Journal of Neurochemistry, 2001, 78, 1153±1161
The caspase-derived C-terminal fragment of bAPP induces caspase-independent toxicity and triggers selective increase of Ab42 in mammalian cells CeÂcile Dumanchin-Njock,* Cristine Alves da Costa,* Luc Mercken,² Laurent Pradier² and FreÂdeÂric Checler* *Institut de Pharmacologie MoleÂculaire et Cellulaire, CNRS, Universite de Nice-Sophia Antipolis, Valbonne, France ²Aventis Pharma, Quai Jules Guesde, Vitry sur Seine, France
Abstract During its physiopathological maturation, the b-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 Ab40 and Ab42, two highly aggregable bAPP-derived fragments generated by combined cleavages by b- and g-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 bAPP 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 staurosporineinduced 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 Ab42 but not Ab40 production by HEK293 cells expressing wild-type bAPP751. Altogether, our data demonstrate that C31 induces a caspase-independent toxicity in TSM1 neurons and potentiates the pathogenic bAPP maturation pathway by increasing selectively Ab42 species in wild type-bAPP-expressing human cells. Keywords: Alzheimer's disease, amyloid, b-amyloid precursor protein, caspases, HEK293 cells TSM1 neurons. J. Neurochem. (2001) 78, 1153±1161.
One of the main histological hallmarks observed in Alzheimer's disease-affected brains is senile plaque (for a review see Selkoe 1997), an extracellular deposit mainly composed of a set of highly insoluble peptides of various lengths (39±43 amino acids) referred to as b-amyloid peptides (Ab). These peptides derive from the combined action of b- and g-secretases, two sets of enzymes responsible for the generation of the N- and C-termini of Ab, respectively (for a review see Haass and Selkoe 1993; Checler 1995; Maury 1995; Octave 1995). Small amounts of Ab could be physiologically produced by various cell types, but the exacerbation of the b/g-secretases-mediated `amyloidogenic pathway' likely contributes to the complex etiology of Alzheimer's disease (Hardy and Higgins 1992). Recently, an alternative bAPP-hydrolysing pathway was reported that involves another set of activities called
caspases (Barnes et al. 1998; Gervais et al. 1999; LeBlanc et al. 1999; Pellegrini et al. 1999; Weidemann et al. 1999; Lu et al. 2000). Caspases belong to a still growing family of thiol proteases involved at various stages in the programmed cell death (for a review see Schwartz and Milligan 1996; Patel et al. 1996). Several studies independently demonstrated that bAPP was proteolysed at its C-terminal cytoplasmic domain (Gervais et al. 1999; Pellegrini et al.
Received April 19, 2001; revised manuscript received June 25, 2001; accepted June 29, 2001. Address correspondence and reprint requests to FreÂdeÂric Checler, IPMC du CNRS, UMR6097, 660 Route des Lucioles, 06560, Valbonne, France. E-mail:
[email protected] Abbreviations used: Ab, b-amyloid peptides; PARP, poly-(ADPribose)-polymerase; PI, propidium iodide.
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1999; Weidemann et al. 1999; LeBlanc et al. 1999; Lu et al. 2000), that bears a classical consensus target sequence for caspases, leading to the formation of a 31 amino-acids peptide referred to thereafter as C31. This cleavage was abolished by caspase inhibitors (Gervais et al. 1999; LeBlanc et al. 1999; Pellegrini et al. 1999) or mutational approach (Weidemann et al. 1999). Furthermore, various recombinant (Gervais et al. 1999; LeBlanc et al. 1999; Pellegrini et al. 1999) or overexpressed (Lu et al. 2000) caspases including caspases 3, 6 and 8 elicit identical bAPP breakdown. Interestingly, a network of immunohistochemical and cell biology studies suggest that apoptosis could at least contribute to Alzheimer's disease neuropathology (Cotman and Anderson 1995; Mattson et al. 1998) and that caspase expressions appear up-regulated in the brains of affected patients (Gervais et al. 1999; Lu et al. 2000). In line with this hypothesis, it was reported that apoptotic stimuli could both potentiate caspase-mediated bAPP hydrolysis and trigger enhanced production of Ab (LeBlanc et al. 1999). Although a link between apoptotic cell response and caspase-mediated bAPP cleavage was clearly suggested, the putative modulation of apoptotic cellular effectors by C31 and the ability of this peptide to control directly Ab40 or Ab42 productions remained to be ®rmly established. Here we show that C31 overexpression drastically lowers viabilities of TSM1 neurons and to a lesser extent that of HEK293 cells. However, hydrolysis of caspase ¯uorimetric substrate and pro-caspase-3 immunoreactivity indicate that the C31-mediated toxic effect was independent of caspase-3 like activities. Furthermore, the overexpression of C31 does not modify several intracellular intermediates usually recruited during apoptotic cell response such as Bax and p53 expressions, poly-(ADP-ribose)-polymerase (PARP) cleavages and cytochrome c translocation. However, we demonstrate that overexpression of C31 leads to selective increase of secreted Ab42 without affecting Ab40 in cells overexpressing wild-type bAPP. Altogether, our data suggest that C31 production is a caspase-mediated cellular response to apoptotic stimuli but does not per se participate to apoptosis. However, C31 could contribute to a more general cellular toxicity mechanism associated with a selective increase in the production of the Ab42 pathogenic species. Materials and methods HEK293 and TSM1 culture and transient transfection Stably transfected HEK293 cells overexpressing wild-type bAPP751 were obtained and cultured as previously described (Ancolio et al. 1999). The TSM1 neuronal cells were cultured and transiently transfected as previously detailed (Alves da Costa et al. 2000) with 0.5 or 1 mg of empty pSV2 or pSV2-C31 expressing vectors (construct provided by Aventis Pharma, Vitry sur Seine, France).
