TAT-Ngn2 Enhances Cognitive Function Recovery and Regulates Caspase-Dependent and Mitochondrial Apoptotic Pathways After Experimental Stroke

Abstract

Neurogenin-2 (Ngn2) is a basic helix-loop-helix (bHLH) transcription factor that contributes to the identification and specification of neuronal fate during neurogenesis. In our previous study, we found that Ngn2 plays an important role in alleviating neuronal apoptosis, which may be viewed as an attractive candidate target for the treatment of cerebral ischemia. However, novel strategies require an understanding of the function and mechanism of Ngn2 in mature hippocampal neurons after global cerebral ischemic injury. Here, we found that the expression of Ngn2 decreased in the hippocampus after global cerebral ischemic injury in mice and in primary hippocampal neurons after oxygen glucose deprivation (OGD) injury. Then, transactivator of transcription (TAT)-Ngn2, which was constructed by fusing a TAT domain to Ngn2, was effectively transported and incorporated into hippocampal neurons after intraperitoneal (i.p.) injection and enhanced cognitive functional recovery in the acute stage after reperfusion. Furthermore, TAT-Ngn2 alleviated hippocampal neuronal damage and apoptosis, and inhibited the cytochrome C (CytC) leak from the mitochondria to the cytoplasm through regulating the expression levels of brain-derived neurotrophic factor (BDNF), phosphorylation tropomyosin-related kinase B (pTrkB), Bcl-2, Bax and cleaved caspase-3 after reperfusion injury in vivo and in vitro. These findings suggest that the downregulation of Ngn2 expression may have an important role in triggering brain injury after ischemic stroke and that the neuroprotection of TAT-Ngn2 against stroke might involve the modulation of BDNF-TrkB signaling that regulates caspase-dependent and mitochondrial apoptotic pathways, which may be an attractive therapeutic strategy for cerebral ischemic injury.

Keywords: apoptosis; brain-derived neurotrophic factor; global cerebral ischemia; neurogenin-2; transactivator of transcription domain.

Figure 1

Expression of neurogenin-2 (ngn2) mRNA and Ngn2 protein in both in vivo and in vitro studies. (A) Expression of Ngn2 at the mRNA level in the cerebral cortex, hippocampus and corpus striatum was quantified using quantitative real-time PCR (qPCR) at 12 h, 24 h and 48 h after reperfusion in the global cerebral ischemic injury model (n = 8 for each group, *P < 0.05 vs. the sham group, the data were analyzed using one-way ANOVA with Dunnett’s test). (B,C) Expression of Ngn2 at the protein level in the hippocampus was quantified using western blotting at 12 h, 24 h and 48 h after reperfusion in the global cerebral ischemic injury model (n = 8 for each group, *P < 0.05 vs. the sham group, the data were analyzed using one-way ANOVA with Dunnett’s test). (D,E) Expression of Ngn2 in hippocampal neurons was detected using western blotting at 12 h, 24 h and 48 h after oxygen glucose deprivation (OGD) injury. Band densities were measured using the ImageJ program and normalized to β-actin (n = 6 for each group, *P < 0.05 vs. the sham group, ^#P < 0.05 vs. 12 h after reperfusion group, the data were analyzed using one-way ANOVA with Dunnett’s test).

Figure 2

Intraperitoneal (i.p.) injection of transactivator of transcription (TAT)-Ngn2 improved cognitive functional recovery. (A) The delivery and distribution of TAT-Ngn2 fusion proteins in the hippocampus. Double immunofluorescence histochemical staining was performed at 6 h and 48 h after i.p. administration of TAT-Ngn2 (250 mg/kg; n = 6 for each group). Scale bar = 50 μm. (B) Total motor scores (TMSs) were recorded in each animal at 48 h after reperfusion (n = 8 for each group, nine serial sections were acquired per mouse in the same area, serial five sections in the middle of nine serial sections were observed and analyzed per animal). (C) The latency in the step-down passive avoidance test was measured in each animal at 24 h after reperfusion (n = 8 for each group). (D) The number of errors in the step-down passive avoidance test was measured in each animal at 48 h after reperfusion (n = 8 for each group). (E) Mean trials to the first avoidance response on the T-maze were measured in each animal at 48 h after reperfusion (n = 8 for each group). (F) Mean trials to criterion on the T-maze were measured in each animal at 48 h after reperfusion (n = 8 for each group). (G) The transfer latency (TL) on the elevated plus maze was measured in each animal at 48 h after reperfusion (n = 8 for each group; *P < 0.05 vs. the sham group, ^#P < 0.05 vs. the bilateral common carotid artery occlusion (BCCAO) group). The data from the behavioral experiments were analyzed by one-way analysis of variance followed by Tukey’s multiple comparison tests.

Figure 3

Effect of TAT-Ngn2 on neuronal damage and degeneration in the CA1 region of the hippocampus of mice after BCCAO. (A) Representative Nissl staining was performed in the CA1 region of the hippocampus 48 h after reperfusion (n = 6 for each group, nine serial sections were acquired per mouse in the same area, serial five sections in the middle of nine serial sections were observed and analyzed per animal). The black arrows indicate the Nissl-positive neurons. Scale bar = 50 μm. (B) Representative neuronal degeneration in the CA1 region of the hippocampus was shown by fluoro-jade C (FJC) staining (green) 48 h after reperfusion (n = 6 for each group; nine serial sections were acquired per mouse in the same area, serial five sections in the middle of nine serial sections were observed and analyzed per animal). The white arrows indicate the FJC-positive neurons. Scale bar = 50 μm. (C) Statistical analysis of the number of Nissl-positive neurons in the CA1 region (*P < 0.05 vs. sham group, *P < 0.05 vs. BCCAO). (D) Statistical analysis of the number of FJC-positive neurons in the CA1 region (*P < 0.05 vs. sham group, ^#P < 0.05 vs. BCCAO). The data were analyzed using Student’s t-test.

