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Autophagosome maturation mediated by Rab7 contributes to neuroprotection of hypoxic preconditioning against global cerebral ischemia in rats.

ABSTRACT: 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.

SUBMITTER: Zhan L

PROVIDER: S-EPMC5550874 | biostudies-other | 2017 Jul

REPOSITORIES: biostudies-other

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Autophagosome maturation mediated by Rab7 contributes to neuroprotection of hypoxic preconditioning against global cerebral ischemia in rats.

Zhan Lixuan L Chen Siyuan S Li Kongping K Liang Donghai D Zhu Xinyong X Liu Liu L Lu Zhiwei Z Sun Weiwen W Xu En E

Cell death & disease 20170720 7

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) ...[more]

PMID: 28726776

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Project description:BackgroundOur previous study indicated that hypoxic preconditioning reduced receptor interacting protein (RIP) 3-mediated necroptotic neuronal death in hippocampal CA1 of adult rats after transient global cerebral ischemia (tGCI). Although mixed lineage kinase domain-like (MLKL) has emerged as a crucial molecule for necroptosis induction downstream of RIP3, how MLKL executes necroptosis is not yet well understood. In this study, we aim to elucidate the molecular mechanism underlying hypoxic preconditioning that inactivates MLKL-dependent neuronal necroptosis after tGCI.MethodsTransient global cerebral ischemia was induced by the four-vessel occlusion method. Twenty-four hours before ischemia, rats were exposed to systemic hypoxia with 8% O2 for 30 min. Western blotting was used to detect the expression of MLKL and interleukin-1 type 1 receptor (IL-1R1) in CA1. Immunoprecipitation was used to assess the interactions among IL-1R1, RIP3, and phosphorylated MLKL (p-MLKL). The concentration of intracellular free calcium ion (Ca2+) was measured using Fluo-4 AM. Silencing and overexpression studies were used to study the role of p-MLKL in tGCI-induced neuronal death.ResultsHypoxic preconditioning decreased the phosphorylation of MLKL both in neurons and microglia of CA1 after tGCI. The knockdown of MLKL with siRNA decreased the expression of p-MLKL and exerted neuroprotective effects after tGCI, whereas treatment with lentiviral delivery of MLKL showed opposite results. Mechanistically, hypoxic preconditioning or MLKL siRNA attenuated the RIP3-p-MLKL interaction, reduced the plasma membrane translocation of p-MLKL, and blocked Ca2+ influx after tGCI. Furthermore, hypoxic preconditioning downregulated the expression of IL-1R1 in CA1 after tGCI. Additionally, neutralizing IL-1R1 with its antagonist disrupted the interaction between IL-1R1 and the necrosome, attenuated the expression and the plasma membrane translocation of p-MLKL, thus alleviating neuronal death after tGCI.ConclusionsThese data support that the inhibition of MLKL-dependent neuronal necroptosis through downregulating IL-1R1 contributes to neuroprotection of hypoxic preconditioning against tGCI.

Project description: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.

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Autophagosome maturation mediated by Rab7 contributes to neuroprotection of hypoxic preconditioning against global cerebral ischemia in rats.

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Autophagosome maturation mediated by Rab7 contributes to neuroprotection of hypoxic preconditioning against global cerebral ischemia in rats.

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