Tau, a microtubule associated protein, has been implicated in the formation of neurofibrillary tangles, a hallmark of Alzheimer’s disease.1 Resulting from the hyperphosphorylation of tyrosine, serine and threonine residues, protein kinases play an intricate role in the pathway of NFT formation.2 While a number of serine/ threonine kinases such as Glycogen Synthase Kinase 3 beta, have been implicated in the hyperphosphorylation of tau, and it is now believed that tyrosine phosphorylation plays a role. It appears that the degree to which tau is phosphorylated, and region in which phosphorylation occurs play a critical role in aggregate formation. Here we present the use of Ferrocene bioconjugates to monitor tyrosine-mediated hyperphosphorylation and aggregation. Electrochemistry, electron microscopy, surface plasmon resonance, Time of Flight Secondary Ion Mass Spectrometry and X-Ray Photoelectron Spectroscopy were used to gain insight into the role of tyrosine phosphorylation on hyperphosphorylation.

Tau, a microtubule associated protein, has been implicated in the formation of neurofibrillary tangles, a hallmark of Alzheimer’s disease.1 Resulting from the hyperphosphorylation of tyrosine, serine and threonine residues, protein kinases play an intricate role in the pathway of NFT formation.2 While a number of serine/ threonine kinases such as Glycogen Synthase Kinase 3 beta, have been implicated in the hyperphosphorylation of tau, and it is now believed that tyrosine phosphorylation plays a role. It appears that the degree to which tau is phosphorylated, and region in which phosphorylation occurs play a critical role in aggregate formation. Here we present the use of Ferrocene bioconjugates to monitor tyrosine-mediated hyperphosphorylation and aggregation. Electrochemistry, electron microscopy, surface plasmon resonance, Time of Flight Secondary Ion Mass Spectrometry and X-Ray Photoelectron Spectroscopy were used to gain insight into the role of tyrosine phosphorylation on hyperphosphorylation.

En 1907, Aloïs Alzheimer publiait, pour la première fois, la description des deux lésions histologiques caractéristiques observées dans le cerveau d'une patiente âgée de 56 ans et atteinte d'une démence présénile qui, par la suite, sera dénommée Maladie d'Alzheimer (MA). Ces deux lésions correspondant à l'accumulation de matériel protéique présentent des caractéristiques différentes de par leur contenu protéique et leur localisation. Ainsi, on distinguera, d'une part, les dépôts amyloïdes, caractérisés par l'accumulation, dans le milieu extracellulaire, d'un peptide appelé peptide amyloïde- . D'autre part, on observera une lésion intra-neuronale appelée dégénérescence neurofibrillaire (DNF) qui correspond à l'accumulation de protéines Tau qui sont retrouvées hyperphosphorylées, anormalement phosphorylées et agrégées sous la forme de structures fibrillaires hélicoïdales singulières appelées PHFs pour " Paired Helical Filaments ". Notre laboratoire s'intéresse tout particulièrement à cette lésion qui est au cœur du processus dégénératif de la MA et d'un ensemble de pathologies démentielles appelées " Tauopathies ". Cependant, si la dérégulation de phosphorylation de Tau semble être au cœur du processus dégénératif de ces pathologies, le rôle exact de celle-ci et les mécanismes mis en jeu restent encore mal connus. Ce travail, se place ainsi dans le cadre général de la recherche des mécanismes moléculaires impliqués dans la mort neuronale liée à Tau et de l'étude de la signification et du rôle de la dérégulation de la phosphorylation de Tau dans son agrégation et au cours de la mort neuronale. Afin de répondre à ces objectifs, nous avons entrepris l'étude de modèles permettant de moduler soit l'état de phosphorylation de Tau par le biais du complexe kinasique p25/Cdk5 soit de moduler le caractère agrégatif de Tau par l'utilisation de mutations pathologiques de Tau connues chez l'homme pour mener à son agrégation et à des syndrômes démentiels (Démences Fronto-Temporales avec syndrôme Parkinsonien liées au chromosome 17). De plus, notre travail a permis la caractérisation d'un modèle in vivo de DNF présentant l'ensemble des caractéristiques de la pathologie Tau observée au cours de la MA. Ce modèle, en raison de l'absence de troubles moteurs, a permis de réaliser des études comportementales, montrant l'existence de troubles de mémoire spatiale chez ces souris. En parallèle à ce modèle pertinent d'un point de vue physiopathologique, le développement et l'analyse d'un modèle cellulaire de type neuronal basé sur la surexpression de protéines Tau mutées ont permis de montrer l'existence de modifications conformationnelles des protéines Tau mutées qui sont associées à un état particulier de phosphorylation. Cependant, dans ce contexte, il apparaît que les mutations, à elles seules ne semblent pas suffisantes pour mener à l'agrégation des protéines Tau sous la forme de PHFs. De plus, l'analyse d'un modèle cellulaire de type neuronal présentant une phosphorylation anormale de Tau ne montre pas non plus l'existence de structures analogues aux PHFs. L'ensemble de ce travail suggère que la phosphorylation anormale ou les mutations de Tau ne sont pas suffisantes pour mener au phénotype pathologique caractéristique de la DNF qui semble nécessiter d'autres événements moléculaires. Nous émettons également l'hypothèse que des voies compensatoires impliquant des systèmes de déphosphorylation des protéines Tau et/ou des systèmes de dégradation protéique pourraient être mis en jeu dans nos modèles cellulaires et être ainsi responsables de l'absence de phénotype pathologique. La diminution d'activité de ces systèmes de compensation au cours du vieillissement chez l'homme et dans les modèles murins pourrait alors concourir à l'apparition de la DNF. Ainsi, l'étude de ces systèmes dans nos différents modèles constitue des perspectives prometteuses pour l'avancée dans la compréhension de l'étiopathologie de la MA et des Tauopathies.

