Urolithin A And Tomatidine Protect Against Ischemic Neuronal Injury Via Autophagy-Related Mechanisms

Stroke is one of the leading causes of death and main cause of permanent adult disability worldwide.Stroke presents a major global burden to patients,their relatives,and whole economies.Ischemic stroke accounts for most stroke issues.Ischemic stroke results in neuronal injury and death that can lead to severe disability and neurological impairment.The cellular and molecular mechanisms underlying cerebral ischemia involve a variety of pathological processes such as oxidative stress,autophagy,apoptosis,inflammatory reactions,and necrosis;these mechanisms influence the prognosis of ischemic neuronal injury.Nevertheless,the exact pathological mechanisms underlying ischemic neuronal injury are not fully elucidated.To date,the only drug approved by FDA for the thrombolytic treatment is recombinant tissue plasminogen activator?rt-PA?.However,the use of rt-PA is extremely limited due to very short therapeutic window and high risk of hemorrhagic complications.Besides rt-PA,a variety of neuroprotectants show promising implication in stroke therapy by rescuing the penumbra,the area that surrounds the ischemic core.Novel and effective neuroprotective strategies might therefore restrain core expansion and/or penumbra survival for later use of reperfusion therapy.Given the complex nature of cerebral ischemia,it is a major challenge to develop neuroprotective drugs for ischemic stroke.Autophagy is a lysosome-mediated intracellular catabolic process that is responsible for the digestion of superfluous or damaged cytoplasmic macromolecules and organelles.During autophagy,bulk cytoplasm and organelles are sequestered into double-membrane vesicles called autophagosomes.Autophagosomes ultimately fuse with lysosomes to generate autophagolysosomes,which provide hydrolases and degrade the luminal contents.Autophagy occurs at a low,basal level in most cells under physiological condition.In the central nervous system?CNS?,autophagy is further activated by various intracellular or extracellular signals including cerebral ischemia,hypoxia,nutrient deprivation,endoplasmic reticulum stress,neurotoxins or excitotoxic stimuli.It has been documented that autophagosomal formation was increased following neonatal hypoxia ischemia?HI?and focal cerebral ischemia in mice.In addition,lysosomal activity was also found increased in ischemic cortical and hippocampal CA3 neurons.Similar alternations were also found in ischemic brain tissue after transient middle cerebral artery occlusion?MCAO?in adult mice.These data indicated the autophagy activation in ischemic brains.Autophagy activating has been considered a defense process that provides cytosolic component turnover to promote cell survival.Studies have suggested that autophagy may protect neurons through degradation of damaged organelles,preventing apoptosis or delaying ionic imbalance to stop ensuing necrosis.Recent studies demonstrated the neuroprotective role of autophagy in ischemic models both in vitro and in vivo.Previous studies showed that autophagy in the reperfusion phase removed damaged mitochondria that would have otherwise possibly induced apoptosis,and thus protected against cerebral I-R injury.In addition,moderate ER stress during reperfusion protected against ischemic brain injury by reinforcing mitophagy.Further researches demonstrated that PARK2-dependent mitophagy is responsible for neuroprotection and reperfusion window extension.Overall,reinforced autophagy shows potential neuroprotective effects against ischemic stroke.These studies implied that autophagy-inducing compounds might serve as a new strategy for treatment of stroke.However,there is a paucity of autophagy-activating chemicals.Although some clinically available drugs,including rapamycin,carbamazepine,tyrosine kinase inhibitors stimulate autophagy,their efficacy for stroke treatment have not been identified.Besides,the potential implications of these drugs for stroke therapy may also be rendered by their prominent adverse effects.Therefore,there is still a need for novel autophagy activators to attenuate ischemic brain injury.Natural compounds,isolated and exploited from plants,animals and microorganisms,have been reported to be involved in the modulation of several biological processes including autophagy.Such compounds may serve as an invaluable source of drug discovery for diverse diseases.For example,Spermidine plays a neuroprotective role in both acute ischemia and during ischemia/reperfusion by restoring Beclin-1-mediated autophagy.Activation of macroautophagy by Ginkgolic acid is beneficial in protecting neuronal cells against toxic stress due to the accumulation of?