Endothelial TIGAR Elicits Protective Effect On Tight Junction Integrity Against Excessive Autophagy During Hypoglycemia Induced Stress

The blood-brain barrier(BBB)is a dynamic and complex interface between the blood and the central nervous system(CNS).The BBB endothelium is characterized by distinctive expression patterns of transmembrane transport systems to regulate traffic of substances in and out of the brain parenchyma.The BBB is comprised of a tightly sealed monolayer of brain microvascular endothelial cells that are characterized by the absence of fenestrations,a low level of pinocytic vesicles and an elaborate junctional complex formed by both tight junctions and adherens junctions.Hypoglycemia is a medical emergency referred by an abnormally low level of blood glucose.Hypoglycemia mediated insult disrupts endothelial tight junctions can lead to the increase in cerebrovascular diseases.Before a new strategy can be developed to counter the adverse effects of hypoglycemia induced endothelial tight junction disruption and neurovascular injury,it is necessary to explain the mechanisms responsible for hypoglycemia mediated blood brain-barrier damage.Autophagy is a catabolic mechanism which involves the lysosomal degradation of cytoplasmic constituents,provides energy as well as structural building blocks for critical cellular processes.In human vascular endothelial cells,autophagy acts primarily as a prosurvival function whereas it can be further stimulated in endothelial cells by various stressors including brain ischemia,glucose deprivation,hypoxia and reactive nitrogenous species.Growing number of evidences suggest that autophagy and apoptosis may exist chronologically,however autophagy deregulation can contribute to cellular dysfunction,or boost the endothelial cell death.LC3 is the key autophagy marker,the cytoplasmic LC3-? become lipidated in LC3-? form and considered as an indicator of patterns of autophagy in endothelial cells.The impairment of autophagic flux in endothelial cells attenuates the secretory granules triggered by thrombosis.In spite of evidences demonstrating that autophagy is up-regulated during brain degenerative diseases,very little understanding about the mechanisms that involve in exaggeration or inhibition of autophagy in brain endothelial cells during hypoglycemic stress.Peroxynitrite(ONOO-)belongs to reactive nitrogen species,generated by reaction of nitric oxide(·NO)and superoxide(O2·-)radicals.ONOO-inactivates prostacyclin synthase via protein tyrosine nitration and induces peroxiredoxin 1 ubiquitination whereas nitrosative stress regulats the autophagic process during ischemic brain injury.Mounting evidences showed that endothelial cells are the prime targets for nitrosative stress in the neurodegenerative complications.Although nitrosative stress is motivating the autophagy and peroxynitrite mediated damage to proteins has been implicated in neurovascular injury and blood-brain barrier damage,however the underlying mechanisms remain unclear.TIGAR(TP53-induced glycolysis and apoptosis regulator)having Phosphofructo-2-kinase activity to inhibit glycolysis by hydrolyzing cellular levels of fructose-2,6-bisphosphate and fructose-1,6-bisphosphate by promoting pentose phosphate pathway flux.TIGAR activates mitochondrial respiratory chain results in regulating redox metabolism for cell survival under hypoxic conditions.TIGAR shows protection in cerebral ischemia by increasing NADPH and promotes antioxidant activity,thereby limiting oxidative stress mediated apoptosis and mitophagy.However,the protective role of TIGAR during hypoglycemic stress mediated endothelial cell tight junction damage and underlying mechanism remain elusive.Although TIGAR activates mitochondrial respiratory chain to regulate redox metabolism and promotes antioxidant activity for cell survival whereas its protective role during hypoglycemia stress mediated neurovascular damage remains unresolved.The present research work aimed to elucidate the molecular mechanism in metabolic stress-induced endothelial tight junction injury,focusing on determining the role of TIGAR in the crosstalk via coupling autophagy and brain endothelial injury.Here in this study,we investigated the role of TIGAR in hypoglycemia induced stress in brain endothelial cells.We found TIGAR as sensitive biosensor