1,721,107 research outputs found
Senescence-related impairment of autophagy induces toxic intraneuronal amyloid-β accumulation in a mouse model of amyloid pathology
No full textAbstract Aging is the main risk factor for Alzheimer’s disease (AD) and other neurodegenerative pathologies, but the molecular and cellular changes underlying pathological aging of the nervous system are poorly understood. AD pathology seems to correlate with the appearance of cells that become senescent due to the progressive accumulation of cellular insults causing DNA damage. Senescence has also been shown to reduce the autophagic flux, a mechanism involved in clearing damaged proteins from the cell, and such impairment has been linked to AD pathogenesis. In this study, we investigated the role of cellular senescence on AD pathology by crossing a mouse model of AD-like amyloid-β (Aβ) pathology (5xFAD) with a mouse model of senescence that is genetically deficient for the RNA component of the telomerase (Terc −/− ). We studied changes in amyloid pathology, neurodegeneration, and the autophagy process in brain tissue samples and primary cultures derived from these mice by complementary biochemical and immunostaining approaches. Postmortem human brain samples were also processed to evaluate autophagy defects in AD patients. Our results show that accelerated senescence produces an early accumulation of intraneuronal Aβ in the subiculum and cortical layer V of 5xFAD mice. This correlates with a reduction in amyloid plaques and Aβ levels in connecting brain regions at a later disease stage. Neuronal loss was specifically observed in brain regions presenting intraneuronal Aβ and was linked to telomere attrition. Our results indicate that senescence affects intraneuronal Aβ accumulation by impairing autophagy function and that early autophagy defects can be found in the brains of AD patients. Together, these findings demonstrate the instrumental role of senescence in intraneuronal Aβ accumulation, which represents a key event in AD pathophysiology, and emphasize the correlation between the initial stages of amyloid pathology and defects in the autophagy flux.SAO-FRAUCLouvain Action de Recherche ConcertéeQueen Elisabeth Medical Foundation http://dx.doi.org/10.13039/100017885Fonds De La Recherche Scientifique - FNRS http://dx.doi.org/10.13039/501100002661FWO-VlaanderenFondation Louvain http://dx.doi.org/10.13039/10000735
Analysis by a highly sensitive split luciferase assay of the regions involved in APP dimerization and its impact on processing
No full textAlzheimer's disease (AD) is a neurodegenerative disease that causes progressive loss of cognitive functions, leading to dementia. Two types of lesions are found in AD brains: neurofibrillary tangles and senile plaques. The latter are composed mainly of the β-amyloid peptide (Aβ) generated by amyloidogenic processing of the amyloid precursor protein (APP). Several studies have suggested that dimerization of APP is closely linked to Aβ production. Nevertheless, the mechanisms controlling APP dimerization and their role in APP function are not known. Here we used a new luciferase complementation assay to analyze APP dimerization and unravel the involvement of its three major domains: the ectodomain, the transmembrane domain and the intracellular domain. Our results indicate that within cells full-length APP dimerizes more than its α and β C-terminal fragments, confirming the pivotal role of the ectodomain in this process. Dimerization of the APP transmembrane (TM) domain has been reported to regulate processing at the γ-cleavage site. We show that both non-familial and familial AD mutations in the TM GXXXG motifs strongly modulate Aβ production, but do not consistently change dimerization of the C-terminal fragments. Finally, we found for the first time that removal of intracellular domain strongly increases APP dimerization. Increased APP dimerization is linked to increased non-amyloidogenic processing.This work was supported by a grant of the Belgian F.N.R.S FRIA (Fonds National pour la Recherche Scientifique) to M.D., Foundation for Research on Alzheimer’s disease (P.K-C.), by the Interuniversity Attraction Poles Programme-Belgian Sate-Belgian Science Policy (IAP-P7/16 and IAP-P7/13) to J-N.O. and P.K-C, and by the NIH (AG027317) to S.O.S. We are grateful to J-F. Paradis and S.W. Michnick (UMontreal) for the leucine zipper split-luciferase plasmids. We greatly acknowledge B. Tasiaux for her excellent technical support
Improvement of synaptic plasticity by pharmacological activation of RXR nuclear receptors is PPARα dependent
