Julie Dunys

p53 Is Regulated by and Regulates Members of the γ-Secretase Complex

Amyloid β-peptides is the generic term for a set of hydrophobic peptides that accumulate in Alzheimer’s disease (AD)-affected brains. These amyloid-β peptide fragments are mainly generated by an enzymatic machinery referred to as γ-secretase complex that is built up by the association of four distinct proteins, namely presenilin 1 (PS1) or PS2, nicastrin, Aph-1 and Pen-2. AD is also characterized by exacerbated cell death that appears linked to the tumor suppressor p53. Interestingly, all members of the γ-secretase complex control p53-dependent cell death. On the other hand, p53 appears to be able to regulate directly or indirectly the expression and transcription of PS1, PS2 and Pen-2. This review will focus on the functional cross-talk between the members of the γ-secretase complex and p53 and will discuss the putative implication of this oncogene in AD pathology.

Neprilysin activity and expression are controlled by nicastrin

We recently demonstrated that the presenilin‐dependent γ‐secretase complex regulates the expression and activity of neprilysin, one of the main enzymes that degrade the amyloid β‐peptide (Aβ) which accumulates in Alzheimers disease. Here, we examined the influence of endogenous nicastrin (NCT), a member of the γ‐secretase complex, on neprilysin physiology. We show that nicastrin deficiency drastically lowers neprilysin expression, membrane‐bound activity and mRNA levels, but it did not modulate the expression of two other putative Aβ‐cleaving enzymes, endothelin‐converting enzyme and insulin‐degrading enzyme. Furthermore, we show that nicastrin restores neprilysin activity and expression in nicastrin‐deficient, but not presenilin‐deficient fibroblasts, indicating that the control of neprilysin necessitates the complete γ‐secretase complex harbouring its four reported components. Finally, we show that NCT expression peaked 24 h after NCT cDNA transfection of wild‐type and NCT–/– fibroblasts, while neprilysin expression drastically increased only after 36 h and was maximal at 48 h. This delayed effect on neprilysin expression correlates well with our demonstration of an indirect γ‐secretase‐dependent modulation of neprilysin at its transcriptional level.

p53-dependent control of transactivation of the Pen2 promoter by presenilins

The senile plaques found in the brains of patients with Alzheimers disease are mainly due to the accumulation of amyloid β-peptides (Aβ) that are liberated by γ-secretase, a high molecular weight complex including presenilins, PEN-2, APH-1 and nicastrin. The depletion of each of these proteins disrupts the complex assembly into a functional protease. Here, we describe another level of regulation of this multimeric protease. The depletion of both presenilins drastically reduces Pen2 mRNA levels and its promoter transactivation. Furthermore, overexpression of presenilin-1 lowers Pen2 promoter transactivation, a phenotype abolished by a double mutation known to prevent presenilin-dependent γ-secretase activity. PEN-2 expression is decreased by depletion of β-amyloid precursor protein (APP) and increased by the APP intracellular domain (AICD). We show that AICD and APP complement for Pen2 mRNA levels in APP/APLP1-2 knockout fibroblasts. Interestingly, overexpression of presenilin-2 greatly increases Pen2 promoter transactivation. The opposite effect triggered by both presenilins was reminiscent of our previous study, which showed that these two proteins elicit antagonistic effects on p53. Therefore, we examined the contribution of p53 on Pen2 transcription. Pen2 promoter transactivation, and Pen2 mRNA and protein levels were drastically reduced in p53–/– fibroblasts. Furthermore, PEN-2 expression could be rescued by p53 complementation in p53- and APP-deficient cells. Interestingly, PEN-2 expression was also reduced in p53-deficient mouse brain. Overall, our study describes a p53-dependent regulation of PEN-2 expression by other members of the γ-secretase complex, namely presenilins.

