A novel approach for studying endogenous abeta processing using cultured primary neurons isolated from APP transgenic mice.
The central component of senile amyloid plaques in Alzheimer's disease (AD) is the beta-amyloid peptide (Abeta), derived from proteolytic processing of the amyloid precursor protein (APP). In this study, we developed an in vitro model to measure and identify soluble Abeta from primary cortical neurons. Neurons were isolated from mice transgenic for human APP695 containing the K670N, M671L double mutation. We characterized soluble Abeta using Western blot and ELISA assays. We found that the Abeta levels in conditioned media from these neurons were readily detectable and almost five times higher than in CSF. The majority of Abeta in the media was Abeta1-40; however, Abeta1-42 was also detectable. When the neurons were exposed to Phorbol 12-myristate 13-acetate (PMA), alpha1-antichymotrypsin, or alpha1-antitrypsin, the alterations of soluble Abeta levels were consistent with other models reported. Most importantly, the soluble Abeta in our model was remarkably stable, and aliquots were unchanged after prolonged incubations or repeated freeze/thaw cycles. The Abeta appeared to be monomeric by Western blot analysis. Soluble Abeta coimmunoprecipitated with endogenous mouse apolipoprotein E from the primary cultures. Taken together, our data demonstrated that using a Western blot assay to detect soluble Abeta from transgenic mouse overexpressing APP695 is sensitive, specific, and reliable and provides an accessible model for examining the neuronal metabolism of APP and Abeta.
Qiu
Z
Z
Alzheimer Research Unit, Massachusetts General Hospital, Boston, MA 02129, USA.
Naten
D L
DL
Liston
J C
JC
Yess
J
J
Rebeck
G W
GW
D016229:Amyloid beta-Peptides / Q000032:analysis / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000737:chemistry; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012680:Sensitivity and Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000514:alpha 1-Antichymotrypsin / Q000494:pharmacology; D000515:alpha 1-Antitrypsin / Q000494:pharmacology
D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D017077:Culture Media, Conditioned; D000514:alpha 1-Antichymotrypsin; D000515:alpha 1-Antitrypsin; D013755:Tetradecanoylphorbol Acetate