Papers by Pascal Kienlen-Campard
Alzheimer's research & therapy, Jun 28, 2024
Molecular Neurobiology, Oct 4, 2021
The β-amyloid peptide (Aβ) is found as amyloid fibrils in senile plaques, a typical hallmark of A... more The β-amyloid peptide (Aβ) is found as amyloid fibrils in senile plaques, a typical hallmark of Alzheimer's disease (AD). However, intermediate soluble oligomers of Aβ are now recognized as initiators of the pathogenic cascade leading to AD. Studies using recombinant Aβ have shown that hexameric Aβ in particular acts as a critical nucleus for Aβ self-assembly. We recently isolated hexameric Aβ assemblies from a cellular model, and demonstrated their ability to enhance Aβ aggregation in vitro. Here, we report the presence of similar hexameric-like Aβ assemblies across several cellular models, including neuronal-like cell lines. In order to better understand how they are produced in a cellular context, we investigated the role of presenilin-1 (PS1) and presenilin-2 (PS2) in their formation. PS1 and PS2 are the catalytic subunits of the γ-secretase complex that generates Aβ. Using CRISPR-Cas9 to knockdown each of the two presenilins in neuronal-like cell lines, we observed a direct link between the PS2-dependent processing pathway and the release of hexameric-like Aβ assemblies in extracellular vesicles. Further, we assessed the contribution of hexameric Aβ to the development of amyloid pathology. We report the early presence of hexameric-like Aβ assemblies in both transgenic mice brains exhibiting human Aβ pathology and in the cerebrospinal fluid of AD patients, suggesting hexameric Aβ as a potential early AD biomarker. Finally, cellderived hexameric Aβ was found to seed other human Aβ forms, resulting in the aggravation of amyloid deposition in vivo and neuronal toxicity in vitro.
bioRxiv (Cold Spring Harbor Laboratory), Dec 23, 2020
Background: The -amyloid peptide (A) plays a key role in Alzheimer's disease. After its product... more Background: The -amyloid peptide (A) plays a key role in Alzheimer's disease. After its production by catabolism of the amyloid precursor protein (APP) through the action of presenilin 1 (PS1)-or presenilin 2 (PS2)-dependent -secretases, monomeric A can assemble in oligomers. In a pathological context, this eventually leads to the formation of fibrils, which deposit in senile plaques. Many studies suggest that Aβ toxicity is related to its soluble oligomeric intermediates. Among these, our interest focuses on hexameric Aβ, which acts as a nucleus for A self-assembly. Methods: Biochemical analyses were used to identify hexameric A in a wide range of models; cell lines, cerebrospinal fluid from cognitively impaired patients and transgenic mice exhibiting human A pathology (5xFAD). We isolated this assembly and assessed both its effect on primary neuron viability in vitro, and its contribution to amyloid deposition in vivo following intracerebral injection. In both cases, we used wild-type mice (C57BL/6) to mimic an environment where hexameric A is present alone and 5xFAD mice to incubate hexameric A in a context where human Aβ species are pre-existing. Using CRISPR-Cas9, we produced stable knockdown human cell lines for either PS1 or PS2 to elucidate their contribution to the formation of hexameric A. Results: In WT mice, we found that neither in vitro or in vivo exposure to hexameric A was sufficient to induce cytotoxic effects or amyloid deposition. In 5xFAD mice, we observed a significant increase in neuronal death in vitro following exposure to 5M hexameric A, as well as a 1.47-fold aggravation of amyloid deposition in vivo. At the cellular level, we found hexameric A in extracellular vesicles and observed a strong decrease in its excretion when PS2 was knocked down by 60%. Conclusions: Our results indicate the absence of cytotoxic effects of cell-derived hexameric A by itself, but its capacity to aggravate amyloid deposition by seeding other A species. We propose an important role for PS2 in the formation of this particular assembly in vesicular entities, in line with previous reports linking the restricted location of PS2 in acidic compartments to the production of more aggregationprone A.
Acta Neuropathologica, Jan 12, 2017
dependent on its catalytically active cysteine, but independent of its noncanonical pathway modul... more dependent on its catalytically active cysteine, but independent of its noncanonical pathway modulated by its N-terminal domain in primary neurons. Otub1 strongly increased AT8-positive Tau and oligomeric Tau forms and increased Tau-seeded Tau aggregation in primary neurons. Finally, we demonstrated that expression of Otub1 but not its catalytically inactive form induced pathological Tau forms after 2 months in Tau transgenic mice in vivo, including AT8positive Tau and oligomeric Tau forms. Taken together, we here identified Otub1 as a Tau deubiquitinase in vitro and in vivo, involved in formation of pathological Tau forms, including small soluble oligomeric forms. Otub1 and particularly Otub1 inhibitors, currently under development for cancer therapies, may therefore yield interesting novel therapeutic avenues for Tauopathies and AD.
