Inactivation Of NSF ATP Ase Leads To Neuronal Injury After Cerebral Ischemia Reperfusion

Ischemic cerebrovascular disease has a high incidence and high disability characteristics,leading patients with long-term neurological deficits,cancer and cardiovascular disease and known as the third major disease that threatens human survival.The main reason for its onset is due to severe intracranial vascular stenosis or blockage,as well as insufficient blood supply to the heart or local brain tissue caused by decreased blood supply and neurological deficit syndrome.Although endovascular treatment of internal carotid artery endarterectomy,endovascular stenting,intracranial and extracranial vascular bypass have been used clinically to restrain the blood supply to the ischemic area,it still can not fundamentally solve the problem of post-ischemic reperfusion of brain injury,is currently the main reason for the impact of ischemic cerebrovascular disease efficacy and prognosis.The first decades of research into ischemic stroke have focused on areas such as neurotoxicity,oxidative stress,neuroinflammation,and the neurovascular unit.However,more and more recent studies have found that protein membrane trafficking pathways after cerebral ischemia play a crucial role in the reperfusion injury of the neurons.NSF ATPase,also known as N-ethyl-maleimide sensitivity factor ATPase,is the most critical factor involved in membrane fusion,enabling the transfer of vesicles from one cell membrane component to another.During this process,the SNARE proteins on the two attached membranes form a tight complex that facilitates the fusion of vesicles with the target membrane.Once membrane fusion occurs,the NSF ATPase uses ATP as an energy source to dissociate these SNARE complexes by hydrolysis,allowing the isolated SNAREs to be recycled for the next membrane fusion.NSF ATPase is the only ATPase effective after membrane fusion to hydrolyze inactive SNARE complexes,and most organisms have only one NSF ATPase except for Drosophila expressing d NSF-1 and d NSF-2.Golgi apparatus-late endosome-lysosome pathway as the only membrane fusion transport pathway that provides lysosomal enzymes,it is used to digest proteins destined to be degraded to maintain the intracellular environment stable.However,a large number of inactivation of NSF ATPase leads to a series of chain reactions in the Golgi-late endosome-lysosome membrane fusion transport pathway,including damaged Golgi,transport vesicles,and late endosomes when ischemic stroke occurs mass accumulation,lethal release of Cathepsin B,mitochondrial membrane permeability(MOMP),eventually leading to delayed neuronal death following reperfusion.In recent years,more and more evidences show that proteolytic enzymes are the very important research objects leading to the irreversibility of cerebral ischemia-reperfusion neurons.In this paper,from the two aspects of "membrane transport pathways" and "lysosome rupture" to explore the possible molecular mechanism that leads to neuronal death in ischemic brain injury and to provide basis for future research on the mechanism of ischemia-reperfusion brain injury and its potential prevention and treatment,further suggesting that selective cathepsin inhibitors may be treatment of stroke and rehabilitation as new therapeutic targets.Our previous study found that ischemic cerebrovascular disease reperfusion mitochondrial oxidative phosphorylation disorder,resulting in decreased intracellular ATP-induced energy metabolism disorder,the cytoplasmic soluble autophagy-related protein NSF aggregation of the inactivation of the process can make the function of vesicle fusion obstacles,which may inhibit autophagic degradation pathway,triggering neuronal damage.In order to further study the important role of NSF ATPase in the Golgi-late endosome-lysosomal membrane protein transport pathway and the mechanism of neuronal death after cerebral ischemia-reperfusion in rats with cerebral ischemia-reperfusion injury caused by massive release of lysosomal cathepsin B.This topic mainly carries on two parts of research:Part Ⅰ Mechanism of membrane protein transport disorder due to inactivation of NSF ATPase after transient cerebral ischemiaObjective: To investigate the inactivation of NSF ATPase induced by 2VO cerebral ischemia-reperfusion injury in rats,which is involved in the mediation of Golgi-late endosome-lysosomal membrane protein transport disorder