γ-Secretase-Mediated Regulation of Neprilysin: Influence of Cell Density and Aging and Modulation by Imatinib

Proteolytic degradation has emerged as a key pathway involved in controlling levels of the Alzheimers disease (AD)-associated amyloid-β peptides (Aβ) in the brain. The ectopeptidase, neprilysin (NEP), has been reported as the major Aβ-degrading enzyme in mice and human brains. We have previously shown that NEP expression and activity are regulated by AICD, the intracellular domain of the amyloid-β protein precursor (AβPP) generated by γ-secretase. Thus, NEP transcription, expression, and enzymatic activity are dramatically reduced in fibroblasts devoid of AβPP (the precursor of AICD) or lacking both presenilin (PS) 1 and 2 (two parent proteins contributing to AICD formation). We demonstrate here that NEP expression and activity are influenced by a number of cell passages and density, and we confirm a drastic reduction of NEP expression and activity in AβPP and PS null fibroblasts examined at similar passages and cell densities. Furthermore, Imatinib (Gleevec), a known tyrosine kinase inhibitor was recently shown to elevate AICD in H4 human neuroglioma cells, and this was accompanied by concomitant increases of NEP protein, mRNA levels, and activity. However, the demonstration of a causal link between Imatinib and AICD levels was still lacking. We show here an Imatinib-dependent effect on NEP expression and activity in murine fibroblasts and establish that Imatinib-induced modulation of NEP was abolished by the depletion of AβPP or its homologues APLP1 and APLP2, thereby confirming that Imatinib-mediated control of NEP could indeed be accounted for its effect on AICD.

p53-dependent Aph-1 and Pen-2 Anti-apoptotic Phenotype Requires the Integrity of the γ-Secretase Complex but Is Independent of Its Activity

The presenilin-dependent γ-secretase activity, which is responsible for the generation of amyloid β-peptide, is a high molecular weight complex composed of at least four components, namely, presenilin-1 (or presenilin-2), nicastrin, Aph-1, and Pen-2. Previous data indicated that presenilins, which are thought to harbor the catalytic core of the complex, also control p53-dependent cell death. Whether the other components of the γ-secretase complex could also modulate the cell death process in mammalian neurons remained to be established. Here, we examined the putative contribution of Aph-1 and Pen-2 in the control of apoptosis in TSM1 cells from a neuronal origin. We show by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and DNA fragmentation analyses that the overexpression of Aph-1a, Aph-1b, or Pen-2 drastically lowered staurosporine-induced cellular toxicity. In support of an apoptosis rather than necrosis process, Aph-1 and Pen-2 also lower staurosporine- and etoposide-induced caspase-3 expression and diminished caspase-3 activity and poly(ADP-ribose) polymerase inactivation. The Aph-1 and Pen-2 anti-apoptotic phenotype was associated with a drastic reduction of p53 expression and activity and lowered p53 mRNA transcription. Furthermore, the Aph-1- and Pen-2-associated reduction of staurosporine-induced caspase-3 activation was fully abolished by p53 deficiency. Conversely, Aph-1a, Aph-1b, and Pen-2 gene inactivation increases both caspase-3 activity and p53 mRNA levels. Finally, we show that Aph-1 and Pen-2 did not trigger an anti-apoptotic response in cells devoid of presenilins or nicastrin, whereas the protective response was still observed in fibroblasts devoid of β-amyloid precursor protein and amyloid precursor protein like-protein 2. Furthermore, Aph-1- and Pen-2-associated protection against staurosporine-induced caspase-3 activation was not affected by the γ-secretase inhibitors N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester and difluoromethylketone. Altogether, our study indicates that Aph-1 and Pen-2 trigger an anti-apoptotic response by lowering p53-dependent control of caspase-3. Our work also demonstrates that this phenotype is strictly dependent on the molecular integrity of the γ-secretase complex but remains independent of the γ-secretase catalytic activity.

Catabolism of endogenous and overexpressed APH1a and PEN2: evidence for artifactual involvement of the proteasome in the degradation of overexpressed proteins.

