Delayed administration of a PTEN inhibitor BPV improves functional recovery after experimental stroke

Abstract

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibitors administered prior to or immediately after experimental stroke confer acute neuroprotection. However, it remains unclear if delayed treatment with a PTEN inhibitor improves long-term functional recovery after stroke. We addressed the issue in this study. Adult male mice were subjected to 1h of middle cerebral artery occlusion (MCAO) followed by treatment with a well-established PTEN inhibitor BPV or saline daily for 14 days, starting at 24h after MCAO. Functional recovery was assessed with behavioral tests and acute infarct volumes were analyzed histologically. Delayed BPV treatment did not reduce infarction during the acute phase, but significantly improved long-term functional recovery after MCAO. Since PTEN is a critical intrinsic inhibitory factor in axonal regeneration, we further examined BPV effects on axonal densities following MCAO using bielschowsky silver staining and immunohistochemistry with antibodies against myelin basic protein. Delayed BPV treatment significantly increased axon densities in the ischemic brain at 14 days after MCAO. Moreover, PTEN expression persistently remained high in the ischemic brain over 14 days after MCAO, and BPV treatment increased post-ischemic activation of Akt and mTOR in the ischemic brain. Akt and mTOR activation are the well-established cascades downstream to PTEN inhibition and have been shown to contribute to post-injury axonal regrowth in response to PTEN inhibition. Consistently, in an in vitro neuronal ischemia model, BPV enhanced axonal outgrowth of primary cortical neurons after oxygen-glucose deprivation and the enhancing effects were abolished by Akt/mTOR inhibition. In conclusion, delayed BPV treatment improved functional recovery from experimental stroke possibly via enhancing axonal growth and Akt/mTOR activation contributed to BPV-enhanced post-stroke axon growth.

Figure 1

Delayed bpv treatment improved functional recovery from MCAO in mice. A: Modified neurological severity scores (mNSS) were significantly lower in bpv-treated vs. saline-treated mice from 11 days following MCAO (n = 12 per group). B: bpv-treated mice performed better in the forelimb placement test from 11 days after MCAO (n = 12 per group). C: Compared to saline-treated mice (n = 11), bpv-treated mice (n = 13) exhibited improved asymmetrical motor deficits at 13 days after MCAO in the elevated body swing test. * p < 0.05, and ** p < 0.01 compared to the saline-treated group.

Figure 2

Delayed bpv treatment did not reduce acute infarction at 4 days after MCAO but increased axonal densities at 14 day after MCAO. Acute infarct damage was analyzed in bpv- and saline-treated mice using TTC histology. Bielschowsky silver and MBP expression were assessed in sham-, saline-treated and bpv-treated MCAO mice. A: Representative images of TTC histology and the bar graph. B: Bielschowsky silver staining (scale bar: 50 μm); B, MBP immunostaining (scale bar: 50 μm); C quantitative data of bielschowsky silver staining; D: quantitative data of MBP immunostaining * p < 0.05 compared to saline-treated mice (n = 8 per group).

Figure 3

PTEN expression persistently remained high levels in the ischemic boundary zones (IBZs) over 14 days after MCAO. A: representative images of western blot analysis of total PTEN and phosphorylated (inactive) PTEN in the IBZs of the cortex and striatum. B: Quantitative data of ratios of phosphorylated/total PTEN in the ischemic cortex (n = 4 per group, left panel) and striatum (n = 4 per group, right panel). Ipsil: ipsilateral sides, Contra: contralateral sides.

Figure 4

The PTEN inhibitor bpv inhibited cerebral PTEN and activated cerebral Akt and mTOR following MCAO at 4 days after MCAO. Compared to saline-injected mice (ns), bpv-treated mice (bpv) displayed elevated activation of AKT (phosphorylation, panel A) and elevated activation of and mTOR (S6 phosphorylation, panel B) in the IBZs of the ipsilateral (Ipsil) striatum. β-actin was used as a loading control. C: quantitative data of ratios of phosphorylated/total Akt (left panel) and phosphorylated/total S6 (right panel). D: Compared to saline, bpv inhibited PTEN expression in the IBZs of the ipsilateral (Ipsil) striatum. **p < 0.01 compared to other groups, n = 3 per group. Ipsil: ipsilateral sides, Contra: contralateral sides.

Figure 5

The PTEN inhibitor bpv enhanced neurite outgrowth of primary cortical neurons following OGD, which was abolished by Akt or mTOR inhibition. A: TUJ1 immunostaining in post-OGD neurons treated with vehicle, bpv, bpv + ly (bpv plus the Akt inhibitor LY294002) and bpv + Rap (bpv plus the mTOR inhibitor rapamycin). Scale bar: 20 μm. B: Quantitative data of neurite outgrowth. ** p < 0.01 compared with the bpv-treated neurons (n = 6 per group).