RPA (replication protein A) is an essential factor for DNA DSB (double-strand break) repair and cell cycle checkpoint activation. The 32 kDa subunit of RPA undergoes hyperphosphorylation in response to cellular genotoxic insults. However, the potential involvement of hyperphosphorylated RPA in DSB repair and check-point activation remains unclear. Using co-immunoprecipitation assays, we showed that cellular interaction of RPA with two DSB repair factors, Rad51 and Rad52, was predominantly mediated by the hyperphosphorylated species of RPA in cells after UV and camptothecin treatment. Moreover, Rad51 and Rad52 displayed higher affinity for the hyperphosphorylated RPA than native RPA in an in vitro binding assay. Checkpoint kinase ATR (ataxia telangiectasia mutated and Rad3-related) also interacted more efficiently with the hyperphosphorylated RPA than with native RPA following DNA damage. Consistently, immunofluorescence microscopy demonstrated that the hyperphosphorylated RPA was able to co-localize with Rad52 and ATR to form significant nuclear foci in cells. Our results suggest that hyperphosphorylated RPA is preferentially localized to DSB repair and the DNA damage checkpoint complexes in response to DNA damage.

A doença de Alzheimer (DA) é uma patologia neuropsiquiátrica caracterizada por perda cognitiva, com marcante déficit de memória desde o estágio inicial, degeneração neuronal progressiva, e constitui a principal causa de demência no mundo. Além disso, a DA é frequentemente agravada pela ocorrência de comorbidades. Dados epidemiológicos apontam para uma importante associação clínica entre a DA e a epilepsia. A ausência de um modelo de estudo relevante, entretanto, tem impedido o avanço do entendimento dos mecanismos moleculares que subjazem essa comorbidade. A cepa Wistar Audiogenic Rat (WAR) tem sido amplamente utilizada como modelo animal para o estudo de epilepsia e, recentemente, déficits de memória foram relatados em animais desta cepa, o que suscitou a possibilidade de constituírem um modelo experimental para o estudo da comorbidade entre DA e epilepsia. No presente estudo, foi avaliado o desempenho de WAR adultos em diferentes idades no teste de memória do labirinto aquático de Morris (LAM). Realizaram-se ainda análises bioquímicas e de imunoistoquímica dos principais biomarcadores celulares da DA, o peptídeo beta-amiloide (A?) e a proteína Tau hiperfosforilada (pTau), no hipocampo e no córtex pré-frontal de WAR e Wistar de idades pareadas. No LAM, os WAR de 9 meses apresentaram déficit de aprendizagem e de retenção de memória (24 horas após o treinamento), quando comparados aos Wistar de idade pareada, enquanto que aos 12 meses, os WAR apresentaram apenas déficit de aprendizagem. Os animais WAR de 12 meses apresentaram também aumento significativo nos níveis de pTau e Tau total em extratos de hipocampo, quando comparados aos Wistar controles de mesma idade, o que não foi observado para os extratos de córtex pré-frontal. Os WAR também apresentaram elevação idade-dependente nos níveis e distribuição anormal de pTau em algumas sub-regiões hipocampais, como avaliado por imunoistoquímica. Não foi possível quantificar A? endógeno por meio das estratégias utilizadas. Quando analisados em conjunto, os dados sugerem que os déficits de memória observados nos WAR podem ser reflexo da hiperfosforilação e consequente distribuição anormal da proteína neuronal Tau, por um mecanismo molecular a ser desvendado. Não foi possível, ainda, determinar se este mecanismo inclui aumento nos níveis de agregados de A?, como esperado em modelos de DA. Portanto, o emprego de cepa WAR como modelo experimental para o estudo dos fenômenos moleculares subjacentes à comorbidade entre epilepsia e doença de Alzheimer, embora promissor, ainda precisa ser melhor caracterizado. / Alzheimer disease (AD) is a neuropsychiatric disorder characterized by cognitive loss, marked memory deficit since the early stage, progressive neuronal degeneration, and it is the leading cause of dementia around the world. In addition, AD is often aggravated by the occurrence of comorbidities. Epidemiological data point to a clinical association between AD and epilepsy. The absence of a relevant model, however, has impaired the advance of the understanding about the molecular mechanisms that underlie this comorbidity. The Wistar Audiogenic Rat (WAR) strain has been widely used as an animal model to the study of epilepsy. Recently, memory deficits have been reported in WARs, which has raised the possibility that this strain may represent an experimental model to the study of comorbidity between AD and epilepsy. Here we evaluated the performance of WARs in the reference memory test Morris water maze (MWM) at different ages. Biochemical and immunohistochemical analyzes of the main AD biomarkers, beta-amyloid peptide (A?) and hyperphosphorylated Tau protein (pTau), were also performed in the hippocampus and prefrontal cortex of age-matched WARs and Wistar controls. In the LAM, middle-aged (9 months) WARs presented learning and memory retention deficits (24 hours after training session) when compared to age-matched Wistar rats, whereas at 12 months, WARs presented only learning deficits. Adult WAR animals showed a significant increase in the levels of pTau and total Tau in hippocampal extracts, when compared to Wistar controls, which was not observed in prefrontal cortex extracts. WARs also showed higher levels and abnormal distribution of pTau in some hippocampal subregions, as assessed by immunohistochemistry. It was not possible to detect endogenous A? by the strategies used. Taken together, these data suggest that memory deficits observed in WARs may be a consequence of hyperphosphorylation and consequent abnormal distribution of Tau triggered by a molecular mechanism yet to be identified. It has not yet been determined whether this mechanism includes increased levels of A? aggregates, as expected in AD models. Therefore, the use of the WAR strain as an experimental model to the study of the molecular phenomena underlying the comorbidity between epilepsy and Alzheimer disease, although promising, still needs to be further characterized.