No full textDeconstruction of Neurotrypsin Reveals a Multi-factorially Regulated Activity Affecting Myotube Formation and Neuronal Excitability
Get PDFNeurotrypsin (NT) is a highly specific nervous system multi-domain serine protease best known for its selective processing of the potent synaptic organizer agrin. Its enzymatic activity is thought to influence processes of synaptic plasticity, with its deregulation causing accelerated neuromuscular junction (NMJ) degeneration or contributing to forms of mental retardation. These biological effects are likely to stem from NT-based regulation of agrin signaling. However, dissecting the exact biological implications of NT-agrin interplay is difficult, due to the scarce molecular detail regarding NT activity and NT-agrin interactions. We developed a strategy to reliably produce and purify a catalytically competent engineered variant of NT called "NT-mini" and a library of C-terminal agrin fragments, with which we performed a thorough biochemical and biophysical characterization of NT enzyme functionality. We studied the regulatory effects of calcium ions and heparin, identified NT's heparin-binding domain, and discovered how zinc ions induce modulation of enzymatic activity. Additionally, we investigated myotube differentiation and hippocampal neuron excitability, evidencing a dose-dependent increase in neuronal activity alongside a negative impact on myoblast fusion when using the active NT enzyme. Collectively, our results provide in vitro and cellular foundations to unravel the molecular underpinnings and biological significance of NT-agrin interactions
Templated misfolding of Tau by prion-like seeding along neuronal connections impairs neuronal network function and associated behavioral outcomes in Tau transgenic mice
No full textPrion-like seeding and propagation of Tau-pathology have been demonstrated experimentally and may underlie the stereotyped progression of neurodegenerative Tauopathies. However, the involvement of templated misfolding of Tau in neuronal network dysfunction and behavioral outcomes remains to be explored in detail. Here we analyzed the repercussions of prion-like spreading of Tau-pathology via neuronal connections on neuronal network function in TauP301S transgenic mice. Spontaneous and GABA(A)R-antagonist-induced neuronal network activity were affected following templated Tau-misfolding using synthetic preformed Tau fibrils in cultured primary neurons. Electrophysiological analysis in organotypic hippocampal slices of Tau transgenic mice demonstrated impaired synaptic transmission and impaired long-term potentiation following Tau-seed induced Tau-aggregation. Intracerebral injection of Tau-seeds in TauP301S mice, caused prion-like spreading of Tau-pathology through functionally connected neuroanatomical pathways. Electrophysiological analysis revealed impaired synaptic plasticity in hippocampal CA1 region 6 months after Tau-seeding in entorhinal cortex (EC). Furthermore, templated Tau aggregation impaired cognitive function, measured in the object recognition test 6 months post-seeding. In contrast, Tau-seeding in basal ganglia and subsequent spreading through functionally connected neuronal networks involved in motor control, resulted in motoric deficits reflected in clasping and impaired inverted grid hanging, not significantly affected following Tau-seeding in EC. Immunostaining, biochemical and electron microscopic analysis in the different models suggested early pathological forms of Tau, including Tau-oligomers, rather than fully mature neurofibrillary tangles (NFTs) as culprits of neuronal dysfunction. We here demonstrate for the first time using in vitro, ex vivo and in vivo models, that prion-like spreading of Tau-misfolding by Tau seeds, along unique neuronal connections, causes neuronal network dysfunction and associated behavioral dysfunction. Our data highlight the potential relevance of this mechanism in the symptomatic progression in Tauopathies. We furthermore demonstrate that the initial site of Tau-seeding thereby determines the behavioral outcome, potentially underlying the observed heterogeneity in (familial) Tauopathies, including in TauP301 mutants.Prion-like seeding and propagation of Tau-pathology have been demonstrated experimentally and may underlie the stereotyped progression of neurodegenerative Tauopathies. However, the involvement of templated misfolding of Tau in neuronal network dysfunction and behavioral outcomes remains to be explored in detail. Here we analyzed the repercussions of prion-like spreading of Tau-pathology via neuronal connections on neuronal network function in TauP301S transgenic mice. Spontaneous