The Function And Regulatory Mechanism Of TBC1D25/LAPTM5 In Cerebral Ischemia-reperfusion Injury

The mitogen-activated protein kinase(MAPK)signaling pathway has been demonstrated to play an important role in the pathophysiology of cerebral ischemia-reperfusion injury(CIRI).We found that TBC1 Domain Family Member 25(TBC1D25)and Lysosomal-associated transmembrane protein 5(LAPTM5),as an upstream regulatory molecule,could regulate CIRI through the MAPK pathway.Then we will explore the role of these two molecules on CIRI in two parts:Part 1 TBC1D25 regulates cerebral ischemia-reperfusion injury through TAK1-JNK/p38 MAPK pathwayObjective We will explore the potential role of TBC1D25 in CIRI.Methods The m RNA and protein expression levels of LAPTM5 were detected both in vivo and in vitro;LAPTM5 knockout(KO)mice were generated,we detected infarct size and assessed neurological impairment in LAPTM5-KO I/R group compared with WT I/R group.Also,we detected the expression of inflammation and apoptosis related molecules in both group;Primary neurons were infected with LAPTM5 knockdown or overexpression adenovirus followed by oxygen and glucose deprivation/reoxygenation(OGD/R)treatment,we detect cell viability,and the expression of inflammation and apoptosis related molecules;Combined RNA-sequencing(RNA-seq)and Western blot verification,the pathways involved in the regulation of CIRI by LAPTM5 were identified;After pharmacological inhibition of transforming growth factor-β-activated kinase 1(TAK1),we detect the expression of MAPK pathway,inflammation and apoptosis related molecules in LAPTM5 knockdown neurons followed by OGD/R treatment.Results We found that the m RNA and protein expression levels of TBC1D25 decreased in mouse brain after I/R injury and primary cortical neurons treated with OGD/R.Then TBC1D25 knockout(KO)mice were applied to demonstrate that TBC1D25 ablation aggravated cerebral I/R-induced neuronal loss and infarct size.In addition,neuronal apoptosis and inflammation were significantly potentiated in the TBC1D25-KO group.In an in vitro OGD/R model,TBC1D25 knockdown can attenuate neuronal cell viability and aggravate the process of inflammation and apoptosis.Conversely,overexpression of TBC1D25 in primary neurons ameliorated the aforementioned processes.Mechanistically,RNA-seq analysis revealed MAPK signaling pathway was the most significant pathway that contributed to TBC1D25-mediated brain I/R injury process.Through experimental verification,TBC1D25 deficiency increased the phosphorylation of the TAK1-c-Jun N-terminal kinase(JNK)/p38 axis in neurons during CIRI.Furthermore,we found that TAK1 blockade abrogated the apoptosis and inflammatory response produced by TBC1D25 knockdown in vitro.Conclusion This study is the first to demonstrate the functional significance of TBC1D25 in the pathophysiology of CIRI,and the protective mechanism of TBC1D25 is dependent on the TAK1-JNK/p38 pathway.Part 2 LAPTM5 regulates cerebral ischemia-reperfusion injury through ASK1-JNK/p38 MAPK pathwayObjective Lysosomal-associated transmembrane protein 5(LAPTM5)has been demonstrated to be involved in the regulation of immunity,inflammation,cell death and autophagy in the pathophysiological process of many diseases.In this study,we will investigate the potential role of LAPTM5 in CIRI.Methods First,the tMCAO model in mouse and the OGD/R model in primary neurons were established,and we detected the LAPTM5 expression both in vivo and in vitro;Then LAPTM5 knockout mice were constructed,and we detected the infarct size and assessed neurological function.Also,we detected the expression levels of apoptosis-and inflammation-related molecules,and RNA-seq was performed to analyze the expression profile of inflammation and apoptosis in LAPTM5-KO mice;Primary neurons were infected with LAPTM5 knockdown or overexpressed adenovirus and then treated with OGD/R,we explore the effect of LAPTM5 on cell viability,inflammation and apoptosis;The main pathways involved in LAPTM5 deficiency on CIRI was revealed by RNA-seq and verified by Western blot;The LAPTM5-ASK1 interaction was confirmed by protein interaction methods such as Co-IP,GST-pull down,and IP-mapping.Results In this study,we found that LAPTM5 expression was dramatically decreased during CIRI both in vivo and in vitro.LAPTM5-KO mice was compared with the control,and they showed a larger infarct size and more serious neurological dysfunction after tMCAO treatment.In addition,inflammatory response and apoptosis was exacerbated in these processes.Furthermore,gain-and loss-of-function investigations in an in vitro model revealed that neuronal inflammation and apoptosis was aggravated by LAPTM5 knockdown but mitigated by its overexpression.Mechanistically,combined RNA sequencing and experimental verification showed that the apoptosis signal-regulating kinase 1(ASK1)-JNK/p38 pathway was mainly involved in the detrimental effects of LAPTM5 deficiency following I/R injury.Specifically,LAPTM5 directly interacts with ASK1,leading to decreased ASK1N-terminal dimerization and the subsequent reduced activation of downstream JNK/p38 signaling.Conclusion LAPTM5 deficiency aggravates CIRI through the ASK1-JNK/p38 pathway.