The Role Of δ-catenin Related GluR2 Internalization On Hypoxic-ischemic Brain Damage In Neonatal Mice And Its Mechanism

Hypoxic-ischemic brain damage(HIBD)is a common cause of neonatal brain injury,which has high incidence and high mortality.HIBD can lead to a variety of severe neurological sequelaes.There is still a lack of effective treatments for neonatal hypoxic-ischemic brain damage.Therefore,it is important that elucidating the pathophysiological mechanism of HIBD for finding new therapeutic targets,increasing the cure rate of HIBD,and improving the prognosis of patients.Neuronal excitotoxicity is a well-known pathological mechanism of hypoxic-ischemic brain damage.Under normal condition,the release,uptake and reabsorption of glutamate in the brain maintain a delicate balance.After hypoxic-ischemic insult,the insufficient energy supply of the brain tissue can disrupt the balance of release and reuptake of glutamate which causes the excess accumulation of glutamate in extracellular fluid.The subsequent overactivation of glutamate receptors induces an overflow,causing intracellular calcium overload,which in turn initiates the process of neuron necrosis and apoptosis.AMPA receptors,one type of ionotropic glutamate receptors that have been implicated in excitotoxicity,are tetraheteromeric structures assembled from GluR1-GluR4 subunits.The calcium permeability of AMPA receptors is mainly determined by the presence or absence of the GluR2 subunit.The AMPA receptors containing GluR2 subunit are calcium impermeable;whereas the AMPA receptors lacking GluR2 subunit are high calcium permeable.After ischemia and hypoxia,the calcium-impermeable AMPA receptors on the cell membrane may be transformed into calcium-permeable AMPA receptors,resulting in a lethal influx of extracellular calcium ion and triggering a range of downstream neurotoxic cascades.δ-catenin,a member of the p120-catenin superfamily,is primarily expressed in the brains of healthy individual.δ-catenin contributes to neuronal development and synapse formation through connections between the cadherins and actin cytoskeleton.There is mounting evidence that δ-catenin is overexpressed in a variety of tumor tissues,which is an important factorpromoting tumor cell survival and proliferation.However,it has not been reported whether δ-catenin can impact neuronal death and survival in the hypoxic-ischemic brain damage.Objective:In the present study,the HIBD model of C57BL/6J mice(postnatal 10days)was used to explore the effect of δ-catenin on neuronal injury and death after HIBD,and observe whether the effect is related to the internalization of GluR2 subunit of AMPA receptors.Methods1.Animal model: HIBD model of C57BL/6J mice aged 10 days was established by left carotid artery ligation followed by hypoxia(7.5% O2 +92.5% N2)for 1.5 hours.2.Groups The C57BL/6J mice pups were divided into the following groups randomly:(1)Sham;HIBD(0 h,6 h,12 h,24 h,48 h).(2)Sham;HIBD;HIBD+si-δ-catenin(HIBD+si-δ-cat);HIBD+negative control(HIBD+NC).3.Detective indicators(1)Western blotting and immunofluorescence was used to detect the levels of the target proteins in brain tissue.(2)TTC staining was used to detect infarct area of mouse brain tissue.(3)Nissl staining was used to detect morphologic changes of neurons.(4)TUNEL was used to detect neuronal apoptosis.(5)Immunocytochemistry and laser confocal fluorescence microscopy were used to detect GluR2 subcellular distribution and co-localization of GluR2 and GRIP1.(6)Co-immunoprecipitation was used to detect the interaction between GluR2 and GRIP1.Results1.The expression of δ-catenin on the ipsilateral brain tissue was significantly reduced at 12 h,24 h and 48 h after HI insult.2.After si RNA interfered with the expression of δ-catenin,the infarct volume of brains and the number of apoptotic neurons further increased,the expressions of pro-apoptotic proteins in the ipsilateral brain tissue further elevated,and the level of anti-apoptotic protein further falls.3.After HI insult,GluR2 expression on the plasma membrane was significantly down-regulated,which coincided with the temporal dynamics ofthe δ-catenin level alteration.4.After si RNA interfered with the δ-catenin expression,the level of GluR2 on the plasma membrane was further down-regulated. 5.After interfering with the δ-catenin expression by si RNA,the interaction between GluR2 and GRIP1 in neurons is further reduced after HI insult.Conclusion In the neonatal mouse hypoxic-ischemic brain damage model,the expression of δ-catenin is down-regulated,leading to brain injury.The underlying mechanism may be that the suppressed δ-catenin expression reduces the binding of δ-catenin,GRIP1 and GluR2,thereby causing the internalization of GluR2 and an increase in the calcium permeability of the AMPA receptors.The subsequent intracellular calcium overload occurence leads to neuronal death.