Frontiers in Cellular Neuroscience, Nov 22, 2018
Aβ peptides, the major components of Alzheimer's disease (AD) amyloid deposits, are released foll... more Aβ peptides, the major components of Alzheimer's disease (AD) amyloid deposits, are released following sequential cleavages by secretases of its precursor named the amyloid precursor protein (APP). In addition to secretases, degradation pathways, in particular the endosomal/lysosomal and proteasomal systems have been reported to contribute to APP processing. However, the respective role of each of these pathways toward APP metabolism remains to be established. To address this, we used HEK 293 cells and primary neurons expressing full-length wild type APP or the β-secretasederived C99 fragment (β-CTF) in which degradation pathways were selectively blocked using pharmacological drugs. APP metabolites, including carboxy-terminal fragments (CTFs), soluble APP (sAPP) and Aβ peptides were studied. In this report, we show that APP-CTFs produced from endogenous or overexpressed full-length APP are mainly processed by γ-secretase and the endosomal/lysosomal pathway, while in sharp contrast, overexpressed C99 is mainly degraded by the proteasome and to a lesser extent by γ-secretase.
medRxiv (Cold Spring Harbor Laboratory), Feb 27, 2024
INTRODUCTION: Amyloid-β (Aβ) and tau are brain hallmarks of Alzheimer's disease (AD) also present... more INTRODUCTION: Amyloid-β (Aβ) and tau are brain hallmarks of Alzheimer's disease (AD) also present in blood as soluble biomarkers or encapsulated in extracellular vesicles (EVs). Our goal was to assess how soluble plasma biomarkers of AD pathology correlate with number and content of EVs. Single-molecule enzyme-linked assays were used to quantify Aβ42/40 and tau in plasma samples and neurally-derived EVs (NDEVs) from a cohort of APOE ε4-and APOE ε4+ cognitively normal individuals (CN) and AD patients. Aβ42/40 ratio is decreased in AD patients compared to CN individuals. The amount and content (Aβ40, Aβ42, tau) of plasma NDEVs were similar between groups. Quantity of soluble biomarkers were negatively correlated to NDEVs number only in CN individuals. DISCUSSION: Soluble Aβ42/40 ratio is the most robust AD plasma biomarker. Analysis of NDEVs and their content pointed toward peculiar mechanisms of Aβ release in AD.
International Journal of Molecular Sciences, Jan 17, 2024
Alzheimer's disease (AD) is characterized by amyloid beta (Aβ) plaques and hyperphosphorylated ta... more Alzheimer's disease (AD) is characterized by amyloid beta (Aβ) plaques and hyperphosphorylated tau in the brain. Aβ plaques precede cognitive impairments and can be detected through amyloid-positron emission tomography (PET) or in cerebrospinal fluid (CSF). Assessing the plasma Aβ42/Aβ40 ratio seems promising for non-invasive and cost-effective detection of brain Aβ accumulation. This approach involves some challenges, including the accuracy of blood-based biomarker measurements and the establishment of clear, standardized thresholds to categorize the risk of developing brain amyloid pathology. Plasma Aβ42/Aβ40 ratio was measured in 277 volunteers without dementia, 70 AD patients and 18 non-AD patients using single-molecule array. Patients (n = 88) and some volunteers (n = 66) were subject to evaluation of amyloid status by CSF Aβ quantification or PET analysis. Thresholds of plasma Aβ42/Aβ40 ratio were determined based on a Gaussian mixture model, a decision tree, and the Youden's index. The 0.0472 threshold, the one with the highest sensitivity, was retained for general population without dementia screening, and the 0.0450 threshold was retained for research and clinical trials recruitment, aiming to minimize the need for CSF or PET analyses to identify amyloid-positive individuals. These findings offer a promising step towards a cost-effective method for identifying individuals at risk of developing AD.
