Autophagy disruption leads to neuronal damage in hypoxic-ischemic brain injury. Rab7, a member of the Rab GTPase superfamily, has a unique role in the regulation of autophagy. Hypoxic preconditioning (HPC) provides neuroprotection against transient global cerebral ischemia (tGCI). However, the underlying mechanisms remain poorly understood. Thus, the current study explored the potential molecular mechanism of the neuroprotective effect of HPC by investigating how Rab7 mediates autophagosome (AP) maturation after tGCI in adult rats. We found that HPC attenuated AP accumulation in the hippocampal CA1 region after tGCI via restoration of autophagic flux. We also confirmed that this HPC-induced neuroprotection was not caused by the increase in lysosomes or the improvement of lysosomal function after tGCI. Electron microscopic analysis then revealed an increase in autolysosomes in CA1 neurons of HPC rats. Moreover, the inhibition of autophagosome-lysosome fusion by chloroquine significantly aggravated neuronal death in CA1, indicating that AP maturation contributes to HPC-induced neuroprotection against neuronal injury after tGCI. Furthermore, the activation of Rab7 was found to be involved in the neuroprotective effect of AP maturation after HPC. At last, the knockdown of ultraviolet radiation resistance-associated gene (UVRAG) in vivo disrupted the interaction between Vps16 and Rab7, attenuated the activation of Rab7, interrupted autophagic flux, and ultimately abrogated the HPC-induced neuroprotection against tGCI. Our results indicated that AP maturation was enhanced by the activation of Rab7 via UVRAG-Vps16 interaction, which further demonstrated the potential neuroprotective role of Rab7 in HPC against tGCI-induced neuronal injury in adult rats.

We previously reported that hypoxic postconditioning (HPC) ameliorated hippocampal neuronal death induced by transient global cerebral ischemia (tGCI) in adult rats. However, the mechanism of HPC-induced neuroprotection is still elusive. Notably, heat shock protein 27 (Hsp27) has recently emerged as a potent neuroprotectant in cerebral ischemia. Although its robust protective effect on stroke has been recognized, the mechanism of Hsp27-mediated neuroprotection is largely unknown. Here, we investigated the potential molecular mechanism by which HPC modulates the posttranslational regulations of Hsp27 after tGCI. We found that HPC increased expression of Hsp27 in CA1 subregion after tGCI. Inhibition of Hsp27 expression with lentivirus-mediated short hairpin RNA (shRNA) abolished the neuroprotection induced by HPC in vivo. Furthermore, pretreatment with cycloheximide, a protein synthesis inhibitor, resulted in a significant decrease in the degradation rate of Hsp27 protein in postconditioned rats, suggesting that the increase in the expression of Hsp27 after HPC might result from its decreased degradation. Next, pretreatment with leupeptin, a lysosomal inhibitor, resulted in an accumulation of Hsp27 after tGCI, indicating that autophagic pathway may be responsible for the degradation of Hsp27. We further showed that the formation of LC3-II and autophagosomes increased after tGCI. Meanwhile, the degradation of Hsp27 was suppressed and neuronal damage was reduced when blocking autophagy with 3-Methyladenine, whereas activating autophagy with rapamycin showed an opposite tendency. Lastly, we confirmed that HPC increased the expression of phosphorylated MAPKAP kinase 2 (MK2) and Hsp27 after tGCI. Also, administration of SB203580, a p38 mitogen-activated protein kinase inhibitor, decreased the expressions of phosphorylated MK2 and Hsp27. Our results suggested that inhibition of Hsp27 degradation mediated by down-regulation of autophagy may induce ischemic tolerance after HPC. Additionally, phosphorylation of Hsp27 induced by MK2 might be associated with the neuroprotection of HPC.

Mitophagy alleviates neuronal damage after cerebral ischemia by selectively removing dysfunctional mitochondria. Phosphatase and tensin homolog (PTEN) induced putative kinase 1 (PINK1)/Parkin-mediated mitophagy is the most well-known type of mitophagy. However, little is known about the role of PINK1/Parkin-mediated mitophagy in ischemic tolerance induced by hypoxic postconditioning (HPC) with 8% O2 against transient global cerebral ischemia (tGCI). Hence, we aimed to test the hypothesis that HPC-mediated PINK1/Parkin-induced mitochondrial ubiquitination and promotes mitophagy, thus exerting neuroprotection in the hippocampal CA1 subregion against tGCI. We found that mitochondrial clearance was disturbed at the late phase of reperfusion after tGCI, which was reversed by HPC, as evidenced by the reduction of the translocase of outer mitochondrial membrane 20 homologs (TOMM20), translocase of inner mitochondrial membrane 23 (TIMM23) and heat shock protein 60 (HSP60) in CA1 after HPC. In addition, HPC further increased the ratio of LC3II/I in mitochondrial fraction and promoted the formation of mitophagosomes in CA1 neurons after tGCI. The administration of lysosome inhibitor chloroquine (CQ) intraperitoneally or mitophagy inhibitor (Mdivi-1) intracerebroventricularly abrogated HPC-induced mitochondrial turnover and neuroprotection in CA1 after tGCI. We also found that HPC activated PINK1/Parkin pathway after tGCI, as shown by the augment of mitochondrial PINK1 and Parkin and the promotion of mitochondrial ubiquitination in CA1. In addition, PINK1 or Parkin knockdown with small-interfering RNA (siRNA) suppressed the activation of PINK1/Parkin pathway and hampered mitochondrial clearance and attenuated neuroprotection induced by HPC, whereas PINK1 overexpression promoted PINK1/Parkin-mediated mitophagy and ameliorated neuronal damage in CA1 after tGCI. Taken together, the new finding in this study is that HPC-induced neuroprotection against tGCI through promoting mitophagy mediated by PINK1/Parkin-dependent pathway.

