Frédéric Checler | Université Nice Sophia Antipolis (original) (raw)

Papers by Frédéric Checler

Research paper thumbnail of Nuclear Factor-κB Regulates βAPP and β- and γ-Secretases Differently at Physiological and Supraphysiological Aβ Concentrations

Journal of Biological Chemistry, 2012

Background: NF-B regulates BACE1 but there is little data suggesting ␤APP and ␥-secretase involve... more Background: NF-B regulates BACE1 but there is little data suggesting ␤APP and ␥-secretase involvement. Results: NF-B differentially regulates A␤ production at physiological and supraphysiological A␤ concentrations by modulating transactivation of ␤APP and ␥-secretase promoters, thereby controlling ␥-secretase activity. Conclusion: Under physiological conditions, NF-B regulates A␤ homeostasis while it contributes in increasing A␤ production in the pathological context. Significance: NF-B may be seen as a potential therapeutic target. * This work was supported by the Fondation pour la Recherche Médicale (FRM), by the Conseil Général des Alpes Maritimes, and by the Ministère de l'enseignement supérieur et de la Recherche. This work has been developed and supported through the LABEX (excellence laboratory, program investment for the future) DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to Alzheimer disease). □ S This article contains supplemental Fig. S1.

Research paper thumbnail of P4-317 Gamma-secretase inhibitors: a critical evaluation of their potential therapeutic use in Alzheimer disease

Neurobiology of Aging, 2004

The role of each protein in the complex and the mechanisms of the enzymatic activity. Methods: Bi... more The role of each protein in the complex and the mechanisms of the enzymatic activity. Methods: Biochemical, cell biological and irnmtmochemical techniques, including immtmocytochernistry, glycerol velocity gradient fractionation, and 2-dimensional blue native gel electrophoresis. Results: we show that APH-1 proteins directly interact with immature and mature forms of presenilins and nicastrin. Furthermore, we report data from several complementary experiments suggest that there exist several distinct presenilin complexes. These complexes are not all associated with functional y-secretase activity and are differentially affected by presenilin mutations and detergents. Conclusions: Our data indicated that APH-1 proteins play a role in the initial assembly and maturation of presenilin-nicastfin complexes and that a low abundance, very high molecular weight species of presenilin-complexes is associated with functional y-secretase activity.

Research paper thumbnail of JLK isocoumarin inhibitors: Selective ?-secretase inhibitors that do not interfere with notch pathway in vitro or in vivo

Journal of Neuroscience Research, 2003

gamma-Secretase activity is involved in the generation of Abeta and therefore likely contributes ... more gamma-Secretase activity is involved in the generation of Abeta and therefore likely contributes to the pathology of Alzheimer's disease. Blocking this activity was seen as a major therapeutic target to slow down or arrest Abeta-related AD progression. This strategy seemed more doubtful when it was established that gamma-secretase also targets other substrates including Notch, a particularly important transmembrane protein involved in vital functions, at both embryonic and adulthood stages. We have described previously new non-peptidic inhibitors able to selectively inhibit Abeta cellular production in vitro without altering Notch pathway. We show here that in vivo, these inhibitors do not alter the Notch pathway responsible for somitogenesis in the zebrafish embryo. In addition, we document further the selectivity of JLK inhibitors by showing that, unlike other described gamma-secretase inhibitors, these agents do not affect E-cadherin processing. Finally, we establish that JLKs do not inhibit beta-site APP cleaving enzymes (BACE) 1 and BACE2, alpha-secretase, the proteasome, and GSK3beta kinase. Altogether, JLK inhibitors are the sole agents to date that are able to prevent Abeta production without triggering unwanted cleavages of other proteins.

Research paper thumbnail of Further characterization of a putative serine protease contributing to the γ-secretase cleavage of β-amyloid precursor protein

Bioorganic & Medicinal Chemistry, 2013

The 3-alkoxy-7-amino-4-chloro-isocoumarins JLK-6 and JLK-2 have been shown to markedly reduce the... more The 3-alkoxy-7-amino-4-chloro-isocoumarins JLK-6 and JLK-2 have been shown to markedly reduce the production of Amyloid b-peptide (Ab) by Amyloid-b Precursor Protein (APP) expressing HEK293 cells by affecting the c-secretase cleavage of APP, with no effect on the cleavage of the Notch receptor. This suggested that these compounds do not directly inhibit the presenilin-dependent c-secretase complex but more likely interfere with an upstream target involved in c-secretase-associated pathway. The mechanism of action of these compounds is unknown and there are high fundamental and therapeutical interests to unravel their target. Isocoumarin compounds were previously shown to behave as potent mechanism-based irreversible inhibitors of serine proteases, suggesting that the JLK-directed target could belong to such enzyme family. To get further insight into structure-activity relationships and to develop more potent isocoumarin derivatives, we have synthesized and evaluated a series of isocoumarin analogues with modifications at positions 3, 4 and 7. In particular, the 7-amino group was substituted with either acyl, urethane, alkyl or aryl groups, which could represent additional interaction sites. Altogether, the results highlighted the essential integrity of the 3-alkoxy-7-amino-4-chloro-isocoumarin scaffold for Ab-lowering activity and supported the involvement of a serine protease, or may be more generally, a serine hydrolase. The newly reported 7-N-alkyl series produced the most active compounds with an IC 50 between 10 and 30 lM. Finally, we also explored peptide boronates, a series of reversible serine protease inhibitors, previously shown to also lower cellular Ab production. The presented data suggested they could act on the same target or interfere with the same pathway as isocoumarins derivatives.