A doença de Alzheimer (DA) é uma patologia neuropsiquiátrica caracterizada por perda cognitiva, com marcante déficit de memória desde o estágio inicial, degeneração neuronal progressiva, e constitui a principal causa de demência no mundo. Além disso, a DA é frequentemente agravada pela ocorrência de comorbidades. Dados epidemiológicos apontam para uma importante associação clínica entre a DA e a epilepsia. A ausência de um modelo de estudo relevante, entretanto, tem impedido o avanço do entendimento dos mecanismos moleculares que subjazem essa comorbidade. A cepa Wistar Audiogenic Rat (WAR) tem sido amplamente utilizada como modelo animal para o estudo de epilepsia e, recentemente, déficits de memória foram relatados em animais desta cepa, o que suscitou a possibilidade de constituírem um modelo experimental para o estudo da comorbidade entre DA e epilepsia. No presente estudo, foi avaliado o desempenho de WAR adultos em diferentes idades no teste de memória do labirinto aquático de Morris (LAM). Realizaram-se ainda análises bioquímicas e de imunoistoquímica dos principais biomarcadores celulares da DA, o peptídeo beta-amiloide (A?) e a proteína Tau hiperfosforilada (pTau), no hipocampo e no córtex pré-frontal de WAR e Wistar de idades pareadas. No LAM, os WAR de 9 meses apresentaram déficit de aprendizagem e de retenção de memória (24 horas após o treinamento), quando comparados aos Wistar de idade pareada, enquanto que aos 12 meses, os WAR apresentaram apenas déficit de aprendizagem. Os animais WAR de 12 meses apresentaram também aumento significativo nos níveis de pTau e Tau total em extratos de hipocampo, quando comparados aos Wistar controles de mesma idade, o que não foi observado para os extratos de córtex pré-frontal. Os WAR também apresentaram elevação idade-dependente nos níveis e distribuição anormal de pTau em algumas sub-regiões hipocampais, como avaliado por imunoistoquímica. Não foi possível quantificar A? endógeno por meio das estratégias utilizadas. Quando analisados em conjunto, os dados sugerem que os déficits de memória observados nos WAR podem ser reflexo da hiperfosforilação e consequente distribuição anormal da proteína neuronal Tau, por um mecanismo molecular a ser desvendado. Não foi possível, ainda, determinar se este mecanismo inclui aumento nos níveis de agregados de A?, como esperado em modelos de DA. Portanto, o emprego de cepa WAR como modelo experimental para o estudo dos fenômenos moleculares subjacentes à comorbidade entre epilepsia e doença de Alzheimer, embora promissor, ainda precisa ser melhor caracterizado. / Alzheimer disease (AD) is a neuropsychiatric disorder characterized by cognitive loss, marked memory deficit since the early stage, progressive neuronal degeneration, and it is the leading cause of dementia around the world. In addition, AD is often aggravated by the occurrence of comorbidities. Epidemiological data point to a clinical association between AD and epilepsy. The absence of a relevant model, however, has impaired the advance of the understanding about the molecular mechanisms that underlie this comorbidity. The Wistar Audiogenic Rat (WAR) strain has been widely used as an animal model to the study of epilepsy. Recently, memory deficits have been reported in WARs, which has raised the possibility that this strain may represent an experimental model to the study of comorbidity between AD and epilepsy. Here we evaluated the performance of WARs in the reference memory test Morris water maze (MWM) at different ages. Biochemical and immunohistochemical analyzes of the main AD biomarkers, beta-amyloid peptide (A?) and hyperphosphorylated Tau protein (pTau), were also performed in the hippocampus and prefrontal cortex of age-matched WARs and Wistar controls. In the LAM, middle-aged (9 months) WARs presented learning and memory retention deficits (24 hours after training session) when compared to age-matched Wistar rats, whereas at 12 months, WARs presented only learning deficits. Adult WAR animals showed a significant increase in the levels of pTau and total Tau in