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ABSTRACT: In physiological conditions, both β-amyloid precursor protein (βAPP) and cellular prion (PrP(c)) undergo similar disintegrin-mediated α-secretase cleavage yielding N-terminal secreted products referred to as soluble amyloid precursor protein-α (sAPPα) and N1, respectively. We recently demonstrated that N1 displays neuroprotective properties by reducing p53-dependent cell death both in vitro and in vivo. In this study, we examined the potential of N1 as a neuroprotector against amyloid β (Aβ)-mediated toxicity. We first show that both recombinant sAPPα and N1, but not its inactive parent fragment N2, reduce staurosporine-stimulated caspase-3 activation and TUNEL-positive cell death by lowering p53 promoter transactivation and activity in human cells. We demonstrate that N1 also lowers toxicity, cell death, and p53 pathway exacerbation triggered by Swedish mutated βAPP overexpression in human cells. We designed a CHO cell line overexpressing the London mutated βAPP (APP(LDN)) that yields Aβ oligomers. N1 protected primary cultured neurons against toxicity and cell death triggered by oligomer-enriched APP(LDN)-derived conditioned medium. Finally, we establish that N1 also protects neurons against oligomers extracted from Alzheimer disease-affected brain tissues. Overall, our data indicate that a cellular prion catabolite could interfere with Aβ-associated toxicity and that its production could be seen as a cellular protective mechanism aimed at compensating for an sAPPα deficit taking place at the early asymptomatic phase of Alzheimer disease.
Journal of Biological Chemistry 12/2011; 287(7):5021-32. · 4.77 Impact Factor
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Cristine Alves da Costa, Claire Sunyach,
Emilie Giaime,
Andrew West,
Olga Corti,
Alexis Brice,
Stephen Safe,
Patrick M Abou-Sleiman,
Nicholas W Wood,
Hitoshi Takahashi,
Mathew S Goldberg,
Jie Shen,
Frédéric Checler
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ABSTRACT: Mutations of the ubiquitin ligase parkin account for most autosomal recessive forms of juvenile Parkinson's disease (AR-JP). Several studies have suggested that parkin possesses DNA-binding and transcriptional activity. We report here that parkin is a p53 transcriptional repressor. First, parkin prevented 6-hydroxydopamine-induced caspase-3 activation in a p53-dependent manner. Concomitantly, parkin reduced p53 expression and activity, an effect abrogated by familial parkin mutations known to either abolish or preserve its ligase activity. ChIP experiments indicate that overexpressed and endogenous parkin interact physically with the p53 promoter and that pathogenic mutations abolish DNA binding to and promoter transactivation of p53. Parkin lowered p53 mRNA levels and repressed p53 promoter transactivation through its Ring1 domain. Conversely, parkin depletion enhanced p53 expression and mRNA levels in fibroblasts and mouse brains, and increased cellular p53 activity and promoter transactivation in cells. Finally, familial parkin missense and deletion mutations enhanced p53 expression in human brains affected by AR-JP. This study reveals a ubiquitin ligase-independent function of parkin in the control of transcription and a functional link between parkin and p53 that is altered by AR-JP mutations.
Nature Cell Biology 10/2009; 11(11):1370-5. · 19.49 Impact Factor
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ABSTRACT: Cellular prion protein (PrP(c)) undergoes a disintegrin-mediated physiological cleavage, generating a soluble amino-terminal fragment (N1), the function of which remained unknown. Recombinant N1 inhibits staurosporine-induced caspase-3 activation by modulating p53 transcription and activity, whereas the PrP(c)-derived pathological fragment (N2) remains biologically inert. Furthermore, N1 protects retinal ganglion cells from hypoxia-induced apoptosis, reduces the number of terminal deoxynucleotidyltransferase-mediated biotinylated UTP nick end labeling-positive and p53-immunoreactive neurons in a pressure-induced ischemia model of the rat retina and triggers a partial recovery of b-waves but not a-waves of rat electroretinograms. Our work is the first demonstration that the alpha-secretase-derived PrP(c) fragment N1, but not N2, displays in vivo and in vitro neuroprotective function by modulating p53 pathway. It further demonstrates that distinct N-terminal cleavage products of PrP(c) harbor different biological activities underlying the various phenotypes linking PrP(c) to cell survival.