Research paper thumbnail of Chronic fornix deep brain stimulation in a transgenic Alzheimer’s rat model reduces amyloid burden, inflammation, and neuronal loss

Brain Structure and Function, 2018

Recent studies have suggested deep brain stimulation (DBS) as a promising therapy in patients wit... more Recent studies have suggested deep brain stimulation (DBS) as a promising therapy in patients with Alzheimer's disease (AD). Particularly, the stimulation of the forniceal area was found to slow down the cognitive decline of some AD patients, but the biochemical and anatomical modifications underlying these effects remain poorly understood. We evaluated the effects of chronic forniceal stimulation on amyloid burden, inflammation, and neuronal loss in a transgenic Alzheimer rat model TgF344-AD, as well as in age-matched control rats. 18-month-old rats were surgically implanted with electrodes in stereotactic conditions and connected to a portable microstimulator for chronic DBS in freely moving rats. The stimulation was continuous during 5 weeks and animals were immediately sacrificed for immunohistochemical analysis of pathological markers. Implanted, but non-stimulated rats were used as controls. We found that chronic forniceal DBS in the Tg-AD rat significantly reduces amyloid deposition in the hippocampus and cortex, decreases astrogliosis and microglial activation and lowers neuronal loss, as determined by NeuN staining. In control animals, the stimulation neither affects neuroinflammation nor neuronal count. In the Tg-F344-AD rat model, 5 weeks of forniceal DBS decreased amyloidosis, inflammatory responses, and neuronal loss in both cortex and hippocampus. These findings strongly suggest a neuroprotective effect of DBS and support the beneficial effects of targeting the fornix in Alzheimer's disease patients.

Research paper thumbnail of Sporadic cerebral amyloid angiopathy presenting as frontal dementia A case with unusual histological features

Research paper thumbnail of The α-Secretase-derived N-terminal Product of Cellular Prion, N1, Displays Neuroprotective Function in Vitro and in Vivo

Journal of Biological Chemistry, 2009

Cellular prion protein (PrP c) undergoes a disintegrin-mediated physiological cleavage, generatin... more 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 ␣-secretasederived 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. * This work was supported by CNRS, the Fédé ration pour la Recherche sur le Cerveau, and the Fondation pour la Recherche Mé dicale.

Research paper thumbnail of alpha-secretase cleavage of APP in the furin-deficient LoVo cell line: Involvement of the prohormone convertase PC7 and the disintegrin ADAM10

Research paper thumbnail of Transcription- and phosphorylation-dependent control of a functional interplay between XBP1s and PINK1 governs mitophagy and potentially impacts Parkinson disease pathophysiology

Autophagy, 2021

Parkinson disease (PD)-affected brains show consistent endoplasmic reticulum (ER) stress and mito... more Parkinson disease (PD)-affected brains show consistent endoplasmic reticulum (ER) stress and mitophagic dysfunctions. The mechanisms underlying these perturbations and how they are directly linked remain a matter of questions. XBP1 is a transcription factor activated upon ER stress after unconventional splicing by the nuclease ERN1/IREα thereby yielding XBP1s, whereas PINK1 is a kinase considered as the sensor of mitochondrial physiology and a master gatekeeper of mitophagy process. We showed that XBP1s transactivates PINK1 in human cells, primary cultured neurons and mice brain, and triggered a pro-mitophagic phenotype that was fully dependent of endogenous PINK1. We also unraveled a PINK1-dependent phosphorylation of XBP1s that conditioned its nuclear localization and thereby, governed its transcriptional activity. PINK1-induced XBP1s phosphorylation occurred at residues reminiscent of, and correlated to, those phosphorylated in substantia nigra of sporadic PD-affected brains. Overall, our study delineated a functional loop between XBP1s and PINK1 governing mitophagy that was disrupted in PD condition.Abbreviations: 6OHDA: 6-hydroxydopamine; baf: bafilomycin A1; BECN1: beclin 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CCCP: carbonyl cyanide chlorophenylhydrazone; COX8A: cytochrome c oxidase subunit 8A; DDIT3/CHOP: DNA damage inducible transcript 3; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FACS: fluorescence-activated cell sorting; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFN2: mitofusin 2; OPTN: optineurin; PD: Parkinson disease; PINK1: PTEN-induced kinase 1; PCR: polymerase chain reaction:; PRKN: parkin RBR E3 ubiquitin protein ligase; XBP1s [p-S61A]: XBP1s phosphorylated at serine 61; XBP1s [p-T48A]: XBP1s phosphorylated at threonine 48; shRNA: short hairpin RNA, SQSTM1/p62: sequestosome 1; TIMM23: translocase of inner mitochondrial membrane 23; TM: tunicamycin; TMRM: tetramethyl rhodamine methylester; TOMM20: translocase of outer mitochondrial membrane 20; Toy: toyocamycin; TP: thapsigargin; UB: ubiquitin; UB (S65): ubiquitin phosphorylated at serine 65; UPR: unfolded protein response, XBP1: X-box binding protein 1; XBP1s: spliced X-box binding protein 1.

