C-terminal binding proteins 1 and 2 in traumatic brain injury-induced inflammation and their inhibition as an approach for anti-inflammatory treatment

Abstract

Traumatic brain injury (TBI) induces an acute inflammatory response in the central nervous system that involves both resident and peripheral immune cells. The ensuing chronic neuroinflammation causes cell death and tissue damage and may contribute to neurodegeneration. The molecular mechanisms involved in the maintenance of this chronic inflammation state remain underexplored. C-terminal binding protein (CtBP) 1 and 2 are transcriptional coregulators that repress diverse cellular processes. Unexpectedly, we find that the CtBPs can transactivate a common set of proinflammatory genes both in lipopolysaccharide-activated microglia, astrocytes and macrophages, and in a mouse model of the mild form of TBI. We also find that the expression of these genes is markedly enhanced by a single mild injury in both brain and peripheral blood leukocytes in a severity- and time-dependent manner. Moreover, we were able to demonstrate that specific inhibitors of the CtBPs effectively suppress the expression of the CtBP target genes and thus improve neurological outcome in mice receiving single and repeated mild TBIs. This discovery suggests new avenues for therapeutic modulation of the inflammatory response to brain injury.

Keywords: CtBP; microglia activation; neuroinflammation; proinflammatory transcription; traumatic brain injury.

Figure 1

CtBP1 and CtBP2 are required for LPS-induced proinflammatory gene expression in microglia and macrophage cell lines. (A and B) Simultaneous knockdown of CtBP1 and CtBP2 suppresses mRNA expression of proinflammatory genes in LPS-activated microglia and macrophages. Murine RAW264.7 (A) and BV2 (B) cells were transfected with siRNAs specific for CtBP1 and CtBP2 or scrambled control siRNAs for a period of 24 h, followed by LPS stimulation (200 ng/ml) for 6 h. Total RNA was extracted and analyzed by the RT-qPCR. Relative mRNA expression was normalized to that of ACTB and depicted as fold changes vs. the scrambled siRNA transfected and non-stimulated control (Untreated). Data are presented as mean ± SD. n = 3; **p<0.01, ***p<0.001. (C) LPS induces increased binding of CtBP1, CtBP2 and p300 to the IL1B, IL6, TNFA and S100A8 gene promoters. Chromatin fractions from control and LPS-treated BV2 cells were precipitated with antibodies specific to CtBP1, CtBP2 and p300. Bars represent fold changes of relative ChIP signals normalized to the respective controls.

Figure 2

Pep1-E1A suppresses the induction of CtBP-regulated proinflammatory genes in LPS-activated mouse primary microglia. (A) Mouse primary microglia were incubated with 20 μM Pep1-E1A or the PLDLS-to-PLDEL mutant (Pep1-E1A^Mut) peptide for 2 h, followed by LPS stimulation (200 ng/ml) for 2 h. Relative mRNA expression was depicted as fold changes vs. the untreated controls. n = 3; *p<0.05, **p<0.01, ***p<0.001. (B) Representative immunostaining images of Pep1-E1A internalization into cultured primary microglia after 1 h incubation with the peptide. Scale bar, 50 μm.

Figure 3

Impact dose- and time-dependent induction of CtBP target genes in brain and peripheral blood leukocytes following single mild TBI. (A and B) Mice (n = 5 per group) were subjected to a single head injury of varying impact energies and the brain (A) and peripheral blood leukocytes (B) were harvested for mRNA analysis 24 h after injury. Results were normalized to the sham group and are presented as mean ± SD. (C) Representative Western blots of the CtBP1, CtBP2, S100A9, NLRP3 proteins of the brain tissues from the dose-response groups. Relative protein expression was normalized to the loading control GAPDH and shown as percent change vs sham under the blots. (D and E) Mice (n = 5 per group) received a single head impact of 0.7 J energy and mRNA expression in brain (D) and blood (E) were analyzed at the indicated time points postinjury. (F) Western blots showing protein expression of the brain tissues from the time course experiment.