and GABA(A)R-antagonist-induced neuronal network activity were affected following templated Tau-misfolding using synthetic preformed Tau fibrils in cultured primary neurons. Electrophysiological analysis in organotypic hippocampal slices of Tau transgenic mice demonstrated impaired synaptic transmission and impaired long-term potentiation following Tau-seed induced Tau-aggregation. Intracerebral injection of Tau-seeds in TauP301S mice, caused prion-like spreading of Tau-pathology through functionally connected neuroanatomical pathways. Electrophysiological analysis revealed impaired synaptic plasticity in hippocampal CA1 region 6 months after Tau-seeding in entorhinal cortex (EC). Furthermore, templated Tau aggregation impaired cognitive function, measured in the object recognition test 6 months post-seeding. In contrast, Tau-seeding in basal ganglia and subsequent spreading through functionally connected neuronal networks involved in motor control, resulted in motoric deficits reflected in clasping and impaired inverted grid hanging, not significantly affected following Tau-seeding in EC. Immunostaining, biochemical and electron microscopic analysis in the different models suggested early pathological forms of Tau, including Tau-oligomers, rather than fully mature neurofibrillary tangles (NFTs) as culprits of neuronal dysfunction. We here demonstrate for the first time using in vitro, ex vivo and in vivo models, that prion-like spreading of Tau-misfolding by Tau seeds, along unique neuronal connections, causes neuronal network dysfunction and associated behavioral dysfunction. Our data highlight the potential relevance of this mechanism in the symptomatic progression in Tauopathies. We furthermore demonstrate that the initial site of Tau-seeding thereby determines the behavioral outcome, potentially underlying the observed heterogeneity in (familial) Tauopathies, including in TauP301 mutants.This work was supported by the Belgian Fonds
National pour la Recherche Scientifique—Fonds de la Recherche
Scientifique (FNRS-FRS; Qualified Researcher, Impulse Financing,
Research Credits), by Interuniversity Attraction Poles ProgrammeBelgian State-Belgian Science Policy, The Belgian Fonds de la
Recherche Scientifique Médicale, by the Institute for the Promotion
of Innovation by Science and Technology (IWT) in Flanders (IWT
O&O), Belgium
P1-033: Amyloid-induced tauopathy contributes to synaptic and cognitive deficits in a transgenic model for Alzheimer's disease
No full textBackground: Evaluation of biomarkers and innovative therapies for Alzheimer's disease (AD) suffers from lack of models close to disease progression in human. Most of transgenic models express supraphysiological levels of APP metabolites to mimic AD lesions such as amyloid plaques and neurofibrillary tangles. Our goal was to develop a modelling strategy by gene transfer with two major objectives: (1) create a relevant mouse model closer to human physiopathology, (2) mimic the early stages of AD and thus allowing characterization of early events. We focused on the amyloid cascade and the APP processing to trigger in vivo the production of neurotoxic peptides such as bCTF and Ab42. Methods: We used Adeno-Associated Viruses (AAVs) to express APP (with Swedish and London mutations) and PS1 (M146L mutation). We made a single stereotactic injection in the hippocampus of wild-type mice, followed by behavioral, biochemical and histological analysis. Results: Our strategy allows expression of human APP and PS1 and leads to bAPP production and its neurotoxic catabolites such as sAPPb, bCTF, Ab38, Ab40 and Ab42, as soon as one month post-injection. This production was stable during at least 12 months, without senile plaque formation. Interestingly, only co-injection of APP and PS1 increased the ratio Ab42/Ab40 and triggered hyperphosphorylation of the murine Tau protein which was correlated with increased levels of GSK3b. We also demonstrated a decrease of Beclin1 and NEP specifically observed in AD patient's brain. Finally, significant behavior impairments (Morris Water Maze and Openfield) appeared from 2.5 months after injection. Conclusions: This strategy induced amyloid pathology within the first month post-injection and overcame two major pitfalls of transgenic models, i.e. continuous expression of transgenes from in utero and limitations to the transfer to other species. Stable and more physiological amount of neurotoxic peptides derived from APP was produced and brought out an early link between the APP processing and Tau pathway
Correlation between beta-amyloid peptide production and human APP-induced neuronal death.