Research paper thumbnail of MT5-MMP controls APP and β-CTF/C99 metabolism through proteolytic-dependent and -independent mechanisms relevant for Alzheimer’s disease

ABSTRACTWe previously discovered the implication of membrane-type 5-matrix metalloproteinase (MT5... more ABSTRACTWe previously discovered the implication of membrane-type 5-matrix metalloproteinase (MT5-MMP) in Alzheimer’s disease AD pathogenesis. Here we shed new light on pathogenic mechanisms by which MT5-MMP controls APP processing and the fate of amyloid beta peptide (Aβ), its precursor C99 and C83. We found in HEK carrying the APP Swedish familial mutation (HEKswe) that MT5-MMP-mediated processing of APP that releases the soluble 95 kDa form (sAPP95), was hampered by the removal of the C-terminal non-catalytic domains of MT5-MMP. Catalytically inactive MT5-MMP variants increased the levels of Aβ and promoted APP/C99 sorting in the endo-lysosomal system. We found interaction of C99 with the C-terminal portion of MT5-MMP, the deletion of which caused a strong degradation of C99 by the proteasome, preventing Aβ accumulation. These findings reveal novel mechanisms for MT5-MMP control of APP metabolism and C99 fate involving proteolytic and non-proteolytic actions mainly mediated by th...

Research paper thumbnail of Pyk2 Overexpression in Postsynaptic Neurons Blocks Aβ1-42-induced Synaptotoxicity in a Microfluidic Co-Culture Model

Recent meta-analyses of genome-wide association studies identified a number of genetic risk facto... more Recent meta-analyses of genome-wide association studies identified a number of genetic risk factors of Alzheimer’s disease; however, little is known about the mechanisms by which they contribute to the pathological process. As synapse loss is observed at the earliest stage of Alzheimer’s disease, deciphering the impact of Alzheimer’s risk genes on synapse formation and maintenance is of great interest. In this paper, we report a microfluidic co-culture device that physically isolates synapses from pre- and postsynaptic neurons and chronically exposes them to toxic amyloid-beta (Aβ) peptides secreted by model cell lines overexpressing wild-type or mutated (V717I) amyloid precursor protein (APP). Co-culture with cells overexpressing mutated APP exposed the synapses of primary hippocampal neurons to Aβ1-42 molecules at nanomolar concentrations and induced a significant decrease in synaptic connectivity, as evidenced by distance-based assignment of postsynaptic puncta to presynaptic pun...

Research paper thumbnail of Aging Increased Amyloid Peptide and Caused Amyloid Plaques in Brain of Old APP/V717I Transgenic Mice by a Different Mechanism than Mutant Presenilin1

The Journal of Neuroscience, 2000

Aging of transgenic mice that overexpress the London mutant of amyloid precursor protein (APP/V71... more Aging of transgenic mice that overexpress the London mutant of amyloid precursor protein (APP/V717I) (Moechars et al., 1999a) was now demonstrated not to affect the normalized levels of ␣or ␤-cleaved secreted APP nor of the ␤-C-terminal stubs. This indicated that aging did not markedly disturb either ␣or ␤-secretase cleavage of APP and failed to explain the origin of the massive amounts of amyloid peptides A␤40 and A␤42, soluble and precipitated as amyloid plaques in the brain of old APP/V717I transgenic mice. We tested the hypothesis that aging acted on presenilin1 (PS1) to affect ␥-secretase-mediated production of amyloid peptides by comparing aged APP/V717I transgenic mice to double transgenic mice coexpressing human PS1 and APP/V717I. In double transgenic mice with mutant (A246E) but not wild-type human PS1, brain amyloid peptide levels increased and resulted in amyloid plaques when the mice were only 6-9 months old, much earlier than in APP/V717I transgenic mice (12-15 months old). Mutant PS1 increased mainly brain A␤42 levels, whereas in aged APP/V717I transgenic mice, both A␤42 and A␤40 increased. This resulted in a dramatic difference in the A␤42/A␤40 ratio of precipitated or plaqueassociated amyloid peptides, i.e., 3.11 Ϯ 0.22 in double APP/ V717I ϫ PS1/A246E transgenic mice compared with 0.43 Ϯ 0.07 in aged APP/V717I transgenic mice, and demonstrated a clear difference between the effect of aging and the effect of the insertion of a mutant PS1 transgene. In conclusion, we demonstrate that aging did not favor amyloidogenic over nonamyloidogenic processing of APP, nor did it exert a mutant PS1-like effect on ␥-secretase. Therefore, the data are interpreted to suggest that parenchymal and vascular accumulation of amyloid in aging brain resulted from failure to clear the amyloid peptides rather than from increased production.

Research paper thumbnail of Proteasome Inhibitors Prevent the Degradation of Familial Alzheimer’s Disease-Linked Presenilin 1 and Potentiate Aβ42 Recovery from Human Cells

Research paper thumbnail of Visualization of specific γ-secretase complexes using bimolecular fluorescence complementation

Journal of Alzheimer's disease : JAD, 2014

γ-Secretase is involved in the regulated intramembrane proteolysis of amyloid-β protein precursor... more γ-Secretase is involved in the regulated intramembrane proteolysis of amyloid-β protein precursor (AβPP) and of many other important physiological substrates. γ-secretase is a multiproteic complex made of four main core components, namely presenilin 1 or 2, APH-1, PEN-2, and Nicastrin. Since APH-1 exists as different variants, combinations of these proteins can theoretically yield distinct γ-secretase complexes. Whether γ-secretase complexes trafficking and targeting to either similar or distinct subcellular compartments depend upon their molecular composition remains unknown. A differential complex-specific distribution may drive a narrow specificity for a subset of substrates that would traffic within the same cellular compartments. Here, we generated bigenic expression vectors to co-express untagged nicastrin or presenilin 1 together with either PEN-2 or distinct variants of APH-1 (aL, aS and b) tagged with complementary fragments of the fluorescent protein Venus. We show that th...

