Jean Sevalle

NFkappaB-dependent control of BACE1 promoter transactivation by Abeta42.

β-Amyloid (Aβ) peptides that accumulate in Alzheimer disease are generated from the β-amyloid precursor protein (βAPP) by cleavages by β-secretase BACE1 and by presenilin-dependent γ-secretase activities. Very few data document a putative cross-talk between these proteases and the regulatory mechanisms underlying such interaction. We show that presenilin deficiency lowers BACE1 maturation and affects both BACE1 activity and promoter transactivation. The specific γ-secretase inhibitor DFK167 triggers the decrease of BACE1 activity in wild-type but not in presenilin-deficient fibroblasts. This decrease is also elicited by catalytically inactive γ-secretase. The overexpression of APP intracellular domain (AICD), the γ/ϵ-secretase-derived C-terminal product of β-amyloid precursor protein, does not modulate BACE1 activity or promoter transactivation in fibroblasts and does not alter BACE1 expression in AICD transgenic brains of mice. A DFK167-sensitive increase of BACE1 activity is observed in cells overexpressing APPϵ (the N-terminal product of βAPP generated by ϵ-secretase cleavage harboring the Aβ domain but lacking the AICD sequence), suggesting that the production of Aβ could account for the modulation of BACE1. Accordingly, we show that HEK293 cells overexpressing wild-type βAPP exhibit a DFK167-sensitive increase in BACE1 promoter transactivation that is increased by the Aβ-potentiating Swedish mutation. This effect was mimicked by exogenous application of Aβ42 but not Aβ40 or by transient transfection of cDNA encoding Aβ42 sequence. The IκB kinase inhibitor BMS345541 prevents Aβ-induced BACE1 promoter transactivation suggesting that NFκB could mediate this Aβ-associated phenotype. Accordingly, the overexpression of wild-type or Swedish mutated βAPP does not modify the transactivation of BACE1 promoter constructs lacking NFκB-responsive element. Furthermore, APP/β-amyloid precursor protein-like protein deficiency does not affect BACE1 activity and expression. Overall, these data suggest that physiological levels of endogenous Aβ are not sufficient per se to modulate BACE1 promoter transactivation but that exacerbated Aβ production linked to wild-type or Swedish mutated βAPP overexpression modulates BACE1 promoter transactivation and activity via an NFκB-dependent pathway.

Aminopeptidase A contributes to the N-terminal truncation of amyloid β-peptide

Several lines of data previously indicated that N‐terminally truncated forms of amyloid‐β (Aβ) peptides are likely the earliest and more abundant species immunohistochemically detectable in Alzheimer’s disease‐affected brains. It is noteworthy that the free N‐terminal residue of full‐length Aβ (fl‐Aβ) is an aspartyl residue, suggesting that Aβ could be susceptible to exopeptidasic attack by aminopeptidase A (APA)‐like proteases. In this context, we have examined whether APA could target Aβ peptides in both cell‐free and cellular models. We first show that the general aminopeptidase inhibitor amastatin as well as two distinct aminopeptidase A inhibitors EC33 and pl302 both significantly increase the recovery of genuine fl‐Aβ peptides generated by cells over‐expressing Swedish‐mutated β amyloid precursor protein (APP) while the aminopeptidase N blocker pl250 did not modify fl‐Aβ recovery. In agreement with this observation, we establish that over‐expressed APA drastically reduces, in a calcium dependent manner, fl‐Aβ but not APP IntraCellular Domain in a cell‐free model of Aβ production. In agreement with the above data, we show that recombinant APA degrades fl‐Aβ in a pl302‐sensitive manner. Interestingly, we also show that EC33 and pl302 lower staurosporine‐stimulated activation of caspase‐3 in wild‐type fibroblasts but not in βAPP/β‐amyloid precursor protein‐like protein 2 (APLP2) double knockout fibroblasts, suggesting that protecting endogenous fl‐Aβ physiological production triggers neuroprotective phenotype. By contrast, EC33 does not modify staurosporine‐induced caspase‐3 activation in wild‐type and Swedish‐mutated βAPP‐HEK293 expressing cells that display exacerbated production of Aβ. Overall, our data establish that APA contributes to the N‐terminal truncation of Aβ and suggest that this cleavage is likely abrogating a protective function associated with physiological but not supraphysiological levels of genuine fl‐Aβ peptides.

