The Study On The Mechanisms Of KIBRA Regulating Amyloid β Metabolism By Controlling Extracellular Vesicles Secretion

Background:Alzheimer’s disease(AD),the most common cause of dementia,is characterized by intracellular accumulation of neurofibrillary tangles and extracellular deposition of amyloid plaques,which are mainly composed of hyper-phosphorylated tau protein and peptide amyloid β(Aβ),respectively.Toxic Aβ peptide is generated from the amyloid precursor protein(APP),which undergoes sequential cleavages by β and y secretases.Although attempts to develop therapies targeting the accumulation of Aβ have not been successful,the amyloid cascade hypothesis is still widely recognized as a plausible explanation of the pathogenesis of AD.It is widely accepted that the homeostasis of APP cleavage and metabolism is critical in the pathogenesis of AD and is related to each organelle of the endolysosomal system,from multivesicular bodies(MVBs)to lysosomes.Under pathological conditions,APP may be internalized into the endosomal-lysosomal system,where APP C-terminal fragment β(APP-CTFβ)and Aβ monomers are generated by β-and γ-secretases,respectively.On one hand,APP-CTFβ/Aβ peptides are sorted to MVBs and eventually release exosomes harboring APP-CTFβ/Aβ into the extracellular space.On the other hand,endocytosed APP and cleaved peptides may also be trafficked to lysosomes for further degradation.Accordingly,the complex APP processing and trafficking network primarily depend on the endosomal-lysosomal-exosomal system,and impaired balance of endosomal-lysosomal trafficking may lead to abnormal Aβ metabolism,eventually triggering AD pathogenesis.However,the key molecule regulating the balance between lysosomal degradation and exosome secretion is largely unknown.The WWC1 gene was first described to be associated with human memory performance in a genome-wide search for single nucleotide polymorphisms(SNPs)and the original name,KIBRA,was given for its predominant expression in the KIdney and BRAin.KIBRA is a scaffolding protein enriched at postsynaptic sites,which can interact with various postsynaptic components,including actin regulatory networks,the atypical protein kinase M ζ,and the synaptic protein dendrin to regulate synaptic plasticity and learning performance.In addition,growing evidence suggest that KIBRA is involved in cell polarity and vesicle transport.WWC1 C2 domain has a lipid binding capacity with preference for phosphatidylinositol-3-phosphate(PI3P),which is enriched on endosomal membranes,suggesting that KIBRA may be involved in the regulation of vesicles trafficking in the endolysosomal system.Our previous research has revealed that KIBRA controls exosome secretion by inhibiting the proteasomal degradation of Rab27a.Given the important role of endosomal-lysosomal-exosomal trafficking in Aβ metabolism,it is reasonable to hypothesize that KIBRA may influence Aβ metabolism by regulating exosome secretion.Aims:1.To analyze the effects of KIBRA on Aβ metabolism.2.To investigate the mechanisms by which KIBRA regulates Aβ metabolism.Methods and results:1.Construction of KIBRA-/-5XFAD mice and KIBRA-KD cell lineTo investigate whether KIBRA can affect Aβ metabolism,we selected 5XFAD transgenic mice.We crossed 5XFAD mice with KIBRA knockout mice,and obtained KIBRA-/-5XFAD mice through genotype identification.In addition,we overexpressed human APPswe gene(including Swedish mutation,K670N and M671L)in mouse hippocampal neuron cell line(HT22)to form an APPswe overexpressing cell line.Then,the KIBRA gene in the APPswe overexpression cell line was knocked down by CRISPR-Cas9 and lentivirus infection,thus the KIBRA knockdown(KIBRA-KD)cells with the APPswe overexpression were constructed.KIBRA knockdown and APP overexpression efficiency were verified by western blotting(WB)and real-time PCR.2.Analyze various forms of Aβ in