The regulatory role of NF-κB in autophagy-like cell death after focal cerebral ischemia in mice

Abstract

Autophagy may contribute to ischemia-induced cell death in the brain, but the regulation of autophagic cell death is largely unknown. Nuclear factor kappa B (NF-κB) is a regulator of apoptosis in cerebral ischemia. We examined the hypothesis that autophagy-like cell death could contribute to ischemia-induced brain damage and the process was regulated by NF-κB. In adult wild-type (WT) and NF-κB p50 knockout (p50(-/-)) mice, focal ischemia in the barrel cortex was induced by ligation of distal branches of the middle cerebral artery. Twelve to 24h later, autophagic activity increased as indicated by enhanced expression of Beclin-1 and LC3 in the ischemic core and/or penumbra regions. This increased autophagy contributed to cell injury, evidenced by terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) co-staining and a protective effect achieved by the autophagy inhibitor 3-methyladenine. The number of Beclin-1/TUNEL-positive cells was significantly more in p50(-/-) mice than in WT mice. Neuronal and vascular cell death, as determined by TUNEL-positive cells co-staining with NeuN or Collagen IV, was more abundant in p50(-/-) mice. Immunostaining of the endothelial cell tight junction marker occludin revealed more damage to the blood-brain barrier in p50(-/-) mice. Western blotting of the peri-infarct tissue showed a reduction of Akt-the mammalian target of rapamycin (mTOR) signaling in p50(-/-) mice after ischemia. These findings provide the first evidence that cerebral ischemia induced autophagy-like injury is regulated by the NF-κB pathway, which may suggest potential treatments for ischemic stroke.

Fig. 1

Ischemia-enhanced Beclin-1 immunoreactivity in the brain of WT and NF-κB p50 knockout mice. Immunohistochemical staining was performed at different time points after barrel cortex ischemia in cortex brain sections. (A, B) Demonstration of the ischemic and non-ischemic regions distinguishable by staining with specific antibodies at early time points of 3 and 6 h after barrel cortex ischemia in WT mice. The NeuN/Hoechst 33342 nuclear staining shows condensed nuclei in the ischemic core. A few TUNEL-positive (green) and Beclin-1-positive (red) cells can be seen in the ischemic and surrounding regions. (C–H) Beclin-1 staining (red) was examined in brain sections of sham control, 12 and 24 h post-ischemia from WT and p50^−/− mice. Hoechst 33342 (blue) staining revealed nuclei of all cells. There was very weak Beclin-1 immunoreactivity in sham-operated mice (C, D). Increased beclin-1-poistive cells were seen 6 (E, F) and 12 h (G, H) after ischemia. (I) The bar graph shows quantitative assessment of Beclin-1-positive cells in the penumbra. Cell counts show increased numbers of Beclin-1-positive cells in WT and p50^−/− mice from 6 h after ischemia. Even higher Beclin-1 staining was seen in p50^−/− mice from 6 h to 3 days after ischemia. N = 5 animals in each group. Data are expressed as mean ± SD. *P < 0.05 compared with sham controls, ^#P < 0.05 compared with WT group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Increased expression of autophagic factors in the ischemic brain of WTand p50^−/− mice. (A, B) Western blot analysis of Beclin-1 and LC3-II/LC3-I was performed in the ischemic/penumbra tissues from WT and p50^−/− mice at 3–72 h following cerebral ischemia. (C and D) The immunoblotting diagrams show quantified results of protein expression of Beclin-1 and LC3-II/LC3-I, respectively. In densitometry analysis for each factor, gray intensity was normalized against β-actin and quantified using ImageJ software. From 12 to 24 h after ischemia, Beclin-1 and LC3 levels were significantly higher in p50^−/− mice than that in WT mice. Beclin-1 expression, however was higher in WT mice 72 h after ischemia. (E, F) Effect of 3-MA on Beclin-1 protein expression in the ischemic brain measured by Western blot assay. Quantified data in the bar graph show significant reduction in Beclin-1 expression by 3-MA (1.2 mg/kg, i.c.v.) 24 h after ischemic and 3-MA treatment. CTL: ischemic control, 3MA: ischemia plus 3-MA treatment. N = 5 animals in each group, mean ± SD. *P < 0.05 compared with the WT group.

