The ubiquitin-proteasome system is an important proteolytic pathway implicated in the formation of neurofibrillary tangles from hyperphosphorylated tau, a pathological hallmark of tauopathies such as Alzheimer’s disease. We hypothesize that proteasome inhibition will result in tau accumulation, leading to the formation of aggregates. We established a combined pharmacological and genetic model of proteasome inhibition in SHSY-5Y neuroblastoma cells. Through lentiviral infection, we expressed a mutant T1A form of the β5 proteasome subunit to impair the chymotryptic proteolytic activity of the proteasome. Cells were then treated with different pharmacological inhibitors of the proteasome to further exacerbate the effects of mutation-mediated proteasome inhibition and tested for changes in tau expression. Contrary to our hypothesis, chronic proteasome inhibition and exposure to mild oxidative stress concurrently resulted in increased tau degradation. Therefore, although chronic proteasome inhibition was insufficient to induce changes in tau turnover, it rendered cells more vulnerable to oxidative insult.

The ubiquitin-proteasome system is an important proteolytic pathway implicated in the formation of neurofibrillary tangles from hyperphosphorylated tau, a pathological hallmark of tauopathies such as Alzheimer’s disease. We hypothesize that proteasome inhibition will result in tau accumulation, leading to the formation of aggregates. We established a combined pharmacological and genetic model of proteasome inhibition in SHSY-5Y neuroblastoma cells. Through lentiviral infection, we expressed a mutant T1A form of the β5 proteasome subunit to impair the chymotryptic proteolytic activity of the proteasome. Cells were then treated with different pharmacological inhibitors of the proteasome to further exacerbate the effects of mutation-mediated proteasome inhibition and tested for changes in tau expression. Contrary to our hypothesis, chronic proteasome inhibition and exposure to mild oxidative stress concurrently resulted in increased tau degradation. Therefore, although chronic proteasome inhibition was insufficient to induce changes in tau turnover, it rendered cells more vulnerable to oxidative insult.

La pathologie Tau est une lésion neuronale commune à plus d’une vingtaine de maladies neurodégénératives. Elle correspond à l’agrégation des protéines Tau anormalement modifiées. Les mécanismes moléculaires impliqués dans l’agrégation de Tau demeurent encore mal compris. Toutefois, parmi les différentes hypothèses étiologiques, celle d’une dérégulation de l’épissage alternatif de Tau nous intéresse tout particulièrement. Ici, nous considérons la dystrophie myotonique de type 1 (DM1) comme maladie « modèle » pour étudier cette relation, puisqu’elle présente à la fois une dérégulation de l’épissage alternatif de Tau et des agrégats Tau. La DM1 est la forme adulte la plus fréquente de dystrophie musculaire. Il s’agit d’une maladie héréditaire à transmission autosomale dominante caractérisée par des répétitions CTGn>50 instables localisées dans la région 3’UTR du gène DMPK. Les mécanismes impliqués supposent un gain de fonction toxique des ARN mutés conduisant à une modification de l’épissage alternatif de nombreux transcrits parmi lesquels Tau. Dans ce contexte, nos objectifs étaient 1) de caractériser le défaut d’épissage de Tau dans le cerveau de plusieurs cas DM1 2) de modéliser ce défaut d’épissage afin d'identifier les facteurs trans-régulateurs impliqués et 3) de proposer une approche visant à restaurer un épissage normal. Le défaut d’épissage de Tau a été observé dans tous les cas analysés. Celui de l’exon 10, en revanche, n’a été rapporté que chez deux cas, qui, de façon intéressante, présentaient également une augmentation de l’expression des protéines CELF, décrites comme protéines régulatrices de l’épissage de Tau. Outre les protéines CELF, nous nous sommes également intéressés à MBNL1. MBNL1 est un facteur d’épissage jouant un rôle essentiel dans la physiopathologie de la DM1 où il a été décrit comme séquestré dans les foci. Peu de choses sont connues sur MBNL1 dans le cerveau et sur son rôle sur l’épissage alternatif des transcrits