the brain tissue of 5XFAD mice after KIBRA knockoutTo investigate the effect of KIBRA on Aβ metabolism,we detected the effect of KIBRA knockout on Aβ in the brain by various antibodies and staining methods.First,we detected Aβ in whole brain tissue,including FA-soluble and TBS-soluble Aβ monomers,Aβ oligomers,and amyloid plaques,by ELISA,dot blotting,thioflavin staining,and WB.In addition,in order to verify whether KIBRA has a direct effect on APP or has an effect on the production of Aβ,we further detected the full-length APP and APP-CTFβ by WB.Our results demonstrate that depletion of KIBRA in the 5XFAD mice significantly reduced Aβmetabolites from monomers,oligomers to plaque deposits in the early stage,without affecting APP protein levelSecond,we quantitatively analyzed the effect of KIBRA knockdown on Aβ in neurons by digital slice scanning technique.Quantitative analysis of 4G8-positive large pyramidal neurons density in the cortex and hippocampal cells revealed no significant change in 3-5-month-old KIBRA-/-5XFAD mice,suggesting that deletion of KIBRA had no effect on intraneuronal APP-CTFs/Aβ in vivo.In addition,compared with the control cells(CTRL)on a background of overexpressing APPswe,intracellular APP,APP-CTFβ,Aβ40,and Aβ42 levels were unchanged in KIBRA-KD cells.Taken together,these results demonstrated that KIBRA knockdown had no significant effect on intracellular APP-CTFs/Aβ,only decreases the extracellular APP-CTFs/Aβ.3.Analyze exosomes and contents of APP-CTFβ/Aβ in KIBRA-KD cell and brain tissue space of KIBRA-/-5XFAD miceTo explore the mechanism of KIBRA on Aβ,we next examined whether KIBRA regulates secretion of EVs harboring APP-CTFβ/Aβ.First,the cell culture supernatant from KIBRA-KD and its control cells was taken for gradient centrifugation and finally a pellet rich in extracellular vesicles(EVs)was obtained.The pellet was resuspended in PBS for NTA detection or in cell lysate containing protease inhibitors for WB detection.We found that knockdown of KIBRA led to a significant decrease of EVs biomarker proteins(e.g.,Alix,CD63,and CD9)with equivalent numbers of cells.To detect Aβ in EVs,we further purified the pellet by sucrose density gradient centrifugation to reduce contamination by soluble proteins and protein aggregates.The Aβ detection was performed on the Single-molecule Array(Simoa)platform.Notably,Aβ40 or Aβ42 peptides and APP-CTFβ levels in EVs secreted by KIBRA-KD cells showed significantly drop compared with equivalent numbers of control cells,demonstrating that depletion of KIBRA decreased the secretion of EVs-associated APP-CTFβ/Aβ in vitro.Second,to verify the role of KIBRA in exosome secretion in a physiological context,we isolated and purified EVs from the extracellular space of the brain of KIBRA-/-5XFAD mice,KIBRA+/-5XFAD mice,and 5XFAD mice counterparts by sucrose density gradient centrifugation.Our results showed a decrease of extracellular EVs(CD63 and CD9)in the brain of KIBRA-/-5XFAD mice compared to 5XFAD mice and Aβ40,Aβ42,and APP-CTFβlevels in EVs from the KIBRA-/-5XFAD mice brain displayed significant decrease.In summary,these results demonstrated that downregulation of KIBRA caused a decrease in the secretion of EVs harboring APP-CTFβ/Aβ both in vivo and in vitro.Moreover,electron microscopy analysis showed that the number of MVBs per cell profiles and intraluminal vesicles(ILVs)per MVB in the hippocampal neurons and cortical neurons of KIBRA-/-5XFAD mice considerably increased compared with the 5XFAD mice.Confocal analysis showed that KIBRA knockout significantly increased colocalization of AβPP/Aβwith CD63 in mice brain neuron.These findings suggested that deletion of KIBRA may further aggravate the effect of Aβ accumulation by increasing MVBs number.4.Detecting the effect of KIBRA knockout on lysosomal functionIn order to explore the mechanism by which KIBRA