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During its physiopathological maturation, the β-amyloid precursor protein undergoes several distinct proteolytic events by activities called secretases. In Alzheimer's disease, the main histological hallmark called senile plaque is clearly linked to the overproduction of the amyloid peptides Aβ40 and Aβ42, two highly aggregable βAPP-derived fragments generated by combined cleavages by β- and γ-secretases. Recently, an alternative hydrolytic pathway was described, involving another category of proteolytic activities called caspases, responsible for the production of a 31 amino acids βAPP C-terminal fragment called C31. C31 was reported to lower the viability of N2a cells but the exact mechanisms mediating C31-toxicity remained to be established. Here we show that the transient transfection of pSV2 vector encoding C31 lowers by about 80% TSM1 neuronal cells viability. Arguing against a C31-stimulated apoptotic response, we demonstrate by combined enzymatic and immunological approaches that C31 expression did not modulate basal or staurosporine-induced caspase 3-like activity and pro-caspase-3 activation. Furthermore, C31 did not modify Bax and p53 expressions, poly-(ADP-ribose)-polymerase cleavage and cytochrome c translocation into the cytosol. However, we established that C31 overexpression triggers selective increase of Aβ42 but not Aβ40 production by HEK293 cells expressing wild-type βAPP751. Altogether, our data demonstrate that C31 induces a caspase-independent toxicity in TSM1 neurons and potentiates the pathogenic βAPP maturation pathway by increasing selectively Aβ42 species in wild type-βAPP-expressing human cells. [ABSTRACT FROM AUTHOR]

gamma-Secretase activity is involved in the generation of Abeta and therefore likely contributes to the pathology of Alzheimer's disease. Blocking this activity was seen as a major therapeutic target to slow down or arrest Abeta-related AD progression. This strategy seemed more doubtful when it was established that gamma-secretase also targets other substrates including Notch, a particularly important transmembrane protein involved in vital functions, at both embryonic and adulthood stages. We have described previously new non-peptidic inhibitors able to selectively inhibit Abeta cellular production in vitro without altering Notch pathway. We show here that in vivo, these inhibitors do not alter the Notch pathway responsible for somitogenesis in the zebrafish embryo. In addition, we document further the selectivity of JLK inhibitors by showing that, unlike other described gamma-secretase inhibitors, these agents do not affect E-cadherin processing. Finally, we establish that JLKs do not inhibit beta-site APP cleaving enzymes (BACE) 1 and BACE2, alpha-secretase, the proteasome, and GSK3beta kinase. Altogether, JLK inhibitors are the sole agents to date that are able to prevent Abeta production without triggering unwanted cleavages of other proteins., (Copyright 2003 Wiley-Liss, Inc.)

We have set up stably transfected HEK293 cells overexpressing the beta-secretases BACE1 and BACE2 either alone or in combination with wild-type beta-amyloid precursor protein (betaAPP). The characterization of the betaAPP-derived catabolites indicates that cells expressing BACEs produce less genuine Abeta1- 40/42 but higher amounts of secreted sAPPbeta and N-terminal-truncated Abeta species. This was accompanied by a concomitant modulation of the C-terminal counterpart products C89 and C79 for BACE1 and BACE2, respectively. These cells were used to set up a novel BACE assay based on two quenched fluorimetric substrates mimicking the wild-type (JMV2235) and Swedish-mutated (JMV2236) betaAPP sequences targeted by BACE activities. We show that BACEs activities are enhanced by the Swedish mutation and maximal at pH 4.5. The specificity of this double assay for genuine beta-secretase activity was demonstrated by means of cathepsin D, a "false positive" BACE candidate. Thus, cathepsin D was unable to cleave preferentially the JMV2236-mutated substrate. The selectivity of the assay was also emphasized by the lack of JMV cleavage triggered by other "secretases" candidates such as ADAM10 (A disintegrin and metalloprotease 10), tumor necrosis alpha-converting enzyme, and presenilins 1 and 2. Finally, the assay was used to screen for putative in vitro BACE inhibitors. We identified a series of statine-derived sequences that dose-dependently inhibited BACE1 and BACE2 activities with IC50 in the micromolar range, some of which displaying selectivity for either BACE1 or BACE2.