Research paper thumbnail of Evidence that the amyloid-β protein precursor intracellular domain, AICD, derives from β-secretase-generated C-terminal fragment

Journal of Alzheimer's disease : JAD, 2012

One of the major pathological hallmarks of brains affected with Alzheimer's disease (AD) is t... more One of the major pathological hallmarks of brains affected with Alzheimer's disease (AD) is the senile plaque, an extracellular deposit mainly composed of a set of highly insoluble peptides of various lengths (39-43 amino acids) referred to as amyloid-β (Aβ) peptides. Aβ peptides are derived from combined proteolytic cleavages undergone on the amyloid-β protein precursor (AβPP) by a set of enzymes called secretases. Several lines of anatomical and biological evidence suggest that Aβ peptides would not account for all pathological stigmata and molecular dysfunctions taking place in AD. In amyloidogenic and non-amyloidogenic pathways, AβPP first undergoes β- or α-secretases-mediated cleavages yielding C99 and C83, respectively. These two membrane-embedded C-terminal fragments are both potential targets of subsequent γ-secretase-mediated proteolysis. The latter cleavage not only generates either p3 or Aβ peptides but similarly gives rise to an AβPP IntraCellular Domain (AICD fragme...

Research paper thumbnail of NACP/alpha-synuclein, NAC, and beta-amyloid pathology of familial Alzheimer's disease with the E184D presenilin-1 mutation: a clinicopathological study of two autopsy cases

Acta neuropathologica, 2002

Approximately 60% of familial and sporadic Alzheimer's disease (AD) cases manifest Lewy bodie... more Approximately 60% of familial and sporadic Alzheimer's disease (AD) cases manifest Lewy bodies (LBs), of which a major component is alpha-synuclein. Although the pathogenic role of alpha-synuclein in AD remains unclear, LB formation might be associated with pathological beta-amyloid (Abeta) overproduction. Here, we present the clinical and pathological characteristics of two affected family members from a pedigree with the E184D mutation of presenilin-1. One case presented with typical clinical features of AD, but the other case also developed clinical characteristics of dementia with Lewy bodies (DLB), including visual hallucinations, delusions, and parkinsonism. In both cases, neuropathological examination revealed numerous neurofibrillary tangles and severe Abeta deposition in senile plaques and amyloid angiopathy, in which Abeta42 rather than Abeta40 was predominant. Furthermore, remarkable alpha-synuclein pathology, including LBs and the accumulation of the non-Abeta compon...

Research paper thumbnail of Métabolisme du précurseur du peptide amyloïde et présénilines

Research paper thumbnail of Neurotensin Receptors in Primary Culture of Neurons

Methods in Neurosciences, 1993

Publisher Summary This chapter describes the main biochemical techniques that are available for n... more Publisher Summary This chapter describes the main biochemical techniques that are available for neurotensin receptor characterization, localization, and regulation on the primary cultures of neurons. These methods can be applied to other cell culture systems if cautions concerning the degradation and internalization of the ligand are taken. The neuromodulatory role of neurotensin is supported by its actions through specific high-affinity binding sites in discrete regions of the mammalian central nervous system. These neurotensin receptors are localized along dopaminergic pathways and have been extensively studied in terms of biochemical, anatomical, functional, and molecular properties. The chapter describes a set of tools and methods to study neurotensin receptors, which bind to dispersed neurons in primary culture, and discusses some techniques that allow the visualization of neurotensin receptors, both in situ and in denaturing conditions by radioautography. It also explains the regulatory mechanisms and the functional relevance of neurotensin receptors present in the neuronal cell system.

Research paper thumbnail of p53, a Molecular Bridge Between Alzheimer’s Disease Pathology and Cancers?

Research and Perspectives in Alzheimer's Disease, 2010

Cancers are characterized by enhanced cell survival and altered differentiation processes whereas... more Cancers are characterized by enhanced cell survival and altered differentiation processes whereas Alzheimer’s disease (AD)-affected brains exhibit exacerbated neuronal loss and cell death. Interestingly, several studies have consistently reported on an inverse relationship between cancer and AD. On the other hand, p53, a tumor-suppressor oncogene, is mutated and inactivated in a majority of human cancers; conversely, several lines of evidence concur to suggest an elevation of p53 and its transcriptional targets in AD brains. Therefore, one could envision p53 as a molecular bridge between cancer and AD pathologies. Although the role of p53 in cancer likely results from its inactivation by somatic mutations, the mechanistic aspects underlying a dysfunction in the control of p53 in AD had not been delineated. Here we survey recent evidence that p53 could control and be controlled by several members of the presenilin-dependent γ-secretase complex, and we briefly discuss the possibility that a functional deficit in presenilins could contribute to the genesis of a subset of tumors.