Most neurodegenerative diseases have the characteristics of protein folding disorders, i.e., they... more Most neurodegenerative diseases have the characteristics of protein folding disorders, i.e., they cause lesions to appear in vulnerable regions of the nervous system, corresponding to protein aggregates that progressively spread through the neuronal network as the symptoms progress. Alzheimer's disease is one of these diseases. It is characterized by two types of lesions: neurofibrillary tangles (NFTs) composed of tau proteins and senile plaques, formed essentially of amyloid peptides (Aβ). A combination of factors ranging from genetic mutations to age-related changes in the cellular context converge in this disease to accelerate Aβ deposition. Over the last two decades, numerous studies have attempted to elucidate how structural determinants of its precursor (APP) modify Aβ production, and to understand the processes leading to the formation of different Aβ aggregates, e.g., fibrils and oligomers. The synthesis proposed in this review indicates that the same motifs can control APP function and Aβ production essentially by regulating membrane protein dimerization, and subsequently Aβ aggregation processes. The distinct properties of these motifs and the cellular context regulate the APP conformation to trigger the transition to the amyloid pathology. This concept is critical to better decipher the patterns switching APP protein conformation from physiological to pathological and improve our understanding of the mechanisms underpinning the formation of amyloid fibrils that devastate neuronal functions.
Journal of Biological Chemistry, May 1, 2002
Alzheimer disease (AD), the most frequent cause of dementia, is characterized by an important neu... more Alzheimer disease (AD), the most frequent cause of dementia, is characterized by an important neuronal loss. A typical histological hallmark of AD is the extracellular deposition of -amyloid peptide (A), which is produced by the cleavage of the amyloid precursor protein (APP). Most of the gene mutations that segregate with the inherited forms of AD result in increasing the ratio of A42/A40 production. A42 also accumulates in neurons of AD patients. Altogether, these data strongly suggest that the neuronal production of A42 is a critical event in AD, but the intraneuronal A42 toxicity has never been demonstrated. Here, we report that the long term expression of human APP in rat cortical neurons induces apoptosis. Although APP processing leads to production of extracellular A1-40 and soluble APP, these extracellular derivatives do not induce neuronal death. On the contrary, neurons undergo apoptosis as soon as they accumulate intracellular A1-42 following the expression of full-length APP or a C-terminal deleted APP isoform. The inhibition of intraneuronal A1-42 production by a functional ␥-secretase inhibitor increases neuronal survival. Therefore, the accumulation of intraneuronal A1-42 is the key event in the neurodegenerative process that we observed. * This work was supported by the Belgian Fonds de la Recherche Scientifique Médicale, Pôles d'attraction interuniversitaires/Services fédéraux des Affaires scientifiques, techniques et culturelles, and the Queen Elisabeth Medical Foundation.
La mort neuronale programmee ou apoptose neuronale est un programme cellulaire permettant de stru... more La mort neuronale programmee ou apoptose neuronale est un programme cellulaire permettant de structurer le systeme nerveux central au cours du developpement. C'est egalement un processus participant a de nombreuses pathologies neurodegeneratives. L'apoptose est controlee in vivo par des facteurs neurotrophiques favorisant la differenciation et la survie neuronale. Une hypothese recente postule que la mort neuronale resulte d'une tentative de reactiver le cycle cellulaire. Nous avons montre qu'une proteine controlant la phase g1 du cycle cellulaire, la cycline d1, est induite lors de l'apoptose du neurone en grain du cervelet. Cependant, la transcription de ce gene est constitutive dans ces cellules. L'accumulation de cd1 depend de l'activite du systeme ubiquitine/proteasome permettant sa degradation. L'activite de ce systeme est entretenue dans les conditions de survie par la depolarisation du neurone et les influx calciques qu'elle genere. Nous proposons un modele ou l'apoptose est un programme latent rapidement execute par le neurone a partir de facteurs preexistants quand les conditions necessaires a la survie ne sont plus satisfaites. Dans ce modele, le systeme ubiquitine/proteasome joue un role neuroprotecteur essentiel. Cette voie de degradation est entretenue par des facteurs neurotrophiques comme le bdnf ou le pacap. Nous avons etudie plus en detail les mecanismes d'action du pacap en caracterisant dans un premier temps les recepteurs de type pr1 dans le neurone en grain. Ces recepteurs sont positivement couples a l'adenylate cyclase et la phospholipase c mais le role neuroprotecteur du pacap repose entierement sur la voie de transduction dependant de l'ampc et de la pka (proteine kinase a). L'inhibition de la voie de degradation ubiquitine/proteasome abolit les effets neuroprotecteurs du pacap et du bdnf. Ceci montre que la degradation specifique de certaines proteines impliquees dans l'apoptose (dont la cd1) est controlee par des effecteurs physiologiques et renforce l'idee que la voie de degradation ubiquitine/proteasome est un mecanisme essentiel et actif dans le maintien de la survie du neurone.
