Amyloid precursor protein of Alzheimer's disease: evidence for a stable, full-length, trans-membrane pool in primary neuronal cultures.
We and colleagues have shown that the amyloid protein precursor of Alzheimer's disease (APP) is distributed along the surface of neurites of fixed but nonpermeabilized neurons in primary culture in a segmental pattern, which shows 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 experiments. Total cellular APP has previously been shown to have a short half-life of approximately 30-60 min. We confirm this in neurons in primary culture in pulse-chase experiments using short labelling times. Additionally, we provide evidence that a separate, stable pool of neuronal APP can be demonstrated in pulse-chase experiments using long labelling times. Experiments involving inhibition of protein synthesis suggest that this corresponds with the surface, segmental pool. Metabolic labelling followed by surface biotinylation and two-stage precipitation demonstrates that the surface APP is trans-membrane and full-length (not carboxyl-terminal truncated), and confirms that the surface APP belongs to the stable pool. This two-stage procedure 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 consistent with a role for APP in stable cell-matrix or cell-cell interactions.
Storey
E
E
Van Cleet/Roet Centre for Nervous Diseases and Department of Neuroscience, Monash University (Alfred Hospital), Prahran, Australia. Elsdon.storey@med.monash.edu.au
Katz
M
M
Brickman
Y
Y
Beyreuther
K
K
Masters
C L
CL
D000544:Alzheimer Disease / Q000378:metabolism*; D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D000818:Animals; D019921:Biotinylation; D001923:Brain Chemistry / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D018604:Epitope Mapping; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009474:Neurons / Q000737:chemistry* / Q000166:cytology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013462:Sulfur Radioisotopes
D016564:Amyloid beta-Protein Precursor; D013462:Sulfur Radioisotopes