Research paper thumbnail of Alzheimer's and prion diseases: distinct pathologies, common proteolytic denominators

Trends in Neurosciences, 2002

Research paper thumbnail of Nuclear Factor-κB Regulates βAPP and β- and γ-Secretases Differently at Physiological and Supraphysiological Aβ Concentrations

Journal of Biological Chemistry, 2012

Background: NF-B regulates BACE1 but there is little data suggesting ␤APP and ␥-secretase involve... more Background: NF-B regulates BACE1 but there is little data suggesting ␤APP and ␥-secretase involvement. Results: NF-B differentially regulates A␤ production at physiological and supraphysiological A␤ concentrations by modulating transactivation of ␤APP and ␥-secretase promoters, thereby controlling ␥-secretase activity. Conclusion: Under physiological conditions, NF-B regulates A␤ homeostasis while it contributes in increasing A␤ production in the pathological context. Significance: NF-B may be seen as a potential therapeutic target. * This work was supported by the Fondation pour la Recherche Médicale (FRM), by the Conseil Général des Alpes Maritimes, and by the Ministère de l'enseignement supérieur et de la Recherche. This work has been developed and supported through the LABEX (excellence laboratory, program investment for the future) DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to Alzheimer disease). □ S This article contains supplemental Fig. S1.

Research paper thumbnail of P4-317 Gamma-secretase inhibitors: a critical evaluation of their potential therapeutic use in Alzheimer disease

Neurobiology of Aging, 2004

The role of each protein in the complex and the mechanisms of the enzymatic activity. Methods: Bi... more The role of each protein in the complex and the mechanisms of the enzymatic activity. Methods: Biochemical, cell biological and irnmtmochemical techniques, including immtmocytochernistry, glycerol velocity gradient fractionation, and 2-dimensional blue native gel electrophoresis. Results: we show that APH-1 proteins directly interact with immature and mature forms of presenilins and nicastrin. Furthermore, we report data from several complementary experiments suggest that there exist several distinct presenilin complexes. These complexes are not all associated with functional y-secretase activity and are differentially affected by presenilin mutations and detergents. Conclusions: Our data indicated that APH-1 proteins play a role in the initial assembly and maturation of presenilin-nicastfin complexes and that a low abundance, very high molecular weight species of presenilin-complexes is associated with functional y-secretase activity.

Research paper thumbnail of JLK isocoumarin inhibitors: Selective ?-secretase inhibitors that do not interfere with notch pathway in vitro or in vivo

Journal of Neuroscience Research, 2003

gamma-Secretase activity is involved in the generation of Abeta and therefore likely contributes ... more gamma-Secretase activity is involved in the generation of Abeta and therefore likely contributes to the pathology of Alzheimer's disease. Blocking this activity was seen as a major therapeutic target to slow down or arrest Abeta-related AD progression. This strategy seemed more doubtful when it was established that gamma-secretase also targets other substrates including Notch, a particularly important transmembrane protein involved in vital functions, at both embryonic and adulthood stages. We have described previously new non-peptidic inhibitors able to selectively inhibit Abeta cellular production in vitro without altering Notch pathway. We show here that in vivo, these inhibitors do not alter the Notch pathway responsible for somitogenesis in the zebrafish embryo. In addition, we document further the selectivity of JLK inhibitors by showing that, unlike other described gamma-secretase inhibitors, these agents do not affect E-cadherin processing. Finally, we establish that JLKs do not inhibit beta-site APP cleaving enzymes (BACE) 1 and BACE2, alpha-secretase, the proteasome, and GSK3beta kinase. Altogether, JLK inhibitors are the sole agents to date that are able to prevent Abeta production without triggering unwanted cleavages of other proteins.

Research paper thumbnail of Further characterization of a putative serine protease contributing to the γ-secretase cleavage of β-amyloid precursor protein

Bioorganic & Medicinal Chemistry, 2013

The 3-alkoxy-7-amino-4-chloro-isocoumarins JLK-6 and JLK-2 have been shown to markedly reduce the... more The 3-alkoxy-7-amino-4-chloro-isocoumarins JLK-6 and JLK-2 have been shown to markedly reduce the production of Amyloid b-peptide (Ab) by Amyloid-b Precursor Protein (APP) expressing HEK293 cells by affecting the c-secretase cleavage of APP, with no effect on the cleavage of the Notch receptor. This suggested that these compounds do not directly inhibit the presenilin-dependent c-secretase complex but more likely interfere with an upstream target involved in c-secretase-associated pathway. The mechanism of action of these compounds is unknown and there are high fundamental and therapeutical interests to unravel their target. Isocoumarin compounds were previously shown to behave as potent mechanism-based irreversible inhibitors of serine proteases, suggesting that the JLK-directed target could belong to such enzyme family. To get further insight into structure-activity relationships and to develop more potent isocoumarin derivatives, we have synthesized and evaluated a series of isocoumarin analogues with modifications at positions 3, 4 and 7. In particular, the 7-amino group was substituted with either acyl, urethane, alkyl or aryl groups, which could represent additional interaction sites. Altogether, the results highlighted the essential integrity of the 3-alkoxy-7-amino-4-chloro-isocoumarin scaffold for Ab-lowering activity and supported the involvement of a serine protease, or may be more generally, a serine hydrolase. The newly reported 7-N-alkyl series produced the most active compounds with an IC 50 between 10 and 30 lM. Finally, we also explored peptide boronates, a series of reversible serine protease inhibitors, previously shown to also lower cellular Ab production. The presented data suggested they could act on the same target or interfere with the same pathway as isocoumarins derivatives.