upon low-glucose(1mM)stress.Proteomic analysis in brain endothelial cells demonstrated that TIGAR upregulated in brain endothelial cells cultured in glucose-free medium conditions accompanied by glucose treatment in concentration(10-0 mM)dependent manner for 24 h.A time dependent increase in TIGAR was observed,followed by a decline after 24 h.The tight junction proteins;Claudin-5 and occludin expression are the crucial factor which reflects brain barrier permeability in cultured brain endothelial cells.Immunocytochemical experiments revealed subcellular localization of TIGAR(red fluorescence)by using anti-TIGAR antibody.Upon induction of lentiviral-GFP vector encoding Claudin-5(GFP-Claudin-5)in cell cultures,we found strong GFP fluorescence in brain endothelial tight junctions.Immunocytochemical image analysis of the brain endothelial cells showed co-localization of TIGAR and Claudin-5.Moreover,breakdown of occludin and JAM-A were observed in a concentration and time-dependent manner during low-glucose induced stress.We also found enhanced phosphorylation of p38 MAP(Thrl80/Tyrl82)kinase,JNK(Thrl83/Tyr185)and upregulation of matrix metalloproteinase 9(MMP-9)in response to low-glucose stress.The time dependent decrease in phosphorylation of AMPKa with increases in ULK-1 phosphorylation showed the energy homeostasis during low-glucose induced insult.Moreover,TIGAR showed endothelial tight junction localization in control conditions whereas TIGAR expression decreased and tight junctions lost their integrity under low-glucose mediated stress for 24 h.We next addressed the role of TIGAR on low glucose-induced brain endothelial cells injury.The human brain endothelial cells transfected with either an empty vector or recombinant human TAT-TIGAR and lentiviral-GFP-TIGAR for 24 h following low-glucose exposure.Overexpression of TIGAR inhibited breakdown products of occludin and JAM-A.Notably,we found concentration-dependent elevation in phosphorylation of JNK(Thr183/Tyr185)and degradation of tight junction proteins in endothelial cells after 3-PO treatment under low-glucose insult.Furthermore,we evaluated the protective effects of TIGAR against low-glucose stress-induced tight junction damage.Immunocytochemical analysis revealed subcellular localization of occludin in human brain endothelial cells.The occludin appeared diffusely distributed across the plasma membrane under low-glucose exposure.TIGAR overexpression blocked the degradation of occludin and protected endothelial cell tight junction integrity.We then used the electron microscopy to reveal the ultrastructural morphology of endothelial cell tight junctions.In control conditions,endothelial junctions normally appeared at the cell-cell contact regions,presenting the kissing points of tight junctions whereas their architecture was disturbed following low-glucose treatment.However,LV-TIGAR overexpression in cell cultures blocked the architectural abnormalities in cell tight junctions.These results showed that TIGAR facilitated protection of tight junction coinciding with its inhibition of autophagy process in glucose starvation.Autophagy is a regulated phenomenon of degradation and recycling of cellular machineries.LC3(microtubule-associated protein 1 light chain 3)is the key marker of autophagic process.During gluose-free medium conditions,conversion of LC3-? to LC3-? was increased with concentration dependent treatment of glucose(10?0 mM).Moreover,we also found a time dependent substantial rise in LC3-? levels during hypoglycemia induced stress for 24h,indicating activation of autophagy.These results indicated that low-glucose mediated stress upset the autophagic flux in brain endothelial cells.To evaluate autophagosome formation,we constructed LC3 with green and red fluorescent probe plasmid(mRFP-GFP-LC3).Autophagy was induced in brain endothelial cells by concentration dependent glucose treatment in aglycemic conditions.A time dependent disturbance of autophagic flux was observed with the increase of LC3-? levels during low-glucose stress for 24h.The mRFP-GFP-LC3 puncta in endothelial cells validated the autophagy induction during low-glucose induced insult which clearly showed the autophagosome formation whereas the chloroquine promoted the mRFP-GFP-LC3 punctate formation.The autophagosome formation at low glucose in presence