Measurements of total Ab or Ab40 and Ab42 Stably transfected HEK293 cells overexpressing wild typebAPP751 were transiently transfected with empty pSV2 or pSV2-C31 vectors. Medium was changed 48 h after transfection then cells were incubated for 7 h in the presence of phosphoramidon (Sigma, St Louis, MO, USA). Media were collected, diluted in 1/10 RIPA 10X buffer (10 mm Tris, pH 7.5 containing 150 mm NaCl, 5 mm EDTA, 0.1% SDS and 1% Nonidet P40) and incubated overnight with a 200-fold dilution of FCA18 (total Ab). Ab42 and Ab40 was performed by sequential immunoprecipitation as described (Ancolio et al. 1999) with fully speci®c and characterized FCA3542 and FCA3340 antibodies, respectively (used at a 200-fold dilution; Barelli et al. 1997). After further incubation for 3 h with protein A-sepharose (Zymed, San Francisco, CA, USA) and centrifugation, pelleted proteins were submitted to 16,5% Tris-tricine gels then western-blotted on hybond C membranes (Amersham Life Science, Piscataway, NJ, USA) for 45 min Nitrocellulose sheets were heated in boiling PBS for 5 min and capped with 5% skim milk in PBS containing 0.05% Tween 20 for 1 h. Membranes were then incubated overnight with WO2 antibody (speci®c for the 5±8 N-terminal region of Ab; Ida et al. 1996) and detected by enhanced chemoluminescence. Immunoprecipitation of sAPPa Conditioned media from wild type-bAPP751-expressing HEK293 cells transiently transfected with empty or C31-encoding pSV2 vectors were incubated with a 700-fold dilution of 207 antibody (recognizes the N-terminus of bAPP and sAPPa) in the presence of pansorbin (Calbiochem, San Diego, CA, USA) as described previously (Marambaud et al. 1997). After centrifugation, pellets were resuspended with loading buffer submitted to a 8% TrisGlycine electrophoresis and western blotted for 2 h. Nitrocellulose membranes were exposed overnight to a 500-fold dilution of the monoclonal 10D5C antibody and revealed with anti-mouse IgGrabbit antibodies coupled to peroxydase as described (Lopez-Perez et al. 1999). Caspase activity Forty-eight hours after transient transfection, TSM1 cells were cultured in 6-well plates as described (Alves da Costa et al. 2000) and then incubated for 2 h at 378C in the presence or absence of 1.0 mm of staurosporine (Sigma). In some cases cells were preincubated with 100 mm Ac-DEVD-al (caspase inhibitor; Neosystem, Strasbourg, France) for 24 h before stimulation. Cells were then rinsed, gently scraped, pelleted by centrifugation, and then resuspended in 40 mL of lysis buffer (25 mm HEPES, pH 7.5, containing 5 mm MgCl2 and dithiothreitol, 2 mm 4-(2-aminoethyl)benzenesulfonyl ¯uoride, 10 mg/mL pepstatin A and leupeptin). Cell lysates were submitted to 2 freezing/thawing cycles and then centrifuged (16 000 g for 5 min). Caspase activity of supernatants (10 mL, 50±100 mg of proteins) was measured in 96-well plates as follows. A 100-mL reaction mixtures containing water, 32 mL of reaction buffer (312 mm HEPES, pH 7.5, 31.25% sucrose, 0.31% Chaps), 10 mL of 100 mm dithiothreitol, and 2 mL of Me2SO were incubated for 1 h with 2 mL of 2.5 mm Ac-DEVD-AMC (caspase substrate, Neosystem). When cells were not pre-incubated 24 h with 100 mm Ac-DEVD-al before staurosporine stimulus, caspase assays were performed in the presence and absence of 2 mL of Ac-DEVD-al (2.5 mm; pre-incubated for 30 min at 378C).
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Fluorimetry was recorded at 360 and 460 nm for excitation and emission wavelenghts, respectively, by means of a microtiter plate reader (Floroskan II; Labsystems, Helsinki, Finland). Caspasespeci®c activity was calculated from the linear part of ¯uorimetry recording and expressed in arbitrary units/ h/mg of proteins (established by the Bio-rad procedure). One arbitrary unit corresponds to 4 nmol of AMC released. Trypan blue exclusion The viability of TSM1 neurons was determined by the measurement of their capacity of exclude the vital dye trypan blue. Brie¯y, TSM1 cells were cultured in 12-well plates and incubated for 2 h at 378C in the presence or absence of 1.0 mm of staurosporine. Then, cells were rinsed, gently scraped, pelleted by centrifugation, resuspended in 500 mL of culture medium containing 0.1% of trypan blue, loaded into a haemocytometer and examined by light microscopy. Viable and non-viable (blue) cells were then counted and the score obtained expressed as the percentage of viable mock-transfected cells. Cytochrome c translocation analysis Mock- and C31-transfected TSM1 cells were grown in a 6.5% CO2 atmosphere in 6-well plates and incubated for 2 h at 378C in the presence or absence of 1.0 mm of staurosporine. Cells were harvested, pelleted by centrifugation at 1000 g for 10 min at 48C, lysed in 1 mL of buffer (HEPES-NaOH 20 mm, pH 7.5, 10 mm NaCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol, 1 mm 4-(2-aminoethyl)benzenesulfonyl ¯uoride containing 250 mm of sucrose) and homogenized with a Dounce homogenizer (100 strokes/300 rpm). Homogenates were then centrifuged at 750 g for 10 min at 48C for recovery of the nuclear fraction followed by a centrifugation at 10 000 