Background: Alzheimer’s disease (AD) is an increasingly prevalent neurodegenerative disease characterized by protein aggregation in the form of amyloid plaques containing beta-amyloid peptides and neurofibrillary tan...Background: Alzheimer’s disease (AD) is an increasingly prevalent neurodegenerative disease characterized by protein aggregation in the form of amyloid plaques containing beta-amyloid peptides and neurofibrillary tangles containing hyperphosphorylated tau protein. The central molecular events underlying AD pathogenesis remain controversial and poorly defined. Drosophila melanogaster has emerged as an important genetic resource for studying the pathology of AD. Many AD models have been created using Drosophila, taking advantage of its short generation times, sophisticated genetic tools, and abundance of homologs to human genes. Purpose: This review summarizes different models for studying AD in Drosophila melanogaster, including the full-length APP, C99, Aβ42 and Tau models, explaining how the models were built and what we have learned from them. Conclusion: Four main AD Drosophila models are introduced in this review, which can serve as a future method to investigate genes and drugs that can modify symptoms.展开更多
In order to investigate the pathogenesis of Alzheimer disease （AD） and study the enzymatic progress of amyloid precursor protein （APP）, the fluorescent eukaryotic expression plasmid of C99 was constructed containi...In order to investigate the pathogenesis of Alzheimer disease （AD） and study the enzymatic progress of amyloid precursor protein （APP）, the fluorescent eukaryotic expression plasmid of C99 was constructed containing APP717 mutation. The fragment encoding the last 99-aa of APP （which was named C99 containing APP717 mutation）, together with the fragment encoding yellow fluorescence protein （which was named YFP） were amplified by PCR. The two fragments （YFP and C99） were inserted into the vector pcDNA3.0. The recombinant plasmid pcDNA3.0-YFP-C99 was accomplished and its authenticity was confirmed by enzyme digestion and sequencing. Then SH-SY5Y cells were transiently transfected with the recombinant plasmid pcDNA3.0-YFP-C99. The expression of the fusion gene was detected by laser confocalmicroscopy. Amyloid-β （Aβ） was detected by both microscopy and immunochemistry. The authenticity of the construct was confirmed by the endonuclease digestion and DNA sequencing. The YFP fluorescence could be seen and proved the expression of fusion gene. Aβ labeled by YFP was detected by confocalmicroscopy and confirmed by immunocytochemistry. It was found that Aβ accumulated and deposited in the intracytoplasm, membrane and outside of the cell. Furthermore, Aβ accumulated mainly within the cell ahead of the deposition in the cell space and the cell shape was rough. It was suggested that Aβ could be generated within the cells. Aβ accumulated in the cell at the early stage before the deposition outside of the cells Intracellular Aβ accumulation induced the secondary damage to the cells and caused the cell shape rough. Taken together, the recombinant plasmid, pcDNA3.0-YFP-C99 could be a useful tool to further study the cleavage mechanism of APP and to explore the pathogenesis of AD.展开更多
Background:We recently demonstrated an endolysosomal accumulation of theβ-secretase-derived APP C-terminal fragment(CTF)C99 in brains of Alzheimer disease(AD)mouse models.Moreover,we showed that the treatment with th...Background:We recently demonstrated an endolysosomal accumulation of theβ-secretase-derived APP C-terminal fragment(CTF)C99 in brains of Alzheimer disease(AD)mouse models.Moreover,we showed that the treatment with theγ-secretase inhibitor(D6)led to further increased endolysosomal APP-CTF levels,but also revealed extracellular APP-CTF-associated immunostaining.We here hypothesized that this latter staining could reflect extracellular vesicle(EV)-associated APP-CTFs and aimed to characterize theseγ-secretase inhibitor-induced APPCTFs.Methods:EVs were purified from cell media or mouse brains from vehicle-or D6-treated C99 or APPswedish expressing cells/mice and analyzed for APP-CTFs by immunoblot.Combined pharmacological,immunological and genetic approaches(presenilin invalidation and C99 dimerization mutants(GXXXG))were used to characterize vesicle-containing APP-CTFs.Subcellular APP-CTF localization was determined by immunocytochemistry.Results:Purified EVs from both AD cell or mouse models were enriched in APP-CTFs as compared to EVs from control cells/brains.Surprisingly,EVs from D6-treated cells not only displayed increased C99 and C99-derived C83 levels but also higher molecular weight(HMW)APP-CTF-immunoreactivities that were hardly detectable in whole cell extracts.Accordingly,the intracellular levels of HMW APP-CTFs were amplified by the exosomal inhibitor GW4869.By combined pharmacological,immunological and genetic approaches,we established that these HMW APP-CTFs correspond to oligomeric APP-CTFs composed of C99 and/or C83.Immunocytochemical analysis showed that monomers were localized mainly to the trans-Golgi network,whereas oligomers were confined to endosomes and lysosomes,thus providing an anatomical support for the selective recovery of HMW APP-CTFs in EVs.The D6-induced APP-CTF oligomerization and subcellular mislocalization was indeed due toγ-secretase blockade,since it similarly occurred in presenilin-deficient fibroblasts.Further,our data proposed that besides favoring APP-CTF oligomerization展开更多
The Bloch constants for quasiregular harmonic mappings and open planar harmonic mappings are considered. Better estimates are obtained. The results, presented in this paper, improve the one made by Chen et al. and Gri...The Bloch constants for quasiregular harmonic mappings and open planar harmonic mappings are considered. Better estimates are obtained. The results, presented in this paper, improve the one made by Chen et al. and Grigoryan.展开更多
文摘Background: Alzheimer’s disease (AD) is an increasingly prevalent neurodegenerative disease characterized by protein aggregation in the form of amyloid plaques containing beta-amyloid peptides and neurofibrillary tangles containing hyperphosphorylated tau protein. The central molecular events underlying AD pathogenesis remain controversial and poorly defined. Drosophila melanogaster has emerged as an important genetic resource for studying the pathology of AD. Many AD models have been created using Drosophila, taking advantage of its short generation times, sophisticated genetic tools, and abundance of homologs to human genes. Purpose: This review summarizes different models for studying AD in Drosophila melanogaster, including the full-length APP, C99, Aβ42 and Tau models, explaining how the models were built and what we have learned from them. Conclusion: Four main AD Drosophila models are introduced in this review, which can serve as a future method to investigate genes and drugs that can modify symptoms.