Research paper thumbnail of Chronic fornix deep brain stimulation in a transgenic Alzheimer’s rat model reduces amyloid burden, inflammation, and neuronal loss

Brain Structure and Function, 2018

Recent studies have suggested deep brain stimulation (DBS) as a promising therapy in patients wit... more Recent studies have suggested deep brain stimulation (DBS) as a promising therapy in patients with Alzheimer's disease (AD). Particularly, the stimulation of the forniceal area was found to slow down the cognitive decline of some AD patients, but the biochemical and anatomical modifications underlying these effects remain poorly understood. We evaluated the effects of chronic forniceal stimulation on amyloid burden, inflammation, and neuronal loss in a transgenic Alzheimer rat model TgF344-AD, as well as in age-matched control rats. 18-month-old rats were surgically implanted with electrodes in stereotactic conditions and connected to a portable microstimulator for chronic DBS in freely moving rats. The stimulation was continuous during 5 weeks and animals were immediately sacrificed for immunohistochemical analysis of pathological markers. Implanted, but non-stimulated rats were used as controls. We found that chronic forniceal DBS in the Tg-AD rat significantly reduces amyloid deposition in the hippocampus and cortex, decreases astrogliosis and microglial activation and lowers neuronal loss, as determined by NeuN staining. In control animals, the stimulation neither affects neuroinflammation nor neuronal count. In the Tg-F344-AD rat model, 5 weeks of forniceal DBS decreased amyloidosis, inflammatory responses, and neuronal loss in both cortex and hippocampus. These findings strongly suggest a neuroprotective effect of DBS and support the beneficial effects of targeting the fornix in Alzheimer's disease patients.

Research paper thumbnail of Sporadic cerebral amyloid angiopathy presenting as frontal dementia A case with unusual histological features

Research paper thumbnail of The α-Secretase-derived N-terminal Product of Cellular Prion, N1, Displays Neuroprotective Function in Vitro and in Vivo

Journal of Biological Chemistry, 2009

Cellular prion protein (PrP c) undergoes a disintegrin-mediated physiological cleavage, generatin... more 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 ␣-secretasederived 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. * This work was supported by CNRS, the Fédé ration pour la Recherche sur le Cerveau, and the Fondation pour la Recherche Mé dicale.

Research paper thumbnail of alpha-secretase cleavage of APP in the furin-deficient LoVo cell line: Involvement of the prohormone convertase PC7 and the disintegrin ADAM10

Research paper thumbnail of Transcription- and phosphorylation-dependent control of a functional interplay between XBP1s and PINK1 governs mitophagy and potentially impacts Parkinson disease pathophysiology

Autophagy, 2021

Parkinson disease (PD)-affected brains show consistent endoplasmic reticulum (ER) stress and mito... more Parkinson disease (PD)-affected brains show consistent endoplasmic reticulum (ER) stress and mitophagic dysfunctions. The mechanisms underlying these perturbations and how they are directly linked remain a matter of questions. XBP1 is a transcription factor activated upon ER stress after unconventional splicing by the nuclease ERN1/IREα thereby yielding XBP1s, whereas PINK1 is a kinase considered as the sensor of mitochondrial physiology and a master gatekeeper of mitophagy process. We showed that XBP1s transactivates PINK1 in human cells, primary cultured neurons and mice brain, and triggered a pro-mitophagic phenotype that was fully dependent of endogenous PINK1. We also unraveled a PINK1-dependent phosphorylation of XBP1s that conditioned its nuclear localization and thereby, governed its transcriptional activity. PINK1-induced XBP1s phosphorylation occurred at residues reminiscent of, and correlated to, those phosphorylated in substantia nigra of sporadic PD-affected brains. Overall, our study delineated a functional loop between XBP1s and PINK1 governing mitophagy that was disrupted in PD condition.Abbreviations: 6OHDA: 6-hydroxydopamine; baf: bafilomycin A1; BECN1: beclin 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CCCP: carbonyl cyanide chlorophenylhydrazone; COX8A: cytochrome c oxidase subunit 8A; DDIT3/CHOP: DNA damage inducible transcript 3; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FACS: fluorescence-activated cell sorting; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFN2: mitofusin 2; OPTN: optineurin; PD: Parkinson disease; PINK1: PTEN-induced kinase 1; PCR: polymerase chain reaction:; PRKN: parkin RBR E3 ubiquitin protein ligase; XBP1s [p-S61A]: XBP1s phosphorylated at serine 61; XBP1s [p-T48A]: XBP1s phosphorylated at threonine 48; shRNA: short hairpin RNA, SQSTM1/p62: sequestosome 1; TIMM23: translocase of inner mitochondrial membrane 23; TM: tunicamycin; TMRM: tetramethyl rhodamine methylester; TOMM20: translocase of outer mitochondrial membrane 20; Toy: toyocamycin; TP: thapsigargin; UB: ubiquitin; UB (S65): ubiquitin phosphorylated at serine 65; UPR: unfolded protein response, XBP1: X-box binding protein 1; XBP1s: spliced X-box binding protein 1.