g for 15 min for obtention of the mitochondrial fraction. The mitochondrial pellets were resuspended in 25±50 mL of the hypotonic buffer and the supernatants further centrifuged at 100 000 g for 1 h at 48C for obtention of the cytosolic fractions. Both fractions (cytosolic and mitochondrial) were then submitted to western blot analysis by means Tris-Tricine gels as previously described (Ancolio et al. 1999). In brief, 25 mg of proteins were separated on 16.5 Tris-Tricine gels, imobilized in nitrocellulose sheets and probed with an anti-cytochrome c (rabbit polyclonal IgG, H104, Santa Cruz Technologies, Santa Cruz, CA, USA) antibody. Immunological complexes were revealed with a goat anti-rabbit IgG-coupled to peroxidase (Immunotech, Marseille, France) and Enhanced chemoluminescence. Flow cytometry analysis Mock- and C31-transfected TSM1 cells were grown in 12-well plates and incubated for 24 h at 378C in the presence or absence of 0.5 mm of staurosporine. Cells were harvested, pelleted by centrifugation at 1000 g for 10 min at 48C, gently resuspended in 500 mL of 0.1% sodium citrate buffer containing 50 mg/mL propidium iodide (PI) and incubated overnight at 48C. The PI ¯uorescence of individual nuclei was measured using a FACS scan ¯ow cytometer (program CellQuest; Becton Dickinson, Franklin Lakes, NJ, USA). Red ¯uorescence due to PI staining of DNA was expressed on a logarithmic scale simultaneously to the forward scatter of the particles. Two hundred thousand events were counted on the scatter gate. All measurements were performed under identical conditions. This technique allows the discrimination of populations of fragmented nuclei from debris and non-viable cells
and also from diploid nuclei that show higher ¯uorescence staining. The number of apoptotic nuclei is expressed as a percentage of the total number of events. Western blot analyses Equal amounts of protein (50 mg) were separated on 12% and 8% SDS-PAGE gels for the detection of p53, active CPP32, Bax and PARP, respectively, western blotted as above and incubated overnight with the following primary antibodies: anti-p53 (mouse monoclonal, Pab 1801; Santa Cruz Biotechnologies), antiProCPP32 (mouse monoclonal IgG1, clone 46; Transduction Laboratories, Lexington, KY, USA), anti-Bax (rabbit polyclonal IgG, Upstate Biotechnology, Lake Placid, NY, USA), and antiPARP (rabbit polyclonal IgG, Upstate Biotechnology). Immunological complexes were revealed either with an anti-rabbit peroxidase (Immunotech) or anti-mouse peroxidase (Amersham Life Science) antibodies depending on the host used for obtention of the primary antibodies above described, followed by electrochemoluminescence (Amersham Pharmacia Biotech). Measurements of bgal activity TSM1 neurons were transiently cotransfected with various amounts of pSV2-encoding b-gal or C31. Forty-eight hours after transfection, b-galactosidase activity was measured using the b-galactosidase Enzyme Assay System according to the manufacturer's recommendations (Promega, Madison, WI, USA). Absorbance was monitored at 420 nm. It should be noted that an identical absorbance (optical density 0.54) was observed when 1 mg of b-galactosidase cDNA was transfected alone or in combination with 1 or 2 mg of empty pSV2 or C31-encoding cDNAs, allowing us to determine the amount of C31 cDNA that would `saturate' TSM1 neurons expression program in subsequent experiments of transient transfection. Statistical analysis Statistical analysis were performed with PRISM software (Graphpad Software, San Diego, CA, USA) using the Newman Keuls multiple comparison test for one way ANOVA. Protein concentrations Protein concentrations were established by the Bio-rad method as previously described (Bradford 1976).
Results The effect of transient transfection of pSV2-C31 or empty vector on the viability of TSM1 neurons is presented in the Fig. 1(a). Cells transfected with 0.5 mg of empty vector exhibit about 80% of viable cells (taken as 100%), a percentage that does not vary according to the transfected dose of construct (Fig. 1a). It should be noted here that basal cell morbidity (20% in the present study) closely agrees with the 22% of cell death observed by Lu et al. after transfection of empty vector in N2a cells (Lu et al. 2000). In contrast, C31 transfection elicits a dose-dependent decrease in TSM1 cell viability, yielding 75±85% of non-viable cells at a 1-mg dose of cDNA (Fig. 1a). Flow cytometry analysis (FACS) indicates that in basal conditions, PI incorporation is only
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Fig. 2 Basal and staurosporine-induced caspase-3-like activity in mock- and C31-transfected TSM1 neuronal cells. TSM1 neurons were transiently transfected with empty pSV2 (M) or pSV2-C31 (C31) vectors. Forty-eight hours after transfection, cells were incubated for 2 h at 378C without (Basal) or with (STS) 1 mM of staurosporine, then caspase activity was assayed as detailed in Materials and methods. Bars correspond to the Ac-DEVD-al-sensitive-Ac-DEVD-7AMC-hydrolysing activity and are the means ^ SEM of duplicate determinations of 4±5 independent determinations.