Journal of Biological Chemistry 10/2009; 284(51):35973-86. · 4.77 Impact Factor
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ABSTRACT: The presenilin-dependent gamma-secretase processing of the beta-amyloid precursor protein (betaAPP) conditions the length of the amyloid beta peptides (Abeta) that accumulate in the senile plaques of Alzheimer's disease-affected brains. This, together with an additional presenilin-mediated epsilon-secretase cleavage, generates intracellular betaAPP-derived fragments named amyloid intracellular domains (AICDs) that regulate the transcription of several genes. We establish that presenilins control the transcription of cellular prion protein (PrP(c)) by a gamma-secretase inhibitor-sensitive and AICD-mediated process. We demonstrate that AICD-dependent control of PrP(c) involves the tumor suppressor p53. Thus, p53-deficiency abolishes the AICD-mediated control of PrP(c) transcription. Furthermore, we show that p53 directly binds to the PrP(c) promoter and increases its transactivation. Overall, our study unravels a transcriptional regulation of PrP(c) by the oncogene p53 that is directly driven by presenilin-dependent formation of AICD. Furthermore, it adds support to previous reports linking secretase activities involved in betaAPP metabolism to the physiology of PrP(c).
Journal of Neuroscience 06/2009; 29(20):6752-60. · 7.11 Impact Factor
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ABSTRACT: Alzheimer's disease (AD) is by far the most common form of dementia in the elderly and concerns one out of three individuals over 85. Like other neurodegenerative disorders such as Parkinson, Hungtington or prion diseases, AD is characterized by the formation of amyloid plaques in the central nervous system. In the brain of AD patients, the main component of these abnormal deposits is an aggregated form of the so-called amyloid beta-peptide (Abeta), which is produced from a large trans-membrane type-1 protein, the beta-amyloid precursor protein (betaAPP), by the sequential action of the beta- and gamma-secretases. Beside these two amyloidogenic proteolytic attacks, betaAPP is targeted by a third enzyme termed alpha-secretase. Of utmost importance, this cleavage, which can be of constitutive or regulated origin, occurs right in the middle of the Abeta sequence, thus precluding its production. For this reason, and because the sAPPalpha secreted fragment derived from this cleavage displays beneficial effects, tremendous efforts have been made recently in order to both identify the proteases involved and the way they are regulated. More recently, it emerged that alpha-secretase was also responsible for the physiological processing of the cellular prion protein (PrP(c)) in the middle of its toxic 106-126 sequence. This review will focus on the recent advances in the alpha-secretase pathways regulation and will discuss the putative therapeutic approaches that could be envisioned concerning the treatment of two apparently distinct diseases that share common denominators according to their metabolism.
Current Alzheimer Research 05/2008; 5(2):202-11. · 3.95 Impact Factor
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Celia J Parkyn,
Esmeralda G M Vermeulen,
Roy C Mootoosamy, Claire Sunyach,
Christian Jacobsen,
Claus Oxvig,
Søren Moestrup,
Qiang Liu,
Guojun Bu,
Angela Jen,
Roger J Morris
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ABSTRACT: The trafficking of normal cellular prion protein (PrPC) is believed to control its conversion to the altered conformation (designated PrPSc) associated with prion disease. Although anchored to the membrane by means of glycosylphosphatidylinositol (GPI), PrPC on neurons is rapidly and constitutively endocytosed by means of coated pits, a property dependent upon basic amino acids at its N-terminus. Here, we show that low-density lipoprotein receptor-related protein 1 (LRP1), which binds to multiple ligands through basic motifs, associates with PrPC during its endocytosis and is functionally required for this process. Moreover, sustained inhibition of LRP1 levels by siRNA leads to the accumulation of PrPC in biosynthetic compartments, with a concomitant lowering of surface PrPC, suggesting that LRP1 expedites the trafficking of PrPC to the neuronal surface. PrPC and LRP1 can be co-immunoprecipitated from the endoplasmic reticulum in normal neurons. The N-terminal domain of PrPC binds to purified human LRP1 with nanomolar affinity, even in the presence of 1 muM of the LRP-specific chaperone, receptor-associated protein (RAP). Taken together, these data argue that LRP1 controls both the surface, and biosynthetic, trafficking of PrPC in neurons.
