Monitoring "de novo" APP synthesis by taking advantage of the reversible effect of cycloheximide

Abstract

By blocking "de novo" protein synthesis using cycloheximide, we previously described a dynamic model system to monitor turnover of a specific population of the Alzheimer's amyloid precursor protein. Here we show that cycloheximide is nontoxic and its effect is reversible, allowing protein synthesis to reinitiate. Upon cycloheximide removal protein synthesis restarted and by 1 hour the amyloid precursor protein- green fluorescent protein could be clearly detected, permitting the monitoring of amyloid precursor protein anterograde transport, particularly the secretory pathway. The consensus NPTY motif in amyloid precursor protein, typically associated with endocytosis, was mutated to NPTF or NPTE to mimic a constitutively dephosphorylated or phosphorylated residue, respectively. Our data reveal that disruption of this motif affects amyloid precursor protein endocytosis, as shown previously, but also its incorporation into trans-Golgi network budding vesicles. Thus, cycloheximide can be a useful tool to study both anterograde and retrograde "in vivo" protein transport. © 2009 Sage Publications.