- Original Articles
- Open Access
Proteasome Inhibitors Prevent the Degradation of Familial Alzheimer’s Disease-Linked Presenilin 1 and Potentiate Aβ42 Recovery from Human Cells
© Picower Institute Press 1998
- Accepted: 26 November 1997
- Published: 1 March 1998
Several lines of evidence suggest that most of the early-onset forms of familial Alzheimer’s disease (FAD) are due to inherited mutations borne by a chromosome 14–encoded protein, presenilin 1 (PS1). This is likely related to an increased production of amyloid β-peptide (A β)42, one of the main components of the extracellular deposits called senile plaques that invade human cortical areas during the disease.
Materials and Methods
We set up stably transfected HEK293 cells overexpressing wild-type (wt) and various FAD-linked mutated PS1. By Western blot analysis, we examined the influence of specific proteasome inhibitors on PS1-like immunoreactivities. Furthermore, by means of metabolic labeling and immunoprecipitation with A β40 and A β42-directed specific antibodies, we assessed the effect of the inhibitors on the production of A βs by wt and mutated PS1-expressing cells transiently transfected with βAPP751.
We show that two distinct proteasome inhibitors, Z-IE(Ot-Bu)A-Leucinal and lactacystin, increase in a time- and dose-dependent manner the immunoreactivities of both wt and mutated PS1. Furthermore, we demonstrate that PS1 is polyubiquitinated in these cells. Other inhibitors, ineffective on the proteasome, fail to protect wt and mutated PS1-like immunoreactivities. We also establish that the FAD-linked mutations of PS1 trigger a selective increased formation of Aβ42 as reflected by higher Aβ42 over total Aβ ratios when compared with wtPS1-expressing cells. Interestingly, this augmentation was further amplified by proteasome inhibitors in cells expressing mutated but not wtPS1.
Altogether, our data indicate that PS1 undergoes polyubiquitination in HEK293 cells and that the proteasome contributes to the degradation of wt and FAD-linked PS1, thereby directly influencing the Aβ production in human cells.
A network of independent studies has led to the suggestion that the presenilins (PS) likely contribute to the physiopathological maturation of the β amyloid precursor protein (βAPP). Thus, consensual data indicate that mutations on PS, which are responsible for agressive early-onset forms of Alzheimer’ s disease (AD), consistently lead to increased formation of the pathogenic amyloid β-peptide (A β)42 species. This was not only shown in the brains of affected patients (1–3) but also evidenced by means of transfected cells (4–7) and transgenic animal models (4,5,6,8). Interestingly, we recently showed that mutations on PS1 could also trigger decreased secretion of the physiological secreted product APPα (9). Although it is not yet demonstrated, it can be postulated that PS likely interfere with or control the routing of βAPP, as these two proteins often colocalize in the central nervous system (10–12) and are able to physically interact (13,14).
We previously established that the proteasome contributes to the α-secretase pathway. Thus, two proteasome inhibitors, lactacystin and Z-IE(Ot-Bu)A-Leucinal, increased constitutive APPα secretion by human cells. Furthermore, the proteasome appears to exert a dual control over protein kinase C (PKC)-regulated APPα secretion by human kidney (HEK)293 cells, as short-term treatment of these cells with proteasome inhibitors led to enhanced recovery of APPα whereas prolonged exposure of the cells triggered decreased APPα recovery (15,16). We established that PKC effectors modified neither the proteasome activity nor its basal phosphorylation state (17) in vitro and in vivo. This indicates that the proteasome does not correspond to α-secretase itself, and it suggests the likely occurrence of another intermediate involved in βAPP processing that should behave as a substrate of this multicatalytic complex.
We present here evidence that wild-type and familial Alzheimer’s disease (FAD)-linked PS1 behave as substrates of the proteasome in stably transfected HEK293 cells. Furthermore, we demonstrate that proteasome inhibitors modulate the production of Aβ secretion by these cells, and particularly, that they exacerbate the phenotypic overproduction of Aβ42 by cells overexpressing the mutated forms of PS1. Combined with our previous studies, these data suggest that the proteasome could control the intracellular levels of PS1, upstream to PS1 and βAPP interaction, thereby influencing the production of physiological and pathogenic catabolites of βAPP maturation.