of inhibitor further induced the elevation of LC3 fluorescent puncta and implicating a defect of fusion of phagosome with lysosome.Further,we tested the ability of autophagy inhibitor in brain endothelial cells under low-glucose exposure,to probe the relationship between autophagic flux and the disruption of endothelial tight junction proteins.BafilomycinAl,an autophagy inhibitor that acts at late steps of the autophagy pathway,was used.There was no change in expressions of LC3-? in absence of BafilomycinA1 whereas the increased levels of LC3-II in presence of bafilomycinAl at 6-24 h,suggesting that autophagy indeed induced.Enhanced occludin breakdown products were found at 12-24 h in endothelial cells in presence of bafilomycinAl.Interestingly,we found that low-glucose stress increased LC3-II turnover at 12 h and 24 h in absence of bafilomycinA1,whereas further increase in accumulation of LC3-? was noted at 6,12 and 24 h of low-glucose treatment in the presence of bafilomycinAl.Similarly,degraded occludin levels were significantly upregulated after treatment of bafilomycinA1.The BafilomycinAl treatment increased LC3-II turnover profoundly at 6,12 and 24 h in presence of PFKFB3 inhibitor,3-PO as compared to LC3-II accumulation which was more at 6 h and 24 h during low-glucose exposure.The degraded occludin and ZO-1 levels were increased markedly in similar manner like LC3-? upon BafilomycinAl and 3-PO treatment whereas less damage to occludin protein was observed at 6 to 24 h in BafilomycinAl absence under low-glucose stress conditions.After 48 h of transfection,siRNA knockdown of Atg5 significantly reduced the accumulation of LC3-II.We observed decrease in breakdown of occludin and phosphorylation of JNK in brain endothelial cells subjected to low-glucose(1mM).Next,we also found that induction of recombinant human TAT-TIGAR significantly inhibited the accumulation of LC3-? in brain endothelial cells under hypoglycemic insult.Moreover,GFP-tag TIGAR plasmid transfected cells subjected to low-glucose for 24 h decreased the phosphorylation of JNK and significantly reduced the LC3-? levels.Further,we found an increase in LC3-? accumulation after low-glucose stress that was significantly amplified after 3-PO treatment.Immunocytochemical analysis revealed that accumulation of RFP-GFP-LC3 vesicles observed in endothelial cells following low-glucose stress and RFP-GFP-LC3 punctate formation increased upon treatment of 3-PO(20?M)after low-glucose exposure.We investigated the hypoglycemia-induced autophagosome formation by using transmission electron microscope.We also observed autophagic vacuoles and distinctive features of autophagosomes in endothelial cells during low-glucose stress induced injury upon microscopic examination whereas TIGAR overexpression inhibited this autophagic vacuole formation.Taken together,these results sturdily suggested that hypoglycemic injury induced autophagy can play damaging role in the tight junction disruption whereas endothelial TIGAR was found to regulate exaggerated autophagic response to low-glucose treatment.We further determined whether nitrosative stress involved in TIGAR dysfunction in brain endothelial cells during low-glucose exposure.In brain endothelial cells,nitrosative stress was activated in very short period of time(20-60 min)after the low-glucose induced injury,as shown by the significantly rise in nitrotyrosine levels by measurements from western blot and Immunofluorescence analysis.We next validated the correlation of low glucose mediated nitrosative stress and autophagy activation.The mRFP-GFP-LC3 punctate formation in response to low-glucose stress was blunted by Calmodulin inhibitor;W7(1?M)and peroxinitrite(ONOO-)scavenger;Melatonin(100nM).Moreover,the peroxinitrite donor 3-morpholinosydnonimine(SIN-1)stimulated the fluorescent puncta in brain endothelial cells resulting in autophagosome formation whereas inhibition of ONOO-with uric acid significantly reduced RFP-GFP-LC3 punctate.To investigate the binding molecule with TIGAR at the outer membrane of tight junction during autophagy,we performed the mass spectroscopic examination.Twenty-nine different proteins were identified with high confidence.Furthermore,immunoprecipitation of cell lysate of Calmodulin-HA and TIGAR-Flag treated HEK-293 cells followed by probing with an