Research paper thumbnail of MT5-MMP controls APP and β-CTF/C99 metabolism through proteolytic-dependent and -independent mechanisms relevant for Alzheimer’s disease

ABSTRACTWe previously discovered the implication of membrane-type 5-matrix metalloproteinase (MT5... more ABSTRACTWe previously discovered the implication of membrane-type 5-matrix metalloproteinase (MT5-MMP) in Alzheimer’s disease AD pathogenesis. Here we shed new light on pathogenic mechanisms by which MT5-MMP controls APP processing and the fate of amyloid beta peptide (Aβ), its precursor C99 and C83. We found in HEK carrying the APP Swedish familial mutation (HEKswe) that MT5-MMP-mediated processing of APP that releases the soluble 95 kDa form (sAPP95), was hampered by the removal of the C-terminal non-catalytic domains of MT5-MMP. Catalytically inactive MT5-MMP variants increased the levels of Aβ and promoted APP/C99 sorting in the endo-lysosomal system. We found interaction of C99 with the C-terminal portion of MT5-MMP, the deletion of which caused a strong degradation of C99 by the proteasome, preventing Aβ accumulation. These findings reveal novel mechanisms for MT5-MMP control of APP metabolism and C99 fate involving proteolytic and non-proteolytic actions mainly mediated by th...

Research paper thumbnail of Pyk2 Overexpression in Postsynaptic Neurons Blocks Aβ1-42-induced Synaptotoxicity in a Microfluidic Co-Culture Model

Recent meta-analyses of genome-wide association studies identified a number of genetic risk facto... more Recent meta-analyses of genome-wide association studies identified a number of genetic risk factors of Alzheimer’s disease; however, little is known about the mechanisms by which they contribute to the pathological process. As synapse loss is observed at the earliest stage of Alzheimer’s disease, deciphering the impact of Alzheimer’s risk genes on synapse formation and maintenance is of great interest. In this paper, we report a microfluidic co-culture device that physically isolates synapses from pre- and postsynaptic neurons and chronically exposes them to toxic amyloid-beta (Aβ) peptides secreted by model cell lines overexpressing wild-type or mutated (V717I) amyloid precursor protein (APP). Co-culture with cells overexpressing mutated APP exposed the synapses of primary hippocampal neurons to Aβ1-42 molecules at nanomolar concentrations and induced a significant decrease in synaptic connectivity, as evidenced by distance-based assignment of postsynaptic puncta to presynaptic pun...

Research paper thumbnail of Aging Increased Amyloid Peptide and Caused Amyloid Plaques in Brain of Old APP/V717I Transgenic Mice by a Different Mechanism than Mutant Presenilin1

The Journal of Neuroscience, 2000

Aging of transgenic mice that overexpress the London mutant of amyloid precursor protein (APP/V71... more Aging of transgenic mice that overexpress the London mutant of amyloid precursor protein (APP/V717I) (Moechars et al., 1999a) was now demonstrated not to affect the normalized levels of ␣or ␤-cleaved secreted APP nor of the ␤-C-terminal stubs. This indicated that aging did not markedly disturb either ␣or ␤-secretase cleavage of APP and failed to explain the origin of the massive amounts of amyloid peptides A␤40 and A␤42, soluble and precipitated as amyloid plaques in the brain of old APP/V717I transgenic mice. We tested the hypothesis that aging acted on presenilin1 (PS1) to affect ␥-secretase-mediated production of amyloid peptides by comparing aged APP/V717I transgenic mice to double transgenic mice coexpressing human PS1 and APP/V717I. In double transgenic mice with mutant (A246E) but not wild-type human PS1, brain amyloid peptide levels increased and resulted in amyloid plaques when the mice were only 6-9 months old, much earlier than in APP/V717I transgenic mice (12-15 months old). Mutant PS1 increased mainly brain A␤42 levels, whereas in aged APP/V717I transgenic mice, both A␤42 and A␤40 increased. This resulted in a dramatic difference in the A␤42/A␤40 ratio of precipitated or plaqueassociated amyloid peptides, i.e., 3.11 Ϯ 0.22 in double APP/ V717I ϫ PS1/A246E transgenic mice compared with 0.43 Ϯ 0.07 in aged APP/V717I transgenic mice, and demonstrated a clear difference between the effect of aging and the effect of the insertion of a mutant PS1 transgene. In conclusion, we demonstrate that aging did not favor amyloidogenic over nonamyloidogenic processing of APP, nor did it exert a mutant PS1-like effect on ␥-secretase. Therefore, the data are interpreted to suggest that parenchymal and vascular accumulation of amyloid in aging brain resulted from failure to clear the amyloid peptides rather than from increased production.

Research paper thumbnail of Proteasome Inhibitors Prevent the Degradation of Familial Alzheimer’s Disease-Linked Presenilin 1 and Potentiate Aβ42 Recovery from Human Cells

Research paper thumbnail of Visualization of specific γ-secretase complexes using bimolecular fluorescence complementation

Journal of Alzheimer's disease : JAD, 2014

γ-Secretase is involved in the regulated intramembrane proteolysis of amyloid-β protein precursor... more γ-Secretase is involved in the regulated intramembrane proteolysis of amyloid-β protein precursor (AβPP) and of many other important physiological substrates. γ-secretase is a multiproteic complex made of four main core components, namely presenilin 1 or 2, APH-1, PEN-2, and Nicastrin. Since APH-1 exists as different variants, combinations of these proteins can theoretically yield distinct γ-secretase complexes. Whether γ-secretase complexes trafficking and targeting to either similar or distinct subcellular compartments depend upon their molecular composition remains unknown. A differential complex-specific distribution may drive a narrow specificity for a subset of substrates that would traffic within the same cellular compartments. Here, we generated bigenic expression vectors to co-express untagged nicastrin or presenilin 1 together with either PEN-2 or distinct variants of APH-1 (aL, aS and b) tagged with complementary fragments of the fluorescent protein Venus. We show that th...