Figure 4

Effect of TAT-Ngn2 on neuronal apoptosis in the CA1 region of the hippocampus of mice after BCCAO. (A) Neuronal apoptosis in the CA1 region of the hippocampus is shown by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining at 48 h after reperfusion. The rectangular highlighted box was the observation area (n = 6 for each group, nine serial sections were acquired per mouse in the same area, serial five sections in the middle of nine serial sections were observed and analyzed per animal). Scale bar = 50 μm. (B) Statistical analysis of the number of TUNEL-positive cells in the CA1 region (*P < 0.05 vs. sham group, ^#P < 0.05 vs. BCCAO). The data were analyzed using Student’s t-test.

Figure 5

Effect of TAT-Ngn2 on the expression of NGF, brain-derived neurotrophic factor (BDNF), the level of phosphorylation of tropomyosin-related kinase B (TrkB) and mitochondrial cytochrome C (CytC) leakage in vivo (n = 6 for each group). (A,C) Western blotting showing NGF and BDNF expression in the hippocampus at 48 h after reperfusion. (B,D) Statistical analysis of NGF and BDNF expression in every group. (E) Western blotting showing the p-TrkB expression in the hippocampus at 48 h after reperfusion. (F) Statistical analysis of p-TrkB expression in every group. (G,I) Western blotting showing the level of CytC in the cytoplasm (C CytC) and CytC in the mitochondria (M CytC) in the hippocampus at 48 h after reperfusion. (H,J) Statistical analysis of C CytC and M CytC levels in every group. Band densities were measured using the ImageJ program and normalized to COX IV or to β-actin (*P < 0.05 vs. sham group, ^#P < 0.05 vs. BCCAO group. The data from the Western blotting studies were analyzed using one-way ANOVA with Dunnett’s test). (K) Electron microscopic image of the ultrastructure of hippocampal neurons at 48 h after reperfusion. Arrowheads indicate swollen mitochondria in the cytoplasm. The mitochondria swelled to a spherical shape. Scale bar = 1 μm. (L) Statistical analysis of the number of swollen mitochondria in every group (n = 6 for each group, serially five sections were observed and analyzed per animal; *P < 0.05 vs. sham group, ^#P < 0.05 vs. BCCAO group). The data were analyzed by one-way analysis of variance followed by Tukey’s multiple comparisons tests.

Figure 6

Effect of TAT-Ngn2 or ngn2 overexpression on cell viability and lactate dehydrogenase (LDH) release after exposure to OGD injury (n = 6 for each group). (A) Transduction effect of TAT-Ngn2 into hippocampal neurons. Triple immunofluorescence staining was performed 24 h after TAT-Ngn2 incubation with hippocampal neurons. The neurons were detected by double-labeling with βIII-tubulin-positive as red and 6× His-positive as green. The nucleus was counterstained with DAPI (blue). Scale bars = 50 μm. (B) The cultured primary hippocampal neurons in all groups under light microscope at 48 h after exposure to OGD. Scale bars = 50 μm. (C) Cell viability was detected by an MTT assay at 48 h after exposure to OGD. The values are presented as the mean ± SEM of each group and were analyzed by one-way analysis of variance followed by Tukey’s multiple comparisons test (*P < 0.05 vs. normal group, ^#P < 0.05 vs. OGD group). (D) LDH release was detected at 48 h after exposure to OGD. The normal group was defined as 100%. The values are presented as the mean (% of normal) ± SEM of each group and were analyzed by one-way analysis of variance followed by Tukey’s multiple comparisons tests (*P < 0.05 vs. normal group, ^#P < 0.05 vs. OGD group).

Figure 7

Effect of TAT-Ngn2 or ngn2 gene overexpression on neuronal apoptosis in neurons exposed to OGD injury (n = 6 for each group). (A–E) Representative tunel staining of hippocampal neurons 48 h after exposure to OGD injury. The TUNEL-positive neurons are represented in green. Scale bar = 100 μm. (F) Statistical analysis of the number of TUNEL-positive cells in each group (*P < 0.05 vs. normal group, ^#P < 0.05 vs. OGD group, ^$P < 0.05 vs. OGD+Ngn2 virus group). The data were analyzed by one-way analysis of variance followed by Tukey’s multiple comparisons test.

Figure 8

Effect of TAT-Ngn2 on the expression of BDNF, the level of phosphorylation of TrkB and mitochondrial CytC leakage in vitro (n = 6 for each group). (A) Western blotting showing BDNF, p-TrkB expression in cultured primary hippocampal neurons 48 h after reperfusion. (B,C) Statistical analysis of BDNF, p-TrkB expression in every group. (D) Western blotting showing the level of C CytC and M CytC in the cultured primary hippocampal neurons at 48 h after reperfusion. (E,F) Statistical analysis of C CytC and M CytC levels in every group. Band densities were measured using the ImageJ program and normalized to COX IV or to β-actin (*P < 0.05 vs. normal group, ^#P < 0.05 vs. OGD group, ^$P < 0.05 vs. OGD+Ngn2 virus group). (G) Representative western blotting of active caspase-3, Bax and Bcl-2 in cultured primary hippocampal neurons 48 h after exposure to OGD injury. (H–J) Quantification of relative changes in active caspase-3, Bax and Bcl-2 expression (*P < 0.05 vs. normal group, ^#P < 0.05 vs. OGD group, ^$P < 0.05 vs. OGD+Ngn2 virus group). The data were analyzed using one-way ANOVA with Dunnett’s test.