Fig. 3

Ischemia-induced autophagy-like cell death in WT and p50^−/− mice. (A–D) Immunostaining of Beclin-1 alone and co-stained with TUNEL in the penumbra region of the ischemic brain from WT and p50^−/− mice. The images show the immunoreactivity of TUNEL-positive (green; A and B) and TUNEL/Beclin-1 (green/red; C and D)-positive cells. Hoechst 33342 (blue) was used to show the nuclei of all cells. (E, F) The bar graphs show cell counts of TUNEL-positive cells and Beclin-1/TUNEL double-positive cells at different times after ischemia. TUNEL-positive cells started to appear from 12 h after ischemia in both analyses and reached a peak around 1–3 days after ischemia. By 7 days after ischemia, general cell death was still relatively high while autophagic death (Beclin-1/TUNEL-positive cells) had already subsided. NF-κB p50 deletion increased cell death in both analyses. N = 5, mean ± SD. *P < 0.05 compared with sham controls, ^#P < 0.05 compared with WT group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Autophagy-like neuronal cell death after ischemia and worsening effect of p50 deletion. (A–F) Immunohistochemical staining of NeuN (green) as well as Beclin-1 (red) staining was performed in the brain sections from WT and p50^−/− mice at different time points before and after ischemia. Blue color was Hoechst 33342 staining to reveal the nuclei of all cells. (G–J) Images show triple staining of NeuN/Beclin 1/TUNEL (arrows), indicative of autophagy-like neuronal death. (K) The bar graph shows cell counts of NeuN/Beclin-1 double-positive cells at different times and the comparison between WT and p50^−/− mice. At each time point from 6 h to 3 days after ischemia, significantly more double-positive cells were detected in p50^−/− mice. The autophagic neuronal death returned to control levels 7 days after ischemia. N = 5 animals in each group, mean ± SD. *P < 0.05 compared with sham controls, ^#P < 0.05 compared with WT group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Autophagic microvessel injury after ischemia and worsening effect of p50 deletion. (A–F) Immunostaining of Beclin-1 (red) and Collagen IV (green) was performed in brain sections of sham-operated and post-ischemia from WT and p50^−/− mice. Nuclei were stained with Hoechst 33342. (G–I) Enlarged images show co-localization of Beclin1 and Collagen IV (arrows). (J, K) Cell counts in bar graphs show gradual reduction of Collagen IV-positive cells after cerebral ischemia. More reduction of Collagen IV staining was seen in p50^−/− mice 24 and 72 h after ischemia. In contrast, Beclin-1/Collagen IV double-positive cells were increased after ischemia and reached to a peak level at 12 h after ischemia in both WT and p50^−/− mice. More Beclin-1/Collagen IV double-positive cells were detected in p50^−/− mice. At 72 h after ischemia, the double-positive cells were already back to control level while significantly higher numbers of these cells were detected in p50^−/− mice. N = 5 animals in each group, mean ± SD. *P < 0.05 compared with sham controls, ^#P < 0.05 compared with the WT group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Ischemia-induced autophagic microvessel deterioration. (A–D) Immunohistochemical triple staining of collagen IV, Beclin-1 and TUNEL in the penumbra region 12 h after ischemia in a brain section from a p50^−/− mouse. Collagen IV (blue) staining show consistent morphology of microvessels. Some of collagen IV-positive cells were co-labeled with TUNEL (green) and Beclin-1 (red) (arrows). The triple-positive staining indicated autophagic vasculature damage. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Autophagic injury to the blood-brain barrier (BBB) and worsening effect of deleting p50. (A–D) BBB integrity was evaluated using immunostaining of occludin (red) in WT and p50^−/− mice 24 h after ischemia. Hoechst 33342 staining (blue) showed nuclei of all cells. Occludin staining exhibited strong occludin expression in the cerebral vasculature of control animals, while the focal ischemia noticeably reduced occluding expression in the penumbra region of WT and p50^−/− mice. Even less occluding immunoreactivity was seen in the p50^−/− brain (B, D). (E–H) In the confocal image panels of triple staining of occluding (green), Becline-1 (red) and Collagen IV (blue), the overlapping of the three markers illustrated autophagic damage to BBB. Representative of five animals in each group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Up-regulation of Akt signaling after ischemia and impaired regulation in p50^−/− mice Western blot analysis of the Akt-mTOR signaling pathway at different time points after cerebral ischemia. (A, B) Representative electropherograms show the expression levels of phospho-Akt (p-Akt) and total Akt in the penumbra region of the WTand p50^−/− brain. Densitometric analysis provided a quantified comparison of the p-Akt/Akt ratio in the two groups of animals at different time points. Gray intensity was normalized against β-actin. The Akt phosphorylation, representative of activation of the pathway, was almost doubled at 12 h after ischemia in WT mice followed by less prominent bust significant enhancement of p-Akt compared to p50^−/− mice up to 72 h after ischemia. The increased Akt activation was completely absent in p50^−/− mice. N = 5 for each group. *P < 0.05 vs. sham; ^#P < 0.05 vs. WT.

Fig. 9

Changes in autophagic signaling in WT and p50^−/− mice after focal ischemia. Western blot analysis of several autophagic signals in the post-ischemic brain of WT and p50^−/− mice. (A, B) Electropherograms show the expression levels of total mTOR substrate S6K1 and phosphorylated S6K1. (C, D) Quantified comparisons are shown in the bar graph of the total S6K1 level (C) and the p-S6K1/S6K1 ratio (D) in the two groups of animals at different time points. Consistent with enhanced autophagic cell death in the area, the total S6K1 level and phosphorylated S6K1 (p-S6K1) were diminishing in the ischemic brain. Even more reduction in p-S6K1 was observed in p50^−/− mice 12–72 h after ischemia. (E, F) mTOR substrate S6R in the post-ischemic WT and p50^−/− brain. Electropherograms show the expression levels of phospho-S6R (p-S6R) and total S6R in the penumbra region of the ischemic brain. G and H. Quantified data of E and F. The total level of S6R gradually declined after ischemia, significant reduction was seen from 12 to 72 h after ischemia in both WTand p50^−/− mice (G). The ratio of p-S6R/S6R in WT animals was about the same until it increased at 72 h after ischemia (H). This ratio was much lower in the p50^−/− brain from 6 to 72 h after ischemia compared to WT controls. Gray intensity was normalized against the loading control of β-actin. N = 5 animals in each group. mean ± SD. *P < 0.05 vs. sham control, ^#P < 0.05 vs. WT group.