The two proteases ␤-secretase and ␥-secretase generate the amyloid ␤ peptide and are drug targets for Alzheimer's disease. Here we tested the possibility of targeting the cellular environment of ␤-secretase cleavage instead of the ␤-secretase enzyme itself. ␤-Secretase has an acidic pH optimum and cleaves the amyloid precursor protein in the acidic endosomes. We identified two drugs, bepridil and amiodarone, that are weak bases and are in clinical use as calcium antagonists. Independently of their calcium-blocking activity, both compounds mildly raised the membrane-proximal, endosomal pH and inhibited ␤-secretase cleavage at therapeutically achievable concentrations in cultured cells, in primary neurons, and in vivo in guinea pigs. This shows that an alkalinization of the cellular environment could be a novel therapeutic strategy to inhibit ␤-secretase. Surprisingly, bepridil and amiodarone also modulated ␥-secretase cleavage independently of endosomal alkalinization. Thus, both compounds act as dual modulators that simultaneously target ␤-and ␥-secretase through distinct molecular mechanisms. In addition to Alzheimer's disease, compounds with dual properties may also be useful for drug development targeting other membrane proteins.

ADAM proteases are type I transmembrane proteins with extracellular metalloprotease domains. As for most ADAM family members, ADAM8 (CD156a, MS2) is involved in ectodomain shedding of membrane proteins and is linked to inflammation and neurodegeneration. To identify potential substrates released under these pathologic conditions, we screened 10-mer peptides representing amino acid sequences from extracellular domains of various membrane proteins using the ProteaseSpotீ system. A soluble ADAM8 protease containing a pro-and metalloprotease domain was expressed in E. coli and purified as active protease owing to autocatalytic prodomain removal. From 34 peptides tested in the peptide cleavage assay, significant cleavage by soluble ADAM8 was observed for 14 peptides representing membrane proteins with functions in inflammation and neurodegeneration, among them the b-amyloid precursor protein (APP). The in vivo relevance of the ProteaseSpotீ method was confirmed by cleavage of full-length APP with ADAM8 in human embryonic kidney 293 cells expressing tagged APP. ADAM8 cleaved APP with similar efficiency as ADAM10, whereas the inactive ADAM8 mutant did not. Exchanging amino acids at defined positions in the cleavage sequence of myelin basic protein (MBP) revealed sequence criteria for ADAM8 cleavage. Taken together, the results allowed us to identify novel candidate substrates that could be cleaved by ADAM8 in vivo under pathologic conditions.

The link between cholesterol and Alzheimer's disease has recently been revealed in Niemann Pick type C disease. We found that NPC1-/- cells show decreased expression of APP at the cell surface and increased processing of APP through the β-secretase pathway resulting in increased C99, sAPPβ and intracellular Aβ40 levels. This effect is dependent on increased cholesterol levels, since cholesterol depletion reversed cell surface APP expression and lowered Aβ/C99 levels in NPC1-/- cells to the levels observed in wt cells. Finding that overexpression of C99, a direct γ-secretase substrate, does not lead to increased intracellular Aβ levels in NPC1-/- cells vs. CHOwt suggests that the effect on intracellular Aβ upon cholesterol accumulation in NPC1-/- cells is not due to increased APP cleavage by γ-secretase. Our results indicate that cholesterol may modulate APP processing indirectly by modulating APP expression at the cell surface and, thus, its cleavage by β-secretase.

ADAM proteases are type I transmembrane proteins with extracellular metalloprotease domains. Like most ADAM family members, ADAM8 (CD156a, MS2) is involved in ectodomain shedding of membrane proteins and is linked to neuroinflammation and neurodegeneration. To identify potential substrates released under these pathologic conditions, we screened 10-mer peptides representing amino acid sequences from extracellular domains of various membrane proteins using the ProteaseSpot™ system. A soluble ADAM8 protease containing pro-and metalloprotease domain was expressed in E. coli and purified as active protease due to autocatalytic prodomain removal. From 34 peptides tested in the peptide cleavage assay, significant cleavage by soluble ADAM8 was observed for 14 peptides representing membrane proteins with functions in inflammation and neurodegeneration, among them the beta amyloid precursor protein (APP) and the Tumor Necrosis Factor alpha Receptor type I (TNFRI). The in vivo relevance of the ProteaseSpot™ method was confirmed by cleavage of full length APP with ADAM8 in human embryonic kidney 293 cells expressing tagged APP. ADAM8 cleaved APP with similar efficiency as ADAM10, whereas inactive ADAM8 mutant did not. Cleavage of TNFRI was assessed in primary cell lines deficient in ADAM8 and revealed a gene dosage-dependent effect. Exchanging amino acids at defined positions in the cleavage sequence of myelin basic protein (MBP) revealed sequence criteria for ADAM8 cleavage. Taken together, we identified novel substrates that could be cleaved by ADAM8 in vivo under pathologic conditions.

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