Phosphorylation of STAT3 at Tyr705 contributes to TFEB-mediated autophagy-lysosomal pathway dysfunction and leads to ischemic injury in rats.

We previously reported that permanent ischemia induces marked dysfunction of the autophagy-lysosomal pathway (ALP) in rats, which is possibly mediated by the transcription factor EB (TFEB). However, it is still unclear whether signal transducer and activator of transcription 3 (STAT3) is responsible for the TFEB-mediated dysfunction of ALP in ischemic stroke. In the present study, we used AAV-mediated genetic knockdown and pharmacological blockade of p-STAT3 to investigate the role of p-STAT3 in regulating TFEB-mediated ALP dysfunction in rats subjected to permanent middle cerebral occlusion (pMCAO). The results showed that the level of p-STAT3 (Tyr705) in the rat cortex increased at 24 h after pMCAO and subsequently led to lysosomal membrane permeabilization (LMP) and ALP dysfunction. These effects can be alleviated by inhibitors of p-STAT3 (Tyr705) or by STAT3 knockdown. Additionally, STAT3 knockdown significantly increased the nuclear translocation of TFEB and the transcription of TFEB-targeted genes. Notably, TFEB knockdown markedly reversed STAT3 knockdown-mediated improvement in ALP function after pMCAO. This is the first study to show that the contribution of p-STAT3 (Tyr705) to ALP dysfunction may be partly associated with its inhibitory effect on TFEB transcriptional activity, which further leads to ischemic injury in rats.

Neuroprotective effect of pseudoginsenoside-F11 on permanent cerebral ischemia in rats by regulating calpain activity and NR2A submit-mediated AKT-CREB signaling pathways.

BACKGROUND: N-methyl-d-aspartate receptors (NMDARs) have been demonstrated to play central roles in stroke pathology and recovery, including dual roles in promoting either neuronal survival or death with their different subtypes and locations. PURPOSE: We have previously demonstrated that pseudoginsenoside-F11 (PF11) can provide long-term neuroprotective effects on transient and permanent ischemic stroke-induced neuronal damage. However, it is still needed to clarify whether NMDAR-2A (NR2A)-mediated pro-survival signaling pathway is involved in the beneficial effect of PF11 on permanent ischemic stroke. MATERIAL AND METHODS: PF11 was administrated in permanent middle cerebral artery occlusion (pMCAO)-operated rats. The effect of PF11 on oxygen-glucose deprivation (OGD)-exposed primary cultured neurons were further evaluated. The regulatory effect of PF11 on NR2A expression and the activation of its downstream AKT-CREB pathway were detected by Western blotting and immunofluorescence in the presence or absence of a specific NR2A antagonist NVP-AAM077 (NVP) both in vivo and in vitro. RESULTS: PF11 dose- and time-dependently decreased calpain1 (CAPN1) activity and its specific breakdown product α-Fodrin expression, while the expression of Ca2+/calmodulin-dependent protein kinase II alpha (CaMKII-α) was significantly upregulated in the cortex and striatum of rats at 24 h after the onset of pMCAO operation. Moreover, PF11 prevented the downregulation of NR2A, p-AKT/AKT, and p-CREB/CREB in both in vivo and in vitro stroke models. Finally, the results indicated treatment with NVP can abolish the effects of PF11 on alleviating the ischemic injury and activating NR2A-mediated AKT-CREB signaling pathway. CONCLUSIONS: Our results demonstrate that PF11 can exert neuroprotective effects on ischemic stroke by inhibiting the activation of CAPN1 and subsequently enhancing the NR2A-medicated activation of AKT-CREB pathway, which provides a mechanistic link between the neuroprotective effect of PF11 against cerebral ischemia and NR2A-associated pro-survival signaling pathway.