Fig. 1 Viability and ¯ow cytometry analysis of Mock- or C31-transfected TSM1 neurons. In panel (a), TSM1 neurons were transiently transfected with the indicated quantities of empty pSV2 vector (M) or pSV2 encoding C31 (C31). Forty-eight hours after transfection, cell viability was determined by Trypan blue exclusion as detailed in Materials and methods and is expressed as the percentage of control (taken as 100) corresponding to cell viability of mock-transfected cells. Bars are the means ^ SEM of six independent counts. NS, non-statistically signi®cant. Panel (b) represents cells staining with propidium iodide (PI) analysed as described in Materials and methods. Each diagram represents the graphical integration of 200,000 nuclei. Percentages of nuclei presented fragmented DNA are indicated.
slightly increased in cells expressing C31 when compared with controls (Fig. 1b, left panel). We examined whether the cell toxicity associated with C31 overexpression was related with some of the cellular effectors mediating `classical' apoptotic response in both basal and staurosporine-stimulated conditions. First, we measured Ac-DEVD-al-sensitive caspase-3-like activity in TSM1 cells. All experimental conditions for measuring such an activity in TSM1 neurons were carefully designed in a previous work where both basal and staurosporine-induced activation of caspase-3 activity were monitored (Alves da Costa et al. 2000). Mock-transfected and C31-expressing cells exhibit an identical basal caspase activity (Fig. 2). This activity was enhanced by staurosporine, but remained virtually identical in both cell lines (Fig. 2). Accordingly,
Fig. 3 PARP, active CPP32, Bax and p53 immunoreactivities in Mock- and C31-transfected TSM1 neurons. TSM1 neurons were transiently transfected with empty pSV2 vector (M) or pSV2 encoding C31 (C31). Forty-eight hours after transfection, cells were incubated for 2 h at 378C without (B) or with (STS) 1 mM of staurosporine, then PARP (a), CPP32 (b), Bax (c) and p53 (d) immuno-reactivities were monitored as detailed in Materials and methods. Bars correspond to the densitometric analyses measured in basal (B) or STS conditions of the indicated proteins expressed as the percentage (taken as 100) of densitometry recorded for Mock±transfected cells in basal conditions and are the means ^ SEM of 3±4 experiments.
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Fig. 4 Effect of C31 overexpression on cytochrome c translocation in TSM1 neurons. TSM1 neurons were transiently transfected with empty pSV2 (M) or pSV2-C31 (C31) vectors. Forty-eight hours after transfection, cells were incubated for 2 h at 378C without (Basal) or with (STS) 1 mM of staurosporine, then cytochrome c-like immunoreactivities in mitochondria and cytosol fractions were monitored as detailed in Materials and methods. Bars correspond to means (^ SEM of 4 independent experiments) of densitometric analysis expressed as the ratio of mitochondrial/cytosol cytochrome c immunoreactivity.
basal and staurosporine-stimulated conditions led to similar pro-caspase 3 and active caspase-3 immunoreactivities in mock-transfected and C31-expressing cells (Fig. 3b). Examination of the immunoreactivities of PARP and its staurosporine-induced proteolytic product (Fig. 3a), Bax (Fig. 3c) and p53 (Fig. 3d), all led to identical patterns in both cell lines. Finally, we measured cytochrome c translocation into the cytosol in response to C31. Figure 4(a) shows that in basal conditions, most of the cytochrome c immunoreactivity was associated with the mitochondrial pellet and that identical label was observed in the cytosol of mock-transfected and C31-expressing TSM1 neurons. Upon staurosporine stimulation, a clear increase in the cytosolic cytochrome c immunoreactivity was observed that was identical whatever the cell type examined (Fig. 4a). Indeed, the ratio of mitochondrial vs. cytosolic immunoreactivities of the cytochrome c remained very similar in both mockand C31-transfected cells (Fig. 4b). We also examined whether the expression of C31 could alter bAPP physiopathological maturation. As Ab is a very minor endogenous catabolite, we used stably transfected HEK293 cells overexpressing wild-type bAPP751 because these cells secrete easily measurable quantities of Ab (Ancolio et al. 1999). As TSM1 neurons, the viability of HEK293 cells is affected by C31 overexpression, although to a lesser extent (data not shown). Our experiments indicate
that expression of C31 does not apparently affect the recovery of total Ab (Figs 5b and d). It should be noted that C31 does not affect bAPP expression (Fig. 5a) and does not modulate the secretion of sAPPa (Fig. 5c and d), the a-secretase-derived product of bAPP maturation. However, as Ab42 is a minor Ab species that usually accounts for about 10% of total Ab, it was not possible to exclude that a modulation of Ab42 production by C31 expression could not be phenotypically re¯ected by total Ab measurements. In this context, we took advantage of fully speci®c antibodies directed towards the C-terminus of Ab (Barelli et al. 1997) to delineate the relative contribution of Ab40 and Ab42 to total Ab recovered. Our experiments indicate that expression of C31 does not apparently affect the recovery of total Ab40 (Fig. 6b) but induces a statistically signi®cant selective increase (about 35% above control value) of secreted Ab42 (Figs 6a and c). We previously documented the fact that the mixed inhibitor Z-IE(Ot-Bu)A-leucinal potentiated the recovery of Ab through the blockade of the proteasome-mediated degradation of presenilins (Marambaud et al. 1998). A recent paper also documented the fact that pepstatin could increase the recovery of Ab from cells expressing wild-type bAPP (GruÈninger-Leitch et al. 2000). We took this opportunity to enhance the recovery of Ab42 and examine the in¯uence of C31 overexpression. When added during secretion time, the cell permeant Z-IE(Ot-Bu)A-leucinal proteasome inhibitor and pepstatin potentiated the Ab42 recovery from mock-transfected bAPP-expressing HEK293 cells (Fig. 7a). This enhanced production of Ab42 was even higher when inhibitors were pre-incubated with cells and left
Fig. 5 Effect of C31 overexpression on total Ab and sAPPa generated by bAPP751-HEK293 expressing cells. Stably transfected HEK293 cells expressing wild type-bAPP751 were obtained as described in Materials and methods and transiently transfected with empty pSV2 (M) or pSV2-C31 (C31) vectors. After forty-eight hours, cells were rinsed and allowed to secrete for further 7 h at 378C. Secretion media were monitored for their total Ab (b) or sAPPa (c) by combined immunoprecipitation and western blot analysis as described in Materials and methods. Full length bAPP751 (a) in cell lysates was monitored with WO2 as described. Bars in (d) correspond to the densitometric analysis of Ab and sAPPa secreted by C31-expressing HEK293 cells (means ^ SEM of 5 independent determinations) and are expressed as the percentage of control (secretions observed with mock-transfected cells).
