BACE1 SUMOylation Increases Its Stability And Aggravates Aβ Toxicity And Cognitive Impairment

Background:Alzheimer’s disease(AD)is the most common neurodegenerative disorder.The deposition of extracellular senile plaques(SP)is one of the important characteristic neuropathological changes of AD.SP is formed by the accumulation of β-amyloid(A(3)and Aβ is formed by the sequential proteolysis of the type 1 membrane protein APP.APP is first cleaved by the β-secretase enzyme(also known as BACE1)to yield a membrane-bound C-terminal fragment called C99.C99 is cut by y-secretase to liberate Aibe/Aiberate ut by brane-bound C-terminal/Aiberate ut by b Evidence exists that BACE-1 expression is significantly enhanced in brains derived from patients with sporadic AD.The reasons for the enhanced BACE-1 expression are currently unclear.SUMOylation is an important post-translational modification.SUMO(small ubiquitin-like modifer)is a ubiquitin-like modifer,including SUMO-1,SUMO-2,SUMO-3 and SUMO-4.Through the actions of enzymes termed E1,E2,and E3,the mature SUMO proteins are attached to to the specific sequence of the target protein:ψ-K-X-D/E(pecific sequence of the target protein:ψ-K,the mature SUMO proteins are attached to)is a ubiquitinhematoxylin binding sequence).Broadly speaking,SUMOylation can have exclusive consequences:the attached SUMO can change the interaction profile of the substrate,either by blocking binding of an interacting protein,or by the attached SUMO acting as a recruitment factor for new proteins.Alternatively,SUMOylation can control substrate stability,or regulate substrate activity,through inducing a change in protein conformation.Given the wide variety of proteins which are SUMOylated or predicted to be SUMOylated in neurons,it is not surprising that dysregulation of this post-translational modification has been found to be involved in AD.A genome-wide association study found a link between variations in a SUMO-related gene and sporadic late-onset AD.Previous study demonstrated SUMO1 immunoreactivity in phospho-tau aggregates in the Tg2576 APP overexpressing transgenic mouse model.We also found tau SUMOylation promotes reciprocally its hyperphosphorylation,which results in reduced tau ubiquitination and degradation with an increased tau aggregation.However,whether SUMO1 is involved in the formation of Aitination and degradation with an increased tau aggregation.model.dysregulation of this post-translational modification has been founSUMO-binding:274LKMD277 and 495DDISLLK501,suggesting that BACE1 may bind to SUMO1.Objective:To explore the effect of BACE1 SUMOylation on the Ao production and cognitive impairment and its potential molecular mechanism,and seek effective target for AD drug development.Methods:HEK293T and N2A-APP cells were cultured and the BACE1,SUMO1 and SUMO-specific conjugating enzyme UBC9 were transiently transfected and AAV-BACE1 was injected into the hippocampus of C57 mice.Whether BACE1 binding with SUMO1 was detected by co-immunoprecipitation method.SUMOylation sites were predicted by SUMOsp2.0,and BACE1 lysine 275 and 501 to arginine(K275R and K501R)were mutated.The mutant or wild-type BACE1 was co-transfected with SUMO1 and UBC9 to HEK293T or N2A-APP cells to detect BACE1 SUMOylation level by immunoprecipitation.BACE1K501R,wild type BACE1 and UBC9 were co-transfected into N2A-APP cells.The effects of BACE1 SUMOylation on the cleavage of APP and Aβproduction were detected by immunoblotting and ELISA,the effect of SUMOylation on the distribution of BACE1 subcellular cells by confocal,the mRNA degree of BACE1 by reverse transcription and RT-PCR,the stability of BACE1 by immunoblotting after treated cells with Cycloheximide(GHX).C57 mice were injected with AAV-Vector,AAV-BACE1WT,and AAV-BACE1K501R.The changes of behavior were observed by open field and Morris water maze test.The expression of MAP2 and subcellular distribution of BACE1 were observed under Confocal laser scanning microscopy.Golgi staining was used to detect the change of dendritic