Figure 4

Pep1-E1A reduces CtBP-dependent proinflammatory gene expression and ameliorates neurological deficits after mild brain injury. Mice (n = 4 per group) received sham, a single 0.8 J TBI, or a single 0.8 J TBI followed by treatment with Pep1-E1A or Pep1-E1A^Mut (2 mg/kg, i.p.) at 1 h and 24 h after injury. (A) Comparison of NSS scores at 1, 24 and 48 h postinjury. Data are presented as mean ± SD from four animals and were analyzed among the three TBI groups by one-way ANOVA followed by Bonferroni's post hoc tests. Asterisk indicates significant difference between TBI + vehicle and TBI + Pep1-E1A mice at 48 h postinjury (p < 0.05; F_(2,9) = 5.33). (B and C) Comparison of mRNA expression of the CtBP target genes in brain (B) and peripheral blood leukocytes (C) at 48 h postinjury. Results (mean ± SD) were normalized to sham and analyzed by t-test. n = 4; *p<0.05, **p<0.01, ***p<0.001.

Figure 5

Post-injury treatment with NSC93597 reduces microglia and astrocyte activation, suppresses transactivation of CtBP target genes and improves neurological outcome. Mice (n = 4 per group) received a single 0.8 J TBI followed by i.p. injections of vehicle or NSC95397 (0.5 mg/kg) at 1 h, 24 h and 48 h postinjury. (A) NSS scores (mean ± SD) analyzed by t-test revealed significant effect of NSC95397 treatment at 48 h (n = 4; *p<0.05) and 72 h (n = 4; ## p<0.01).(B and C) Relative mRNA expression levels (mean ± SD) in brain (B) and peripheral blood leukocytes (C) at 72 h postinjury were normalized to sham and analyzed by t-test. n = 4; ***p<0.001. (D) Representative images of microglia visualized by Iba1 immunostaining (red) in the optic tract at 72 h postinjury. Nuclei were stained by DAPI. Scale bar, 100 μm. The three panels on the right are higher magnification images of the respective framed regions within the Iba1-stained panels on the left. (E) Representative images of astrocytes in the corpus callosum visualized by immunostaining for GFAP (green) as described in (D). (F and G) Quantitation of the microglia and astrocyte response by counting the number of Iba1-positive (F) and GFAP-positive (G) cells per mm² in the optic tract and corpus callosum regions, respectively. Data are presented as mean ± SD and analyzed by t-test. n = 3; **p<0.01.(H) Representative images of GFAP-positive astrocytes in the hippocampal CA1 region 72 h postinjury. Scale bar, 100 μm. Higher magnification images of the respective framed regions within the GFAP-stained panels on the left are shown in panels on the right.

Figure 6

NSC95397 and MTOB attenuate neuroinflammation caused by repetitive mild TBI. (A) Experimental timeline. Mice (n = 4 per group) received a single head impact of 0.5 J energy (1xTBI), or two 0.5 J impacts (2xTBI) spaced 24 h apart. The CtBP inhibitor-treated groups were given an i.p. injection of MTOB (860 mg/kg) or NSC95397 (1.5 mg/kg) at 1 h and 18 h after the first injury. (B) LRR durations (mean ± SD) following the first and second head injury were analyzed by t-test. n = 4; *p<0.05, **p<0.01, ***p<0.001. (C) CtBP inhibitors improved neurological deficits in mice receiving repeated mTBI. NSS assessment at 1 h and 18 h was given prior to the administration of the CtBP inhibitors. NSS scores (mean ± SD) were analyzed by two-way ANOVA with Bonferroni post hoc test of significance between individual groups. Asterisk indicate a significant difference between 1xTBI and 2xTBI (***p<0.001) mice; hash symbol indicates a significant difference between 2xTBI and 2xTBI with MTOB treatment, (^#p<0.01, ^##p<0.01, ^###p<0.001); plus symbol indicates a significant difference between the 2xTBI and 2xTBI + NSC95397 groups (⁺p<0.01, ⁺⁺⁺p<0.001). (D) NSC95397 and MTOB prevent a further increase in the mRNA expression levels of CtBP target genes in the animal brains experiencing repeated TBI. Brain tissues were collected for mRNA analysis at 72 h after the first injury; results (mean ± SD) were normalized to sham and analyzed by t-test. n = 4; **p<0.01, ***p<0.001. (E) A model for CtBP-mediated transactivation in inflammatory response and immune activation.