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Fig. 6 Effect of C31 overexpression on Ab40 and Ab42 generated by bAPP751-HEK293 expressing cells. HEK293 cells expressing wild type-bAPP751 were obtained and transfected with empty (M) or C31-encoding cDNA (C31). Secretion media were monitored for their Ab42 (a) and Ab40 (b) content by sequential immunoprecipitation using FCA3542 and FCA3340 antibodies and western blot with WO2 as described in the methods. In panels (a) and (b), two typical experiments are shown. Bars are the mean densitometric analysis (^ SEM of 5 independent experiments). Controls taken as 100 correspond to Ab42 and Ab40 recovered with mock-transfected bAPP751-expressing HEK293 cells (white bars).
during secretion time (Fig. 7a). Interestingly, C31 expression further increased Ab42 recovery in all experimental conditions (Fig. 7b). The Z-IE(Ot-Bu)A-leucinal also enhanced Ab40 recovery but to a similar extent in mockand C31-transfected cells (not shown). Altogether, these data con®rm the selective increase in Ab42 triggered by C31 in bAPP-expressing HEK293 cells.
Discussion Several studies reported on the alternative enzymatic attack of bAPP by secretases-independent activities called caspases (Gervais et al. 1999; LeBlanc et al. 1999; Pellegrini et al. 1999; Weidemann et al. 1999; Lu et al. 2000). Although the characterization of the caspase candidate involved in bAPP processing is still a matter of discussion, consensual data led to the observation that bAPP was cleaved at its C-terminal intracytoplasmic tail and more particularly, after the aspartyl residue of a canonical caspase-substrate sequence. This leads to a 31 amino acidC-terminal APP fragment and its N-terminal counterpart referred to as NcasAPP (Pellegrini et al. 1999) APPDC31 (Lu et al. 2000) or DC-APP (Gervais et al. 1999). Activation of caspases is a general phenomenon implying a cascade of events ultimately leading to cell death (Cohen 1997). This activation can be triggered by a series of stimuli
Fig. 7 Effect of inhibitors on C31-induced Ab42 production by bAPP751-HEK293 expressing cells. HEK293 cells expressing wild type-bAPP751 were obtained and transfected with empty pSV2 (M) or pSV2-C31 (C31) vectors as in Fig. 6. Cells were pre-incubated for 12 h and allowed to secrete for further 7 h at 378C. Z-IE(OtBu)A-leucinal and pepstatin (10 mM) were added either during secretion or during both secretion and pre-incubation periods. `None' condition corresponds to control carried out with adequate DMSO concentrations. Ab42 was monitored as in Fig. 6 with FCA3542 (panel a). Bars in (b) correspond to the densitometric analysis of Ab42 (one typical experiment) expressed as the percentage of control Ab42 recovered in identical conditions with mock-transfected cells.
(for a review see Henderson 1996; Los et al. 2000) and as expected, most of these stimuli indeed increase caspasemediated bAPP-cleavage. As a previous work indicated that wild-type, but not mutant bAPP, could confer protection against p53-mediated apoptosis (Xu et al. 1999), caspase cleavage could be seen as a cell mean to abolish wild-type bAPP-mediated inhibitory control of cell death. Another study however, suggested that bAPP was pro-apoptotic when overexpressed in neuronal cells (Lu et al. 2000). In this case, the possibility that bAPP could behave as a precursor of C31 that would be liberated upon caspase attack and able to act as a cell `toxin' could be envisaged. Whether C31 production could be linked to, or responsible for, a selective increased production of Ab, thereby modulating cell responsiveness to apoptotic stimuli remained questionable. In the present study, we show that the transient transfection of various amounts of cDNA encoding C31 lowered TSM1 neuronal cell viability. It should be noted
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that our efforts to visualize C31 immunoreactivity was unsuccessful, even after employing several immunoprecipitating antibodies or a cocktail of protease inhibitors although the ef®ciency of transfection was undoubtful as established by b-gal analysis after co-transfection experiments. This indicates that C31 is either extremely short-lived in TSM1 cells or rapidly interacts with sequestrating cellular protein partners. These observations are consistent with the fact that the bAPP C-terminal fragments derived from g-secretase (C57) or caspases (C31) cleavages are usually not recovered from cell lysates or brain homogenates (Pinnix et al. 2001). This also explains why authors examining the functional in¯uence of caspase-mediated bAPP cleavage generally employed the N-terminal counterpart of C31 (Gervais et al. 1999; Lu et al. 2000). However, this fragment remains embedded in the membrane while C31 is theoretically released in the cytosol where its pharmacological spectrum could be distinct from that of its N-terminal counterpart. It should be noted that our co-transfections and quanti®cations with b-gal encoding cDNA (see Methods and materials) together with the fact that the effect of C31 on cell viability was dose-dependent and particularly drastic at a 1-mg dose (about 80 ^ 3% of decrease in cell viability while corresponding amounts of empty vector was ineffective) indicates that C31 transfection was indeed very ef®cient. TSM1 are neuronal cells from neocortical origin that are particularly useful to study apoptotic response. This cell system was used previously in our laboratory to evidence a control of basal and agonist-stimulated apoptotic responses by wild-type and mutated a-synucleins (Alves da Costa et al. 2000). Here we con®rm that TSM1 cells exhibit a basal Ac-DEVD-al-sensitive caspase-3-like activity that is enhanced by staurosporine (Fig. 2). However, expression of C31 does not modify basal and staurosporine-induced caspase activity and did not affect pro-caspase3 conversion into active enzyme (Figs 2 and 3b). As the caspase-3 activation is a common ®nal step of a complex molecular cascades, we examined whether C31 could modulate other cellular effectors. Thus, PARP cleavage, Bax and p53 immunoreactivities (Figs 3a, c and d) as well as cytochrome c translocation into the cytosol (Fig. 4) remained unchanged upon C31 expression in both basal and staurosporinestimulated conditions. These data clearly suggest that C31-induced decrease of TSM1 viability (Fig. 1a) is not directly linked to caspase-mediated activation of apoptotic response. More likely, C31 behaves as a agent triggering accute toxic insult thereby lowering cell viability as that occuring during necrosis. Several lines of evidences have indicated that Ab production could be modulated by apoptotic stimuli (LeBlanc 1995). Furthermore, Gervais et al. showed that neuronal NT2 cells undergoing apoptosis after serum deprivation display substantially higher amounts of Ab, the production of which appeared sensitive to caspase
