The Roles And Mechanisms Of MLKL Regulates Cx43 Ubiquitination Degradation In Secondary Thalamic Damage After Cortical Cerebral Infarction In Adult Rats

Objective As the main type of stroke,ischemic stroke accounts for about70%of all strokes.In recent years,although significant progress has been made in the treatment of cerebral infarction,about 25%of the survivors are still unable to take care of themselves.Therefore,there is an urgent need for the treatment that can reduce the neurological deficit of patients with cerebral infarction.The neurological damage caused by cerebral infarction is not only at primary infarct site,but also in the non-occlusive blood supply regions and the remote brain areas which have fibrous association with the primary infarct site.For example,thalamus,hippocampus,substantia nigra,corpus striatum,spinal cord,corticospinal tract,peripheral nerves and muscles will suffer from secondary neurodegeneration and be progressively exacerbated,which have a serious effect on the patient’s neurological rehabilitation.Therefore,the study on the pathogenesis of secondary damage after cerebral infarction will have an important guiding significance for the neurological rehabilitation of patients with cerebral infarction.However,the pathological mechanism of secondary damage after focal cerebral infarction is not completely understood.Recent studies have confirmed that it may be related to axonal degeneration,neuroinflammation,apoptosis,excitatory neurotoxicity,peripheral immune cell infiltration,blood-brain barrier destruction,autophagy,abnormal protein aggregation and so on.Recent studies showed that as a newly discovered programmed cell death mode,necroptosis has been confirmed to play an important role in the occurrence and progression of ischemic stroke.As the main executor of necroptosis,mixed lineage kinase domain-like protein（MLKL）is implicated in the pathophysiological process of cerebral ischemia.So far,the molecular mechanism of MLKL and its downstream signal molecules mediating necroptosis is still unclear.What is the role of MLKL and its downstream signal molecules in secondary damage after cerebral infarction?It is still unclear.Cx43is one of the most important gap junction proteins in cell membrane,participating in the formation of intercellular gap junctions and maintaining intercellular communication.Under physiological conditions,most of the gap junction hemichannels composed of Cx43 are closed,and only a few gap junction hemichannels can be activated in the resting state.Opening of connexin hemichannels has been associated with cerebral ischemia,as well as inflammation,oxygen glucose deprivation.This may cause extracellular Ca²⁺influx,disruption of the resting membrane potential,leading to cell rupture and death.Furthermore,recent studies have confirmed the up-regulation of Cx43 is closely related to cell necroptosis.Interestingly,our molecular docking results showed that Cx43 carboxyl terminal Lys303 interacts with MLKL at Ser454 by hydrogen bonds.Meanwhile,we used bioinformatics methods to predict that Von Hippel-Lindau（VHL）might be one of the E3 ubiquitin ligases regulating the polyubiquitination of Cx43 at lysine 303.Based on the above evidence,we first established the distal middle cerebral artery occlusion（dMCAO）model of adult male SD rats to confirm the occurrence of secondary damage in the VPN region of ipsilateral thalamus.The expressions of MLKL and Cx43 in VPN region of thalamus after dMCAO were observed in adult SD rats,and the possible relationship between MLKL and Cx43 was explored.Secondly,we successfully established the necroptosis cell model by TSZ induction in SH-SY5Y cell line.Finally,we used molecular docking technology to predict the binding site of MLKL and Cx43,and bioinformatics methods to predict the E3 ubiquitin ligase of Cx43.On this basis,we applied