-syn aggregates.Glycyrrhizic acid had the ability to up-regulate the conversion of LC3B II/I and the expression of Beclin-1,and induce autophagy in SH-SY5Y cells.Thus,it has efficient activity against neurotoxicity in SH-SY5Y cells.Nevertheless,these compounds have some limitations for their potential application for stroke.E.g.spermidine causes potentially cytotoxicity.Ginkgo leaf extract might increase the risk of liver and thyroid cancers.Glycyrrhizic acid can cause tachycardia and cardiac arrest.Therefore,some metabolites from naturally derived compounds may have advantages in aspects of drug ability and adverse effects.Urolithins are naturally occurring metabolites formed by the gut microbiota following the consumption of foods rich in ellagitannins and ellagic acid.Urolithin A?Uro-A?has been reported to have many beneficial health effects,including anti-inflammatory properties,anti-cancer,antioxidant as well as maintaining and improving mitochondrial and muscle function.Urolithin A was found to induce autophagy in human colorectal cancer cells and macrophages.Uro-A can cross the blood-brain barrier and has been shown to protect neuronal cell in the context of Alzheimer's disease.Emerging data indicated Uro-A attenuated ischemic injury in myocardial cells.More recently,Uro-A was demonstrated to induce mitophagy in C.elegans and murine cell lines.Tomatidine is a steroidal alkaloid and the aglycone of alpha-tomatine,an abundant glycoalkaloid in tomato plants?Solanaceae family?.When consumed by animals,alpha tomatine is hydrolyzed by stomach acid and intestinal bacteria to tomatidine,which is absorbed by the gut.It has been documented that tomatidine activates autophagic degradation in cells of multiple species,including nematodes,rats,and humans and in C.elegans.In addition,tomatidine reduced the formation of foam cell in human monocyte-derived macrophages and has beneficial effect on atherogenesis in ApoE-deficient mice.Furthermore,tomatidine protected the SH-SY5Y cells from H₂O₂-induced oxidative stress by increasing the anti-oxidant enzyme activity as well as inhibiting apoptosis.The precursors of urolithin A and tomatidine are found in pomegranate and tomato respectively.There are studies indicated that both Uro-A and tomatidine activate autophagy in several biological systems.Uro-A has been reported to prevent the degeneration of muscle cells whilst tomatidine activates autophagic degradation in cells of multiple species.Although their benefits have been documented,their potential neuroprotection against ischemic neuronal injury remains undetermined.In the first part of the present study,we determined the efficacy of Uro-A on autophagy induction and neuroprotection by using oxygen-glucose deprivation?OGD?models in vitro and mouse model of middle cerebral artery occlusion?MCAO?in vivo.Mice were randomly grouped into a sham,sham+Uro-A,MCAO and MCAO+Uro-A groups.Mice were given an intraperitoneal injection of 2.5 or 5.0 mg/kg of Uro-A for 24 h and 1 h before MCAO.Mice in the sham group were injected with the same volume of saline.The neurological deficit was determined and the cerebral infarct size was examined by TTC.The N2a cells and primary cultured mice cortical neurons were subjected to OGD and reperfusion?OGD/R?to mimic ischemia/reperfusion like conditions in vitro.The N2a cells and primary cultured mice cortical neurons were pretreated with?3?30?M?of urolithin A and then subjected to OGD for 4 h and1.5 h,respectively.Cells were then subjected to 24 h reperfusion and cell viability was determined by MTT and LDH assay.Autophagosomes were visualized by transfecting mCherry-LC3.Protein levels of LC3-II,p62,TIMM23,and COX4I1 were detected by western blot.The ER stress markers,ATF6 and C/EBP homologous protein?CHOP?were determined by RT-PCR.Our data indicated that N2a cell and primary cultured neurons dramatically reduced the cell viability after OGD/R,while Uro?A pretreatment significantly reversed the viability loss dose dependently with a maximal effect of 10?M of Uro?A.Likewise,2.5 and 