inhibitors (Gervais et al. 1999). More directly, these authors show that overexpressing DC-APP led to increased recovery of Ab (x-40). It remained unclear as to whether C31 itself could modulate Ab recovery (instead of its membraneembedded N-terminal counterpart DC-APP) and if so, whether this effect could be selective or not of one given Ab species. The HEK293 cells overexpressing wild-type bAPP751 were used to assess the in¯uence of C31 on bAPP physiopathological maturation because genuine and N-terminally intact endogenous Ab is poorly recovered in TSM1 cells. Importantly, HEK293 cells also display lower viability upon C31 overexpression. C31 does not modify bAPP expression and did not affect sAPPa recovery indicating that its expression did not alter constitutive secretory process. Total Ab recovery was not modi®ed but careful examination of Ab40 and Ab42 productions indicated that C31 selectively increased the recovery of the latter. As Ab42 accounts for about 10% of total Ab and as there was a 35±40% increase, this explains why no change was detectable in total Ab recovery. To further con®rm the C31-stimulatory effect on Ab42 production, we took advantage of our data showing that proteasome inhibitors, by preventing presenilins degradation (Marambaud et al. 1998) and thereby exacerbating their associated phenotypes, led to increased Ab recovery in various cell lines (for a review see Checler et al. 1999). As would have been expected from the above observations, inhibitors enhanced the recovery of Ab42 that was further potentiated upon C31 overexpression. Altogether, our data indicate that the caspase-derived bAPP fragment C31 triggers selective increase of Ab42 and induce cell toxicity through caspase-independent cellular response. It is interesting to underline the fact that several studies demonstrated that DC-APP (and therefore likely its C-terminal counterpart C31) was drastically increased in Alzheimer's disease brain and that this was accompanied by an increased caspase-like immunoreactivity. Furthermore, recent studies indicated that in Alzheimer's disease brains, neurons show increased intracellular Ab42 or x-42 labelling (Gouras et al. 2000; Russo et al. 2000) that is frequently associated with apoptosis stigmata (Chui et al. 2001). Our data here suggest that Ab42 is more likely concommittant to apoptosis in neurons rather than directly responsible for the observed cell death. A general scheme could be that upon various apoptotic stimuli, caspase activation leads to enhanced bAPP cleavage, thereby liberating substantially higher amounts of C31 mediating selective Ab42 increase responsible for subsequent non-apoptotic cellular insult. The mechanism by which C31 triggers selective Ab42 increase is obviously of interest but still remains a matter of speculation. It was reported that several proteins interact with the C-terminus of bAPP and more particularly at the level of the 759±762 (APP770 numbering) NPTY sequence
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of bAPP (Chow et al. 1996; GueÂnette et al. 1996; Zambrano et al. 1997; Sastre et al. 1998; Yang et al. 1998). It is thought that such interaction increases bAPP stability, modify its traf®cking and leads to altered production of Ab (Borg et al. 1998; Yang et al. 1998; GueÂnette et al. 1999; Sabo et al. 1999; Mueller et al. 2000). This is the case for proteins of the X11 family that were shown to lower the production of Ab. In this context, it is easy to envision that caspase cleavage, occuring at the C-terminus of bAPP prevents these interactions, and thereby, indirectly abolishes the inhibitory tonus on Ab production. Alternatively the generation of C31 could be seen as a dominant negative regulatory mechanism. C31 that corresponds to the bAPP 740±770 (so that includes the NPTY sequence) would compete for X11 binding to bAPP, leading to increased Ab42 recovery. It should be noted that such an endogenous regulation has been evidence in the case of X11 by Lee et al. (2000) who reported on the control of X11 activity by an X11-binding protein called XB51. This speculative model of a C31/X11 interaction still remains to be ®rmly demonstrated because X11 lowers both Ab40/42 while C31 only augments Ab42. However, one can not exclude the occurence of other unknown protein partners of bAPP, more selectively involved in the production of Ab42 (for instance by routing bAPP to a cell compartment permissive for Ab42-generating secretase), that would be displaced by C31. Altogether, our data strongly suggest that a stategy aimed at preserving the bAPP cytoplasmic tail could be seen, beside others, as a putative way to down regulate Ab42 production in Alzheimer's disease. Acknowledgements We are indebted to Stephanie Hughes for initiating us to FACS analysis. We wish to acknowledge Drs T. Hartmann and K. Beyreuther (Heidelberg, Germany) for WO2. Drs M. Savage and D. Schenk are thanked for giving us 207 and 10D5C antibodies, respectively. CDN is the recipient of a Fellowship from the association `France Alzheimer et troubles apparenteÂs'. CAC is supported by Aventis Pharma. This work was supported by the CNRS and INSERM.
