The C-terminal fragment of the Alzheimer's disease amyloid protein precursor is degraded by a proteasome-dependent mechanism distinct from γ-secretase

The beta -amyloid protein (A beta) is derived by proteolytic processing of the amyloid protein precursor (APP). Cleavage of APP by beta -secretase generates a C-terminal fragment (APP-CTF beta), which is subsequently cleaved by gamma -secretase to produce A beta. The aim of this study was to examine the cleavage of APP-CTF beta by gamma -secretase in primary cortical neurons from transgenic mice engineered to express the human APP-CTF beta sequence. Neurons were prepared from transgenic mouse cortex and proteins labelled by incubation with [S-35]methionine and [S-35]cysteine. Labelled APP-CTF beta and A beta were then immunoprecipitated with a monoclonal antibody (WO2) specific for the transgene sequences. Approximately 30% of the human APP-CTF beta (hAPP-CTF beta) was converted to human A beta (hA beta), which was rapidly secreted. The remaining 70% of the hAPP-CTF beta was degraded by an alternative pathway. The cleavage of hAPP-CTF beta to produce hA beta was inhibited by specific gamma -secretase inhibitors. However, treatment with proteasome inhibitors caused an increase in both hAPP-CTF beta and hA beta levels, suggesting that the alternative pathway was proteasome-dependent. A preparation of recombinant 20S proteasome was found to cleave a recombinant cytoplasmic domain fragment of APP (APP(cyt)) directly. The study suggests that in primary cortical neurons, APP-CTF beta is degraded by two distinct pathways, one involving gamma -secretase, which produces A beta, and a second major pathway involving direct cleavage of APP-CTF beta within the cytoplasmic domain by the proteasome. These results raise the possibility that defective proteasome function could lead to an increase in A beta production in the AD brain.