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Comments on Paper and Primary News |
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Comment by: Hiroshi Mori, ARF Advisor
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 I recommend this paper
"This presents a crucial result on the localization and function for BACE but the question remains open whether all of APP is not digested by BACE in distal Golgi membrane, ER, endosome or plasma membrane."  View all comments by Hiroshi Mori |
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Comments on Related Papers |
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Related Paper: Endoplasmic reticulum and trans-Golgi network generate distinct populations of Alzheimer beta-amyloid peptides.
Comment by: Hiroshi Mori, ARF Advisor
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Two amyloid species (Abeta40 and Abeta42/43) are enerated at TGN and ER, respectively. The question is how they change to an insoluble form in brain tissue in an age-dependent and pathological manner. View all comments by Hiroshi Mori |
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Related Paper: Stimulation of beta-amyloid precursor protein trafficking by insulin reduces intraneuronal beta-amyloid and requires mitogen-activated protein kinase signaling.
Comment by: Dean Hartley
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More glucose, I mean, more food for thought. The role of insulin in the brain is still emerging and this interesting paper by Gasparini and colleagues presents a novel mechanism by which insulin affects the trafficking of A-beta. They find that insulin, through a tyrosine kinase/MAP kinase kinase pathway, accelerates the APP and A-beta trafficking from the trans-Golgi network to the plasma membrane, but not by generating more A-beta through APP cleavage. This data suggests that alterations of insulin signaling can influence A-beta distribution, which may turn out to be very interesting based on the observations that insulin and A-beta are competing for similar receptors and degrading enzymes. In addition to this novel role, they add to the AD controversy concerning how brain cells might be injured by A-beta by suggesting that a decrease in insulin signaling may enhance the intracellular accumulation of A-beta, which may occur in the AD or aging brain. Even though there is no direct evidence showing intrace. View all comments by Dean Hartley |
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Related Paper: Stimulation of beta-amyloid precursor protein trafficking by insulin reduces intraneuronal beta-amyloid and requires mitogen-activated protein kinase signaling.
Comment by: Gunnar K. Gouras
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We appreciate the analysis/comments of our paper by Dean Hartley, but want to stress that this paper is on the effect of insulin on Aß trafficking/degradation and signal transduction regulation thereof, and does not directly assess the role of intracellular versus extracellular Aß toxicity in AD pathogenesis. Aß toxicity via intracellular and/or extracellular Aß protofibrils was beyond the scope of this paper. We regret not to have cited the very interesting work from his group on Aß protofibrils (among the 55 references we cited). We completely agree with Dean Hartley that the potential role of intraneuronal Aß accumulation in AD pathogenesis is intriguing and requires further investigation. - comments by Gunnar Gouras and Huaxi Xu View all comments by Gunnar K. Gouras |
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REAGENTS/MATERIAL:
SHEP cells, Neuro-2a cells or HEK-293 cells.
Antibodies against g-adaptin, EEA1, GM130 and syntaxin 6 were purchased from Transduction Laboratory (Lexington, KY);
antibody against p58 was purchased from Sigma (St. Louis, MO)
antibodies 22C11 and anti-HA (clone 3F10) were purchased from Roche (Indianapolis, IN)
antibodies 4G8 and 6E10 were purchased from Senetek PLC (St. Louis, MO).
For expressing GFP chimeric proteins, PCR amplified products were inserted in frame between BamHI and EcoRI sites within the vector pEGFP-N3 (Clontech, Palo Alto, CA).
For the other expression constructs, the mammalian expression pCDNA3.1 vector (Invitrogen, Carlsband, CA) was used.
By using the GCG Peplot program, four peptides were selected and synthesized for producing peptide antibodies.
B278 which corresponds to the sequence YLRPVEDVATSQD from 366-378;
B279 which corresponds to the sequence LRLPKKVFEAAVKSIK from 295-310;
B280 which corresponds to the sequence DDSLEPFFDSLVKQTHV from 191-207
B690 which corresponds to the sequence CLRQQHDDFADDISLLK from 485-501 of BACE1 protein
They were individually conjugated with keyhole limpet hemocyanin (KLH) via a cysteine residue according to the instructions provided (Pierce, Rockford, IL).
The protocol for generating the peptide antibody and injection of conjugated peptides into rabbits was conducted by Covance (Denver, PA).
FUTURE DIRECTION:
Since transmembrane domain is necessary for BACE1 activity in vivo and that while the catalytic domains may confer substrate specificity, the transmembrane domain is essential for BACE1 to access its natural substrate, APP, the mechanisms for regulating retention of membrane proteins in specific organelles needs further elucidation. It is possible that TM domain contains targeting signals and/or may be important for membrane partitioning and oligomerization.
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