hippocampal extracts, when compared to Wistar controls, which was not observed in prefrontal cortex extracts. WARs also showed higher levels and abnormal distribution of pTau in some hippocampal subregions, as assessed by immunohistochemistry. It was not possible to detect endogenous A? by the strategies used. Taken together, these data suggest that memory deficits observed in WARs may be a consequence of hyperphosphorylation and consequent abnormal distribution of Tau triggered by a molecular mechanism yet to be identified. It has not yet been determined whether this mechanism includes increased levels of A? aggregates, as expected in AD models. Therefore, the use of the WAR strain as an experimental model to the study of the molecular phenomena underlying the comorbidity between epilepsy and Alzheimer disease, although promising, still needs to be further characterized.

No description available.

La maladie d’Alzheimer est une maladie neurodégénérative déterminée par deux caractéristiques : les plaques extracellulaires composées d’amyloïde-β et l’accumulation intracellulaire de tau hyperphosphorylée, appelée enchevêtrements neurofibrillaires. Malgré le nombre important d’études, la nature de la toxicité des espèces tau hyperphosphorylée et hypophosphorylée reste mal connue. Notre projet de recherche vise à caractériser quel état de phosphorylation de la tau contribue le plus à la toxicité neuronale ainsi que d’identifier les mécanismes sous-jacents. Pour répondre à ces objectifs, nous avons généré des modèles transgéniques de C. elegans exprimant soit une tau hyperphosphorylée humaine (12 glutamates pour mimer l’hyperphosphorylation de la tau trouvée chez des patients Alzheimer), une tau sauvage, ou une tau hypophosphorylée (12 alanines pour mimer l’hypophosphorylation), dans les motoneurones GABAergiques. Ensuite, pour caractériser nos modèles, nous avons mesuré leur comportement principalement avec des tests de locomotion en utilisant le logiciel WormLab. Nos résultats ont montré que la tau phosphorylée est l’espèce la plus toxique car la souche hyperP a montré une perturbation du système locomoteur se traduisant par une neurodégénérescence ainsi que des problèmes développementaux (longueur des vers). Puis nous avons testé certains médicaments utilisés dans des modèles de tauopathies, afin d’identifier des voies biologiques impliquées dans la toxicité de la tau hyperphosphorylée. Pour conclure, nos modèles vont être des outils utiles pour identifier des modificateurs génétiques et pharmacologiques dans la toxicité de la tau. / Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by two hallmarks: extracellular plaques composed of amyloid-β (Aβ) deposits and intraneuronal accumulation of hyper and abnormal phosphorylated tau, also called neurofibrillary tangles (NFT). Despite many decades of research, the nature hypophosphorylated or hyperphosphorylated Tau toxicity remains ill understood. Our research project aims to characterize which state of Tau phosphorylation contributes to neuronal toxicity and identify the underlying mechanisms. To assess these objectives, we generated transgenic C. elegans models expressing either a human hyperphosphorylated tau (incorporation of 12 glutamate residues to mimic Tau hyperphosphorylation found in AD’s patients) human wild type Tau, or a human hypophosphorylated tau (incorporation of 12 alanine residues to mimic Tau hypophosphorylation) in the GABAergic motoneurons. Then, to characterize our models, we measured their behavior mainly with locomotion’s test using WormLab software. Our results showed that hyperphosphorylation of tau is the most toxic species for our models because hyperP strain showed an impair in the locomotor system translating into neurodegeneration, as well as developmental problems such as worm length. Then we tested some drugs used in taupathies C. elegans models to see if we could identify some biological pathways implicated in the toxicity. To conclude, our models may be a useful tool to identify genetic and pharmacological modifiers of tau toxicity.

