The role of phosphorylated glucocorticoid receptor in mitochondrial functions and apoptotic signalling in brain tissue of stressed Wistar rats

Abstract

Mitochondrial dysfunction is increasingly recognized as a key component in compromised neuroendocrine stress response and, among other etiological causes, it may also involve action of glucocorticoid hormones. In the current study we followed glucocorticoid receptor and identified its mitochondrial phosphoisophorms in hippocampus and prefrontal brain cortex of Wistar male rats subjected to acute, chronic and combined neuroendocrine stresses. In both brain structures chronic social isolation caused marked increase in mitochondrial glucocorticoid receptor that was preferentially phosphorylated at serine 232 compared to serine 246 or serine 171. This increase corresponded with the decreased expression of mitochondrially encoded cytochrome oxidase subunits 1 and 3 in hippocampus, and with their increased expression in prefrontal brain cortex. Prefrontal brain cortex appeared to be more sensitive to chronic stress, since it exibited higher levels of mitochondrial Bax and cytoplasmic Bcl2 compared to hippocampus. Chronic stress also altered the response of both brain structures to subsequent acute stress according to the studied parameters. Therefore, prolonged social isolation may cause susceptibility to mitochondria triggered proapototic signalling, which at least in part may be mediated by the glucocorticoid receptor dependent mechanism.

Fig. 1

Serum corticosterone concentration (ng/ml) in control, acute, chronic and combined group of Wistar rats presented as mean ± SEM and measured individually. Statistically significant differences at the level of p < 0.05 also include p < 0.01 and 0.001 (*stress vs. control and ^$chronic vs. combined).

Fig. 2

(A) Western blot experiments demonstrating the effects of acute immobilization chronic isolation and combined stress on the level of total glucocorticoid receptor (tGR) and its phosphoisoforms in mitochondria of hippocampus and prefrontal cortex. Mitochondrial lysates were resolved by SDS-PAGE and probed with antibodies against tGR, pGR171, pGR232, pGR246 or mHsp70 as a loading control. (B) Immunoreactivities of mitochondrial tGR, pGR171, pGR232, pGR246 (normalized to mHsp70) in hippocampus and prefrontal cortex. The ratios of pGR232/171 and pGR232/246 (normalized to GR and mHsp70) are expressed as mean ± SEM and presented as a percent of control (as described under Section 2). Asterisk indicates significant differences between treated groups: acute (A), chronic (C) and combined (CA) obtained from one-way ANOVA followed by Tukey post hoc test (*p < 0.05, stress vs. control; ^%p < 0.05, acute vs. combined; ^$p < 0.05 chronic vs. combined). (C) The purity of subcellular fractions was assayed using specific antibodies against α-tubulin for cytoplasmic, NBS1 for nuclear, and mHsp70 for mitochondrial fraction. (D) Western blot of GR probed with antibody specific for its phosphorylated T171 isoform in the absence (lane 1) or presence (lane 4, PEP) of specific peptide used as antigen for this antibody. Membrane strips were also incubated with preimmune serum (lane 2, PI) or with non-specific IgG (lane 3, NS) (top panel). Blot was stripped of primary antibodies and probed with GR specific antibody (middle panel). Lower part of this blot was probed with actin antibody.

Fig. 3

Real time RTqPCR analysis of relative changes in cytochrome oxidase subunit1 (COX 1) and cytochrome oxidase subunit 3 (COX 3) mRNA levels under acute (A), chronic (C) and combined stress (CA) in rat hippocampus and prefrontal cortex. Calculations were performed using ΔΔC_T method. Data are presented as mean ± SEM as a percent of control and statistical analyses was performed using one-way ANOVA followed by Tukey post hoc test (*p < 0.05, stress vs. control; ^%p < 0.05, acute vs. combined; ^$p < 0.05 chronic vs. combined).

Fig. 4

(A) Western blot experiment demonstrating the effect of stress on mitochondrial and cytoplasmic Bcl2 and Bax levels in rat hippocampus and prefrontal cortex. (B) Immunorectivities of Bcl2 and Bax (normalized to mHsp70) in mitochondrial extracts and cytoplasm (normalized to β-actin) of hippocampus or prefrontal cortex under acute (A), chronic (C) and combined (CA) stress and the ratio of mitochondrial to cytoplasmic Bcl2 and Bax are given as mean ± SEM, as a percent of control (as described under Section 2). Asterisk indicates significant differences obtained from one-way ANOVA followed by Tukey post hoc test (*p < 0.05, stress vs. control; ^%p < 0.05, acute vs. combined; ^$p < 0.05 chronic vs. combined).

Fig. 5

Measurement of DNA fragments with the diphenylamine (DPA) colorimetric assay. Results are presented as a percent of control fragmentation following formula % of fragments = (OD_600 nmT/OD_600 nm(T + B)) × 100, T = fragmented DNA, B = intact DNA obtained from rats exposed to acute (A), chronic (C) and combined (CA) stress. Statistical analyses was performed using One-way ANOVA followed by Tukey post hoc test (*p < 0.05, stress vs. control; ^%p < 0.05, acute vs. combined; ^$p < 0.05 chronic vs. combined).