Uncovering a new model of intracellular processing of the amyloid precursor protein: Towards a test of the amyloid hypothesis

The deposition of amyloid-beta peptides (Abeta) in senile plaques (SPs) is a central pathological feature of Alzheimer's disease (AD). Abeta is produced by proteolytic cleavage of the amyloid precursor protein (APP) by activities termed beta- and gamma-secretase. The goals of this dissertation are to develop insights into the intracellular pathways that lead to the production of Abeta from APP. The studies described here are driven by the hypothesis that perturbations in APP processing that increase Abeta production promote the accumulation of SPs in the AD brain and that SPs initiate an inflammatory and neurotoxic cascade leading to neuronal death. Testing of this hypothesis requires identification of the intracellular processing pathways for Abeta production, determination of the regulation of these pathways, understanding of the mechanisms that underlie plaque formation, and methods to inhibit and monitor plaque formation in vivo. Towards these ends, this dissertation provides evidence that Abeta1-42, the major constituent of SPs, is produced in the endoplasmic reticulum and accumulates over time in an insoluble form that may serve as a nidus for plaque generation. Moreover, we found that the exclusive production of Abeta1-42 in the endoplasmic reticulum results from the expression of a distinct gamma-secretase in this organelle. We demonstrate that this gamma-secretase competes with the proteasome for cleavage of APP carboxyl-terminal fragments. In addition, we examined the mechanism by which protein kinase-C (PKC) activation increases the non-amyloidogenic alpha-secretase cleavage of APP. We found that PKC activation increases alpha-secretase cleavage not by changing APP trafficking but rather by activating specific metalloproteases responsible for alpha-secretase activity. Significantly, these proteases are localized in the trans-Golgi network, another known site of Abeta generation. This colocalization results in competition between non-amyloidogenic (alpha-secretase) and amyloidogenic (beta-secretase) processing of APP. Finally, we generated an amyloid-binding probe (BSB) and showed that it is specific and sensitive for SPs and that it crosses the blood-brain barrier. BSB detected SPs in a mouse model of amyloidogenesis and may therefore be useful for the diagnosis and evaluation of AD patients. The preceding studies further our understanding of intracellular processing of APP, and provide data useful for the generation of specific inhibitors of Abeta production. The combined use of inhibitors of Abeta production with radiological monitoring of plaque deposition in AD patients will allow testing of the links between Abeta production, plaque burden, and clinical dementia.