Journal of Cell Science 04/2008; 121(Pt 6):773-83. · 6.11 Impact Factor
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ABSTRACT: Alzheimer's disease (AD) is by far the most common form of dementia in the elderly and concerns one out of three individuals over 85. Like other neurodegenerative disorders such as Parkinson, Hungtington or prion diseases, AD is characterized by the formation of amyloid plaques in the central nervous system. In the brain of AD patients, the main component of these abnormal deposits is an aggregated form of the so-called amyloid β-peptide (Aβ), which is produced from a large trans-membrane type-1 protein, the β-amyloid precursor protein (βAPP), by the sequential action of the β- and γ-secretases. Beside these two amyloidogenic proteolytic attacks, βAPP is targeted by a third enzyme termed α- secretase. Of utmost importance, this cleavage, which can be of constitutive or regulated origin, occurs right in the middle of the Aβ sequence, thus precluding its production. For this reason, and because the sAPPα secreted fragment derived from this cleavage displays beneficial effects, tremendous efforts have been made recently in order to both identify the proteases involved and the way they are regulated. More recently, it emerged that α-secretase was also responsible for the physiological processing of the cellular prion protein (PrPc) in the middle of its toxic 106-126 sequence. This review will focus on the recent advances in the α-secretase pathways regulation and will discuss the putative therapeutic approaches that could be envisioned concerning the treatment of two apparently distinct diseases that share common denominators according to their metabolism.
Current Alzheimer Research 03/2008; 5(2):202-211. · 3.95 Impact Factor
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ABSTRACT: Amyloid beta-peptide (Abeta), which plays a central role in Alzheimer Disease, is generated by presenilin-dependent and presenilin-independent gamma-secretase cleavages of beta-amyloid precursor protein (betaAPP). We report that the presenilins (PS1 and PS2) also regulate p53-associated cell death. Thus, we established that PS deficiency, catalytically inactive PS mutants, gamma-secretase inhibitors and betaAPP 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 betaAPP-deficient mice or in brains where both PS had been invalidated by double conditional knock out. AICDC59 and AICDC50, the gamma- and epsilon-secretase-derived C-terminal fragments of betaAPP, 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.
Current Alzheimer Research 10/2007; 4(4):423-6. · 3.95 Impact Factor
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ABSTRACT: The cellular prion protein (PrP(c)) undergoes a physiological processing yielding the N-terminal fragment referred to as N1, the production of which can be constitutive or protein kinase C regulated. We show that activation of endogenous muscarinic receptors by carbachol and by the M1-selective agonist AF267B increases N1 recovery in an atropine-sensitive manner, in mouse embryonic primary neurons. To identify the muscarinic receptor subtype involved, we used human embryonic kidney HEK293 (HEK) cells stably overexpressing M1, M2, M3, or M4 receptor subtype. Carbachol and the selective M1 agonist AF267B dose dependently increased N1 release by HEK-M3 and HEK-M1 cells, respectively, whereas carbachol did not modify N1 production by HEK-M2 or HEK-M4 cells. We demonstrate that the increase of N1 was not attributable to modified trafficking to the membrane of either PrP(c) or the disintegrin metalloproteases ADAM10 or ADAM17. Furthermore, we establish that carbachol affects the overall phosphorylation of ADAM17 on its threonine and tyrosine but not serine residues, whereas levels of phosphorylated ADAM9 were not affected. Interestingly, carbachol also increases the hydrolysis of the fluorimetric substrate JMV2770, which mimicked the sequence encompassing the N1 site cleavage and was shown previously to behave as an ADAM protease substrate. Mutations of threonine 735 but not of tyrosine 702 of the ADAM17 cytoplasmic tail abolishes the carbachol-induced increase of N1, ADAM17 phosphorylation, and JMV2770-hydrolyzing activity in M1- and M3-expressing HEK293 cells. Thus, our data provide strong evidence that muscarinic receptor activation increases the physiological processing of PrP(c) by upregulating the phosphorylation state and activity of ADAM17 protease.
