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

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