neuronaux. Ici, nous montrons que le niveau d’expression cérébrale de MBNL1 ne varie pas entre les cas DM1 et contrôles. En revanche, nous montrons que son épissage alternatif est dérégulé dans le cerveau. Notre étude de relation entre la structure et la fonction de la protéine suggère que ce changement d’épissage favorise sa séquestration dans les foci en modifiant sa localisation nucléaire, son activité de facteur d’épissage et ses propriétés d’oligomérisation. Le changement d’épissage de MBNL1 n’influence pas celui de Tau. Cependant, sa perte de fonction reproduit un profil d’épissage similaire à celui observé dans les cerveaux DM1. De plus, nous montrons que la surexpression de MBNL1, en présence des répétitions CTG suffit à restaurer un épissage normal de Tau et de plusieurs autres transcrits dérégulés dans la DM1. Enfin, des expériences complémentaires réalisées avec des protéines tronquées non fonctionnelles en tant que facteur d’épissage suggèrent que la restauration d’un profil d’épissage normal dans la DM1 serait due à la saturation des sites de liaisons CUG, ce qui permettrait de libérer les protéines MBNL1 séquestrées. Ces constructions semblent donc présenter un potentiel intérêt pour inverser les changements d’épissage observés dans la DM1 et sont actuellement en cours d’études. / Tau pathology is a brain lesion common to more than twenty neurodegenerative disorders. It consists of the abnormal aggregation of the microtubule-associated protein Tau into neurofibrillary tangles. Mechanisms underlying Tau aggregation are not fully understood yet. However, among the different etiological hypothesises, the one of a relationship between Tau mis-splicing and Tau aggregates particularly interests us. Here, we proposed a disease model, being myotonic dystrophy type I (DMI), in which Tau mis-splicing and Tau aggregate occur. DM1 is the most common adult form of muscular dystrophy. It is an inherited autosomal disorder characterised by a dynamic instable CTG repeats (over 50) in the 3’UTR of DMPK gene. DM1 pathogenesis is suggested to result from a RNA toxic gain of function whereby mutant transcripts modify the splicing machinery activity leading thus to a mis-splicing of several pre-mRNA targets including Tau. In this context, our objectives were to 1) characterize Tau mis-splicing in several DM1 brain patients 2) Model it and identify the trans-regulating splicing factors likely involved and 3) Propose a therapeutic approach to reverse it. Tau mis-splicing was always observed for both exons 2 and 3 in human adult DM1 brain and consisted of a reduced inclusion. Tau exon 10 splicing was seldom mis-regulated and associated with an increase of the CELF proteins family. CELF proteins are splicing factors previously described to regulate alternative splicing of Tau exons 2, 3 and 10. In addition to the CELF proteins, we also investigated the potential role of the splicing factor MBNL1, which was shown to play an essential role in DM1 physiopathology through its sequestration by the CUG repeats. MBNL1’s brain expression was ill-defined. Here, we report that MBNL1’s expression level was not altered but its splicing modified in adult DM1 brain. In addition, we provide evidences by a relationship study between the structure and the function of MBNL1 that this mis-splicing event favored its sequestration to the foci by modifying its cell-localization, splicing activity and oligomerization properties. MBNL1 mis-splicing does not influence Tau mis-splicing. However its loss of expression reproduced the mis-splicing of Tau exons 2/3 as observed in DM1 brain. Interestingly, the overexpression of MBNL1 in the presence of the CTG repeats partially restored a normal splicing of Tau as well as several other mis-regulated pre-mRNA targets. Further experiments performed with different molecular constructs lead us to hypothezied that the reversal of the abnormal splicing events observed in DM1 was mediated by a saturation of the CUG binding sites that lead to the release of a free pool of MBNL1, recovering thus its splicing function. This work leads us to design a new molecular tool that might be of interest to reverse the pathological events observed in DM1.

