Protective Effects Of N-stearyltyrosine On Caspase-independent Cell Death And Their Mechanism

As common central nervous systerm disorder, cerebral ischemia isoften accompanied by profound cognitive dysfunction, resulting in asignificant proportion of neurons loss, which has been attributed tonecrosis, apoptosis and autophagy. A number of studies have revealedthat many neurons in the penumbra zone undergo apoptosis severalhours or days after ischemic, and they could be potentially recoverablefor some time after the onset of cerebral ischemia. Therefore, theneuronal apoptosis in the penumbra are the current targets in stroketreatment studies. Cerebral ischemia triggers two apoptosis pathways:caspase-dependent and caspase-independent cell death pathways. Thetreatments targeted both cell death cascades could lead to more effectiveneuroprotection for stroke therapy.We have developed a series of N-fatty-acyl amino acids as AEA analogues and found that N-stearoyltyrosine (NsTyr) with goodneuroprotective activity could prevent the neuronal loss in hippocampalCA1subfield in transient global cerebral ischemia in gerbils. In this study,we investigated the effects of NsTyr on caspase-independent cell deathand detailed the underlied mechanisms in vitro and in vivo models.NsTyr protects against glutamate-induced oxidative toxicity throughinhibiting AIF nuclear translocation in PC12cellsGlutamate, a major excitatory neurotransmitter in the central nervoussystem, plays a critical role in many neurological disorders such asstroke/cerebral ischemia. The excessive glutamate can result in thecellular oxidative toxicity through a caspase-independent cell deathpathway. In this study, we explored the protective effects of NsTyr onglutamate neurotoxicity in PC12cells and investigated its underlyingmechanisms. Our results revealed that NsTyr treatment attenuatedglutamate-induced oxidative toxicity in PC12cells and primary corticalneurons in a dose-dependent manner and10μΜ NsTyr exhibited thebest performance. The possible mechanisms involved AIF release,mitochondrial dysfunction, intracellular ROS level and MAPK pathwaywere investigated. The data showed that NsTyr suppressed glutamate-induced intracellular ROS elevation and Bid increase in themitochondria, and prevented the loss of mitochondria membraneintegrity, consequently inhibited AIF translocation into the nucleus. Inaddition, we also found that NsTyr was able to inhibit ERK/MAPKover-activation involved in glutamate-induced AIF release. These resultsdemonstrated for the first time that NsTyr could prevent againstglutamate-induced caspase-independent cell death, and the possiblemechanisms may involve the decrease of intracellular ROS production aswell as the blockade of AIF release from mitochondria via preventingBid-mediated mitochondrial damage and ERK/MAPK overactivation.NsTyr protects cortical neurons from OGD-induced injury throughblocking caspase-dependent and caspase-independent cell deathpathways.Oxygen glucose deprivation (OGD) has been widely used to mimic theenvironmental conditions present during cerebral ischemia injury. In thisstudy, the protective effects of NsTyr on OGD-induced neuronalapoptosis and potential mechanisms were investigated. Both in the earlyand late phases of reoxygenation, pretreatment of the cells with NsTyrfor1h following exposure to OGD resulted in a significant elevation of cell viability determined by MTT assay, and reduced the number ofapoptotic cells using Hoechst staining and flow cytometric assessment.At4h of reoxygenation, NsTyr reduced OGD-induced cyto-c releaseand the activation of caspase3, suggesting NsTyr blockedcaspase-dependent apoptotic pathway. At24h of reoxygenation, NsTyrblocked both caspase-dependent and caspase-independent pathwaythrough decreasing cyto-c release, the level of active caspase3and AIFneuclear translocation. Further investigation revealed that NsTyr couldregulate the pro-apoptotic protein Bax and/or anti-apoptotic proteinBcl-2, in turn mitigate the mitochondrial membrane potential decline,which is the initiating event for apoptosis and the critical upstreamregulator for cyto-c release and AIF nuclear translocation. In addition,OGD and ischemia injury could result in the overactivation of PARP-1and calpain-μ, which subsequently enhance the AIF release. NsTyrreversed the reduction of NAD+and ATP levels and decreased thecleavage of α-Fodrin, suggesting that NsTyr inhibited the overactivationof PARP-1and calpain-μ，which may be related with its inhibitory effecton AIF translocation. Protective effects of NsTyr on cognitive impairment in rats oftransient focal cerebral ischemia and involved mechanismA variety of animal models have been developed for modeling cerebralischemic/stroke. The middle cerebral artery occlusion (MCAO) modelhas been utilized extensively, especially in rodents. In this part, weinvestigated the protective effects of NsTyr on cognitive impairment inrats of MCAO and explored the mechanism. In vivo studies revealed that10mg/kg NsTyr could reverse memory impairment assessedbehaviorally by Morris water maze. Neuropathological changes wereexamined using hematoxylin-eosin (HE) and TUNEL staining. Theresult showed that NsTyr attenuated ischemic neuronal damage andreduced the neuronal apoptosis in hippocampus CA1region and corticalareas. The possible protective mechanism is that NsTyr could decreasecyto-c release, reduce the level of active caspase3, and inhibit AIFneuclear translocation in hippocampal and cortical areas in the modelrats, suggesting NsTyr blocked caspase-dependent andcaspase-independent cell death induced by ischemia-reperfusion injury.In summary, we demonstrated for the first time that NsTyr couldelicite protective effects through blocking caspase-dependent andcaspase-independent cell death pathway using in vitro and in vivomodels. Our results suggested that NsTyr might be of therapeutic value for the treatment of cerebral ischemia.