Molecular Cloning of PS1 and Mutagenesis
PS1 was cloned as previously described (18). A polymerase chain reaction (PCR)-derived probe was obtained from a human kidney cDNA library constructed in the λZAPII vector. This PCR probe was used to screen the above cDNA library, leading to the isolation of four PS1 cDNA clones, the sequences of which correspond to full-length PS1. The Metl46Val, His163Arg, and Glu280Ala mutations (18) and the ΔE9-PS1 cDNA (19) were obtained as previously documented.
Stable Transfections in HEK293 Cells
HEK293 cells were stably transfected by calcium phosphate precipitation with 1 µg of pcDNA3 containing either wild-type (wt) PS1, ΔE9-PS1, or one of the above mutated PS1 sequences. Transfectants were identified after protein electrophoresis and Western blot analysis by means of αPS1Loop antibody (19).
Effect of Inhibitors on PS Immunoreactivity in Stably Transfected HEK293 Cells
Stably transfected cells were exposed to a series of inhibitors targeting the proteasome or other proteases. Cells were then washed, lysed in Tris buffer saline (TBS; NaCl, 140 mM; Tris, 20 mM; pH 7.5) containing 2% sodium dodecyl sulfate (TBS-SDS), then 20 µg of protein was diluted twice in the loading buffer, electrophoresed on a 12% SDS-PAGE, and Western blotted for 3 hr. Nitrocellulose sheets were capped for 45 min with skim milk (5% in TBS) and exposed over-night to a 1000-fold dilution of αPS1Loop antibody or to a 5000-fold dilution of an anti-N-terminal-PS1 antibody (B14.5 provided by Bart De Strooper, Leuven, Belgium). The nitrocellulose was rinsed with TBS then incubated with adequate anti-IgGs, revealed, and quantified as previously described (15).
Immunoprecipitation of PS1 and Immunodetection of Ubiquitin
HEK293 cells overexpressing wtPS1 were grown in 35-mm dishes. Before use, cells were washed with phosphate buffer saline (PBS) and lysed in 1 ml of radioimmunoprecipitation assay (RIPA) buffer. wtPS1 was immunoprecipited with αPS1Loop antibody (1/1000 dilution) and 10 mg of protein A-Sepharose (Sigma). Immunoprecipitates were rinsed then submitted to an 8% SDS-PAGE and Western blotted onto a nitrocellulose membrane (Hybond C, Amersham) for 3 hr. Nitrocellulose sheets were capped for 45 min with skim milk (5% in TBS) and exposed overnight to a 500-fold dilution of monoclonal anti-ubiquitin antibodies (Ubi-1). The nitrocellulose was rinsed with TBS then incubated with peroxidase-coupled anti-mouse IgGs and revealed as above.
Transient Transfections, Metabolic Labeling, and Detection of Aβ40 and Aβ42
Stable transfectants overexpressing wild-type, ΔE9, or mutated PS1 were transiently transfected by calcium phosphate precipitation with 2 µg of wild-type βAPP751 cDNA. Transfection efficiency was checked by Western blot with mAb22C11 antibody (15).
After 2 days, cells were treated with proteasome inhibitors for 15 hr then metabolically labeled for 6 hr as described earlier (20). Conditioned media were collected, diluted in a one-tenth volume of RIPA 10× buffer, then submitted to sequential immunoprecipitation procedures by means of FCA3542 and FCA3340 antibodies (21). Briefly, the above diluted media were incubated overnight with a 350-fold dilution of FCA3542 then further incubated for 5 hr with protein A-Sepharose. After centrifugation, the resulting supernatant was further incubated for 15 hr with a 350-fold dilution of FCA3340 and treated as above. Both pellets were resuspended with loading buffer, submitted to 16.5% Tris-tricine electrophoresis, and radioautographed as described earlier (9). Densitometric analysis was performed by phosphorImager (Fuji).
FCA3340 and FCA3542 specifically interact with Aβ40 and Aβ42, respectively (21). αPS1Loop recognizes the hydrophilic loop of PS1 located between its predicted sixth and seventh transmembrane domain (19). Antibody B14.5 is directed toward the N-terminus of PS1. mAb22C11 was from Boehringer, and Ubil was purchased from Zymed (San Francisco, CA).