Research Square (Research Square), Jan 8, 2021
Background The β-amyloid peptide (Aβ) plays a key role in Alzheimer's disease. After its producti... more Background The β-amyloid peptide (Aβ) plays a key role in Alzheimer's disease. After its production by catabolism of the amyloid precursor protein (APP) through the action of presenilin 1 (PS1)-or presenilin 2 (PS2)dependent γ-secretases, monomeric Aβ can assemble in oligomers. In a pathological context, this eventually leads to the formation of brils, which deposit in senile plaques. Many studies suggest that Aβ toxicity is related to its soluble oligomeric intermediates. Among these, our interest focuses on hexameric Aβ, which acts as a nucleus for Aβ self-assembly. Methods Biochemical analyses were used to identify hexameric Aβ in a wide range of models; cell lines,
Neurodegenerative Diseases, 2017
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Alzheimers & Dementia, Jul 1, 2017
Biochemical and Biophysical Research Communications, Sep 1, 2007
The APP intracellular domain (AICD) could be involved in signaling via interaction with the adapt... more The APP intracellular domain (AICD) could be involved in signaling via interaction with the adaptor protein Fe65, and with the histone acetyl transferase Tip60. However, the real function of AICD and Fe65 in regulation of transcription remains controversial. In this study, the human APPGal4 fusion protein was expressed in CHO cells and the transcriptional activity of AICDGal4 was measured in a luciferase-based reporter assay. AICDGal4 was stabilized by expression of Fe65 and levels of AICDGal4 controlled luciferase activity. On the contrary, when human APP was expressed in CHO cells, coexpression of Fe65 increased luciferase activity without affecting the amount of AICD fragment. AICD produced from APP was protected from degradation by orthophenanthroline, but not by lactacystine, indicating that AICD is not a substrate of the chymotryptic activity of the proteasome. It is concluded that Fe65 can control luciferase activity without stabilizing the labile AICD fragment.
Journal of Neuroinflammation, Jan 27, 2016
Background: The proinflammatory cytokine interleukin-1β (IL-1β) is overexpressed in Alzheimer dis... more Background: The proinflammatory cytokine interleukin-1β (IL-1β) is overexpressed in Alzheimer disease (AD) as a key regulator of neuroinflammation. Amyloid-β (Aβ) peptide triggers activation of inflammasomes, protein complexes responsible for IL-1β maturation in microglial cells. Downregulation of NALP3 (NACHT, LRR, and PYD domainscontaining protein 3) inflammasome has been shown to decrease amyloid load and rescue cognitive deficits in a mouse model of AD. Whereas activation of inflammasome in microglial cells has been described in AD, no data are currently available concerning activation of inflammasome in astrocytes, although they are involved in inflammatory response and phagocytosis. Here, by targeting the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD domain), we investigated the influence of activation of the inflammasome on the phagocytic activity of astrocytes. Methods: We used an ASC knockout mouse model, as ASC is a central protein in the inflammasome, acting as an adaptor and stabilizer of the complex and thus critical for its activation. Lipopolysaccharide (LPS)-primed primary cultures of astrocytes from newborn mice were utilized to evaluate Aβ-induced inflammasome activation by measuring IL-1β release by ECLIA (electro-chemiluminescence immunoassay). Phagocytosis efficiency was measured by incorporation of bioparticles, and the release of the chemokine CCL3 (C-C motif ligand 3) was measured by ECLIA. ASC mice were crossbred with 5xFAD (familial Alzheimer disease) mice and tested for spatial reference memory using the Morris water maze (MWM) at 7-8 months of age. Amyloid load and CCL3 were quantified by thioflavine S staining and quantitative real-time polymerase chain reaction (qRT-PCR), respectively.
Experimental Gerontology, Sep 1, 2000
One of the major neuropathological hallmarks of Alzheimer's disease is the presence of senile pla... more One of the major neuropathological hallmarks of Alzheimer's disease is the presence of senile plaques in vulnerable regions of CNS. These plaques are formed of aggregated amyloid peptide. Amyloid peptide is released by the cleavage of its precursor (APP). The establishment of cell lines expressing human APP allowed to characterize both amyloidogenic and non-amyloidogneic pathways of APP catabolism and to identify some of the proteins involved in this processing (known as secretases). This led to a better comprehension of amyloid peptide production, which needs to be further characterized since g -secretase is as yet not identi®ed; moreover, we still lack a clear overview of the interactions between APP and other proteins promoting Alzheimer's disease (tau, presinilins¼). An important limitation of these cell lines for studying the mechanisms involved in Alzheimer's disease is supported by the observation that human APP expression does not modify transfected cells survival. The infection of primary neuronal cultures with full-length human APP indicates that APP expression induces neuronal apoptosis by itself; this neurotoxicity does not rely on extracellular production of APP derivatives (secreted APP, amyloid peptide). It is now essential to understand, in neuronal models, the production, localization and involvement of amyloid peptide in neurodegenerative processes.
