Amyloid precursor protein of Alzheimer's disease: evidence for a stable, full-length, trans-membrane pool in primary neuronal cultures

We and others have shown that the amyloid protein precursor of Alzheimer's disease (APP) is distributed along the surface of neurites of fixed but non permeabilized neurons in primary culture in a segmental pattern, which show s colocalization with some markers of adhesion patches. This is in contrast to the diffuse pattern of immunoreactivity seen after permeabilization. We have also recently demonstrated that the APP in these surface, patches is likely to be integral to the membrane rather than secreted and re-adsorbed, based on alkali stripping experiments and on soluble APP adsorption experi ments. Total cellular APP has previously been shown to have a short half-li fe of approximate to 30-60 min. We confirm this in neurons in primary cultu re in pulse-chase experiments using short labelling times. Additionally, we provide evidence that a separate, stable pool of neuronal APP can be demon strated in pulse-chase experiments using long labelling times. Experiments involving inhibition of protein synthesis suggest that this corresponds wit h the surface, segmental pool. Metabolic labelling followed by surface biot inylation and two-stage precipitation demonstrates that the surface APP is trans-membrane and full-length (not carboxyl-terminal truncated), and confi rms that the surface APP belongs to the stable pool. This two-stage procedu re is necessary as the surface APP appears to be present in low copy number , and is difficult to detect by direct labelling. This information is consi stent with a role for APP in stable cell-matrix or cell-cell interactions.