Transient global cerebral ischemia (tGCI) causes excessive release of glutamate from neurons. Astrocytic glutamate transporter-1 (GLT-1) and glutamine synthetase (GS) together play a predominant role in maintaining glutamate at normal extracellular concentrations. Though our previous studies reported the alleviation of tGCI-induced neuronal death by hypoxic preconditioning (HPC) in hippocampal Cornu Ammonis 1 (CA1) of adult rats, the underlying mechanism has not yet been fully elaborated. In this study, we aimed to investigate the roles of GLT-1 and GS in the neuroprotection mediated by HPC against tGCI and to ascertain whether these roles can be regulated by connexin 43 (Cx43) and cellular-Src (c-Src) activity. We found that HPC decreased the level of extracellular glutamate in CA1 after tGCI via maintenance of GLT-1 expression and GS activity. Inhibition of GLT-1 expression with dihydrokainate (DHK) or inhibition of GS activity with methionine sulfoximine (MSO) abolished the neuroprotection induced by HPC. Also, HPC markedly upregulated Cx43 and inhibited p-c-Src expression in CA1 after tGCI, whereas inhibition of Cx43 with Gap26 dramatically reversed this effect. Furthermore, inhibition of p-c-Src with 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo (3, 4-d) pyrimidine (PP2) decreased c-Src activity, increased protein levels of GLT-1 and Cx43, enhanced GS activity, and thus reduced extracellular glutamate level in CA1 after tGCI. Collectively, our data demonstrated that reduced extracellular glutamate induced by HPC against tGCI through preventing the reduction of GLT-1 expression and maintaining GS activity in hippocampal CA1, which was mediated by upregulating Cx43 expression and inhibiting c-Src activity.

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Human embryonic stem cells-derived neural progenitor cells (hESCs-NPCs) transplantation holds great potential to treat stroke. We previously reported that delayed secondary degeneration occurs in the ventroposterior nucleus (VPN) of ipsilateral thalamus after distal branch of middle cerebral artery occlusion (dMCAO) in adult male Sprague-Dawley (SD) rats. In this study, we investigate whether hESCs-NPCs would benefit the neural recovery of the secondary damage in the VPN after focal cerebral infarction. Permanent dMCAO was performed with electrocoagulation. Rats were randomized into Sham, dMCAO groups with or without hESCs-NPCs treatment. HESCs-NPCs were engrafted into the peri-infarct regions of rats at 48 h after dMCAO. The transplanted hESCs-NPCs survive and partially differentiate into mature neurons after dMCAO. Notably, hESCs-NPCs transplantation attenuated secondary damage of ipsilateral VPN and improved neurological functions of rats after dMCAO. Moreover, hESCs-NPCs transplantation significantly enhanced the expression of BDNF and TrkB and their interaction in ipsilateral VPN after dMCAO, which was reversed by the knockdown of TrkB. Transplantated hESCs-NPCs reconstituted thalamocortical connection and promoted the formation of synapses in ipsilateral VPN post-dMCAO. These results suggest that hESCs-NPCs transplantation attenuates secondary damage of ipsilateral thalamus after cortical infarction, possibly through activating BDNF/TrkB pathway, enhancing thalamocortical projection, and promoting synaptic formation. It provides a promising therapeutic strategy for secondary degeneration in the ipsilateral thalamus post-dMCAO.

Transplantation of bone marrow stromal cells (BMSCs) is a promising therapy for ischemic stroke. Previously, we had reported that the secondary degeneration occurred in the ipsilateral substantia nigra (SN) after permanent distal branch of middle cerebral artery occlusion (dMCAO) in Sprague-Dawley rats. However, whether BMSCs have neurorestorative effects on the secondary damage in the SN after focal cerebral infarction has not known. In this study, rats were subjected to dMCAO followed by intravenous administration of BMSCs 1 day later. We found that transplanted BMSCs survived and migrated to cortical infarct areas and ipsilateral SN. Furthermore, BMSCs promoted neurogenesis through proliferation and differentiation in the SN after dMCAO. Rats implanted with BMSCs showed significant improvement in their performance of modified neurological severity scores and adhesive-removal test. Engrafted BMSCs enhanced survival of dopaminergic neuron, reduced gliosis in the ipsilateral SN, and increased contents of dopamine (DA) and its metabolites in the ipsilateral striatum after dMCAO. With pseudorabies virus-152 as a retrograde tracer, we also demonstrated that BMSCs could effectively enhance the cortico-striatum-nigral connections. These results suggest that BMSCs transplantation exerts neurorestorative effects after cortical infarction through promoting endogenous neurogenesis, increasing contents of DA and its metabolites, alleviating the secondary neuronal damage in the SN, enhancing the cortico-striatum-nigral projections pathway, and finally improving the neurological functional outcome.