the necroptosis model of SH-SY5Y cells,and used co-immunoprecipitation,ubiquitination assay and Western blot to investigate the effect of interaction between MLKL and Cx43 on the ubiquitination of Cx43 and necroptosis.Methods In this study,adult male SD rats were prepared using the bipolar electrocoagulation method for dMCAO model.But in sham operated rats,only the middle cerebral artery was exposed without electrocoagulation of blood vessels.The research methods include:1.TCC staining was used to determine the infarction location and volume.Nissl staining,NeuN and DAPI double staining were used to examine the neurodegenerative neuronal survival in the ipsilateral thalamic VPN area at 1w,2w,3w,4w after dMCAO.Western blot was used to detect MLKL,Cx43,MLKL oligomer and p-MLKL oligomer protein expression at 1-4w after dMCAO.Meanwhile,co-immunoprecipitation was used to detect the interaction between MLKL and Cx43.Triple-labeled immunofluorescence assay was used to determine the localization of MLKL in the cells between sham operated group and 2w after dMCAO group.2.SH-SY5Y cell line was used to establish the necroptosis cell model by TNF-α（human）/Smac Mimetics/Z-Vad（OH）-FMK induction.Western blot was used to detect MLKL,p-MLKL and Cx43 protein expression at 8h,10h,12h after TSZ induction.CCK8 assay,LDH release assay and Annexin V/PI double staining assay were performed to examine cell viability and LDH release level of SH-SY5Y cell line at 8h,10h,12h after TSZ induction.3.Plasmids such as MLKL（WT）,MLKL（MT）,Cx43（WT）,Cx43（MT）and VHL were constructed and verified by Western blot using SH-SY5Y cell line.Molecular docking method was used to predict the binding site of MLKL and Cx43.Co-immunoprecipitation was also performed to verify the interaction site of MLKL and Cx43 using SH-SY5Y cell line.Bioinformatics methods were performed to predict the E3 ubiquitin ligase regulating the polyubiquitination of Cx43 at lysine 303.Co-immunoprecipitation was also used to confirm the E3ubiquitin ligase regulating the polyubiquitination of Cx43 at lysine 303 using SH-SY5Y cell line.We set up four experimental groups using SH-SY5Y cell line:Control+TSZ group,Vehicle+TSZ group,MLKL（MT）+Cx43+TSZ group,MLKL（WT）+Cx43+TSZ group.Western blot was used to detect the protein expression changes of MLKL,p-MLKL,VHL and Cx43 in each group.Co-immunoprecipitation was used to detect the interaction between Cx43 and MLKL,p-MLKL,VHL,as well as the polyubiquitination,K48polyubiquitination and K63 polyubiquitination of Cx43.Ethidium bromide（EB）uptake assay and Fluo-4AM staining assay were used to examine the Cx43hemichannels and intracellular calcium concentration in each group by laser confocal microscopy,respectively.CCK8 assay,LDH release assay and Annexin V/PI double staining assay were performed to examine cell viability and LDH release level of SH-SY5Y cell line in each group.Statistical analyses were performed with SPSS 22.0 package.All data were presented as mean±standard error.The measurement data between two groups were compared by t test.Comparison of measurement data between multiple groups used multi-factor or one way ANOVA.Pairwise comparisons between groups were performed using LSD or Tamhane’s T2 test.p<0.05 was considered statistically significant.Results 1.Expression changes of MLKL and Cx43 in the VPN region of thalamus after dMCAO in adult SD rats（1）Compared to sham operated group,Nissl staining indicated that neuronal loss in the ipsilateral thalamic VPN area was most significant in the first 1 w after dMCAO（p<0.05）.Compared with Sham group,a large number of Neuronal nuclei（NeuN）positive cells were lost in the ipsilateral thalamic VPN region of SD rats from 1 to 4w after dMCAO,and the number of neurons decreased significantly（p<0.05）.