Les protéines Tau sont impliquées dans plusieurs maladies neurodégénératives dénommées tauopathies, dont la plus fréquente est la maladie d’Alzheimer. Ces maladies se caractérisent par une accumulation intracellulaire de protéines Tau hyper- et anormalement phosphorylées sous forme de filaments. Ces lésions, dont l’origine et le rôle exact restent mal connus, sont au cœur d’un processus dégénératif conduisant à de nombreux troubles cognitifs et/ou moteurs et aboutissant le plus souvent à un syndrome de démence. Les protéines Tau appartiennent à la famille des protéines associées aux microtubules. Elles sont principalement neuronales et majoritairement localisées dans les axones où elles modulent l’assemblage et la stabilisation des microtubules. La mise en évidence d’autres localisations au sein des neurones, notamment dans le noyau, suggère néanmoins que Tau pourrait être une protéine multifonctionnelle. Cependant, bien que Tau soit observée dans le noyau des neurones, sa fonction n’a jamais été étudiée. Des études in vitro ont montré que la protéine Tau purifiée est capable de se lier et de stabiliser l’ADN en le protégeant de la dégradation par les DNAses ainsi que des altérations provoquées par les radicaux libres. Les travaux présentés dans cette thèse ont eu pour objectif d’étudier in situ, la fonction de Tau nucléaire sur l’intégrité de l’ADN en condition de stress. Dans ce but, nous avons développé et caractérisé des modèles dans lesquels un stress thermique ou un stress oxydant, un mécanisme précocement impliqué dans la maladie d’Alzheimer, modulent la quantité de Tau dans le noyau de neurones. Nos résultats indiquent qu’en réponse à une hyperthermie, stress non toxique pour les cellules, Tau est déphosphorylée et s’accumule dans le noyau des neurones où elle se lie à l’ADN. Afin d’étudier le rôle de Tau nucléaire, nous avons analysé par Comet assay l’effet de l’hyperthermie sur l’intégrité de l’ADN dans des neurones sauvages ou déficients en Tau. Les résultats ont montré que ce type de stress entraîne des dommages à l’ADN spécifiquement dans les neurones déficients en Tau. Dans ces neurones, l’expression à l’aide de vecteurs adénoviraux de la Tau humaine possédant ou non une séquence de localisation nucléaire pour cibler Tau dans le compartiment nucléaire, prévient les dommages induits par le stress. Inversement, une hypothermie induit une hyperphosphorylation de Tau et prévient son accumulation dans le noyau. Dans ce contexte, nous avons observé la présence de dommages à l’ADN dans les neurones sauvages. L’ensemble de ces résultats suggèrent que l’accumulation de Tau dans les noyaux protège l’ADN neuronal des dommages induits par un stress. En conclusion, ce travail montre, pour la première fois, un nouveau rôle de Tau en tant qu’acteur essentiel de la réponse précoce à un stress dans le neurone où la protéine Tau protège l’intégrité de l’ADN. Dans les tauopathies, l’altération pathologique de Tau pourrait avoir un impact délétère sur sa fonction neuroprotectrice de l’ADN et contribuer ainsi à la physiopathologie de ces maladies. / Tau proteins are involved in several neurodegenerative disorders, named tauopathies. Alzheimer’s disease (AD) is the most common tauopathy. These diseases are characterized by an intracellular accumulation of abnormally and hyperphosphorylated Tau into filaments. The etiology and exact contribution of these lesions are poorly understood but they induce degenerative process leading to cognitive and/or motor troubles and in several cases, dementia. Tau proteins belong to the family of microtubule associated proteins. They are mainly expressed in neurons and strongly localized in axons. Tau’s function is to promote assembly and stabilization of microtubules. However, the observation of additional neuronal localizations suggests that Tau could be a multifunctional protein. Indeed, Tau has been visualized in the nucleus of neurons, but its nuclear function has never been studied. In vitro studies have shown that purified Tau protein can bind to DNA. Tau-DNA complex could stabilize DNA, protecting it from DNAse’s degradation and from damages induced by hydroxyl free radicals. Thus, the aim of this work was to study, in situ, the function of nuclear Tau on DNA integrity in stress condition. In this purpose, we developed and characterized models in which thermal stress or oxidative stress, an early mechanism