PS (presenilin)-dependent gamma-secretase occurs as a high-molecular-mass complex composed of either PS1 or PS2 associated with Nct (nicastrin), PEN2 (presenilin enhancer 2 homologue) and APH1 (anterior pharynx defective 1 homologue). Numerous reports have documented the very complicated physical and functional cross-talk between these proteins that ultimately governs the biological activity of the gamma-secretase, but very few studies examined the fate of the components of the complex. We show that, in both HEK-293 cells and the TSM1 neuronal cell line, the immunoreactivities of overexpressed myc-tagged-APH1a and -PEN2 were enhanced by the proteasome inhibitors ZIE and lactacystin, whereas a broad range of protease inhibitors had no effect. By contrast, proteasome inhibitors were totally unable to affect the cellular expression of endogenous APH1aL and PEN2 in HEK-293 cells, TSM1 and primary cultured cortical neurons. To explain this apparent discrepancy, we examined the degradation of myc-tagged-APH1a and -PEN2, in vitro, by cell extracts containing endogenous proteasome and by purified 20S proteasome. Strikingly, myc-tagged-APH1a and -PEN2 resist proteolysis by endogenous proteasome and purified 20S proteasome. We also show that endogenous PEN2 expression was drastically higher in wild-type than in PS- and Nct-deficient fibroblasts and was enhanced by proteasome inhibitors only in the two deficient cell systems. However, here again, purified 20S proteasome appeared unable to cleave endogenous PEN2 present in PS-deficient fibroblasts. The levels of endogenous APH1aL-like immunoreactivity were not modified by proteasome inhibitors and were unaffected by PS deficiency. Altogether, our results indicate that endogenous PEN2 and APH1aL do not undergo proteasomal degradation under physiological conditions in HEK-293 cells, TSM1 cells and fibroblasts and that the clearance of PEN2 in PS- and Nct-deficient fibroblasts is not mediated by 20S proteasome. Whether the 26S proteasome participates to PEN2 proteolysis in deficient fibroblasts remains to be established.

TMP21 regulates Aβ production but does not affect caspase-3, p53, and neprilysin

The presenilin (PS)-dependent gamma-secretase activity refers to a high molecular mass-complex including, besides PS1 or PS2, three other proteins recently identified, namely nicastrin, Aph-1, and Pen-2. This proteolytic complex has been shown to contribute to both gamma- and epsilon-cleavages of the beta-amyloid precursor protein (betaAPP), thereby generating beta-amyloid peptides (Abeta) and the APP intracellular domain (AICD), respectively. TMP21, a member of the p24 cargo protein family, was recently shown to interact with PS complexes. Interestingly, TMP21 modulates gamma-secretase-mediated Abeta production but does not regulate epsilon-secretase-derived AICD formation [F. Chen, H. Hasegawa, G. Schmitt-ulms, T. Kawarai, C. Bohm, T. Katayama, Y. Gu, N. Sanjo, M. Glista, E. Rogaeva, Y. Wakutami, R. Pardossi-Piquard, X. Ruan, A. Tandon, F. Checler, P. Marambaud, K. Hansen, D. Westaway, P. St. George-Hyslop, P. Fraser, TMP21 is a presenilin complex component that modulates gamma- but not epsilon-secretase activities, Nature 440 (2006) 1208-1212]. Here we investigate the functional incidence of the over-expression or depletion of TMP21 on both intracellular and secreted Abeta recoveries and AICD-associated phenotypes. First we confirm that TMP21 depletion yields increased levels of secreted Abeta40. However, we demonstrate that both staurosporine-stimulated caspase-3 activation, p53 and neprilysin expression and activity were not affected by TMP21 over-expression or depletion. Overall, our functional data further reinforce the view that TMP21 behaves as a regulator of gamma- but not epsilon-cleavages generated by PS-dependent gamma-secretase complex.