（2）Compared with sham operated group,the protein expression level of MLKL in the ipsilateral VPN region of the thalamus in the rats detected by Western blot was gradually upregulated at 1 w after dMCAO（p<0.05）,peaked at 2 w after dMCAO,and showed a downward trend at 3 w after dMCAO,but it was still significantly upregulated compared with that of the Sham group（p<0.05）.Meanwhile,the protein expression level of Cx43 in the ipsilateral thalamic VPN region was also up-regulated（p<0.05）,and the change trend was basically consistent with MLKL protein expression.Compared to sham operated group,the protein expression level of MLKL oligomer in the ipsilateral VPN region of the thalamus in the rats detected by Western blot was gradually upregulated at 1 w after dMCAO,peaked at 2 w after dMCAO,and showed a downward trend at 3 w after dMCAO,was also expressed at 4 w after dMCAO.Compared to the contralateral VPN region of the thalamus,the protein expression level of MLKL oligomer in the ipsilateral VPN region of the thalamus in the rats detected by Western blot was increased significantly at 1-4w after dMCAO.Compared to sham operated group,the protein expression level of p-MLKL oligomer in the ipsilateral VPN region of the thalamus in the rats detected by Western blot was gradually upregulated at 1 w after dMCAO,peaked at 3 w after dMCAO,and showed a downward trend at 4 w after dMCAO.Compared to the contralateral VPN region of the thalamus,the protein expression level of p-MLKL oligomer in the ipsilateral VPN region of the thalamus in the rats detected by Western blot was increased significantly at 1-4w after dMCAO.（3）According to triple labeling with immunofluorescence assay,MLKL was mainly expressed in the nucleus of neurons in the sham group,and was not co-expressed with GFAP and CD68.However,MLKL was co-expressed with GFAP-labeled astrocytes and CD68-labeled activated microglia in addition to co-expressed living neurons at 2w after dMCAO.（4）Co-immunoprecipitation results showed that Cx43 interacted with MLKL directly in the ipsilateral VPN region of the thalamus in the rats after dMCAO.2.Establishment of necroptosis cell model of SH-SY5Y cell line.（1）Compared with the control group,the protein expression level of MLKL in SH-SY5Y cells detected by Western blot was gradually increased significantly with the prolongation of TSZ induction time（p<0.05）,and the protein expression level of p-MLKL began to be up-regulated in TSZ 10h group（p<0.05）,while there was no significant change compared with TSZ 12h group（p>0.05）.With the prolongation of TSZ induction time,Cx43 protein expression level was gradually increased significantly compared with the control group（p<0.05）.（2）CCK-8 assay results showed that with the prolongation of TSZ induction time,the survival rate of SH-SY5Y cells was decreased significantly compared to the control group,especially from TSZ 10h（p<0.05）.（3）LDH release assay results showed that with the prolongation of TSZ induction time,the release level of LDH in SH-SY5Y cells was increased significantly compared to the control group,especially from TSZ 10h（p<0.05）.（4）Annexin V/PI double staining assay showed that with the prolongation of TSZ induction time,the number of necroptotic SH-SY5Y cells was gradually increased significantly,especially from TSZ 10h（p<0.05）.3.The mechanism of MLKL regulating Cx43 ubiquitination and degradation in necroptosis cell model（1）Molecular docking showed that Cx43 carboxyl terminal Asn302 and Lys303 interacted with MLKL at Ser454 through hydrogen bonds.Co-immunoprecipitation assay results showed that Cx43 interacted with MLKL and VHL directly using SH-SY5Y cell line.Co-immunoprecipitation assay results also showed that Cx43 interacted with MLKL（WT）and MLKL（S454A）directly in SH-SY5Y cell line,and the interaction between MLKL（WT）and Cx43 was stronger than that between MLKL（S454A）and Cx43.