We previously established that a 1-µM concentration of two proteasome inhibitors lactacystin (22) and Z-IE(Ot-Bu)A-Leucinal (23) fully abolished all the chymotryptic-like activity present in a homogenate of HEK293 cells and that all this activity could be ascribed to the proteasome as it was immunoprecipitated by specific proteasome antibodies (17). Furthermore, we recently demonstrated that a 30-min treatment of HEK293 cells with 25 µM of Z-IE(Ot-Bu)A-Leucinal fully abolished intracellular proteasome activity (16), indicating that this agent was cell permeant.
Effect of Z-IE(Ot-Bu)A-Leucinal on wild-type and FAD-linked PS1 immunoreactivity in distinct, independent transfectant clones
Number of Experiments
% of Control
276 ± 69
520 ± 73
301 ± 52
442 ± 81
262 ± 55
350 ± 68
211 ± 42
399 ± 74
293 ± 37
442 ± 85
280 ± 35
425 ± 59
249 ± 45
460 ± 79
440 ± 110
Effect of Z-IE(Ot-Bu)A-Leucinal on A β secretion by independent clones expressing wild-type and FAD-linked PS1
Aβ42/Aβ total Ratio
0.18 ± 0.007
0.16 ± 0.025
0.10 ± 0.001
0.13 ± 0.010 (100)
0.24 ± 0.017
0.32 ± 0.015 (246)
0.22 ± 0.020
0.27 ± 0.021 (208)
0.25 ± 0.007
0.34 ± 0.040 (262)
0.26 ± 0.030
0.41 ± 0.029 (315)
0.24 ± 0.030
0.46 ± 0.032 (354)
0.28 ± 0.024
0.45 ± 0.040 (346)
0.23 ± 0.020
0.40 ± 0.032 (308)
We have set up stable transfectants overexpressing wild-type and mutated PS1. By means of an antibody directed toward the extracellular hydrophylic loop of PS1, we detect the overexpression of a protein doublet at 45–48 kDa (referred to as LMW-PS1 and HMW-PS1, respectively) as was previously described in several other studies (4,6,24). The same type of labeling is observed with an antibody directed towards the N-terminal part of PS1 (see Fig. 5B), confirming the identity of the detected protein as genuine PS1. It appears unlikely that these two proteic bands correspond to immature and mature PS1 species since treatment of cell lysates with endoglycosidase H does not modify the immunoreactivity pattern (not shown) in agreement with previous studies showing the absence of glycosylation of PS1 (12,25). We also observe an additional product of about 25 kDa (CTF-PS1) that likely corresponds to the C-terminal product of PS1 maturation. In line of such hypothesis, it should be noted that the overexpression of ΔE9-PS1, a nonmaturated PS1 construction (19), does not lead to the detection of such CTF-PS1 immunoreactivity (Fig. 1). The fact that the molecular weight of CTF-PS1 appears to be slightly higher than those previously reported could reflect a high phosphorylation state of this product in our conditions (26,27).
Two specific inhibitors of the proteasomal multicatalytic complex, lactacystin and Z-IE(Ot-Bu)A-Leucinal, elicit time- and dose-dependent protection of wtPS1 immunoreactivity. These agents also trigger identical protection of FAD-linked M146V-PS1, H163R-PS1, and E280A-PS1. This is not accompanied by a concommitant decrease in CTF-PS1 immunoreactivity, thereby indicating that the inhibitors more likely prevent a proteasomal contribution to PS1 degradation but not maturation. Accordingly, the same protection was observed with the cells expressing the maturation-resistant ΔE9-PS1 construction.
Interestingly, both LMW-PS1 and HMW-PS1 immunoreactivity is affected by proteasome inhibitors, although the extent of inhibition appears to be clearly higher for LMW-PS1. The nature of LMW-PS1 and HMW-PS1 is not yet clear. However, they do not correspond to distinct glycosylation states (not shown), which is in agreement with the fact that the main subcellular immunohistochemical localization of PS includes cell compartments involved early in biosynthetic pathways, such as endoplasmic reticulum and early Golgi (10–12). The current knowledge of proteasome specificity indicates that this catalytic complex triggers the degradation of intracellular polyubiquitinated proteins (28). In this context, it is interesting to note that PS1 can undergo polyubiquitination in HEK293 cells (Fig. 4), as was shown for PS2 (29). Several lines of evidence now suggest that although mainly cytosolic, the proteasome could also be detected in other cell compartments (30,31), including the endoplasmic reticulum and early Golgi (32), where it could contribute to the unconventional degradation of various endoplasmic reticulum-associated proteins (33).