anti-TIGAR and anti-Calmodulin antibodies,revealed an increased amount of TIGAR precipitated with Calmodulin in TIGAR-Flag/Calmodulin-HA co-transfected HEK293 cells.To monitor in real time kinetics of Calmodulin binding to TIGAR,we developed a FRET-based assay using CFP-labeled Calmodulin(CaM-CFP)and YFP-labeled TIGAR(TIGAR-YFP).Before and after YFP photobleaching,CFP images were taken to analyze variations in the acceptor fluorescence.Our data demonstrated that there were no significant differences in fluorescence among the groups in relation to fluorescence of acceptor.Furthermore,images were collected to evaluate alterations in donor fluorescence.HEK-293 cells co-expressing CaM-CFP with TIGAR-YFP for 24 h resulted in a significant increase in donor fluorescence up to 18%more than positive control(14%)that was caused by bleaching of YFP(acceptor).Additionally,we looked for interaction of HKII with TIGAR by FRET assay and immunoprecipitation but found no specific binding between two proteins.Next,to validate whether Calmodulin-dependent nitrosative stress is involved in autophagy activation and tight junction disruption,the HBMECs were treated with Calmodulin inhibitor,W7(1?M)for 24 h following low-glucose insult.These findings showed that inhibition of Calmodulin reduced LC3-II accumulation.In line with this data,we found that W7(1?M)treatment also reduced occludin and ZO-1 breakdown.Therefore,our data provide justification that Calmodulin recruitment to TIGAR is associated with excessive autophagy activation and tight junction damage.We speculated that in brain endothelial cell model,nitrosative stress induced the TIGAR dysfunction due to tyrosine residues nitration.In fact,we identified Y92(92YGVVEGK98)potential tyrosine nitration site of TIGAR with high score by mass spectroscopy.The lentivirus mediated TIGAR overexpression protected the occludin by increasing NADPH levels whereas mutant TIGAR(Y92A)lost its efficiency to preserve endothelial tight junctions.The role of TIGAR in autophagy regulation during low-glucose insult was further validated by inhibition of autophagosomes while mutant TIGAR transfection exaggerated the mRFP-GFP-LC3 punctate as compared to fluorescent puncta in endothelial cell model of hypoglycemia induced stress for 24h.Taken together,our results indicated that TIGAR shielded the brain barrier through preserving endothelial tight junction integrity and regulating autophagy against low-glucose induced stress.At last,we also demonstrated the protective role of TIGAR in mice model of insulin induced hypoglycemia.The effect of TIGAR overexpression against hypoglycemic stress in mice was evaluated by positron emission tomography.The FDG uptake was enhanced in wild type mice whereas the standard uptake value(SUV)in TIGAR transgenic mice was minimal.The lesser FDG uptake in TIGAR-tg mice as compared to wild type mice revealed that TIGAR overexpression corrected the increased uptake by preserving the endothelial tight junctions following hypoglycemia.Moreover,TIGAR-tg mice exhibited the vasoprotection by maintenance of microvessel endothelium architecture through inhibition of Claudin-5 and Laminin degradation as compared to animals following hypoglycemia induced injury.Similar results were obtained upon electron microscopic examination of mice brain cortices,where the vascular damage in hypoglycemic mice model was reversed in TIGAR-tg mice.The TIGAR overexpression in the intact mice protected the brain microvessels and tight junction integrity against insulin induced hypoglycemia.This study identifies an intracellular association between nitrosative stress and endothelial TIGAR signaling in response to autophagy following hypoglycemia mediated stress.Our findings explain the low-glucose stress induced downregulation of TIGAR promoted endothelial tight junctions injury whereas TIGAR overexpression protects their integrity via limiting excessive autophagy.TIGAR inactivation by tyrosine nitration represents the unique phenomenon governed by low-glucose induced nitrosative stress subsequently leading to endothelial tight junction damage.Our data thus illustrate a TIGAR-regulated autophagy pathway via increasing NADPH yield that shows neurovascular protection against nitrosative stress and propose a novel target for prevention and treatment of neurovascular diseases.