p53-Dependent control of cell death by nicastrin: lack of requirement for presenilin-dependent γ-secretase complex

Nicastrin (NCT) is a component of the presenilin (PS)‐dependent γ‐secretase complexes that liberate amyloid β‐peptides from the β‐Amyloid Precursor Protein. Several lines of evidence indicate that the members of these complexes could also contribute to the control of cell death. Here we show that over‐expression of NCT increases the viability of human embryonic kidney (HEK293) cells and decreases staurosporine (STS)‐ and thapsigargin (TPS)‐induced caspase‐3 activation in various cell lines from human and neuronal origins by Akt‐dependent pathway. NCT lowers p53 expression, transcriptional activity and promoter transactivation and reduces p53 phosphorylation. NCT‐associated protection against STS‐stimulated cell death was completely abolished by p53 deficiency. Conversely, the depletion of NCT drastically enhances STS‐induced caspase‐3 activation and p53 pathway and favored p53 nuclear translocation. We examined whether NCT protective function depends on PS‐dependent γ‐secretase activity. First, a 29‐amino acid deletion known to reduce NCT‐dependent amyloid β‐peptide production did not affect NCT‐associated protective phenotype. Second, NCT still reduces STS‐induced caspase‐3 activation in fibroblasts lacking PS1 and PS2. Third, the γ‐secretase inhibitor DFK167 did not affect NCT‐mediated reduction of p53 activity. Altogether, our study indicates that NCT controls cell death via phosphoinositide 3‐kinase/Akt and p53‐dependent pathways and that this function remains independent of the activity and molecular integrity of the γ‐secretase complexes.

Aβ42 oligomers modulate β-secretase through an XBP-1s-dependent pathway involving HRD1

The aspartyl protease β-site APP cleaving enzyme, BACE1, is the rate-limiting enzyme involved in the production of amyloid-β peptide, which accumulates in both sporadic and familial cases of Alzheimer’s disease and is at the center of gravity of the amyloid cascade hypothesis. In this context, unravelling the molecular mechanisms controlling BACE1 expression and activity in both physiological and pathological conditions remains of major importance. We previously demonstrated that Aβ controlled BACE1 transcription in an NFκB-dependent manner. Here, we delineate an additional cellular pathway by which natural and synthetic Aβ42 oligomers enhance active X-box binding protein XBP-1s. XBP-1s lowers BACE1 expression and activity indirectly, via the up-regulation of the ubiquitin-ligase HRD1 that acts as an endogenous down-regulator of BACE1. Thus, we delineate a novel pathway by which cells could compensate for Aβ42 oligomers production and thus, associated toxicity, by triggering a compensatory mechanism aimed at lowering BACE-1-mediated Aβ production by a molecular cascade involving XBP-1s and HRD1. It thus identifies HRD1 as a potential target for a novel Aβ-centered therapeutic strategy.

Study on the putative contribution of caspases and the proteasome to the degradation of Aph-1a and Pen-2.

The presenilin-dependent γ-secretase complex is mainly composed of four distinct proteins, namely presenilin 1 or presenilin 2, nicastrin, anterior pharynx defective-1 (Aph-1) and presenilin enhancer (Pen-2). The mechanisms by which the complex is assembled, how its stochiometry is controlled and how its catalytic activity is regulated are poorly understood. Recent studies indicated that Aph-1 and Pen-2 undergo proteolysis by the proteasome. We have examined the susceptibility of endogenous and overexpressed Aph-1a and Pen-2 to proteolysis by endogenous and purified proteasome as well as by recombinant caspases. We show that endogenous Aph-1a and Pen-2 resist proteolysis by caspases and by the proteasome. Furthermore, we show that unexpected interference of proteasome inhibitors with the cmv promoter region driving expression of Aph-1a and Pen-2 led to artifactual enhancement of overexpressed Aph-1a and Pen-2-like immunoreactivities but that these proteins also resist to in vitro degradation by endogenous and purified proteasome.