Study On The Mechanism Of PERK-mediated Unfolded Protein Response In Myelin Demyelinatio

Background:Multiple sclerosis（MS）is the most common chronic inflammatory,demyelinating disease of the central nervous system in young adults.The disorder is a common cause of severe physical disability in young adults,especially in women.Although the etiology of MS is still unclear,accumulated researches over the past few decades suggests that MS may be the result of the interaction of an individual’s genetic susceptibility and numerous environmental factors.The onset of MS is between the ages of 20 and 40,and about 50%of patients require permanent wheelchair use 25 years after diagnosis.Currently,a variety of drugs are available for the treatment of relapsing-remitting multiple sclerosis（RRMS）,however,there are no effective clinical drugs for promoting myelin repair and axon regeneration.Although there are a large number of oligodendrocyte precursor cells（OPC）recruited in MS demyelinating lesions,these cells may be affected by the microenvironment and fail to differentiate and mature into myelinating oligodendrocytes,which in turn leads to the inability to fundamentally improve neurological dysfunction.The neurological deficits associated with MS,the high cost of treatment,and low job retention rates for patients and their caregivers place a huge financial burden on individuals,families,and society.Therefore,there is an urgent need to establish new strategies for MS treatment to promote myelination and axonal regeneration.However,reversing this unfavorable situation requires a deeper and more comprehensive understanding of the underlying mechanisms of demyelination and remyelination failure in MS.In the present work,the acute demyelination model induced by neurotoxin cuprizone（CPZ）with good reproducibility and relatively stable lesion location was used to analyze the molecular mechanism of demyelination and remyelination involved in MS,which will lay the foundation for the repair and treatment of neurological impairment in neuroinflammation-mediated demyelinating diseases.Objective:The present study aims to elucidate the molecular mechanism by which endoplasmic reticulum stress is involved in oligodendrocyte apoptosis and demyelination and identify the key targets leading to demyelination so as to provide new therapeutic strategies for demyelinating disorders represented by MS.Methods:Firstly,immunofluorescence staining was conducted for assessment demyelination,the composition of oligodendrocyte lineage cells,astrogliosis,and microglia/macrophages activation in the corpus callosum from CPZ-induced acute demyelination model mice at different time points.Then,whole brain cells of mice were isolated at three time points（Baseline,CPZ-5W,and CPZ-5W-2W）for single-cellRNA sequencing（scRNA-seq）and bioinformatics analysis.In addition,we usedRNAscope in situ hybridization（RNAscope-ISH）to evaluate the expression level of Atf4,Ddit3and Trib3 in oligodendrocyte lineage cells at the sequencing time points to preliminarily verify the sequencing results.Secondly,mice were feed with diet containing 0.2%CPZ and simultaneously treated with GSK2656157（GSK）,a protein kinase R-like endoplasmic reticulum kinase（PERK）inhibitor.Western blot was used to assessment levels of PERK-mediated unfolded protein response（UPR）,AKT1 signaling,and mitochondria-mediated intrinsic apoptosis pathway-related proteins.RNAscope-ISH was applied to evaluate the expression level of Atf4,Ddit3,and Trib3 in oligodendrocyte lineage cells.Meanwhile,the integrity of myelin was assessed by LFB staining,transmission electron microscopy analysis,and immunofluorescence staining against myelin basic protein（MBP）.Additionally,the composition of oligodendrocyte lineage cells,astrogliosis,and microglia/macrophages activation were evaluated using immunofluorescence staining.Thirdly,TRIB3 knockout(Trib3^-/-)mice and wild-type（WT）mice were feed with CPZ for 5 weeks.Then,LFB staining were applied to evaluated the myelin integrity in the corpus callosum.Astrogliosis and microglia/macrophages activation in the corpus callosum were measured using immunofluorescence staining,and levels of Ddit3 and Trib3 in the corpus callosum oligodendrocyte lineage cells were assessed byRNAscope-ISH.Western blot was used to compare expression levels of PERK-mediated UPR,AKT1 signaling,and mitochondria-mediated intrinsic apoptosis pathway-related proteins.Fourthly,in situ injection of lentivirus were conducted to overexpress TRIB3 in the corpus callosum oligodendrocytes,followed by induction of acute demyelination 5 days later.5 weeks after CPZ ingestion,normal mice（not injected with virus and not ingested with CPZ）,mice injected with Lenti-Control or Lenti-TRIB3 were immunofluorescence stained to evaluate the expression level of TRIB3 in the corpus callosum.LFB staining was used to evaluate the myelin integrity of the corpus callosum in three groups of mice.Western blot was carried out assessment levels of PERK-mediated UPR,AKT1 signaling,mitochondria-mediated intrinsic apoptosis pathway-related proteins.Finally,to evaluate the effect of PERK-mediated UPR on neuroinflammation-mediated demyelination,GSK were intraperitoneally（i.p.）injected start from 3 d to 35d after immunization with MOG_35-55,and clinical scores were recorded daily.Immunofluorescence staining was used to evaluate the myelin integrity,astrogliosis,and microglia/macrophages activation in the spinal cord.Results:The evaluation of CPZ-induced acute demyelination model in mice showed that the extent of demyelination,the number of total OPCs（Oligodendrocyte precusor cells）,the number of Ki67⁺OPCs,astrogliosis,and microglia/macrophages activation in the corpus callosum all showed trend of"first increase and then decrease"in response to CPZ-induced demyelination.However,the dynamic response patterns of astrocytes and microglia/macrophages were not synchronized.The results from scRNA-seq showed that demyelination-responsive oligodendrocyte populations existed in the mouse brain at the peak of demyelination.Several genes related to PERK-mediated UPR and mitochondria-mediated intrinsic apoptosis were upregulated in demyelination-responsive oligodendrocyte populations,which confirmed byRNAscope-ISH targeting UPR-related genes（Atf4,Ddit3,and Trib3）plus oligodendrocyte lineage cells marker（Sox10）.After PERK-mediated UPR was restricted by GSK,levels of TRIB3,Puma,Bax,cleaved caspase-3,and cleaved PARP1 were reduced in the corpus callosum of CPZ-treated mice,and levels of p-AKT1 and p-Fox O1 were increased,and the degree of demyelination was alleviated.Compared with wild-type mice,Trib3^-/-mice showed higher levels of p-AKT1,p-Fox O1,and Bcl-2,and lower levels of Puma,Bax,Cleaved Caspase-3,and Cleaved PARP1,and demyelination.After overexpression of TRIB3,CPZ-treated mice represented down-regulated p-AKT1 and Bcl-2,up-regulated of cleaved Caspase-3 and Cleaved PARP1,and increased extent of demyelination in the corpus callosum.After PERK-mediated UPR was restricted by GSK,EAE mice displayed significantly reduction in clinical scores,extent of demyelination,astrogliosis,and microglia/macrophages activation at the peak of the disease.Conclusion:The present work discovered the molecular mechanism by which endoplasmic reticulum stress was involved in demyelination.After suffering from CPZ intoxication,CPZ triggered persistent ER stress in oligodendrocytes and activates PERK-mediated terminal UPR.Persistently UPR leads to upregulation of its downstream target TRIB3,a pseudokinase,which directly inactivated the pro-survival signal of AKT1 and initiated the mitochondria-mediated intrinsic apoptotic pathway.It ultimately caused oligodendrocyte apoptosis and demyelination.TRIB3 is a key pathogenic molecule linking endoplasmic reticulum stress and mitochondria-mediated intrinsic apoptosis.Strategies targeting TRIB3 have potential clinical application prospects in demyelinating diseases and endoplasmic reticulum stress-related diseases.