NFκB-dependent Control of BACE1 Promoter Transactivation by Aβ42

β-Amyloid (Aβ) peptides that accumulate in Alzheimer disease are generated from the β-amyloid precursor protein (βAPP) by cleavages by β-secretase BACE1 and by presenilin-dependent γ-secretase activities. Very few data document a putative cross-talk between these proteases and the regulatory mechanisms underlying such interaction. We show that presenilin deficiency lowers BACE1 maturation and affects both BACE1 activity and promoter transactivation. The specific γ-secretase inhibitor DFK167 triggers the decrease of BACE1 activity in wild-type but not in presenilin-deficient fibroblasts. This decrease is also elicited by catalytically inactive γ-secretase. The overexpression of APP intracellular domain (AICD), the γ/ϵ-secretase-derived C-terminal product of β-amyloid precursor protein, does not modulate BACE1 activity or promoter transactivation in fibroblasts and does not alter BACE1 expression in AICD transgenic brains of mice. A DFK167-sensitive increase of BACE1 activity is observed in cells overexpressing APPϵ (the N-terminal product of βAPP generated by ϵ-secretase cleavage harboring the Aβ domain but lacking the AICD sequence), suggesting that the production of Aβ could account for the modulation of BACE1. Accordingly, we show that HEK293 cells overexpressing wild-type βAPP exhibit a DFK167-sensitive increase in BACE1 promoter transactivation that is increased by the Aβ-potentiating Swedish mutation. This effect was mimicked by exogenous application of Aβ42 but not Aβ40 or by transient transfection of cDNA encoding Aβ42 sequence. The IκB kinase inhibitor BMS345541 prevents Aβ-induced BACE1 promoter transactivation suggesting that NFκB could mediate this Aβ-associated phenotype. Accordingly, the overexpression of wild-type or Swedish mutated βAPP does not modify the transactivation of BACE1 promoter constructs lacking NFκB-responsive element. Furthermore, APP/β-amyloid precursor protein-like protein deficiency does not affect BACE1 activity and expression. Overall, these data suggest that physiological levels of endogenous Aβ are not sufficient per se to modulate BACE1 promoter transactivation but that exacerbated Aβ production linked to wild-type or Swedish mutated βAPP overexpression modulates BACE1 promoter transactivation and activity via an NFκB-dependent pathway.

ER-stress-associated functional link between Parkin and DJ-1 via a transcriptional cascade involving the tumor suppressor p53 and the spliced X-box binding protein XBP-1

Summary Parkin and DJ-1 are two multi-functional proteins linked to autosomal recessive early-onset Parkinsons disease (PD) that have been shown to functionally interact by as-yet-unknown mechanisms. We have delineated the mechanisms by which parkin controls DJ-1. Parkin modulates DJ-1 transcription and protein levels via a signaling cascade involving p53 and the endoplasmic reticulum (ER)-stress-induced active X-box-binding protein-1S (XBP-1S). Parkin triggers the transcriptional repression of p53 while p53 downregulates DJ-1 protein and mRNA expressions. We show that parkin-mediated control of DJ-1 is fully p53-dependent. Furthermore, we establish that p53 lowers the protein and mRNA levels of XBP-1S. Accordingly, we show that parkin ultimately upregulates XBP-1 levels. Subsequently, XBP-1S physically interacts with the DJ-1 promoter, thereby enhancing its promoter trans-activation, mRNA levels and protein expression. This data was corroborated by the examination of DJ-1 in both parkin- and p53-null mice brains. This transcriptional cascade is abolished by pathogenic parkin mutations and is independent of its ubiquitin-ligase activity. Our data establish a parkin-dependent ER-stress-associated modulation of DJ-1 and identifies p53 and XBP-1 as two major actors acting downstream of parkin in this signaling cascade in cells and in vivo. This work provides a mechanistic explanation for the increase in the unfolded protein response observed in PD pathology, i.e. that it is due to a defect in parkin-associated control of DJ-1.

The γ /η-Secretase-Derived APP Intracellular Domain Fragments Regulate p53

Amyloid β-peptide (Aβ), which plays a central role in Alzheimer Disease, is generated by presenilin-dependent and presenilin-independent γ-secretase cleavages of β-amyloid precursor protein (βAPP). We report that the presenilins (PS1 and PS2) also regulate p53-associated cell death Thus, we established that PS deficiency, catalytically inactive PS mutants, γ-secretase inhibitors and βAPP or APLP2 depletion reduced the expression and activity of p53, and lowered the transactivation of its promoter and mRNA levels. p53 expression was also reduced in the brains or βAPP-deficient mice or in brains where both PS had been invalidated by double conditional knock out. AICDC59 and AICDC50, the γ- and η- secretase-derived C-terminal fragments of βAPP, respectively, trigger the activation of caspase-3, p53-dependent cell death, and increase p53 activity and mRNA. Finally, HEK293 cells expressing PS1 harboring familial AD (FAD) mutations or FAD-affected brains, all display enhanced p53 activity and p53 expression. Our studies demonstrate that AICDs control p53 at a transcriptional level, in vitro and in vivo and unravel a still unknown function for presenilins.