<p>3’ end cleavage and polyadenylation are important steps in the maturation of eukaryotic mRNAs. Poly(A) polymerase (PAP), the enzyme catalysing the addition of adenosine residues, exists in multiple isoforms. In this study the functional significance of multiple poly(A) polymerases have been investigated. It is concluded (i) that at least three mechanisms generate the multiple isoforms i.e. gene duplication, post-translational modification and alternative mRNA processing and (ii) that the different isoforms of poly(A) polymerases have different catalytic properties. The study highlights regulation of poly(A) polymerase activity through modulation of its affinity for the substrate as visualised by the K_M parameter. We suggest that trans-acting factors modulating the K_M of poly(A) polymerase will play important roles in regulating its activity.</p><p>A new human poly(A) polymerase (PAPγ) encoded by the PAPOLG gene was identified. PAPγ is 65% homologous to the previously identified PAP. In human cells three isoforms of poly(A) polymerases being 90, 100 and 106 kDa in sizes are present. These native isoforms were purified. The PAPOLA gene encoded the 100 and 106 kDa isoforms while the 90 kDa isoform was encoded by the PAPOLG gene. Native PAPγ was found to be more active than 100 kDa PAP while the hyperphosphorylated 106 kDa PAP isoform was comparably inactive due to a 500-fold decrease in affinity for the RNA substrate. </p><p>The PAPOLG gene was shown to encode one unique mRNA while the PAPOLA gene generated five different PAP mRNAs by alternative splicing of the last three exons. The PAPOLA encoded mRNAs were divided into two classes based on the composition of the last three exons. Poly(A) polymerases from the two classes were shown to differ in polyadenylation activities. These differences revealed two novel regulatory motifs in the extreme C-terminal end of PAP, one being inactivating and the other activating for polyadenylation activity.</p>

3’ end cleavage and polyadenylation are important steps in the maturation of eukaryotic mRNAs. Poly(A) polymerase (PAP), the enzyme catalysing the addition of adenosine residues, exists in multiple isoforms. In this study the functional significance of multiple poly(A) polymerases have been investigated. It is concluded (i) that at least three mechanisms generate the multiple isoforms i.e. gene duplication, post-translational modification and alternative mRNA processing and (ii) that the different isoforms of poly(A) polymerases have different catalytic properties. The study highlights regulation of poly(A) polymerase activity through modulation of its affinity for the substrate as visualised by the KM parameter. We suggest that trans-acting factors modulating the KM of poly(A) polymerase will play important roles in regulating its activity. A new human poly(A) polymerase (PAPγ) encoded by the PAPOLG gene was identified. PAPγ is 65% homologous to the previously identified PAP. In human cells three isoforms of poly(A) polymerases being 90, 100 and 106 kDa in sizes are present. These native isoforms were purified. The PAPOLA gene encoded the 100 and 106 kDa isoforms while the 90 kDa isoform was encoded by the PAPOLG gene. Native PAPγ was found to be more active than 100 kDa PAP while the hyperphosphorylated 106 kDa PAP isoform was comparably inactive due to a 500-fold decrease in affinity for the RNA substrate. The PAPOLG gene was shown to encode one unique mRNA while the PAPOLA gene generated five different PAP mRNAs by alternative splicing of the last three exons. The PAPOLA encoded mRNAs were divided into two classes based on the composition of the last three exons. Poly(A) polymerases from the two classes were shown to differ in polyadenylation activities. These differences revealed two novel regulatory motifs in the extreme C-terminal end of PAP, one being inactivating and the other activating for polyadenylation activity.