after ischemic cerebrovascular disease,mediate Cathepsin B lethal release and delayed neuronal death in order to find new clues and therapeutic targets for the future treatment of ischemia-reperfusion injury in neurons.Methods: A transient cerebral ischemia(2VO)/ reperfusion model of bilateral common carotid artery ligation was established in male Wistar rats weighing 280-320 g.Bilateral common carotid arteries were occluded for 20 minutes under anesthesia,and then the cerebral blood flow was re-infused.Brain tissue was infused 0.5,4,24 or 72 hours after reperfusion respectively.The experiment was divided into two groups: sham operation group and cerebral ischemia group.HE staining,immunohistochemical staining,Western Blot and confocal microscopy were used to detect the histopathology of brain tissue infused with different blood flow recovery time,and to explore the mechanism of cerebral ischemia-reperfusion injury after Golgi-late endosome-lysosome,the amount level of protein expression,spatial distribution and localization of related proteins in the membrane transport pathway.Results: The results of HE staining showed that with the reperfusion time prolonging,the number of neuronal deaths in 20-minute transient ischemic group gradually increased,no matter in CA1,CX or DG area,and much higher than that in sham operation group Compared with DG,the neuronal damage in Cx and CA1 groups was more obvious.The delayed neuronal death increased significantly at 24 hours after reperfusion and peaked at 72 hours.Osmium,uranium-lead staining TEM results showed that the Cx neuronal Golgi fragments,transport vesicles,late endosomes and protein aggregates accumulated significantly in cerebral ischemic group,while there was no significant change in control group.Immunofluorescence histochemical staining and confocal microscopy analysis showed that NSF protein aggregates were stained 24 hours after transient ischemic reperfusion of 20 minutes,the Golgi apparatus completely cracked into fluorescent spots,and CTSB protein expression was prolonged with perfusion time Increased significantly,especially 72 hours after reperfusion,and approximately 90% occurred in CA1.Western-blot results showed that the NSF protein band was significantly decreased at 72 h after reperfusion;33k Da LE CTSB increased significantly,while 46 k Da pro CTSB and 24-25 k Da CTSB decreased accordingly,the above results were statistically significant(p<0.05).Conclusions: A series of chain reactions triggered by ischemic cerebral infarction,including the inactivation of NSF ATPase,cause massive accumulation of damaged Golgi apparatus,transport vesicles,and late endosomal organelles,resulting in a lethal CTSB release following transient cerebral ischemia and delayed neuronal death.Part Ⅱ Involvement of key protein NSF ATPase in membrane protein trafficking during ATP depletion during cerebral ischemiaObjective: To determine whether ATP deficiency is the main trigger of NSF inactivation after cerebral ischemia and whether NSF overexpression can protect cells from ATP depletion-induced cell injury in vitro.Methods: The ATP depletion and recovery model of CHO cells was established in vitro.Cell damage situation was detected by LDH and MTT assay.Different protein fractions were obtained using linear glycerol gradient centrifugation and differential centrifugation.Western Blot and immunofluorescence staining were used to detect the NSF protein expression of different components.Results: ATP depletion induced NSF deposition in protein depletion models in CHO cells to form protein aggregates and cellular damage.NSF overexpression protects CHO cells from cell damage due to ATP depletion.In contrast,GS28,syntaxin 4,and syntaxin 6 did not change significantly in this model,suggesting that depletion of ATP resulted in NSF autodeposition rather than deposition of the NSF-SNARE complex.Transient expression of NSF E329 Q ATP binding deficient mutant replicates ATP depletion-induced cell damage whereas overexpression of wildtype NSF decreased ATP depletion-induced cell damage.Conclusions: ATP depletion during ischemia leads to the deposition and inactivation of NSF,a soluble and membrane-associated protein in the cytoplasm,leading to membrane fusion disorders during vesicular trafficking;NSF overexpression overcomes NSF inactivation during and after ATP depletion and retains functional NSF as new method to prevent ischemic brain damage.