基金This project was supported by the Key Project of Chinese Ministry of Education (No 10420)
文摘In order to investigate the pathogenesis of Alzheimer disease （AD） and study the enzymatic progress of amyloid precursor protein （APP）, the fluorescent eukaryotic expression plasmid of C99 was constructed containing APP717 mutation. The fragment encoding the last 99-aa of APP （which was named C99 containing APP717 mutation）, together with the fragment encoding yellow fluorescence protein （which was named YFP） were amplified by PCR. The two fragments （YFP and C99） were inserted into the vector pcDNA3.0. The recombinant plasmid pcDNA3.0-YFP-C99 was accomplished and its authenticity was confirmed by enzyme digestion and sequencing. Then SH-SY5Y cells were transiently transfected with the recombinant plasmid pcDNA3.0-YFP-C99. The expression of the fusion gene was detected by laser confocalmicroscopy. Amyloid-β （Aβ） was detected by both microscopy and immunochemistry. The authenticity of the construct was confirmed by the endonuclease digestion and DNA sequencing. The YFP fluorescence could be seen and proved the expression of fusion gene. Aβ labeled by YFP was detected by confocalmicroscopy and confirmed by immunocytochemistry. It was found that Aβ accumulated and deposited in the intracytoplasm, membrane and outside of the cell. Furthermore, Aβ accumulated mainly within the cell ahead of the deposition in the cell space and the cell shape was rough. It was suggested that Aβ could be generated within the cells. Aβ accumulated in the cell at the early stage before the deposition outside of the cells Intracellular Aβ accumulation induced the secondary damage to the cells and caused the cell shape rough. Taken together, the recombinant plasmid, pcDNA3.0-YFP-C99 could be a useful tool to further study the cleavage mechanism of APP and to explore the pathogenesis of AD.
基金This work has been developed and supported through the LABEX(excellence laboratory,program investment for the future)DISTALZ(Development of lnnovative Strategies for a Transdisciplinary approach to ALZheimers disease)and by Fondation Alzheimer.A.Be and A.Bo were granted from DISTALZ.
文摘Background:We recently demonstrated an endolysosomal accumulation of theβ-secretase-derived APP C-terminal fragment(CTF)C99 in brains of Alzheimer disease(AD)mouse models.Moreover,we showed that the treatment with theγ-secretase inhibitor(D6)led to further increased endolysosomal APP-CTF levels,but also revealed extracellular APP-CTF-associated immunostaining.We here hypothesized that this latter staining could reflect extracellular vesicle(EV)-associated APP-CTFs and aimed to characterize theseγ-secretase inhibitor-induced APPCTFs.Methods:EVs were purified from cell media or mouse brains from vehicle-or D6-treated C99 or APPswedish expressing cells/mice and analyzed for APP-CTFs by immunoblot.Combined pharmacological,immunological and genetic approaches(presenilin invalidation and C99 dimerization mutants(GXXXG))were used to characterize vesicle-containing APP-CTFs.Subcellular APP-CTF localization was determined by immunocytochemistry.Results:Purified EVs from both AD cell or mouse models were enriched in APP-CTFs as compared to EVs from control cells/brains.Surprisingly,EVs from D6-treated cells not only displayed increased C99 and C99-derived C83 levels but also higher molecular weight(HMW)APP-CTF-immunoreactivities that were hardly detectable in whole cell extracts.Accordingly,the intracellular levels of HMW APP-CTFs were amplified by the exosomal inhibitor GW4869.By combined pharmacological,immunological and genetic approaches,we established that these HMW APP-CTFs correspond to oligomeric APP-CTFs composed of C99 and/or C83.Immunocytochemical analysis showed that monomers were localized mainly to the trans-Golgi network,whereas oligomers were confined to endosomes and lysosomes,thus providing an anatomical support for the selective recovery of HMW APP-CTFs in EVs.The D6-induced APP-CTF oligomerization and subcellular mislocalization was indeed due toγ-secretase blockade,since it similarly occurred in presenilin-deficient fibroblasts.Further,our data proposed that besides favoring APP-CTF oligomerization
基金supported by the Research Foundation for Doctor Programme (Grant No. 20050574002)the National Natural Science Foundation of China (Grant No. 10471048)
文摘The Bloch constants for quasiregular harmonic mappings and open planar harmonic mappings are considered. Better estimates are obtained. The results, presented in this paper, improve the one made by Chen et al. and Grigoryan.