APH1 polar transmembrane residues regulate the assembly and activity of presenilin complexes

Complexes involved in the γ/ϵ-secretase-regulated intramembranous proteolysis of substrates such as the amyloid-β precursor protein are composed primarily of presenilin (PS1 or PS2), nicastrin, anterior pharynx defective-1 (APH1), and PEN2. The presenilin aspartyl residues form the catalytic site, and similar potentially functional polar transmembrane residues in APH1 have been identified. Substitution of charged (E84A, R87A) or polar (Q83A) residues in TM3 had no effect on complex assembly or activity. In contrast, changes to either of two highly conserved histidines (H171A, H197A) located in TM5 and TM6 negatively affected PS1 cleavage and altered binding to other secretase components, resulting in decreased amyloid generating activity. Charge replacement with His-to-Lys substitutions rescued nicastrin maturation and PS1 endoproteolysis leading to assembly of the formation of structurally normal but proteolytically inactive γ-secretase complexes. Substitution with a negatively charged side chain (His-to-Asp) or altering the structural location of the histidines also disrupted γ-secretase binding and abolished functionality of APH1. These results suggest that the conserved transmembrane histidine residues contribute to APH1 function and can affect presenilin catalytic activity.

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.

Parkin differently regulates presenilin-1 and presenilin-2 functions by direct control of their promoter transcription

We previously established that besides its canonical function as E3-ubiquitin ligase, parkin also behaves as a transcriptional repressor of p53. Here we show that parkin differently modulates presenilin-1 and presenilin-2 expression and functions at transcriptional level. Thus, parkin enhances/reduces the protein expression, promoter activity and mRNA levels of presenilin-1 and presenilin-2, respectively, in cells and in vivo. This parkin-associated function is independent of its ubiquitin-ligase activity and remains unrelated to its capacity to repress p53. Accordingly, physical interaction of endogenous or overexpressed parkin with presenilins promoters is demonstrated by chromatin immunoprecipitation assays (ChIP). Furthermore, we identify a consensus sequence, the deletion of which abolishes parkin-dependent modulation of presenilins-1/2 and p53 promoter activities. Interestingly, electrophoretic mobility shift assays (EMSA) revealed a physical interaction between this consensus sequence and wild-type but not mutated parkin. Finally, we demonstrate that the RING1-IBR-RING2 domain of parkin harbors parkins potential to modulate presenilins promoters. This transcriptional control impacts on presenilins-associated phenotypes, since parkin increases presenilin-1-associated γ-secretase activity and reduces presenilin-2-linked caspase-3 activation. Overall, our data delineate a promoter responsive element targeted by parkin that drives differential regulation of presenilin-1 and presenilin-2 transcription with functional consequences for γ-secretase activity and cell death.

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.

Pharmacological evidences for DFK167-sensitive presenilin-independent γ-secretase-like activity

Amyloid‐β (Aβ) peptides production is thought to be a key event in the neurodegenerative process ultimately leading to Alzheimer’s disease (AD) pathology. A bulk of studies concur to propose that the C‐terminal moiety of Aβ is released from its precursor β‐amyloid precursor protein by a high molecular weight enzymatic complex referred to as γ‐secretase, that is composed of at least, nicastrin (NCT), Aph‐1, Pen‐2, and presenilins (PS) 1 or 2. They are thought to harbor the γ‐secretase catalytic activity. However, several lines of evidence suggest that additional γ‐secretase‐like activities could potentially contribute to Aβ production. By means of a quenched fluorimetric substrate (JMV2660) mimicking the β‐amyloid precursor protein sequence targeted by γ‐secretase, we first show that as expected, this probe allows monitoring of an activity detectable in several cell systems including the neuronal cell line telencephalon specific murine neurons (TSM1). This activity is reduced by DFK167, N‐[N‐(3,5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT), and LY68458, three inhibitors known to functionally interact with PS. Interestingly, JMV2660 but not the unrelated peptide JMV2692, inhibits Aβ production in an in vitroγ‐secretase assay as expected from a putative substrate competitor. This activity is enhanced by PS1 and PS2 mutations known to be responsible for familial forms of AD and reduced by aspartyl mutations inactivating PS or in cells devoid of PS or NCT. However, we clearly establish that residual JMV2660‐hydrolysing activity could be recovered in PS‐ and NCT‐deficient fibroblasts and that this activity remained inhibited by DFK167. Overall, our study describes the presence of a proteolytic activity displaying γ‐secretase‐like properties but independent of PS and still blocked by DFK167, suggesting that the PS‐dependent complex could not be the unique γ‐secretase activity responsible for Aβ production and delineates PS‐independent γ‐secretase activity as a potential additional therapeutic target to fight AD pathology.