5.0 mg/kg of Uro-A treatments significantly decreased the volume of infarction and neurological deficit score.These results indicated neuroprotective effects of Uro-A against ischemic injury.We found significantly increased quantity and area of mCherry-LC3 positive puncta in N2a cells and primary neurons.Furthermore,Uro-A reinforced OGD/R-upregulated LC3-II,which was further accumulated with the incubation of chloroquine.Consistently,Uro-A significantly increased the LC3-II and reduced the p62 protein level in MCAO model.These data indicated that Uro-A activated autophagy in ischemic brains.Furthermore,Uro-A-conferred protection was abolished by 3-methyladenine,suggesting the requirement of autophagy for neuroprotection.Nevertheless,Uro-A failed to increase mito-GFP and mCherry-LC3 puncta overlap.Similarly,Uro-A treatment did not further reduce the COX 4I1 and TIMM23 in OGD/R-treated N2a cells and primary cultured neurons and in MCAO model.These data suggested that Uro-A did not reinforce mitophagy in ischemic neuronal cells.Alternatively,our data indicated that treatment with Uro-A significantly decreased the upregulations of ATF6and CHOP in a dose-dependent manner,either in vitro or in vivo.Furthermore,our result showed that 3-MA significantly reversed the mRNA levels of ATF6 and CHOP,suggesting Uro-A-induced autophagy alleviated ER stress in ischemic neurons.Taken together,this part of our study demonstrated that Uro-A protected against ischemic neuronal injury by reinforcing autophagy rather than mitophagy.Autophagy activation by Uro-A attenuated ischemic neuronal death by suppressing ER stress.In the second part of the present investigation,we determined the efficacy tomatidine on autophagy induction and neuroprotection by using OGD models in vitro.N2a cells and primary cultured mice cortical neurons were pretreated with tomatidine?1,3,10?M?and then subjected to OGD for 4 h and 1.5 h,respectively and cells were treated by 24 h reperfusion.Cell viability was determined by MTT and LDH assay.Autophagosomes and autolysosomes were visualized by transfecting mCherry-GFP-tandem fluorescent LC3.The protein levels of LC3-II,Cathepsin D,Cathepsin B,and TFEB were detected by western blot.Lysosomes were stained with LysoTracker Red and DQ-BSA red.The results showed that both N2a cells and primary cultured neurons dramatically reduced the cell viability after OGD/R treatment.Tomatidine treatment significantly reversed the viability loss dose-dependently with a maximal effect of 10?M of tomatidine.Interestingly,tomatidine treatment attenuated,rather than reinforced,the OGD/R-elevated LC3-II in a concentration dependent manner,which can be reversed by lysosome inhibitor.Furthermore,OGD/R treatment increased both yellow and red puncta than the control group,whilst treatment with tomatidine decreased the number of yellow puncta but increase the number of red puncta compared with OGD/R group.These data indicated that tomatidine may enhance autophagic flux by activating lysosomal function rather than merely promoting autophagosomes biogenesis.Indeed,tomatidine increased the lysosome number,proteolytic activities,as well as the expression of Cathepsin D and Cathepsin B.Moreover,tomatidine increased the expression and nucleus translocation of transcription factor EB?TFEB?.Consistently,the neuroprotective effects of tomatidine were abolished by lysosome inhibitors BafA1 and CQ,but not by wortmannin,which blocks autophagosome generation.In addition,we found that tomatidine treatment failed to decrease mito-GFP positive area and increase mito-GFP and mCherry-LC3 puncta overlap after OGD.Furthermore,tomatidine treatment did not further reduce the COX 4I1 and TIMM23 in OGD/R-treated N2a cells,suggesting tomatidine has no impact on mitophagy activation in ischemic neuronal cells.Overall,this part of our study revealed the neuroprotection of tomatidine against ischemic injury by promoting lysosomal activity,possibly with the involvement of TFEB-related mechanisms.Taken together,we identified the neuroprotective effects of Uro-A and tomatidine,two naturally derived compounds with autophagy activating activity,against ischemia insults.Uro-A conferred its neuroprotection by attenuating ischemia-induced ER stress while tomatidine reinforced lysosomal activity.Our results provided rationale for the potential implication of these compounds for stroke therapy.