Journal of Ethnopharmacology, Oct 1, 2010
Biochemical and Biophysical Research Communications, Jun 1, 2007
Phosphorylation of human APP695 at Thr668 seems to be specific to neuronal tissue and could affec... more Phosphorylation of human APP695 at Thr668 seems to be specific to neuronal tissue and could affect Ab production. Metabolism of APP mutated at Thr668 residue was analyzed in CHO cell line and primary cultures of rat cortical neurons. By site-directed mutagenesis, T668A or T668D substitutions were introduced in wild-type APP695. In CHO cells, wild-type APP695 was very slightly phosphorylated at Thr668 and produced similar levels of extracellular Ab40 as compared to APPT668A. On the contrary, APPT668D was more efficiently cleaved by b-secretase. However, accumulated bCTF were less cleaved by c-secretase and less extracellular Ab40 was produced. Decreased susceptibility to cleavage by c-secretase was confirmed upon expression of C99T668D. In neurons, part of APP695 was phosphorylated at Thr668. Following neuronal expression of APPT668A, extracellular Ab40 production was increased. In conclusion, phosphorylation of human APP695 at Thr668 increases APP b-cleavage but decreases its c-cleavage and extracellular Ab40 production.
Alzheimers & Dementia, Jul 1, 2008
Revue Neurologique, Oct 1, 2009
ABSTRACT Introduction Bien que le métabolisme cellulaire du précurseur du peptide amyloïde (APP) ... more ABSTRACT Introduction Bien que le métabolisme cellulaire du précurseur du peptide amyloïde (APP) ait été étudié en détail, la fonction précise de la protéine reste inconnue. Le domaine intracellulaire de l’APP (AICD) pourrait contrôler l’expression de plusieurs gènes. Cependant, l’identité de certains gènes cibles de l’APP fait l’objet de controverses, et les mécanismes par lesquels l’APP régule leur transcription est inconnue. Le but de notre travail était de confirmer que l’APP est capable de contrôler l’expression de certains gènes, et d’étudier les mécanismes moléculaires impliqués. Méthodes Nous avons identifié des gènes cibles de l’APP en comparant les profils d’expression de gènes dans les fibroblastes embryonnaires de souris (MEFs) exprimant ou non l’APP (APP +/+ et APP -/-). Etant donné que l’AICD est libéré de l’APP par une activité γ-sécrétase preseniline- dépendante, nous avons réalisé des expériences identiques en utilisant des MEFs exprimant ou non les présénilines 1 et 2 (PS +/+ et PS -/-). Résultats Parmi les gènes différemment exprimés dans ces deux modèles cellulaires, nous nous sommes particulièrement intéressés aux gènes codant l’aquaporine 1 (AQP1) et CXCL5, dont les expressions sont diminuées dans les MEFs APP-/- et PS -/-. Nous avons montré, par qRT-PCR, immunotransfert et ELISA, que l’expression d’AQP1 et de CXCL5 est fortement diminuée dans les cellules n’exprimant pas l’APP ou PS2. Dans ces cellules, l’expression d’AQP1 et de CXCL5 est retrouvée suite à l’expression stable d’APP ou de PS2 humains. Nous avons ensuite analysé l’activité transcriptionnelle des promoteurs des gènes AQP1 et CXCL5, clonés en amont du gène rapporteur de la luciférase. L’activité transcriptionnelle des promoteurs des gènes AQP1 et CXCL5 était identique dans les MEFs APP+/+ et APP -/-. De la même manière, la stabilité des mRNA AQP1 et CXCL5, mesurée en présence d’actinomycine D, était identique dans les MEFs APP+/+ et APP-/-. L’utilisation de trichostatine A, un inhibiteur spécifique des histones déacétylases (HDACs) de type I et II, a démontré que la régulation de l’expression d’AQP1 et de CXCL5 par l’APP était sensible à l’acétylation des histones. Nous avons finalement démontré une co-immunoprécipitation d’activité HDAC avec le domaine intracellulaire de l’APP. Conclusions Ce travail démontre que, dans des MEFs, l’expression des gènes codant l’AQP1 et CXCL5 est régulée par l’APP et PS2, sans modification de l’activité transcriptionnelle des promoteurs mesurée à l’aide d’un gène rapporteur, ni de la stabilité des mRNAs. Une diminution d’AQP1 rénale et de CXCL5 sérique, mise en évidence dans des souris APP-/-, suggère un rôle essentiel de l’APP dans le contrôle épigénétique de l’expression de ces deux gènes.
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Papers by Pascal Kienlen-Campard