Journal of Neuroscience 05/2007; 27(15):4083-92. · 7.11 Impact Factor
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Neuron 03/2007; 53(4):483-6. · 14.74 Impact Factor
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ABSTRACT: The cellular prion protein (PrP(c)) undergoes various endopro-teolytic attacks within its N-terminal domain, leading to the production of C-terminal fragments (C) tethered to the plasma membrane and soluble N-terminal peptides (N). One of these cleavages occurs at position 110/111, thereby generating C1 and N1 products. We have reported that disintegrins ADAM-10, -9, and -17 participate either directly or indirectly to this proteolytic event. An alternative proteolytic event taking place around residue 90 yields C2 and N2 fragments. The putative function of these proteolytic fragments remained to be established. We have set up two novel human embryonic kidney 293 cell lines stably overexpressing either C1 or C2. We show that C1 potentiates staurosporine-induced caspase-3 activation through a p53-dependent mechanism. Thus, C1 positively controls p53 transcription and mRNA levels and increases p53-like immunoreactivity and activity. C1-induced caspase-3 activation remained unaffected by the blockade of endocytosis in HEK 293 cells and was abolished in p53-deficient fibroblasts. Conversely, overexpression of the C2 fragment did not significantly sensitize HEK 293 cells to apoptotic stimuli and did not modify p53 mRNA levels or activity. Therefore, the nature of the proteolytic cleavage taking place on PrP(c) yielded C-terminal catabolites with distinct function and could be seen as a switch mechanism controlling the function of the PrP(c) in cell survival.
Journal of Biological Chemistry 02/2007; 282(3):1956-63. · 4.77 Impact Factor
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ABSTRACT: Presenilins (PSs) are part of the gamma-secretase complex that produces the amyloid beta-peptide (Abeta) from its precursor [beta-amyloid precursor protein (betaAPP)]. Mutations in PS that cause familial Alzheimer's disease (FAD) increase Abeta production and trigger p53-dependent cell death. We demonstrate that PS deficiency, catalytically inactive PS mutants, gamma-secretase inhibitors, and betaAPP or amyloid precursor protein-like protein 2 (APLP2) depletion all reduce the expression and activity of p53 and lower the transactivation of its promoter and mRNA expression. p53 expression also is diminished in the brains of PS- or betaAPP-deficient mice. The gamma- and epsilon-secretase-derived amyloid intracellular C-terminal domain (AICD) fragments (AICDC59 and AICDC50, respectively) of betaAPP trigger p53-dependent cell death and increase p53 activity and mRNA. Finally, PS1 mutations enhance p53 activity in human embryonic kidney 293 cells and p53 expression in FAD-affected brains. Thus our study shows that AICDs control p53 at a transcriptional level, in vitro and in vivo, and that FAD mutations increase p53 expression and activity in cells and human brains.
Journal of Neuroscience 07/2006; 26(23):6377-85. · 7.11 Impact Factor
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ABSTRACT: beta-Amyloid peptide accumulates in the brain of patients affected by sporadic or familial forms of Alzheimer's disease. It derives from the proteolytic attacks of the beta-amyloid precursor protein (betaAPP) by beta- and gamma-secretase activities. An additional epsilon cleavage taking place a few residues C-terminal to the gamma-site has been reported, leading to the formation of an intracellular fragment referred to as APP intracellular domain C50. This epsilon cleavage received particular attention because it resembles the S3 Notch cleavage generating Notch intracellular domain. Indeed, APP intracellular domain, like its Notch counterpart, appears to mediate important physiological functions. gamma and epsilon cleavages on betaAPP appear spatio-temporally linked but pharmacologically distinct and discriminable by mutagenesis approaches. As these cleavages could be seen as either deleterious (gamma-site) or beneficial (epsilon-site), it appears of most interest to set up models aimed at studying these activities separately, particularly to design specific and bioavailable inhibitors. On the other hand, it is important to respect the topology of the substrates in order to examine physiologically relevant cleavages. Here we describe the obtention of cells overexpressing APPepsilon, the epsilon-secretase-derived N-terminal fragment of betaAPP. Interestingly, this N-terminal fragment of betaAPP was shown by biochemical and immunohistochemical approaches to behave as a genuine membrane-bound protein. APPepsilon undergoes constitutive and protein kinase C-regulated alpha-secretase cleavages. Furthermore, APPepsilon is targeted by the beta-secretase beta-site APP-cleaving enzyme and is subsequently cleaved by gamma-secretase. The resulting beta-amyloid peptide production is fully prevented by various gamma-secretase inhibitors. Altogether, our study shows that APPepsilon is a relevant betaAPP derivative to study gamma-secretase activities and to design specific inhibitors without facing any rate-limiting effect of epsilon-secretase-derived cleavage.