No full textThe production of amyloid peptide (Abeta) from its precursor (APP) plays a key role in Alzheimer's disease (AD). However, the link between Abeta production and neuronal death remains elusive. We studied the biological effects associated with human APP expression and metabolism in rat cortical neurons. Human APP expressed in neurons is processed to produce Abeta and soluble APP. Moreover, human APP expression triggers neuronal death. Pepstatin A, an inhibitor of aspartyl proteases that reduces Abeta production, protects neurons from APP-induced neurotoxicity. This suggests that Abeta production is likely to be the critical event in the neurodegenerative process of AD
O5-04-01: Molecular mechanisms of Abeta-induced Tau-pathology: Analysis of cross-seeding of Abeta and Tau and its role in prion-like propagation of Tau-pathology in vitro and in vivo
No full texttissue homogenates with exogenous Ab1-42 under multiple conditions. Samples were analyzed using acid urea gels followed by Western blotting. Results:The PDAPP mice study revealed Ab42 to Ab40 conversion over time reaching equilibrium by 72hr. Acid urea gel analyses demonstrated that over half of the Ab1-42 peptide administered was converted to Ab1-40. Furthermore, ELISA results from rat studies showed similar conversion rates from Ab42 to Ab40 regardless of the route of administration, centrally or peripherally. Ex-vivo studies using rat tissue homog-enates incubated with exogenous Ab1-42 peptide also exhibited Ab conversion. This conversion was present in all tissues tested, cortex, kidney, liver, pancreas, and spleen, and was exacerbated when the pH was lowered to pH5 from pH7. The rate of conversion to Ab40 was diminished when a c-terminal antibody or pro-tease inhibitor was incorporated into the in-vivo/ex-vivo studies. Conclusions: We have identified in-vivo processing of the carboxyl-terminus of Ab in rodents. The extent of in-vivo processing is exacerbated when an Ab antibody extends the half-life of the peptide. The conversion of Ab1-42 to Ab1-40 occurs both centrally and peripherally. Although the potential for this conversion in human is unknown, these results suggest additional biology after secretase liberation of the Ab from the APP may be important for the overall Ab ratios being measured in CNS and periphery. Background: Combined genetic, pathological and clinical data provided the basis for the amyloid cascade hypothesis, which is further supported by biomarker data and remains the major hypothesis for development of therapeutic strategies. In vitro and preclinical in vivo models have robustly recapitulated amyloid induced Tau-pathology providing support to the amyloid cascade hypothesis and providing tools to understand this event, generally considered to be crucial in the pathogenesis of AD. Methods: We previously reported a preclinical model with robust amyloid induced Tau-pathology , providing an experimental window for analysis of mechanisms of ABeta-induced Tau-pathology in vivo. In this model, we demonstrated the induction of Tau-pathology along functional connections , in regions relatively spared of amyloid pathology. The striking similarity between abeta-induced Tau-pathology in pre-clinical models and in our recently reported Tau-seeding model, provided the basis for our current analysis of Abeta-induced Tau-fi-brillization by cross-seeding and its subsequent propagating potential , in vitro and in vivo. Cell-free assays were used to analyze Ta
Etude des rôles des facteurs de transcription Onecut dans la différenciation des neurones moteurs de la moelle épinière
No full textAu cours du développement embryonnaire, les neurones moteurs (MN) sont générés à partir de cellules progénitrices localisées dans la partie ventrale de la moelle épinière. Les nouveaux MN sont post-mitotiques et se diversifient en différents sous-types spécifiquement dédiés au contrôle des muscles squelettiques (MN somatiques) ou des organes viscéraux (MN viscéraux). La famille des facteurs de transcription Onecut comporte trois membres chez les mammifères nommés Hepatocyte Nuclear Factor–6 (HNF-6), Onecut-2 (OC-2) et OC-3. Ces activateurs transcriptionnels sont exprimés de façon transitoire dans les premières étapes de la différenciation des MN médullaires. Le but de mon travail est d’étudier les rôles des facteurs de transcription Onecut au cours de la diversification des MN médullaires.
L’analyse d’embryons mutants Onecut, combinée avec des expériences en gain de fonction dans la moelle embryonnaire de poulet, démontre que cette famille d’activateurs transcriptionnels régule la diversification des MN à tous les niveaux de l’axe rostro-caudal de la moelle épinière. En effet, en absence des facteurs Onecut, la proportion entre MN somatiques et viscéraux est altérée au niveau thoracique, un excès de MN viscéraux étant généré au détriment des MN somatiques. Dans la région des membres, les MN responsables de l’innervation des muscles fléchisseurs perdent leur identité au profit de celle des MN assurant l’innervation des muscles extenseurs.
De plus, nous avons démontré qu’Isl1 est une cible transcriptionnelle directe des facteurs Onecut qui sont, dès lors, essentiels pour maintenir son expression durant la diversification des MN médullaires. Finalement, nous avons identifié un nouveau marqueur des MN viscéraux, Smad-interacting-protein (Sip)-1, qui pourrait agir de manière opposée à celle des protéines OC dans la production de la population viscérale.
Toutes ces observations indiquent que les facteurs OC sont impliqués dans des réseaux géniques au sein desquels ils coopèrent avec de multiples régulateurs afin de contrôler différents aspects de la diversification des MN dans la moelle épinière en développement.(SBIM 3) -- UCL, 201
D\ue9r\ue9gulation du cycle cellulaire dans les pathologies du syst\ue8me nerveux central : cas particulier de Cdk1
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