involved in AD, modulates Tau level in the nucleus of neurons. Our results indicate that, in response to heat stress, a non toxic cellular stress, Tau is dephosphorylated and accumulates into the nucleus of neurons. Tau binds to DNA and heat stress increases Tau-DNA complex formation. To study the role of nuclear Tau, we analyzed the effects of heat stress on DNA integrity by Comet assay in wild type or Tau deficient neurons. Results showed that this stress causes DNA damages specifically in Tau deficient neurons. In these neurons, the expression of Tau with adenoviral vector encoding for hTau with or without a nuclear localization sequence to target Tau in the nuclear compartment prevents heat stress-induced DNA damages. Conversely, cold stress induces Tau hyperphosphorylation and prevents its accumulation into the nucleus. In this context, we observed DNA damages in wild type neurons. All these results suggest that nuclear accumulation of Tau protects neurons from stress-induced DNA damages. In conclusion, this study enlightens, for the first time, a new role of Tau as an essential actor in the early response to cellular stress in neurons where Tau has a neuroprotective function on DNA in stress condition. In tauopathies, pathologic Tau alteration could lead to a loss of its neuroprotective function on DNA, that could likely contribute to the pathophysiology of the disease.

A total of 61 human brain specimens were analyzed with both ELISA and Western Blot using the ALZ50 monoclonal antibody. The brain specimens included: Alzheimer's Disease (AD, n=31), AD/Down's (n=2), Normal (n=14), Parkinson's Disease (n=7), Huntington's chorea (n=2), Wernicke-Korsakov's Encephalopathy (n=3), and Motor Neuron Disease (n=2). The non-AD cases (n=28) had no detectable A68 by ELISA, and showed no A68 bands by Western blot. The AD cases (n=33), all were positive for A68 by the ELISA, but only 31 of 33 had visible A68 band by Western blot. Additionally, a method for solubilization of A68 is reported.

No description available.

Tesis presentada a la Universidad de Chile para optar al grado académico de Magíster en Bioquímica, área de especialización en Bioquímica de Proteínas y Biotecnología, y Memoria para optar al título profesional de Bioquímico / La Enfermedad de Alzheimer (EA) es una enfermedad neurodegenerativa, de evolución lenta y que se caracteriza por ser la forma más común de demencia. En el transcurso de la EA, la proteína tau comienza a hiperfosforilarse irreversiblemente en múltiples sitios, y se autoensambla en estructuras anómalas filamentosas denominadas filamentos helicoidales pareados (PHFs), para dar lugar finalmente a los ovillos neurofibrilares (NFTs). Se ha demostrado que los oligómeros de tau son neurotóxicos y más interesante aún, correlacionan con el deterioro cognitivo. Se discute sobre la naturaleza precisa de las especies que causan dicho efecto. Por lo tanto, la inhibición de la agregación de tau en estructuras oligoméricas o filamentosas se muestra como un blanco terapéutico para el tratamiento de la EA. Se ha descrito previamente que moléculas de la familia de quinolinas y polifenoles afectarían la agregación de tau in vitro. En este contexto, la hipótesis de este trabajo se enmarca en que nuevos compuestos derivados de quinolinas y polifenoles como el ácido fúlvico, tendrían capacidad inhibitoria en la agregación de tau. El objetivo central es desarrollar una metodología para el estudio de moléculas con propiedades antiagregantes de tau y sus posibles mecanismos de la inhibición. Para ello se ha purificado htau recombinante y el fragmento 4RMBD desde E. coli y se indujo la polimerización in vitro con heparina. Se caracterizaron los agregados formados mediante técnicas de espectroscopia de fluorescencia, dynamic light scattering (DLS), microscopia electrónica y microscopia de fuerza atómica (AFM). A su vez, se estableció una metodología para el screening de moléculas con capacidad antiagregante de tau. Mediante estas técnicas, hemos determinado que solo el compuesto THQ-55 de las quinolinas ensayadas tendría un potencial efecto antiagregante de tau a altas concentraciones. A su vez, el ácido fúlvico presenta un efecto inhibitorio en la agregación de tau y desensamblaría los agregados preformados de ésta. Hacia el futuro se espera dilucidar el mecanismo mediante el cual el ácido fúlvico afecta los procesos de agregación proteica. / Alzheimer's