Journal of Neurochemistry 06/2006; 97(3):807-17. · 4.06 Impact Factor
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ABSTRACT: Parkinson disease is the second most frequent neurodegenerative disorder after Alzheimer disease. A subset of genetic forms of Parkinson disease has been attributed to alpha-synuclein, a synaptic protein with remarkable chaperone properties. Synphilin-1 is a cytoplasmic protein that has been identified as a partner of alpha-synuclein (Engelender, S., Kaminsky, Z., Guo, X., Sharp, A. H., Amaravi, R. K., Kleiderlein, J. J., Margolis, R. L., Troncoso, J. C., Lanahan, A. A., Worley, P. F., Dawson, V. L., Dawson, T. M., and Ross, C. A. (1999) Nat. Gen. 22, 110-114), but its function remains totally unknown. We show here for the first time that synphilin-1 displays an antiapoptotic function in the control of cell death. We have established transient and stable transfectants overexpressing wild-type synphilin-1 in human embryonic kidney 293 cells, telecephalon-specific murine 1 neurons, and SH-SY5Y neuroblastoma cells, and we show that both cell systems display lower responsiveness to staurosporine and 6-hydroxydopamine. Thus, synphilin-1 reduces procaspase-3 hydrolysis and thereby caspase-3 activity and decreases poly(ADP-ribose) polymerase cleavage, two main indicators of apoptotic cell death. Furthermore, we establish that synphilin-1 drastically reduces p53 transcriptional activity and expression and lowers p53 promoter transactivation and mRNA levels. Interestingly, we demonstrate that synphilin-1 catabolism is enhanced by staurosporine and blocked by caspase-3 inhibitors. Accordingly, we show by transcription/translation assay that recombinant caspase-3 and, to a lesser extent, caspase-6 but not caspase-7 hydrolyze synphilin-1. Furthermore, we demonstrate that mutated synphilin-1, in which a consensus caspase-3 target sequence has been disrupted, resists proteolysis by cellular and recombinant caspases and displays drastically reduced antiapoptotic phenotype. We further show that the caspase-3-derived C-terminal fragment of synphilin-1 was probably responsible for the antiapoptotic phenotype elicited by the parent wild-type protein. Altogether, our study is the first demonstration that synphilin-1 harbors a protective function that is controlled by the C-terminal fragment generated by its proteolysis by caspase-3.
