Role Of Receptor Interacting Protein 3 And Lysosomal Membrane Permeability On Necroptosis Of Hippocampd CA1 Neurons Induced By Cerebral Ischemia Injury

BackgroundProgrammed cell death (PCD) plays a prominent role in the process of biological growth, development and diseases. Based on morphological and biochemical features, PCD is classified into three types at present:apoptosis, programmed necrosis and autophagic cell death. Apoptosis, the first discovered PCD, subdivided into two categories according to the molecules involved:caspase-dependent and caspase-independent pathway. Most of caspase-independent cell death pathways act on mitochondria, and apoptosis inducing factor (AIF) is considered as the main effector because of nuclear translocation. Programmed necrosis, also termed necroptosis, is another kind of caspase-independent PCD. It was first identified by Degterev et al used tumor necrosis factor alpha and Fas ligand in different cell lines. Necroptosis exhibits the morphological features of necrosis, such as nuclear condensation and organelle swelling, but it can be controlled by many molecules. The formation of receptor-interacting protein 1 (RIP1) and RIP3 complexes (called necrosomes) is the key initiation step for necroptosis. DNA alkylating agents induced caspase-independent PCD is another kind of programmed necrosis study paradigm, in which pathway AIF is found to be the key molecule. Our previous study found that the characters of hippocampal CAl neuronal death induced by global cerebral ischemia reperfusion (I/R) injury conformed to programmed necrosis, rather than the traditional apoptosis pattern. REP3 increased and transferred to nucleus after global cerebral I/R injury. However, the mechanism of neuronal necroptosis caused by RIP3 nucleus translocation and the relationship between RIP3 and AIF were not clear.In addition, we also observed nuclear membrane disappeared, karyopyknosis fragmentation, the integrity of the lysosomal membrane lossed and lysosomal hydrolases released into the cytoplasm in rat hippocampal neurons through the electron microscope. The release of lysosomal enzymes is traditionally thought to cause necrosis, while many studies suggest that it also plays a critical role in programmed cell death, such as apoptosis, depending on the degree of lysosomal membrane damage and the amount of enzymes released. According to the electron microscope results, we consider that the necroptosis was linked with lysosomal enzyme release, but the lack of in-depth study. Cathepsin B is the most abundant cysteine protease in the brain and is the major neuronal lysosomal enzyme; it has important functions in physiological and pathological neuronal processes. Regulated Cathepsin-B release has been demonstrated after apoptosis-inducing stimuli, while severe oxidative stress can damage lysosomal membranes and induce Cathepsin-B leakage. CA074-me, a selective and widely-used inhibitor of Cathepsin-B, administered intracerebroventricularly has neuroprotective against middle cerebral artery occlusion (MCAO) injury. While the role in global ischemia and the effect of lysosomal membrane permeability (LMP) is unclear.ObjectivesWe used the rat global cerebral I/R-induced injury model to perform the following investigations:1. To explore the role of RIP3 nucleus translocation in the process of neuronal necroptosis induced by I/R injury.2. To explore the effect of different signaling pathways inhibitor on hippocampal neurons of necroptosis.3. To investigate the role of LMP changes in neuronal necroptosis.PartⅠThe interaction of Receptor-interacting protein 3 and Apoptosis inducing factor and nuclear translocation synchronously play key roles in necroptosis of rat global cerebral ischemiaMethods:Twenty minutes global cerebral I/R injury was conducted on male Sprague-Dawley (SD) rats weighing 280-350g using four-vessel occlusion (4-VO) method. Necroptosis inhibitor Necrostatin-1 (Nec-1), autophagy inhibitor 3-Methyladenine (3-MA) and caspase-3 inhibitor Ac-DMQD-CHO were administrated by intracerebroventricular injection 1 hour before ischemia respectively. Rat hippocampus was rapidly isolated on ice at 2 days,3 days or 7 days of reperfusion, used for paraffin embedding. Hematoxylin eosin (HE) stain and TUNEL staining were performed to count survival neurons. Double immunofluorescenceas follow:REP3 double staining with β-Tubulin-Ⅲ(neuron specific markers) or AIF; Neun (neuron cell nuclear markers) double staining with cleaved caspase-3 or caspase-8; Caspase-8 double staining with Iba-1 (respectively) or GFAP (astrocyte specific marker). The expression and spatial distribution of the corresponding proteins were observed.Results:AIF and RIP3 in the hippocampus after I/R injury were found in the nucleus and co-located. Western blot study found that the expression of AIF and MLKL in the hippocampus after I/R injury did not change, but the expression of LC3-II increased. HE test showed that Nec-1 and 3-MA had protective effects on neuron. TUNEL staining indicated these two inhibitors could reduce the DNA fracture. And Nec-1 and 3-MA can prevent RIP3 entering into the nucleus to have interaction with AIRConclusions:These data suggested that RIP3 and AIF co-localized in the nucleus and the interaction is the key to necroptosis of neurons induced by I/R injury. Nec-1 and 3-MA have neuroprotective effect against I/R injury by inhibited REP3 interaction with AIF.Part II Protective mechanisms of CA074-me against necroptosis induced by global cerebral ischemia/reperfusion injuryMethods:Rat hippocampal CA1 neuronal death was evaluated after 20-min global cerebral I/R injury. CA074-me were given by intracerebroventricular injection 1 h before ischemia or 1 h post reperfusion. Rat hippocampus were rapidly isolated on ice at 7 days or 30 days of reperfusion, used for paraffin embedding, and samples were stained with HE to count survival neurons. Cathepsin-B, Lysosomal associated membrane protein-1 (LAMP-1), and RIP3 were investigated using immunofluorescence. The expression of heat shock protein 70 (HSP70) was assessed by western blot. The changes of NAD+in hippocampus at 1 day after reperfusion were detected by NAD+/NADH quantification colorimetric kit. Then we cultured primary rat hippocampal neurons in vitro and used DND-153 staining to observe the effect of CA074-me on the lysosomal membrane integrity and the pH changes induced by enzyme release after oxygen-glucose deprivation (OGD).Results:Hippocampal CA1 neuronal necroptosis induced by global cerebral I/R injury was prevented by CA074-me both pre-treatment and post-treatment. Diffuse cytoplasmic Cathepsin-B and LAMP-1 immunostaining synchronized with the pyknotic nuclear changes 2 days post reperfusion, and a rise of lysosomal pH with the leakage of DND-153, a dye of lysosomes, after oxygen-glucose deprivation (OGD) was detected. Both of these changes demonstrated the rupture of lysosomal membrane and the leakage of Cathepsin-B, and this was strongly inhibited by CA074-me pre-treatment. The overexpression and nuclear translocation of RIP3 and the reduction of NAD+level after I/R injury were also inhibited, while the upregulation of HSP70 was strengthened by CA074-me pre-treatment.Conclusions:Delayed fulminant leakage of Cathepsin-B due to lysosomal rupture is a critical harmful factor in neuronal programmed necrosis induced by 20-min global I/R injury. In addition to being an inhibitor of Cathepsin-B, CA074-me may have an indirect neuroprotective effect by maintaining lysosomal membrane integrity and protecting against lysosomal rupture. CA074-me can also inhibit the RIP3 nuclear translocation and enhanced the expression of protective protein HSP70.