Alzheimer's disease is pathologically defined by accumulation of extracellular amyloid-β(Aβ). Approximately 25 mutations in β-amyloid precursor protein(APP) are pathogenic and cause autosomal dominant Alzheimer...Alzheimer's disease is pathologically defined by accumulation of extracellular amyloid-β(Aβ). Approximately 25 mutations in β-amyloid precursor protein(APP) are pathogenic and cause autosomal dominant Alzheimer's disease. To date, the mechanism underlying the effect of APP mutation on Aβ generation is unclear. Therefore, investigating the mechanism of APP mutation on Alzheimer's disease may help understanding of disease pathogenesis. Thus, APP mutations(A673T, A673 V, E682 K, E693 G, and E693Q) were transiently co-transfected into human embryonic kidney cells. Western blot assay was used to detect expression levels of APP, beta-secretase 1, and presenilin 1 in cells. Enzyme-linked immunosorbent assay was performed to determine Aβ_(1–40) and Aβ_(1–42) levels. Liquid chromatography-tandem mass chromatography was used to examine VVIAT, FLF, ITL, VIV, IAT, VIT, TVI, and VVIA peptide levels. Immunofluorescence staining was performed to measure APP and early endosome antigen 1 immunoreactivity. Our results show that the protective A673 T mutation decreases Aβ_(42)/Aβ_(40) rate by downregulating IAT and upregulating VVIA levels. Pathogenic A673 V, E682 K, and E693 Q mutations promote Aβ_(42)/Aβ_(40) rate by increasing levels of CTF99, Aβ_(42), Aβ_(40), and IAT, and decreasing VVIA levels. Pathogenic E693 G mutation shows no significant change in Aβ_(42)/Aβ_(40) ratio because of inhibition of γ-secretase activity. APP mutations can change location from the cell surface to early endosomes. Our findings confirm that certain APP mutations accelerate Aβ generation by affecting the long Aβ cleavage pathway and increasing Aβ_(42/40) rate, thereby resulting in Alzheimer's disease.展开更多
Identified as the pathogenic genes of Alzheimer's disease(AD),APP,PSEN1,and PSEN2 mainly lead to early-onset AD,whose course is more aggressive,and atypical symptoms are more common than sporadic AD.Here,a novel m...Identified as the pathogenic genes of Alzheimer's disease(AD),APP,PSEN1,and PSEN2 mainly lead to early-onset AD,whose course is more aggressive,and atypical symptoms are more common than sporadic AD.Here,a novel missense mutation,APP E674Q(also named“Shanghai APP”),was detected in a Chinese index patient with typical late-onset AD(LOAD)who developed memory decline in his mid-70s.The results from neuroimaging were consistent with AD,where widespread amyloidβdeposition was demonstrated in 18 F-florbetapir Positron Emission Tomography(PET).APP E674Q is close to theβ-secretase cleavage site and the well-studied Swedish APP mutation(KM670/671NL),which was predicted to be pathogenic in silico.Molecular dynamics simulation indicated that the E674Q mutation resulted in a rearrangement of the interaction mode between APP and BACE1 and that the E674Q mutation was more prone to cleavage by BACE1.The in vitro results suggested that the E674Q mutation was pathogenic by facilitating the BACE1-mediated processing of APP and the production of Aβ.Furthermore,we applied an adeno-associated virus(AAV)-mediated transfer of the human E674Q mutant APP gene to the hippocampi of two-month-old C57Bl/6 J mice.AAV-E674Q-injected mice exhibited impaired learning behavior and increased pathological burden in the brain,implying that the E674Q mutation had a pathogenicity that bore a comparison with the classical Swedish mutation.Collectively,we report a strong amyloidogenic effect of the E674Q substitution in AD.To our knowledge,E674Q is the only pathogenic mutation within the amyloid processing sequence causing LOAD.展开更多
基金funded by the National Natural Science Foundation of China,No.81671268(to HQ)partially supported by a grant from the Ministry of Science and Technology of China,No.2013YQ03059514(to HQ)a grant from Key Laboratory for Neurodegenerative Disease of Ministry of Education of China,No.2015SJBX05(to HQ),2015SJZS01(to HQ)
文摘Alzheimer's disease is pathologically defined by accumulation of extracellular amyloid-β(Aβ). Approximately 25 mutations in β-amyloid precursor protein(APP) are pathogenic and cause autosomal dominant Alzheimer's disease. To date, the mechanism underlying the effect of APP mutation on Aβ generation is unclear. Therefore, investigating the mechanism of APP mutation on Alzheimer's disease may help understanding of disease pathogenesis. Thus, APP mutations(A673T, A673 V, E682 K, E693 G, and E693Q) were transiently co-transfected into human embryonic kidney cells. Western blot assay was used to detect expression levels of APP, beta-secretase 1, and presenilin 1 in cells. Enzyme-linked immunosorbent assay was performed to determine Aβ_(1–40) and Aβ_(1–42) levels. Liquid chromatography-tandem mass chromatography was used to examine VVIAT, FLF, ITL, VIV, IAT, VIT, TVI, and VVIA peptide levels. Immunofluorescence staining was performed to measure APP and early endosome antigen 1 immunoreactivity. Our results show that the protective A673 T mutation decreases Aβ_(42)/Aβ_(40) rate by downregulating IAT and upregulating VVIA levels. Pathogenic A673 V, E682 K, and E693 Q mutations promote Aβ_(42)/Aβ_(40) rate by increasing levels of CTF99, Aβ_(42), Aβ_(40), and IAT, and decreasing VVIA levels. Pathogenic E693 G mutation shows no significant change in Aβ_(42)/Aβ_(40) ratio because of inhibition of γ-secretase activity. APP mutations can change location from the cell surface to early endosomes. Our findings confirm that certain APP mutations accelerate Aβ generation by affecting the long Aβ cleavage pathway and increasing Aβ_(42/40) rate, thereby resulting in Alzheimer's disease.
基金supported by grants from the Ministry of Science and Technology of the People's Republic of China(No.2021ZD020180)the Natural Science Foundation of China(No.81971068,81922021,81773635,82073765).
文摘Identified as the pathogenic genes of Alzheimer's disease(AD),APP,PSEN1,and PSEN2 mainly lead to early-onset AD,whose course is more aggressive,and atypical symptoms are more common than sporadic AD.Here,a novel missense mutation,APP E674Q(also named“Shanghai APP”),was detected in a Chinese index patient with typical late-onset AD(LOAD)who developed memory decline in his mid-70s.The results from neuroimaging were consistent with AD,where widespread amyloidβdeposition was demonstrated in 18 F-florbetapir Positron Emission Tomography(PET).APP E674Q is close to theβ-secretase cleavage site and the well-studied Swedish APP mutation(KM670/671NL),which was predicted to be pathogenic in silico.Molecular dynamics simulation indicated that the E674Q mutation resulted in a rearrangement of the interaction mode between APP and BACE1 and that the E674Q mutation was more prone to cleavage by BACE1.The in vitro results suggested that the E674Q mutation was pathogenic by facilitating the BACE1-mediated processing of APP and the production of Aβ.Furthermore,we applied an adeno-associated virus(AAV)-mediated transfer of the human E674Q mutant APP gene to the hippocampi of two-month-old C57Bl/6 J mice.AAV-E674Q-injected mice exhibited impaired learning behavior and increased pathological burden in the brain,implying that the E674Q mutation had a pathogenicity that bore a comparison with the classical Swedish mutation.Collectively,we report a strong amyloidogenic effect of the E674Q substitution in AD.To our knowledge,E674Q is the only pathogenic mutation within the amyloid processing sequence causing LOAD.