References Alves da Costa C., Ancolio K. and Checler F. (2000) Wild-type but not Parkinson's disease-related Ala53Thr-a-synuclein protect neuronal cells from apoptotic stimuli. J. Biol. Chem. 275, 24065±24069. Ancolio K., Dumanchin C., Barelli H., Warter J. M., Brice A., Campion D., FreÂbourg T. and Checler F. (1999) Unusual phenotypic alteration of b amyloid precursor protein (bAPP) maturation by a new Val-.Met bAPP-770 mutation responsible for probable early-onset Alzheimer's disease. Proc. Natl Acad. Sci. USA 96, 4119±4124. Barelli H., Lebeau A., Vizzavona J., Delaere P., Chevallier N., Drouot C., Marambaud P., Ancolio K., Buxbaum J., Khorkova
O., Heroux J., Sahasrabudhe S., Martinez J., Warter J.-M., Mohr M. and Checler F. (1997) Characterization of new polyclonal antibodies speci®c for 40 and 42 amino acids-long amyloid b peptides: their use to examine the cell biology of presenilins and the immunohistochemistry of sporadic Alzheimers' disease and cerebral amyloid angiopathy cases. Mol. Med. 3, 695±707. Barnes N. Y., Li L., Yoshikawa K., Schwartz L. M., Oppenheim R. W. and Milligan C. E. (1998) Increased production of amyloid precursor protein provides a substrate for caspase-3 in dying motoneurons. J. Neurosci. 18, 5869±5880. Borg J.-P., Yang Y., De Taddeo-Borg M., Margolis B. and Turner R. S. (1998) The X11a protein slows cellular amyloid precursor protein processing and reduces Ab40 and Ab42 secretion. J. Biol. Chem. 273, 14761±14766. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248±259. Checler F. (1995) Processing of the b-amyloid precursor protein and its regulation in Alzheimer's disease. J. Neurochem. 65, 1431±1444. Checler F., Alves da Costa C., Ancolio K., Lopez-Perez E. and Marambaud P. (1999) Role of the Proteasome in Alzheimer's disease. Biochim. Biophys. Acta 1502, 133±138. Chow N., Korenberg J. R., Chen X.-N. and Neve R. L. (1996) APP-BP1, a novel protein that binds to the carboxy-terminal region of the amyloid precursor protein. J. Biol. Chem. 271, 11339±11346. Chui D.-H., Dobo E., Makifuchi T., Akiyama H., Kawakatsu S., Petit A., Checler F., Araki W., Takahashi K. and Tabira T. (2001) Apoptotic neurons in Alzheimer's disease frequently show intracellular Ab42 labelling. J. Alzheimer Dis. 3, 231±249. Cohen G. M. (1997) Caspases: the executioners of apoptosis J. Biochem. 326, 1±16. Cotman C. W. and Anderson A. J. (1995) A potential role for apoptosis in neurodegeneration and Alzheimer's disease. Mol. Neurobiol. 10, 19±45. Gervais F. G., Xu D., Robertson G. S., Vaillancourt J. P., Zhu Y., Huang J., LeBlanc A., Smith D., Rigby M., Shearman M. S., Clarke E. E., Zheng H., Van Der Ploeg L. H. T., Ruffolo S. C., Thornberry N. A., Xanthoudakis S., Zamboni R. J., Roy S. and Nicholson D. W. (1999) Involvement of caspases in proteolytic cleavage of Alzheimer's amyloid b precursor protein and amyloidogenic Ab peptide formation. Cell 97, 395±406. Gouras G. K., Tsai J., Naslund J., Vincent B., Edgar M., Checler F., Green®eld J. P., Haroutunian V., Buxbaum J. D., Xu H., Greenghard P. and Relkin N. R. (2000) Intraneuronal Ab42 accumulmation in human brain. Am. J. Pathol. 156, 15±20. GruÈninger-Leitch F., Berndt P., Langen H., Nelboeck P. and DoÈbeli H. (2000) Identi®cation of b-secretase-like activity using a mass spectrometry-based assay system. Nat. Biotech. 18, 66±70. GueÂnette S. Y., Chen J., Jondro P. D. and Tanzi R. E. (1996) Association of a novel human FE65-like protein with the cytoplasmic domain of the b-amyloid precursor protein. Proc. Natl Acad. Sci. USA 93, 10832±10837. GueÂnette S., Chen J., Ferland A., Haass C., Capell A. and Tanzi R. (1999) hFE65L in¯uences amyloid protein maturation and secretion. J. Neurochem. 73, 985±993. Haass C. and Selkoe D. J. (1993) Cellular processing of b-amyloid precursor protein and the genesis of amyloid b-peptide. Cell 75, 1039±1042. Hardy J. A. and Higgins G. A. (1992) Alzheimer's disease: the amyloid cascade hypothesis. Science 256, 184±185. Henderson C. E. (1996) Programmed cell death in the developing nervous system. Neuron 17, 579±585. Ida N., Johannes H., Pantel J., SchroÈder J., Zerfass R., FoÈrstl H.,
q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 1153±1161
C31-mediated toxicity and Ab42 product 1161