Disease (AD) is a neurodegenerative disorder of slow progress that affects the brain. AD is the most common form of dementia. In the course of AD, tau protein begins to be irreversibly hyperphosphorylated at multiple sites, and selfassembles into pathological filamentous structures called paired helical filaments (PHFs), and these filaments assemble into neurofibrillary tangles (NFTs). It has been shown that tau oligomers are neurotoxic and correlate with cognitive decline, although the precise nature of the species that cause this effect is unknown. In this context, the inhibition of tau aggregation in oligomeric or filamentous structures appears as a promising therapeutic target for the treatment of AD. Previously, it has been described that the family of molecules of quinolines and polyphenols would affect the aggregation of tau in vitro. In this context, the hypothesis of this research is based on the activity of quinolines compounds and polyphenols such as fulvic acid, and their inhibitory action on tau aggregation. The central goal is the development of an approach for the study of molecules with anti-tau aggregation properties and its possible mechanisms of inhibition. Htau and the 4RMBD fragment have been purified from E. coli and the in vitro aggregation was induced with heparin. Aggregates formed were characterized by fluorescence spectroscopy techniques, dynamic light scattering (DLS), electron microscopy and atomic force microscopy (AFM). Moreover, a methodology for the screening of molecules with antiaggregation capacity of tau was established. Using these techniques, we determined that of the quinolines tested only the THQ-55 compound has an antiaggregation potential on tau at high concentrations. On the other hand, fulvic acid has a significant inhibitory effect on tau aggregation and disassembles tau preformed aggregates. Towards the future, we hope to elucidate the mechanism by which the fulvic acid affects protein aggregation processes.

While confirmations of the validity of the Standard Model at low and high energies (reached by present accelerators) continue to be produced, the high-energy physics community is turning towards the search for physics beyond the Standard Model. In chapter 2 we review the evidence for the existence of three neutrino flavors. We proceed by quickly summarizing their properties in the Standard Model and examine the present limits set by experiments on each neutrino's mass. We then move on to some theoretical considerations on neutrino masses, mainly based on cosmology and Grand Unified Theories and the chapter closes by illustrating the remarkable possibility of neutrino oscillations. Interestingly, at the end of 1997 it is in the neutrino physics domain that most inconsistencies with the Standard Model are found. Not in experiments using neutrinos to study the weak and strong interactions, as we see at the start of chapter 3, but definitely when probing the solar and atmospheric neutrino fluxes. This is detailed later in that chapter, without forgetting the only neutrino oscillation appearance evidence found so far (LSND). From the results presented it seems to be clear that neutrinos have opened a window towards new physics. However, solving all the present problems (cosmology, solar, atmospheric, LSND) with our simple neutrino oscillation interpretation seems impossible or difficult and definitely not very elegant. So maybe this window we are opening is really much larger than we think and pushing more could yield some fascinating results. The existing constraints are definitely such that experiments should be built to search for appearance at specific oscillation parameter values. When Chorus was designed and started datataking, these constraints were quite a bit weaker. However, the parameter space area which is being explored is still very relevant in the light of a model solving the Hot Dark Matter and solar problems while satisfying the very elegant see-saw mechanism. We briefly describe the experiment in chapter 4, explaining the chosen tau neutrino detection strategy and the proposed sensitivity to Vµ -> Vtau oscillations. In chapter 5 we start by describing the trackers: purpose, geometry and readout. This is followed by an explanation of the event reconstruction algorithm and its efficiency. Our main contribution to the reconstruction in the Target Trackers consists in their