（2）Bioinformatics methods were used to predict that VHL might be the E3ubiquitin ligase regulating Cx43 polyubiquitination at lysine 303,and co-immunoprecipitation assay result showed that VHL was the E3 ubiquitin ligase regulating Cx43 polyubiquitination at lysine 303 using SH-SY5Y cell line.（3）According to the Western blot results,compared with Control+TSZ group,the protein expression level of MLKL,p-MLKL and Cx43 were significantly up-regulated in MLKL（MT）+Cx43+TSZ group and MLKL（WT）+Cx43+TSZ group（p<0.05）.Compared to MLKL（MT）+Cx43+TSZ group,the protein expression level of MLKL,p-MLKL and Cx43 were significantly up-regulated in MLKL（WT）+Cx43+TSZ group（p<0.05）.Compared with Control+TSZ group,the protein expression level of VHL was significantly down-regulatedinMLKL（MT）+Cx43+TSZgroupand MLKL（WT）+Cx43+TSZ group（p<0.05）.Compared to MLKL（MT）+Cx43+TSZ group,the protein expression level of VHL was significantly down-regulated in MLKL（WT）+Cx43+TSZ group（p<0.05）.Compared with Control+TSZ group,the protein expression level of MLKL oligomer and p-MLKL oligomer were significantly up-regulated in MLKL（MT）+Cx43+TSZ group and MLKL（WT）+Cx43+TSZ group（p<0.05）.Compared to MLKL（MT）+Cx43+TSZ group,the protein expression level of MLKL oligomer and p-MLKL oligomer were significantly up-regulated in MLKL（WT）+Cx43+TSZ group（p<0.05）.（4）Co-immunoprecipitation assay results showed that MLKL interacted with Cx43 at Ser454 through hydrogen bonds,which inhibited Cx43polyubiquitin,especially Cx43 K48 polyubiquitin.Compared with Control+TSZ group,the interaction between Cx43 and MLKL,p-MLKL were significantly stronger in MLKL（MT）+Cx43+TSZ group and MLKL（WT）+Cx43+TSZ group.Compared with MLKL（MT）+Cx43+TSZ group,the interaction between Cx43 and MLKL,p-MLKL were significantly stronger in MLKL（WT）+Cx43+TSZ group.Compared with Control+TSZ group,the interaction between Cx43 and VHL was significantly stronger in MLKL（MT）+Cx43+TSZ group and MLKL（WT）+Cx43+TSZ group.However,compared with MLKL（MT）+Cx43+TSZ group,the interaction between Cx43 and VHL was significantly weaker in MLKL（WT）+Cx43+TSZ group.（5）CCK8 assay results showed that compared with Control+TSZ group,the survival rate of SH-SY5Y cells in MLKL（MT）+Cx43+TSZ group and MLKL（WT）+Cx43+TSZ group were significantly decreased（p<0.05）.Compared with MLKL（MT）+Cx43+TSZ group,the survival rate of SH-SY5Y cells in MLKL（WT）+Cx43+TSZ group was significantly decreased（p<0.05）.（6）According to the LDH release assay results,compared with Control+TSZ group,LDH release level of SH-SY5Y cells in MLKL（MT）+Cx43+TSZ group and MLKL（WT）+Cx43+TSZ group were significantly increased（p<0.05）.Compared with MLKL（MT）+Cx43+TSZ group,LDH release level of SH-SY5Y cells in MLKL（WT）+Cx43+TSZ group was significantly increased（p<0.05）.（7）According to the Annexin V/PI double staining results,compared with Control+TSZ group,the number of necroptotic SH-SY5Y cells in MLKL（MT）+Cx43+TSZ group and MLKL（WT）+Cx43+TSZ group were significantly increased（p<0.05）.Moreover,compared with MLKL（MT）+Cx43+TSZ group,the number of necroptotic SH-SY5Y cells in MLKL（WT）+Cx43+TSZ group was significantly increased（p<0.05）.Conclusion 1.After focal cortical infarction in adult male SD rats,secondary damage occurred in the VPN region of ipsilateral thalamus,followed the up-regulation of MLKL,Cx43,MLKL oligomer and p-MLKL oligomer,abnormal distribution of MLKL.MLKL could interact with Cx43 directly.2.The necroptosis cell model was successfully established by TSZ induction in SH-SY5Y cell line.3.Cx43 could interact with MLKL at Ser454 through hydrogen bonds.Interaction between MLKL and Cx43 can inhibit the polyubiquitination of Cx43at Lys 303 through VHL,prevented Cx43 from entering into the ubiquitin proteasome pathway for degradation,caused the opening of a large number of connexin 43 hemichannels in the cell membrane,resulted in a large amount of Ca²⁺influx and intracellular calcium overload,promoted the occurrence of cell necroptosis.