. 3

Representative microphotographs for IDH1 immunohistochemistry surrounding the hematoma. Low level of IDH1 was detected in the sham group (a, b). At day 2 after ICH, the contralateral group showed no significant difference in IDH1 (c, d) compared with the sham ones, while the ipsilateral group (e, f) showed increased IDH1 expression. No positive signals were found in the negative control (g). The number of IDH1 positive cells was largely increased comparing the ipsilateral group with the sham and contralateral groups (h). Asterisk denotes P < 0.05. Scale bar left column, 50 μm; right columns, 20 μm

Fig. 4

The colocalization of IDH1 with different cellular markers by double immunofluorescent staining. In the adult rat caudate within 3 mm distance from the hematoma at the second day after ICH, horizontal sections were labeled with IDH1 (green, a, e, i), different cell markers (red, b, f, j), such as neuronal marker (NeuN), astrocyte marker (GFAP) and microglia marker (CD11b). The yellow color visualized in the merged images represents the colocalization of IDH1 with different specific phenotype markers (c, g, k). The colocalizations of IDH1 with different specific phenotype markers are also shown in the sham-control group (d, h, l). No positive signals were found in the negative control (m, n). The number of NeuN-positive cells expressing IDH1 (%) was remarkably increased in the ICH group compared with the sham group (o). Asterisk means P < 0.05. Scale bars 20 μm (a–n) (Color figure online)

Fig. 5

Association of IDH1 with cell apoptosis after ICH. The expression of active caspase-3 and Bax increased, peaked at day 2 following ICH (a). The expression of Bcl-2 decreased after ICH and reached valley at 2 days (a). The bar graphs indicated the relative density of active caspase-3, Bax, and Bcl-2 versus GAPDH at each time point (b). Data are presented as mean ± SEM. (^{*, &, $} P < 0.05,significantly distinct from the sham group). Immunofluorescent staining showed co-staining of NeuN (green) and IDH1 (green) with active caspase-3 (red) in rat brain around hematoma (c). Scale bar 20 μm (c)

Fig. 6

Modulation of IDH1 on cell apoptosis in vitro. PC12 cells were incubated with hemin at 100 μmol/L for different times. IDH1, 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 IDH1, active caspase-3, Bax, and Bcl-2 versus GAPDH at each time point (b). Western blot showed that Knocking IDH1 down induced decreasing levels of active caspase-3 and Bax and had up-regulation of Bcl-2 expression (c). The bar chart indicated the density of IDH1, active caspase-3, Bax, and Bcl-2 versus GAPDH (d). The data are mean ± SEM(^{#, $, &, ^} P < 0.05, significantly different from the control group)

Fig. 7

The relationship of IDH1 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 IDH1 siRNA was used, these changes were reduced. The yellow color in the merged images represented co-localization of IDH1 (green) with active caspase-3 (red). Scale bars 50 μm (a). Cytotoxicity were estimated after the addition of reagents by means of LDH release assay as described under ‘‘Materials and Methods’’ section (b). The data are mean ± SEM (*P < 0.05, significantly different from the nonspecific siRNA-treated group) (Color figure online)