A novel approach for studying endogenous A beta 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 (A beta), derived from proteolytic processing of the amyloid precursor protein (APP). In this study, we developed an in vitro model to measure and identify soluble A beta from primary cortical ne urons. Neurons were isolated from mice transgenic for human APP695 containi ng the K670N, M671L double mutation. We characterized soluble A beta using Western blot and ELISA assays. We found that the A beta levels in condition ed media from these neurons were readily detectable and almost five times h igher than in CSF. The majority of A beta in the media was A beta1-40; howe ver, A beta1-42 was also detectable. When the neurons were exposed to Phorb ol 12-myristate 13-acetate (PMA), alpha1-antichymotrypsin, or alpha1-antitr ypsin, the alterations of soluble A beta levels were consistent with other models reported. Most importantly, the soluble A beta in our model was rema rkably stable, and aliquots were unchanged after prolonged incubations or r epeated freeze/thaw cycles. The A beta appeared to be monomeric by Western blot analysis. Soluble A beta coimmunoprecipitated with endogenous mouse ap olipoprotein E from the primary cultures. Taken together, our data demonstr ated that using a Western blot assay to detect soluble A beta from transgen ic mouse overexpressing APP695 is sensitive, specific, and reliable and pro vides an accessible model for examining the neuronal metabolism of APP and A beta. (C) 2001 Academic Press.