Pseudoginsenoside F11 ameliorates the dysfunction of the autophagy-lysosomal pathway by activating calcineurin-mediated TFEB nuclear translocation in neuron during permanent cerebral ischemia

Abstract

We have previously found that transcription factor EB (TFEB), as a master regulator of autophagy and lysosome biogenesis, provides neuroprotective effects on cerebral ischemia-induced neuronal damage by activation of autophagy-lysosomal pathway (ALP). We have also reported that Pseudoginsenoside F11 (PF11), an ocotillol-type saponin isolated from Panax quinquefolium L., significantly attenuates the ischemic injury of rats subjected to permanent middle cerebral artery occlusion (pMCAO), possibly by alleviating the autophagic/lysosomal defects. The present study aims to investigate whether the beneficial effect of PF11 on ALP dysfunction induced by permanent ischemic stroke is based on its regulation of TFEB nuclear translocation in pMCAO rats and the oxygen-glucose-deprived (OGD) primary neurons. Meanwhile, the role of calcineurin, a serine/threonine protein phosphatase, during this process in which PF11 regulated TFEB transcriptional activity was also explored. The data showed that PF11 exerted significant protective effects on pMCAO-induced injury and decreased OGD-induced neuronal death. The nuclear localization of TFEB was decreased at 24 h after pMCAO. Notably, PF11 (6, 12 mg/kg, i.v.) significantly increased TFEB nuclear expression and Tfeb mRNA level at 24 h following pMCAO. OGD treatment promoted TFEB aggregation and nuclear translocation until 6 h, and the nuclear localization of TFEB was decreased at 12 h. Similarly, PF11 (30, 100 μM) could also promote the translocation of TFEB into nuclear in primary neurons at 12 h after OGD treatment. Moreover, PF11 attenuated OGD-induced lysosomal dysfunction and abnormal accumulation of autophagosomes and substrates. These in vitro effects could be abolished by neuronal-specific knocking down of TFEB via transfecting primary neurons with lentivirus encoding shTfeb. Further studies indicated that cyclosporine (10 μM), an inhibitor of calcineurin, could significantly diminish the effects of PF11 on TFEB nuclear translocation and ALP dysfunction in OGD-treated neurons. In summary, these results demonstrate that PF11 attenuates the dysfunction of ALP in permanent cerebral ischemia by promoting the calcineurin-mediated nuclear translocation of TFEB and further identifies an autophagic mechanism of PF11 against cerebral ischemia.

Keywords: Autophagy-lysosomal pathway; Calcineurin; Permanent cerebral ischemia; Pseudoginsenoside F11; Transcription factor EB.