Sandbrink R., Masters C. L. and Beyreuther K. (1996) Analysis of heterogeneous bA4 peptides in human cerebrospinal ¯uid and blood by a newly developed sensitive western blot assay. J. Biol. Chem. 271, 22908±22914. LeBlanc A. (1995) Increased production of a 4kDa amyloid b peptide in serum deprived human primary neuron cultures: possible involvement of apoptosis. J. Neurosci. 15, 7837±7846. LeBlanc A., Liu H., Goodyer C., Bergeron C. and Hammond J. (1999) Caspase-6 role in apoptosis of human neurons, amyloidogenesis, and Alzheimer's disease. J. Biol. Chem. 274, 23426±23436. Lee D.-S., Tomita S., Kirino Y. and Suzuki T. (2000) Regulation of X11L-dependent amyloid precursor protein metabolism by XB51, a novel X11L-binding protein. J. Biol. Chem. 275, 23134±23138. Lopez-Perez E., Seidah N. and Checler F. (1999) A proprotein convertase activity contributes to the processing of the Alzheimer's b-amyloid precursor protein in human cells: evidence for a role of the prohormone convertase PC7 in the constitutive a-secretase pathway. J. Neurochem. 73, 2056±2062. Los M., Wesselborg S. and Schulze-Osthoff K. (2000) The role of caspases in development, immunity, and apoptotic signal transduction: Lessons from knockout mice. Immunity 10, 629±639. Lu D. C., Rabizadeh S., Chandra S., Shayya R. F., Ellerby L. M. YeX., Salvesen G. S., Koo E. H. and Bredesen D. E. (2000) A second cytotoxic proteolytic peptide derived from amyloid b-protein precursor. Nat. Med. 6, 397±404. Marambaud P., Lopez-Perez E., Wilk S. and Checler F. (1997) Constitutive and protein kinase C-regulated secretory cleavage of Alzheimer's b amyloid precursor protein: different control of early and late events by the proteasome. J. Neurochem. 69, 2500±2505. Marambaud P., Ancolio K., Lopez-Perez E. and Checler F. (1998) Proteasome inhibitors prevent the degradation of familial Alzheimer's disease-linked presenilin 1 and trigger increased Ab42 secretion by human cells. Mol. Med. 4, 147±157. Mattson M. P., Guo Q., Furukawa K. and Pedersen W. A. (1998) Presenilins, the endoplasmic reticulum, and neuronal apoptosis in Alzheimer's disease. J. Neurochem. 70, 1±14. Maury C. P. J. (1995) Molecular pathogenesis of b-amyloidosis in Alzheimer's disease and other cerebral amyloidoses Lab. Invest. 72, 4±16. Mueller H. T., Borg J.-P., Margolis B. and Turner R. S. (2000) Modulation of amyloid precursor protein metabolism by X11a/ Mint-1. A deletion analysis of protein±protein interaction domains. J. Biol. Chem. 275, 39302±39306. Octave J. N. (1995) The amyloid peptide and its precursor in Alzheimer's disease. Rev. In Neurosci. 6, 287±316.
Patel T., Gores G. and Kaufman S. H. (1996) The role of proteases during apoptosis. FASEB 10, 587±597. Pellegrini L., Passer B. J., Tabaton M., Ganjei J. K. and D'Adamio L. (1999) Alternative, non-secretase processing of Alzheimer's b-amyloid precursor protein during apoptosis by caspase-6 and-8. J. Biol. Chem. 274, 21011±21016. Pinnix I., Musunuru U., Tun H., Sridharan A., Golde T., Eckman C., Ziani-Cherif C., Onstead L. and Sambamurti K. (2001) A novel g-secretase assay based on detection of the putative C-terminal fragment-g of amyloid b protein precursor. J. Biol. Chem. 276, 481±487. Russo C., Schettini G., Saido T. C., Hulette C., Lippa C., Lannfelt L., Ghetti B., Gambetti P., Tabaton M. and Teller J. K. (2000) Presenilin-1 mutations in Alzheimer's disease. Nature 405, 531±532. Sabo S. L., Lanier L. M., Ikin A. F., Khorkova O., Sahasrabudhe S., Greengard P. and Buxbaum J. D. (1999) Regulation of b-amyloid secretion by FE65, an amyloid protein precursor-binding protein. J. Biol. Chem. 274, 7952±7957. Sastre M., Turner R. S. and Levy E. (1998) X11 interaction with b-amyloid precursor protein modulates its cellular stabilization and reduces amyloid b-protein secretion. J. Biol. Chem. 273, 22351±22357. Schwartz L. M. and Milligan C. E. (1996) Cold thoughts of death: the role of ICE proteases in neuronal cell death. Trends Neurosci. 19, 555±562. Selkoe D. J. (1997) Alzheimer's disease: Genotypes, phenotypes and treatments. Science 275, 630±631. Weidemann A., Paliga K., DuÈrrwang U., Reinhard F. B. M., Schuckert O., Evin G. and Masters C. L. (1999) Proteolytic processing of the Alzheimer's disease amyloid precursor protein within its cytoplasmic domain by caspase-like proteases. J. Biol. Chem. 274, 5823±5829. Xu X., Yang D., Wyss-Coray T., Yan J., Gan L. and Mucke L. (1999) Wild-type but not alzheimer-mutant amyloid precursor protein confers resistance against p53-mediated apoptosis. Proc. Natl Acad. Sci. USA 22, 7547±7552. Yang Y., Turner S. and Gaut J. R. (1998) The chaperone BipP/GRP78 binds to amyloid precurosr protein and decrease Ab40 and Ab42 secretion. J. Biol. Chem. 273, 25552±25555. Zambrano N., Buxbaum J. D., Minopoli G., Fiore F., De Candia P., De Renzis S., Faraonio R., Sabo S., Cheetham J., Sudol M. and Russo T. (1997) Interaction of the phosphotyrosine interaction/phosphotyrosine binding-related domains of Fe65 with wild-type and mutant Alzheimer's b-amyloid precursor protein. J. Biol. Chem. 272, 6399±6405.
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