Research paper thumbnail of Evidence that the amyloid-β protein precursor intracellular domain, AICD, derives from β-secretase-generated C-terminal fragment

Journal of Alzheimer's disease : JAD, 2012

One of the major pathological hallmarks of brains affected with Alzheimer's disease (AD) is t... more One of the major pathological hallmarks of brains affected with Alzheimer's disease (AD) is the senile plaque, an extracellular deposit mainly composed of a set of highly insoluble peptides of various lengths (39-43 amino acids) referred to as amyloid-β (Aβ) peptides. Aβ peptides are derived from combined proteolytic cleavages undergone on the amyloid-β protein precursor (AβPP) by a set of enzymes called secretases. Several lines of anatomical and biological evidence suggest that Aβ peptides would not account for all pathological stigmata and molecular dysfunctions taking place in AD. In amyloidogenic and non-amyloidogenic pathways, AβPP first undergoes β- or α-secretases-mediated cleavages yielding C99 and C83, respectively. These two membrane-embedded C-terminal fragments are both potential targets of subsequent γ-secretase-mediated proteolysis. The latter cleavage not only generates either p3 or Aβ peptides but similarly gives rise to an AβPP IntraCellular Domain (AICD fragme...

Research paper thumbnail of NACP/alpha-synuclein, NAC, and beta-amyloid pathology of familial Alzheimer's disease with the E184D presenilin-1 mutation: a clinicopathological study of two autopsy cases

Acta neuropathologica, 2002

Approximately 60% of familial and sporadic Alzheimer's disease (AD) cases manifest Lewy bodie... more Approximately 60% of familial and sporadic Alzheimer's disease (AD) cases manifest Lewy bodies (LBs), of which a major component is alpha-synuclein. Although the pathogenic role of alpha-synuclein in AD remains unclear, LB formation might be associated with pathological beta-amyloid (Abeta) overproduction. Here, we present the clinical and pathological characteristics of two affected family members from a pedigree with the E184D mutation of presenilin-1. One case presented with typical clinical features of AD, but the other case also developed clinical characteristics of dementia with Lewy bodies (DLB), including visual hallucinations, delusions, and parkinsonism. In both cases, neuropathological examination revealed numerous neurofibrillary tangles and severe Abeta deposition in senile plaques and amyloid angiopathy, in which Abeta42 rather than Abeta40 was predominant. Furthermore, remarkable alpha-synuclein pathology, including LBs and the accumulation of the non-Abeta compon...

Research paper thumbnail of Métabolisme du précurseur du peptide amyloïde et présénilines

Research paper thumbnail of Neurotensin Receptors in Primary Culture of Neurons

Methods in Neurosciences, 1993

Publisher Summary This chapter describes the main biochemical techniques that are available for n... more Publisher Summary This chapter describes the main biochemical techniques that are available for neurotensin receptor characterization, localization, and regulation on the primary cultures of neurons. These methods can be applied to other cell culture systems if cautions concerning the degradation and internalization of the ligand are taken. The neuromodulatory role of neurotensin is supported by its actions through specific high-affinity binding sites in discrete regions of the mammalian central nervous system. These neurotensin receptors are localized along dopaminergic pathways and have been extensively studied in terms of biochemical, anatomical, functional, and molecular properties. The chapter describes a set of tools and methods to study neurotensin receptors, which bind to dispersed neurons in primary culture, and discusses some techniques that allow the visualization of neurotensin receptors, both in situ and in denaturing conditions by radioautography. It also explains the regulatory mechanisms and the functional relevance of neurotensin receptors present in the neuronal cell system.

Research paper thumbnail of p53, a Molecular Bridge Between Alzheimer’s Disease Pathology and Cancers?

Research and Perspectives in Alzheimer's Disease, 2010

Cancers are characterized by enhanced cell survival and altered differentiation processes whereas... more Cancers are characterized by enhanced cell survival and altered differentiation processes whereas Alzheimer’s disease (AD)-affected brains exhibit exacerbated neuronal loss and cell death. Interestingly, several studies have consistently reported on an inverse relationship between cancer and AD. On the other hand, p53, a tumor-suppressor oncogene, is mutated and inactivated in a majority of human cancers; conversely, several lines of evidence concur to suggest an elevation of p53 and its transcriptional targets in AD brains. Therefore, one could envision p53 as a molecular bridge between cancer and AD pathologies. Although the role of p53 in cancer likely results from its inactivation by somatic mutations, the mechanistic aspects underlying a dysfunction in the control of p53 in AD had not been delineated. Here we survey recent evidence that p53 could control and be controlled by several members of the presenilin-dependent γ-secretase complex, and we briefly discuss the possibility that a functional deficit in presenilins could contribute to the genesis of a subset of tumors.

Research paper thumbnail of Alzheimer's and prion diseases: distinct pathologies, common proteolytic denominators

Trends in Neurosciences, 2002