The cellular prion protein (PrP c ) is physiologically cleaved in the middle of its 106 -126 amino acid neurotoxic region at the 110/ 1112112 peptidyl bond, yielding an N-terminal fragment referred to as N1. We recently demonstrated that two disintegrins, namely ADAM10 and ADAM17 (TACE, tumor necrosis factor alpha converting enzyme) participated in both constitutive and protein kinase C-regulated generation of N1, respectively. These proteolytic events were strikingly reminiscent of those involved in the socalled "␣-secretase pathway" that leads to the production of secreted sAPP␣ from ␤APP. We show here, by transient and stable transfection analyses, that ADAM9 also participates in the constitutive secretion of N1 in HEK293 cells, TSM1 neurons, and mouse fibroblasts. Decreasing endogenous ADAM9 expression by an antisense approach drastically reduces both N1 and sAPP␣ recoveries. However, we establish that ADAM9 was unable to increase N1 and sAPP␣ productions after transient transfection in fibroblasts depleted of ADAM10. Accordingly, ADAM9 is unable to cleave a fluorimetric substrate of membrane-bound ␣-secretase activity in ADAM10 ؊/؊ fibroblasts. However, we establish that co-expression of ADAM9 and ADAM10 in ADAM10-deficient fibroblasts leads to enhanced membrane-bound and released fluorimetric substrate hydrolyzing activity when compared with that observed after ADAM10 cDNA transfection alone in ADAM10 ؊/؊ cells. Interestingly, we demonstrate that shedded ADAM10 displays the ability to cleave endogenous PrP c in fibroblasts. Altogether, these data provide evidence that ADAM9 is an important regulator of the physiological processing of PrP c and ␤APP but that this enzyme acts indirectly, likely by contributing to the shedding of ADAM10. ADAM9 could therefore represent, besides ADAM10, another potential therapeutic target to enhance the breakdown of the 106 -126 and A␤ toxic domains of the prion and ␤APP proteins.Spongiform encephalopathies are fatal neurodegenerative diseases involving a highly protease-resistant protein referred to as prion scrapie (PrP sc ) 4 (1, 2). Human prion diseases include, among others, Creutzfeldt-Jakob disease, familial transmissible spongiform encephalopathies, new variant of Creutzfeldt-Jakob disease, as well as a few cases of iatrogenic transmission (3-5). PrP sc corresponds to the abnormal conformation of an ubiquitous glycosylphosphatidylinositol-anchored protein called cellular prion (PrP c ) that is mainly synthesized in the central nervous system (6, 7). The mechanisms by which PrP c is converted into PrP sc are not fully understood. However, it appears clearly that inoculation of PrP sc triggers pathology and ultimately cell death in mice only when PrP c is present in the host animal. Thus, PrP c null mice resist infection and toxicity exhibited by scrapie-enriched inoculates (8 -10). On the other hand, it appears that depletion of PrP c is relatively innocuous (11,12). Therefore, all strategies aimed at lowering endogenous PrP c are potential therapeutic approaches. Clas...

We have set up stably transfected HEK293 cells overexpressing the ␤-secretases BACE1 and BACE2 either alone or in combination with wild-type ␤-amyloid precursor protein (␤APP). The characterization of the ␤APP-derived catabolites indicates that cells expressing BACEs produce less genuine A␤1-40/42 but higher amounts of secreted sAPP␤ and N-terminal-truncated A␤ 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) ␤APP 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 ␤-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 ␣-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 IC 50 in the micromolar range, some of which displaying selectivity for either BACE1 or BACE2.

1 The b-secretase BACE1 is one of the enzymes that contribute to the production of the Ab peptide, in vitro and in vivo. JMV1195 was previously shown to inhibit BACE activity in vitro but was unable to block cellular BACE activity. We have designed a new permeable inhibitor, JMV2764 that corresponds to a derivative of JMV1195 to which a penetratin sequence had been added at its N-terminus. We have assessed the ability of JMV2764 to affect BACE1 activity in vitro, and to modify Ab production in various cell systems. 2 Endogenous b-secretase or BACE1 activities were monitored in vitro by means of two distinct fluorimetric substrates in HEK293 extracts of cells expressing either wild-type bAPP, Swedish mutated bAPP or SPA4CT constructs. Ab40 recovery was monitored by immunoprecipitation and Western blot analysis. 3 JMV2764 and JMV1195 inhibited endogenous b-secretase activity of HEK293 cellular homogenates with IC 50 s of 0.8 and 6.6 mM, respectively. Interestingly, JMV2764 also inhibited bsecretase activity after preincubation with intact cells while JMV1195 was inactive, indicating that unlike JMV1195, JMV2764 could penetrate into the cells. 4 JMV2764 but not JMV1195 also prevented Ab production by HEK293 cells overexpressing wildtype and Swedish-mutated bAPP. However, JMV2764 was unable to affect Ab production from cells expressing SPA4CT, a bAPP-derived sequence that does not need b-secretase to produce Ab. 5 Altogether, we have designed a new cell-permeable BACE1 inhibitor that allows to envision to prevent Ab production in vivo. Work is in progress to assess the potential of these compounds to prevent Ab production in transgenic mice overproducing Ab.