Journal of Biological Chemistry 05/2006; 281(17):11515-22. · 4.77 Impact Factor
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ABSTRACT: β-Amyloid peptide accumulates in the brain of patients affected by sporadic or familial forms of Alzheimer's disease. It derives from the proteolytic attacks of the β-amyloid precursor protein (βAPP) by β- and γ-secretase activities. An additional ε cleavage taking place a few residues C-terminal to the γ-site has been reported, leading to the formation of an intracellular fragment referred to as APP intracellular domain C50. This ε cleavage received particular attention because it resembles the S3 Notch cleavage generating Notch intracellular domain. Indeed, APP intracellular domain, like its Notch counterpart, appears to mediate important physiological functions. γ and ε cleavages on βAPP appear spatio-temporally linked but pharmacologically distinct and discriminable by mutagenesis approaches. As these cleavages could be seen as either deleterious (γ-site) or beneficial (ε-site), it appears of most interest to set up models aimed at studying these activities separately, particularly to design specific and bioavailable inhibitors. On the other hand, it is important to respect the topology of the substrates in order to examine physiologically relevant cleavages. Here we describe the obtention of cells overexpressing APPε, the ε-secretase-derived N-terminal fragment of βAPP. Interestingly, this N-terminal fragment of βAPP was shown by biochemical and immunohistochemical approaches to behave as a genuine membrane-bound protein. APPε undergoes constitutive and protein kinase C-regulated α-secretase cleavages. Furthermore, APPε is targeted by the β-secretase β-site APP-cleaving enzyme and is subsequently cleaved by γ-secretase. The resulting β-amyloid peptide production is fully prevented by various γ-secretase inhibitors. Altogether, our study shows that APPε is a relevant βAPP derivative to study γ-secretase activities and to design specific inhibitors without facing any rate-limiting effect of ε-secretase-derived cleavage.
Journal of Neurochemistry 03/2006; 97(3):807 - 817. · 4.06 Impact Factor
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ABSTRACT: The cellular prion protein (PrP(c)) is physiologically cleaved in the middle of its 106-126 amino acid neurotoxic region at the 110/111 downward arrow112 peptidyl bond, yielding an N-terminal fragment referred to as N1. We recently demonstrated that two disintegrins, namely ADAM10 and ADAM17 (TACE, tumor necrosis factor alpha converting enzyme) participated in both constitutive and protein kinase C-regulated generation of N1, respectively. These proteolytic events were strikingly reminiscent of those involved in the so-called "alpha-secretase pathway" that leads to the production of secreted sAPPalpha from betaAPP. We show here, by transient and stable transfection analyses, that ADAM9 also participates in the constitutive secretion of N1 in HEK293 cells, TSM1 neurons, and mouse fibroblasts. Decreasing endogenous ADAM9 expression by an antisense approach drastically reduces both N1 and sAPPalpha recoveries. However, we establish that ADAM9 was unable to increase N1 and sAPPalpha productions after transient transfection in fibroblasts depleted of ADAM10. Accordingly, ADAM9 is unable to cleave a fluorimetric substrate of membrane-bound alpha-secretase activity in ADAM10(-/-) fibroblasts. However, we establish that co-expression of ADAM9 and ADAM10 in ADAM10-deficient fibroblasts leads to enhanced membrane-bound and released fluorimetric substrate hydrolyzing activity when compared with that observed after ADAM10 cDNA transfection alone in ADAM10(-/-) cells. Interestingly, we demonstrate that shedded ADAM10 displays the ability to cleave endogenous PrP(c) in fibroblasts. Altogether, these data provide evidence that ADAM9 is an important regulator of the physiological processing of PrP(c) and betaAPP but that this enzyme acts indirectly, likely by contributing to the shedding of ADAM10. ADAM9 could therefore represent, besides ADAM10, another potential therapeutic target to enhance the breakdown of the 106-126 and Abeta toxic domains of the prion and betaAPP proteins.
Journal of Biological Chemistry 01/2006; 280(49):40624-31. · 4.77 Impact Factor
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Raphaëlle Pardossi-Piquard,
Agnès Petit,
Toshitaka Kawarai, Claire Sunyach,
Cristine Alves da Costa,
Bruno Vincent,
Sabine Ring,
Luciano D'Adamio,
Jie Shen,
Ulrike Müller,
Peter St George Hyslop,
Frédéric Checler
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ABSTRACT: Amyloid beta-peptide (Abeta), which plays a central role in Alzheimer's disease, is generated by presenilin-dependent gamma-secretase cleavage of beta-amyloid precursor protein (betaAPP). We report that the presenilins (PS1 and PS2) also regulate Abeta degradation. Presenilin-deficient cells fail to degrade Abeta and have drastic reductions in the transcription, expression, and activity of neprilysin, a key Abeta-degrading enzyme. Neprilysin activity and expression are also lowered by gamma-secretase inhibitors and by PS1/PS2 deficiency in mouse brain. Neprilysin activity is restored by transient expression of PS1 or PS2 and by expression of the amyloid intracellular domain (AICD), which is cogenerated with Abeta, during gamma-secretase cleavage of betaAPP. Neprilysin gene promoters are transactivated by AICDs from APP-like proteins (APP, APLP1, and APLP2), but not by Abeta or by the gamma-secretase cleavage products of Notch, N- or E- cadherins. The presenilin-dependent regulation of neprilysin, mediated by AICDs, provides a physiological means to modulate Abeta levels with varying levels of gamma-secretase activity.
