Pannexins in ischemia-induced neurodegeneration

Abstract

Pannexin 1 (Px1, Panx1) and pannexin 2 (Px2, Panx2) form large-pore nonselective channels in the plasma membrane of cells and were suggested to play a role in the pathophysiology of cerebral ischemia. To directly test a potential contribution of pannexins in ischemia-related mechanisms, we performed experiments in Px1(-/-), Px2(-/-), and Px1(-/-)Px2(-/-) knockout mice. IL-1β release, channel function in astrocytes, and cortical spreading depolarization were not altered in Px1(-/-)Px2(-/-) mice, indicating that, in contrast to previous concepts, these processes occur normally in the absence of pannexin channels. However, ischemia-induced dye release from cortical neurons was lower, indicating that channel function in Px1(-/-)Px2(-/-) neurons was impaired. Furthermore, Px1(-/-)Px2(-/-) mice had a better functional outcome and smaller infarcts than wild-type mice when subjected to ischemic stroke. In conclusion, our data demonstrate that Px1 and Px2 underlie channel function in neurons and contribute to ischemic brain damage.

Fig. 1.

IL-1β production does not depend on Px1 and Px2. (A) LPS- and ATP-induced release of IL-1β from macrophages did not depend on Px1 and Px2. Peritoneal macrophages from Px1^+/+Px2^+/+ or Px1^−/−Px2^−/− mice were stimulated with LPS (100 μg/mL) for 4 h and then with ATP (5 mM) for 30 min. IL-1β was measured in the medium. Values are mean ± SEM (n = 5–9). (B) After middle cerebral artery occlusion, total IL-1β was higher in the ischemic (I) than in the nonischemic (N) hemispheres but did not differ significantly between wild-type Px1^+/+Px2^+/+, double-knockout Px1^−/−Px2^−/−, and double-heterozygous Px1^+/−Px2^+/− mice. Total IL-1β, including pro–IL-1β and mature IL-1β, was determined in brain extracts by ELISA. Values are mean ± SEM (n = 5). (C) In the same samples pro–IL-1β (31 kDa) was determined by Western blot analysis. Quantification revealed no significant difference in pro–IL-1β normalized to actin between hemispheres and genotypes (two-way ANOVA). Mean values ± SEM out of two to three experiments are shown above a representative blot.

Fig. 2.

Channel activity in astrocytes does not depend on Px1 and Px2. (A) Expression of Px1 and Px2 mRNA in primary cortical neurons (PCN), macrophages (macroph), and astrocytes. mRNA was detected by RT-PCR. (B) BzATP-induced ATP release in primary astrocytes was blocked by carbenoxolone (Cbx, 100 μM) but was not affected by the deficiency of Px1 and Px2. Bz-ATP, 300 μM. Values are mean ± SEM. Two-way ANOVA revealed a significant effect of BzATP treatment, F(2/169) = 44.37, P < 0.0001. ***P < 0.001 (Bonferroni post hoc test). NS, not significantly different compared with untreated control cells. (C) BzATP-induced calcein green dye release from astrocytes was independent of Px1 and Px2. Calcein green fluorescence intensity was measured 750 s after stimulation by BzATP and expressed as percentage of untreated controls (Ctrl). Values are mean ± SEM. Two-way ANOVA revealed a significant effect of BzATP treatment, F(1/108) = 142.6, P < 0.0001. ***P < 0.001 (Bonferroni post hoc test). NS, not significantly different compared with Px1^+/+Px2^+/+ group. (D) Outward current induced by a voltage ramp (−60 to +100 mV) did not differ between astrocytes of Px1^+/+Px2^+/+ and Px1^−/−Px2^−/− mice. (Left) Example recordings from single astrocytes. (Right) Quantification showing median values ± IQR of current amplitude at increasing voltages.

Fig. 3.

In cortical neurons Px1 and Px2 are required for channel activity induced by metabolic inhibition. Calcein green-loaded primary cortical neurons were subjected to NaCN (2 mM) and fluorescence was imaged for 750 s. Decrease of fluorescence intensity indicated dye release through pannexons. (A) Representative image of Px1^+/+Px2^+/+ and Px1^−/−Px2^−/− neurons before and 750 s after NaCN. Intensity is color coded. (Scale bar, 100 μm.) (B) Carbenoxolone (Cbx, 100 μM) inhibited dye release induced by NaCN from wild-type neurons. The time course of fluorescence intensity expressed as a percentage of the starting value (F₀) in a single experiment is shown. (C) Dye release in response to NaCN was inhibited in Px1^−/−Px2^−/− neurons. Values of a single experiment with 91 Px1^+/+Px2^+/+ and 52 Px1^−/−Px2^−/− cells are shown. (D) Mean values of the fluorescence change (F₇₅₀–F₀) × 100/F₀ in five to seven independent experiments 750 s after applying NaCN. The total number of cells evaluated per condition is given in the bars. One-way ANOVA, F(5/1,690) = 12.66, P < 0.0001; *P < 0.05, ***P < 0.001 in comparison with Px1^+/+Px2^+/+ cells (Bonferroni post hoc test).

Fig. 4.

Double-knockout Px1^−/−Px2^−/− mice were protected in a stroke model. (A, B, D, and E) Compared with Px1^+/+Px2^+/+ littermates, neurological deficits did not differ in Px1^−/− and Px2^−/− mice after MCAO as evaluated by the corner test (A and B). When measuring latency to move, Px1^−/− mice showed a trend (D) and Px2^−/− a significantly shorter latency than wild-type littermates (E), F(1/58) = 9.7, P < 0.01. **P < 0.01 (repeated-measures ANOVA). The dashed line indicates the expected behavior without a side preference in 12 trials of the corner test. Mice were investigated before and 24 h after MCAO. Values are mean ± SEM (n = 6–14). (G and H) Px1^−/− and Px2^−/− mice showed no statistically significant differences in infarct volume compared with Px1^+/+Px2^+/+ littermates. Values are mean ± SEM (n = 15–18). (C and F) Double-knockout Px1^−/−Px2^−/− mice had a smaller neurological deficit 24 h after MCAO than Px1^+/+Px2^+/+. In the corner test (C), the pathological preference of Px1^+/+Px2^+/+ littermates to turn to the right side was significantly reduced in Px1^−/−Px2^−/− mice 24 h after MCAO. F(1/12) = 6.7, P < 0.05. *P < 0.05 (repeated-measures ANOVA, n = 5–9). In the latency to move (F), Px1^−/−Px2^−/− mice needed significantly less time to move compared with their Px1^+/+Px2^+/+ littermates. F(1/12) = 5.9, P < 0.01. **P < 0.01 (repeated-measures ANOVA, n = 5–9). (I) Double deletion of both Px1 and Px2 (Px1^−/−Px2^−/−) significantly reduced the infarct volume compared with the Px1^+/+Px2^+/+ littermates. **P < 0.01 (t test, n = 6–10).