affects Aβ metabolism,we further examined the function of another intracellular transport pathway of MVB,namely lysosomes.First,we analyzed the fusion efficiency of MVB and lysosomes in neurons of KIBRA-/-5XFAD and 5XFAD mice by confocal colocalization.Confocal analysis revealed a dramatic increase in the co-localization of CD63 with the lysosome marker Lamp2 in the cortical and hippocampal region of KIBRA-/-5XFAD mice compared with their control mice.Secondly,WB analysis showed that the expression level of lysosome membrane protein Lamp2 and maturity of hydrolase cathepsin D(CatD)in the brain tissues of KIBRA-/-5XFAD and 5XFAD mice were markedly increased.Finally,confocal colocalization showed that there was significantly more co-localization of AβPP/Aβ(stained by antibody 4G8)and lysosomal marker Lamp2 in the cortical pyramidal neurons of 3-5-month-old KIBRA-/-5XFAD mice,and KIBRA knockout showed increased colocalization of AβPP/Aβ with CatD in neurons.These findings support the view that KIBRA knockout indirectly increases MVB-lysosome fusion and facilitates the APP-CTFβ/Aβ degradation by lysosomes.5.Investigate whether inhibition of lysosome function in KIBRA-KD cells reverses exosome secretionIn order to further verify that KIBRA knockout compensatorily increases the lysosomal degradation for APP-CTFβ/Aβ,we inhibited the function of lysosomes in KIBRA-KD and control cells,and whether the effects of KIBRA knockdown on exosome secretion can be reversed.First,we used the lysosomal inhibitor Bafilomycin A(BafAl)to inhibit the hydrogen ion pump and decrease the lysosomal degradation function.Our results showed that the effect of inhibition of lysosomes function by BafAl rescue decrease of EVs secretion from KIBRA depletion.Second,to exclude the non-specific effect of lysosomal inhibitors on MVB-lysosome fusion,we used siRNA technology to knock down the expression of Rab7.Rab7 knockdown rescued EVs secretion in KIBRA KD cells.More importantly,the APP-CTFβ level,Aβ40,and Aβ42 peptides level in the EVs significantly increased in KIBRA KD cells after treatment with BafAl or siRNA targeting Rab7.These findings suggest that KIBRA regulates Aβ metabolism mainly by controlling secretion of EVs-associated APP-CTFβ/Aβ,and inhibiting the fusion of excessive MVBs to lysosomes rescued EVs-harboring APP-CTFβ/Aβ secretion.6.Detect the relationship between KIBRA SNP polymorphism and plasma Aβ in the populationTo explore whether KIBRA gene SNPs are associated with plasma Aβ levels in humans,we sequenced KIBRA gene Single Nucleotide Polymorphism(SNP)risk loci and exon region in the subsample derived from MIND-China study(n=1419).Plasma Aβ40 and Aβ42 levels were measured on a Simoa platform with Human Neurology 3-Plex A assay(N3PA).In nine missense mutations(SNPs)identified within the KIBRA coding region,rs28421695 was significantly associated with plasma Aβ40 and Aβ42 levels.The results showed that plasma Aβ40 and Aβ42 in subjects with T-allele of rs28421695(A/T)was dramatically lower compared to subjects with A/A,and the Aβ42/Aβ40 ratio was higher in persons with A/T compared to those with A/A.These results demonstrate that KIBRA SNP rs28421695 was significantly associated with reduced plasma Aβ levels in humans,which may correlate the loss function of KIBRA with lower EVs secretion and Aβ metabolites.Conclusions:1.Multiple forms of Aβ were dramatically attenuated in KIBRA knockout mouse brain,including monomers,oligomers,and extracellular deposition.2.KIBRA depletion also decreased APP C-terminal fragment β(APP-CTFβ)/Aβ-associated EVs secretion.3.KIBRA knockout increased APP-CTFβ/Aβ degradation from lysosome and inhibition of lysosomal function rescued secretion of APP-CTFβ/Aβ-associated EVs.4.Whole exon sequencing of KIBRA in a large population-based cohort identified the association of rs28421695 polymorphism with plasma Aβ levels.