Neuron 06/2005; 46(4):541-54. · 14.74 Impact Factor
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ABSTRACT: We have previously established that cellular prion PrP(c) elicited p53-dependent caspase 3 activation in various transfected cells and primary cultured neurons. Although we showed that PrP(c) modulates p53 expression at both transcriptional and post-transcriptional levels, it remained unclear as to whether cellular prion signals at the membrane to trigger intracellular messages or if prion proapoptotic activity necessitated its translocation into the cytoplasm. Here, we compare the processing and cell death-related functions of PrP(c) with those of a mutated PrP(c) protein (N-3F4 MoPrP(c)) in which three basic N-terminal residues responsible for PrP(c) internalization had been mutated. As expected, N-3F4 MoPrP(c) remains exclusively located at the membrane, whereas PrP(c) partitions between membrane-associated and intracellular compartments, but both, proteins undergo constitutive and protein kinase C-regulated disintegrin-mediated proteolysis, leading to N1 fragment production. Unlike PrP(c), N-3F4 MoPrP(c) expression does not induce caspase 3 activation after stimulation by staurosporine and was inert on p53 expression and promoter transactivation in both human cells and TSM1 mouse neurons. Interestingly, PrP(c)-induced caspase 3 activation was closely linked to its endocytosis. This phenotype was enhanced by proteasomal inhibition and prevented by sucrose treatment. Accordingly, immunohistochemical analysis showed that protection towards degradation increased intracellular PrP(c)-like immunoreactivity, while sucrose treatments fully abolished PrP(c) intracellular expression and co-localization with transferrin. Altogether, we, establish here, using combined biochemical, mutational and cell biology approaches, that the caspase 3 activation associated with cellular prion is closely related to its ability to undergo endocytosis. This is, to our knowledge, the first direct description of an endocytosis-dependent PrP(c)-associated function.
Journal of Neurochemistry 04/2005; 92(6):1399-407. · 4.06 Impact Factor
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ABSTRACT: We assessed the contribution of the cellular prion protein (PrPc) in the control of neuronal apoptosis by examining cell death in both human cells and murine primary cultured neurons. We first confirmed our previous finding that staurosporine-induced caspase activation is increased by PrPc overexpression in HEK293 cells. We show here that this phenotype is fully dependent on p53 and that the control of p53 activity by PrPc occurs at both transcriptional and post-transcriptional levels in human cells. Of most interest, we demonstrate that neuronal endogenous PrPc also controls a p53-dependent pro-apoptotic phenotype. Thus, DNA fragmentation and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling)-positive cells were lower in primary cultured neurons derived from Zrch-1 mice embryos in which PrPc has been abrogated than in wild-type neurons. PrPc knock-out neurons also displayed drastically diminished caspase-3-like activity and immunoreactivity together with reduced p53 expression and transcriptional activity, a phenotype complemented in part by PrPc transfection. Interestingly, p53 expression was also reduced in the brain of adult Prnp-/- mice. Neuronal PrPc likely controls p53 at a post-transcriptional level because the deletion of cellular prion protein is accompanied by a higher Mdm2-like immunoreactivity and reduced phosphorylated p38 MAPK expression. We therefore propose that the physiological function of endogenous cellular prion could be to regulate p53-dependent caspase-3-mediated neuronal cell death. This phenotype likely occurs through up-regulation of p53 promoter transactivation as well as downstream by controlling p53 stability via Mdm2 expression.
Journal of Biological Chemistry 01/2004; 279(1):612-8. · 4.77 Impact Factor
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Journal of Biological Chemistry 01/2004; 279(january 2):612-618. · 4.77 Impact Factor
