TIGAR Protects Against Ischemic Brain Injury By Correcting Redox Imbalance Via A Pentose Phosphate Pathwayindependent Manner

Ischemic stroke,also termed cerebral ischemia,is an acute cerebrovascular disease.Ischemic stroke is caused by insufficient cerebral blood flow(CBF),which leads to irreversible neuronal death and neurological dysfuction.Globally,stroke has became the second leading cause of death.The pathological mechanisms underlying cerebral ischemia are far from being fully elucidated,and there is paucity of drugs for stroke therapy.Therefore,it is urgent to further clarify the mechanisms underlying cerebral ischemia and discover new strategies for stroke treatment.Neurons are vulnerable to oxidative damage.Massive reactive oxygen species(ROS)are generated in ischemic brains,which lead to oxidative stress and further accelerate brain injury.Therefore,neutralizing oxidative stress has been accepted as a promising strategy to alleviate ischemic injury.However,a variety of mechanisms are involved in the ROS generation in ischemic brains.It remains not fully elucidated how to restore the redox balance in ischemic brains with distinct duration of ischemia.Nicotinamide adenine dinucleotide phosphate(NADPH)serves as the main antioxidant in ischemic brain,which is metabolized into reduced glutathione(GSH)by glutathione reductase,glutathione peroxidase and thioredoxin system.GSH neutralizes oxidative stress in neurons.NADPH is mainly generated by glucose metabolism though pentose phosphate pathway in neurons.TP53-induced glycolysis and apoptosis regulator(TIGAR)catalyzes Fructose 2,6-diphosphate into Fructose 6-phosphate,which channels glucose metabolism from glycolysis into pentose phosphate pathway.It is reported that overexpression of TIGAR alleviates short-term ischemia-caused oxidative stress in neurons and protects against ischemic injury,suggeting that activation of TIGAR-mediated pentose phosphate pathway can be a potential strategy for stroke therapy.However,most stroke patients suffer from prolonged ischemia.It has been documented that glucose is exhausted and 6-phosphofructo-2-kinase/Fructose-2,6-biphosphatate-3(Pfkfb3),a key enzyme in glycolysis,is accumulated with extended ischemia,which shuts off pentose phosphate pathway.Therefore,NADPH level dramatically decreases along with prlonged ischemia.Paradoxically,TIGAR is upregulated even in brains experienced with prolonged ischemia.However,it is still unknown whether and how TIGAR exerts the antioxidant activity and neuroprotection in prolonged ischemic brains.In this study,middle cerebral artery occlussion-reperfusion(MCAO-Rep)in mice and oxygen glucose deprivationreperfusion(OGD-Rep)in neuronal cells were carried out to mimic cerebral ischemia.Furthermore,we employed gene overexpression,gene knockdown,pharmacological intervention and Cre-Lox P specific gene knockout approaches,which were comibined with immunoblotting,immunohistochemistry,live cell imaging,real-time PCR(RT-PCR)analysis to explore whether and how TIGAR offers the antioxidant activity and neuroprotection in the context of prolonged cerebral ischemia.In the study,we documented the significant upregulation of TIGAR that was proportional to the duration of ischemia in mice brains with 0.5 h,1 h or 2 h of MCAO.However,NADPH level was continuously decreased.Furthermore,overexpression of TIGAR upregulated NADPH level in mice with 0.5 h and 1 h of MCAO,but not 2 h of MCAO.These data suggested that glucose metabolism though pentose phosphate pathway in brains was shutted down along with prolonged ischemia.Intriguingly,Overexpression of TIGAR still reduced oxidative stress,infarct volumes and neurological deficit score in mice with 2 h of MCAO.Unexpectedly,although overexpression of TIGAR could not further enhance NADPH level but alleviated oxidative stress and cell death in prolonged OGD-treated neurons and SH-SY5 Y cells.These data supported that TIGAR,via a pentose phosphate pathway independent manner,alleviated oxidative stress and neuronal injury after prolonged ischemia.Inhibition of pentose phosphate pathway by silencing glucose 6-phosphate dehydrogenase cannot cancel the antioxidantion and neuroprotection of TIGAR in SH-SY5 Y cells with prolonged OGD.Furthermore,overexpressing TIGAR mutation(H11A,E102 A,H198A;which lacks enzymatic activity)still retained the antioxidantion and neuroprotection of TIGAR in OGD-treated SH-SY5 Y cells.Together,the results indicated that TIGAR,via the pentose phosphate pathway independent mechanism,alleviated oxidative stress and neuronal injury with prolonged ischemia.TIGAR regulates autophagy in ischemic neurons,which eliminates damaged mitochondria and peroxisomes,and alleviates oxidative stress.We showed that silence of TIGAR decreased LC3B-II level and increased p62 level in prolonged OGD-treated neuronal cells,which suggested that TIGAR inhibited autophagy.On the contrary,overexpression of TIGAR or TIGAR mutation(H11A,E102 A,H198A)activated autophagy.Additionally,inhibition of autophagy in neurons,either by autophagy inhibitor(wortmannin,chlroqunine)or specific knockout of Atg7,reversed the antioxidation and neuroprotection of TIGAR,suggesting that TIGAR,via the pentose phosphate pathway independent manner,activated autophagy and neutralized oxidative stress with prolonged ischemia.However,overexpression of TIGAR did not further induce mitophagy or pexophagy,suggesting that TIGAR-activated autophagy,but not mitophagy or pexophagy,alleviated oxidative stress.We further clarified that TIGAR neutralized oxidative stress in the pentose phosphate pathway independent manner.The key transcription factors,nuclear factor erythroid 2-related factor 2(Nrf2),regulates cellular redox balance.Kelch-like ECHAssociating Protein 1(KEAP1)combines with Nrf2 and inhibits the activation of Nrf2.TIGAR overexpression degraded KEAP1 and promoted Nrf2 translocate to nucleus,upregulating the m RNA levels of heme-oxygenase-1 and glutamate-cysteine ligase catalytic subunit,which was cancelled by wortmannin,inhibitor of autophagy.It suggested that TIGAR-induced autophagic degradation of KEAP1 and activated the transcriptions of Nrf2-regulated antioxidant enzymes with prolonged ischemia.Noteworthy,the antioxidant capacity and neuroprotection of TIGAR was abolished by ML385 or Nrf2 knockdown in neurons.Furthermore,Nrf2 silencing abolished the neuroprotection of TIGAR in mice with prolonged MCAO.These data suggested that TIGAR-activated autophagy,through the pentose phosphate pathway independent manner,activited Nrf2 and alleviated oxidative stress.Taken together,the present study found for the first time that TIGAR,via the pentose phosphate pathway independent antioxidant manner,protects against neuronal injury with prolonged ischemia.Furthermore,TIGAR induces autophagy and activates Nrf2 antioxidant pathway.This study reveals a new antioxidant mechanism of TIGAR,which refines the theory of how TIGAR correct cellular redox balance in the context of cerebral ischemia.This previously unexplored mechanism of TIGAR may partially explain how neurons maintain antioxidant capacity under the condition of insufficient glucose supply and closure of pentose phosphate pathway.Furthermore,it provides phamological experimental basis for the research and development of anti-ischemic stroke drugs.