Beta-secretase cleavage represents the first step in the generation of Abeta polypeptides and initiates the amyloid cascade that leads to neurodegeneration in Alzheimer's disease. By comparative Western blot analysis, we show a 2.7-fold increase in protein expression of the beta-secretase enzyme BACE in the brain cortex of Alzheimer's disease patients as compared to age-matched controls. Similarly, we found the levels of the amyloid precursor protein C-terminal fragment produced by beta-secretase to be increased by nearly twofold in Alzheimer's disease cortex.

Biometals play an important role in Alzheimer disease, and recent reports have described the development of potential therapeutic agents based on modulation of metal bioavailability. The metal ligand clioquinol (CQ) has shown promising results in animal models and small phase clinical trials; however, the actual mode of action in vivo has not been determined. We now report a novel effect of CQ on amyloid ␤-peptide (A␤) metabolism in cell culture. Treatment of Chinese hamster ovary cells overexpressing amyloid precursor protein with CQ and Cu 2؉ or Zn 2؉ resulted in an ϳ85-90% reduction of secreted A␤-(1-40) and A␤-(1-42) compared with untreated controls. Analogous effects were seen in amyloid precursor protein-overexpressing neuroblastoma cells. The secreted A␤ was rapidly degraded through up-regulation of matrix metalloprotease (MMP)-2 and MMP-3 after addition of CQ and Cu 2؉ . MMP activity was increased through activation of phosphoinositol 3-kinase and JNK. CQ and Cu 2؉ also promoted phosphorylation of glycogen synthase kinase-3, and this potentiated activation of JNK and loss of A␤-(1-40). Our findings identify an alternative mechanism of action for CQ in the reduction of A␤ deposition in the brains of CQ-treated animals and potentially in Alzheimer disease patients. Alzheimer disease (AD)4 is characterized by progressive neuronal dysfunction, reactive gliosis, and the formation of amyloid plaques in the brain. The major constituent of AD plaques is the amyloid ␤-peptide (A␤), which is cleaved from the membrane-bound amyloid precursor protein (APP) (1). Aggregated or oligomeric A␤ can induce neurotoxicity through pathways involving free radical production and increased neuronal oxidative stress (2). Among the factors capable of promoting A␤ aggregation in vivo, recent evidence supports a central role for biometals such as Cu 2ϩ and Zn 2ϩ in this process (3). An important factor in controlling A␤ accumulation in AD patients is the activity of A␤-degrading enzymes. Recent studies have identified several candidate proteases that may contribute to catabolism of A␤ in the brain. Neprilysin, insulin-degrading enzyme, angiotensin-converting enzyme, and matrix metalloproteases (MMPs) have all demonstrated A␤-degrading activity in vitro and/or in vivo (4 -6). Reduced activity of these or other A␤-degrading proteases with age may play a role in promoting accumulation and deposition of A␤ in AD patients. Development of strategies to enhance clearance of A␤ may lead to novel therapeutic treatments for AD patients.Promoting A␤ clearance may be achieved through modulating metal sequestration or metal-protein interactions. 5-Chloro-7-iodo-8-hydroxyquinoline or clioquinol (CQ), a disused antibiotic, has received considerable attention as a potential metal ligand in AD and Parkinson disease patients (7-9). Preliminary studies revealed that CQ rapidly and potently dissolved aggregates of synthetic or AD brain-derived A␤ in vitro (10). In subsequent animal studies, a 9-week oral treatment with CQ resulted in a 49% reduction of...

Proteolytic processing of the transmembrane domain of the amyloid precursor protein (APP) is a key component of Alzheimer's disease pathogenesis. Using C-terminally tagged APP derivatives, we have identified by amino-terminal sequencing a novel cleavage site of APP, at Leu-49, distal to the gamma-secretase site. This was termed -cleavage. Brefeldin A treatment and pulse-chase experiments indicate that this cleavage occurs late in the secretory pathway. The level of -cleavage is decreased by expression of presenilin-1 mutants known to impair Abeta formation, and it is sensitive to the gamma-secretase inhibitors MDL28170 and L-685,458. Remarkably, it shares similarities with site 3 cleavage of Notch-1: membrane topology, cleavage before a valine, dependence on presenilins, and inhibition profile.

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Amyloid precursor protein (APP) processing is of major interest in Alzheimer's disease research, since sequential cleavages by ␤-and ␥-secretase lead to the formation of the 4-kDa amyloid A␤ protein peptide that accumulates in Alzheimer's disease brain. The processing of APP involves proteolytic conversion by different secretases leading to ␣-, ␤-, ␥-, ␦-, and ⑀-cleavages. Since modulation of these cleavages represents a rational therapeutic approach to control amyloid formation, its interference with the processing of the members of the APP gene family is of considerable importance. By using C-terminally tagged constructs of APLP-1 and APLP-2 and the untagged proteins, we have characterized their proteolytic C-terminal fragments produced in stably transfected SH-SY5Y cells. Pharmacological manipulation with specific protease inhibitors revealed that both homologues are processed by ␣-and ␥-secretase-like cleavages, and that their intracellular domains can be released by cleavage at ⑀-sites. APLP-2 processing appears to be the most elaborate and to involve alternative cleavage sites. We show that APLP-1 is the only member of the APP gene family for which processing can be influenced by N-glycosylation. Additionally, we were able to detect p3-like fragments of APLP-1 and p3-like and A␤-like fragments of APLP-2 in the media of stably transfected SH-SY5Y cells.

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The b-amyloid protein (Ab) is derived by proteolytic processing of the amyloid protein precursor (APP). Cleavage of APP by b-secretase generates a C-terminal fragment (APPCTFb), which is subsequently cleaved by g-secretase to produce Ab. The aim of this study was to examine the cleavage of APP-CTFb by g-secretase in primary cortical neurons from transgenic mice engineered to express the human APP-CTFb sequence. Neurons were prepared from transgenic mouse cortex and proteins labelled by incubation with [ Approximately 30% of the human APP-CTFb (hAPPCTFb) was converted to human Ab (hAb), which was rapidly secreted. The remaining 70% of the hAPP-CTFb was degraded by an alternative pathway. The cleavage of hAPP-CTFb to produce hAb was inhibited by specific g-secretase inhibitors. However, treatment with proteasome inhibitors caused an increase in both hAPP-CTFb and hAb 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-CTFb is degraded by two distinct pathways, one involving g-secretase, which produces Ab, and a second major pathway involving direct cleavage of APP-CTFb within the cytoplasmic domain by the proteasome. These results raise the possibility that defective proteasome function could lead to an increase in Ab production in the AD brain.

Mutations in the presenilin genes are associated with early onset familial Alzheimer's disease and lead to increased accumulation of beta A4 peptide, the proteolytic product of the amyloid precursor protein (APP). To test whether presenilins interfere with APP metabolism, presenilin-2 (PS2) was coexpressed with APP in mammalian cells. Analysis of PS2 immunoprecipitates revealed that a fraction of APP was associated with the PS2 immunocomplexes. This non-covalent association was specific for the APP family of proteins and restricted to immature forms, occurring probably during transit through the endoplasmic reticulum. Additionally, coexpression with PS2 resulted in a decrease of APP secretion, suggesting a direct participation of presenilins in the intracellular sorting, trafficking and processing of APP molecules.