Inhibitors targeting thiol (E64) or serine (AEBSF) proteases were unable to affect PS1 immunoreactivity. Furthermore, pepstatin A also appears ineffective, indicating that the overall increase in PS1 immunoreactivity was not due to the blockade of acidic proteases located in a lysosomal compartment where they would be responsible for final catabolic processes. Finally, two more specific blocking agents were examined. Phosphoramidon, which appears to protect Aβ from intracellular degradation (34), and Z-L-Leucinal, a calpain inhibitor, were unable to modify PS1 immunoreactivity. None of the above inhibitors affect the recovery of the CTF, indicating that this product does not undergo subsequent cleavage by proteases covered by the inhibitory spectrum of these inhibitors.
PS1 mutations trigger an increased Aβ42 formation that is likely responsible for FAD-linked PS1 pathogenicity (for reviews, see refs. 35–37). Thus, we examined whether proteasome inhibitors could lead to phenotypic enhanced secretion of Aβ42 in transfected cells or could modify the ratio of Aβ42 to total Aβ (Aβ40 + Aβ42) recoveries. Our experiments show that in mock transfected cells, proteasome inhibitors lead to increased production of both Aβ40 and Aβ42 species, as indicated by identical ratios observed in control and inhibition conditions. Overexpression of wtPS1 leads to increased Aβ40 and Aβ42 recovery with a higher magnitude observed for the Aβ40 species. In this cell system, proteasome inhibitors induce a nonselective enhancement of both Aβ species, as indicated by the similar Aβ42 to total Aβ ratio. By contrast, mutations on PS1 lead to increased production of both Aβ40 and Aβ42 but clearly favor the production of the latter species as reflected by the increased Aβ42 to total Aβ ratio. Proteasome inhibitors protect mutated PS1 from degradation, thereby exacerbating Aβ42 production as reflected by an increase of this ratio. Altogether, our study suggests that the proteasome participates in the degradation of wt and mutated PS1 and directly influences Aβ production. It should be noted that direct involvement of the proteasome in Aβ degradation is unlikely, as we previously demonstrated that the purified enzyme was unable to cleave synthetic Aβ in vitro (15).
We previously established that proteasome inhibitors could increase the constitutive secretion of APPα (16), the physiological product of βAPP maturation (38). These data, together with the present study strongly suggest that the proteasome influences both α- and β/γ-secretase pathways, probably through the degradation of PS1. These catabolic events likely occur upstream to the contribution of PS1 to the maturation and/or routing of βAPP. Furthermore, the selective increased secretion of Aβ42 observed with cells expressing FAD-linked PS1 strongly supports a dysfunction in the βAPP routing brought about by these mutations. A recent study seems to indicate that the proteasome could also contribute to the degradation of wild-type presenilin 2 (29).
Whether the proteasome could serve as therapeutic target remains to be established. It is interesting to note, however, that several natural endogenous activators of the proteasome have been described (for reviews, see refs. 39,40). In the case of early-onset forms of Alzheimer’s disease linked to mutations on PS1, it is thus possible to envision that selective enhancers of the proteasome activity could diminish Aβ42 formation through increased degradation of mutated PS1 and therefore slow down or arrest the neurodegenerative process that is likely related to overexpression of such Aβ42 species.
We thank Drs. M. Savage and B. Greenberg (Cephalon, West Chester, PA) for generously providing us with 207 antibody and Dr. B. De Strooper (Center for Human Genetics, Leuven, Belgium) for the generous gift of the antibody (B14) directed toward the N-terminal part of PS1. We sincerely thank Dr. Sherwin Wilk for the generous gift of Z-IE(Ot-Bu)A-Leucinal and Z-L-Leucinal. We sincerely thank Drs. G. Thinakaran and S. Sisodia for generously providing us with αPS1Loop antibody and ΔE9-PS1 cDNA. We also thank J. Kervella for expert secretarial assistance, and Franck Aguila for the artwork. This work was supported by the Institut National de la Santé et de la Recherche Médicale and the Centre National de la Recherche Scientifique.
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