spine,immunoblotting to detect the changes in synaptic proteins:Synaptotagmin,Synapsin-1,Synaptophysin,PSD93,PSD95 and GluRl and APPβ,BACE1.AAV-Vector,AAV-BACE1WT,AAV-BACE1K501R were injected with APP/PS1 mouse hippocampus,and the behavioral changes were detected by open field and Contextual fear condition.Adition fearere observed by thioflavan staining and Confocal laser scanning microscopy.The primary hippocampal neuronal cells were given 0,40,400 nM A,1-42.24 hours later,the level of BACE1 SUMOylation was detected by immunoblotting and immunoprecipitation.Finally,the levels of BACE1 SUMOylation in 3,6,12-month-aged APP/PS1 mice were detected by immunoblotting and immunoprecipitation methods.And BACE1 and SUMO1 co-localization were observed by confocal laser scanning microscopy.Results:Co-transfection of BACE1,SUMO-1,UBC9 in HEK293T or N2A-APP cells,BACE1 and SUMO1 binding was detected by immunoprecipitation.BACE1 SUMOylation was also confirmed in hippocampus of C57 mice injected with AAV-BACE1.In the BACE1K275R or K501R transfected cell lines,the level of BACE1 SUMOylation was significantly lower than that of wild type,so it was clear that K275 and K501 were BACE1 SUMO-modifying sites.We observed that APP-β and Aβ40/42 levels were significantly reduced in the N2A-APP cells transfected with SUMOylation site mutated BACE1 than wild-type.We also observed that mutations in SUMOylation site of BACE1 contributed to the degradation of BACH1,reduced the activity of the enzyme,but did not alter transcription levels.Immunofluorescence results showed that SUMOylation of BACE1 at K501 could regulate its subcellular localization,improve its stability and increase the co-localization of BACE1 and APP.AAV-Vector,AAV-BACEI wild-type or AAV-BACE1K501R virus were injected in CA1 area of C57 mice for 1 month.The BACE1 wild type group produced more severe learning and memory impairment compared to the control group,but the SUMOylation site mutation group had a significant improvement compared with the wild type group.Immunofluorescence,immunoblotting and Golgi staining showed that the dendrites and dendritic spine density and the expression of synaptic proteins were significantly decreased in the wild type group compared with the vehicle group,but the mutant group was more effective than the wild type group.In vitro culture of embryonic hippocampal neurons,we found that Aβ1-42 can increase the level of BACE1 SUMOylation,and in a concentration-dependent manner.In the APP/PS1 mouse hippocampus,we also observed that the BACEI SUMOylation was improved correlating to aging.Conclusion:1.BACE1 can be SUMOylated in vitro and in vivo,and the modification sites are K275 and K501.2.BACE1 SUMOylation can improve its protein stability and enzyme activity.3.BACE1 SUMOylation increases APPcrcleavage and Ageione its p.4.BACE1 SUMOylation aggravates neuronal dendritic injury and learning and memory impairment.5.BACE1 SUMOylation is correlated with aging.Terreterpenoid A(1)and B(2).two meroterpenoids with unprecedented carbon skeletons featuring an unusual biocyclo[3.3.1]nonane carbon scaffold were obtained from Aspergillus terreus.The absolute structure of 1 was identified by X-ray diffraction technique and that of compound 2 was elucidated by calculated electronic circular dichroism.Compounds 1 and 2 exhibited significant inhibitory activities against β-screatase in vitro with IC50 values of 78 nM and 59 nM.respectively(positive control LY2811376 =260 nM),which were the most active natural BACE1 inhibitors until now.In vivo investigation revealed that compound 1 exhibited better activity against Alzheimer’s disease in 3xTg AD mice than LY2811376,one of the most effective BACE1 inhibitor in phase Ⅲ clinical trials.These findings suggest that 1 is likely the first meroterpenoid exhibiting effective BACE1 inhibitory activity with a novel carbon skeleton.Moreover,1 represents a potential lead compound and a versatile starting structure for the development of drugs for the treatment of Alzheimer disease.