alignment: after introducing a few concepts we give the details of the alignment procedure, the trackers' degrees of freedom and the accuracy obtained for each of these. Two more paragraphs say a few more words on the problem of separation of variables for minimization and the detector stability over time. Next, in chapter 6, we discuss our work on the tagging of muons with the Chorus detector. After explaining the various algorithms used we come to the efficiencies, understanding the found results through event kinematics. A study of non-tagged muons shows that the software efficiency is very close to maximal, and physics is the cause of nearly all the losses. The bulk of our study lies in the determination of the scanning efficiencies. Chapter 7 starts with the description of the interface emulsion sheets and the proof of a method we developed to extract the real scanning efficiency for these sheets from the data itself. We proceed to compare the found results with the results obtained by our simulation and comment on the possible causes for the observed discrepancy. Moving on, we explain how the search for the primary vertex and a possible decay kink is conducted and give the corresponding efficiencies we found using our Monte-Carlo. To close the chapter, we elaborate on the video image simulation, a tool we developed to help improve and determine the efficiency of video image analysis programs. These programs will be used intensively in the future to search for kinks. Last but not least, chapter 8 combines all these numbers with the present scanning status to put a new limit on Vµ -> Vtau oscillations. It also includes a few words on backgrounds and the results from a study of systematic uncertainties. We conclude with a new exclusion plot.

A search for the Higgs boson decaying to tau tau using 7.8 fb^-1 of pp collisions at 1.96 TeV collected with CDF II detector is presented. The search is sensitive to four production mechanisms of the Higgs boson: ggH, WH, ZH and VBF. Modes where one tau decay leptonically, and another decay, hadronically, are considered. Two novel techniques are developed and used in the search. A Probabilistic Particle Flow Algorithm is used for energy measurements of the hadronic tau candidates. The signal is discriminated from backgrounds by the Missing Mass Calculator, which allows for full invariant mass reconstruction of tau tau pair. The data are found to be consistent with the background only hypothesis. Therefore a 95% confidence level upper limit on the Standard Model Higgs boson cross section was set. At M_H=120 GeV/c^2 observed limit is 14.9 x sigma_SM x Br(H -> tau tau).

Tau protein is one of the microtubule-associated proteins (MAPs) and mainly expressed in neuronal cells. It hasbeen demonstrated that Tau may play an important role in regulating microtubule dynamic in neurons. Structurally and functionally, Tau protein composed of regulatory projection domain in N-terninus and microtubule-binding domain in C-terminus. It has been shown that the biological function of Tau protein was regulated by phosphorylation and dephosphorylation. In Alzheimer¡¦s disease (AD) brain, hyperphosphorylated Tau caused by over active kinases may contribute to the disassociation of Tau from microtubule and form the pathologically hallmarker, paired helical filaments (PHFs). The reason to study cdc2 and GSK3£] is two folds. First, both cdc2 and GSK3£] activities are raised abbrently in AD brain. Second, the phosphorylation sites of cdc2 and GSK3£] have been identified as those in PHFs.These prompted us to regard cdc2 and GSK3£] as candidates that hyperphosphorylated Tau in AD. In the following study, we used immunofluorescence analysis, co-immunoprecipitation and GST-fusion protein pull down assay to clarify the subcellular localization of Tau. We also shown that the interaction between tubulin withfull length Tau (Tau WT) and some Tau mutants that we found that not only Tau WT, but also N-terminus of Tau (Tau-N) and C-terminus of Tau (Tau-C) can bind to tubulin. Surprisingly, we observed that a fragment of N-terminus, Tau 122-244, localized in nucleus. Furthermore, we used tubulin assembly assay to test if tau or its mutants can promote tubulin assembly in vitro. Results showed that only Tau WT can promote tubulin assembly in vitro but not Tau-N or Tau-C. Although Tau-N or Tau-C can bind to tubulin in vivo and in vitro, these mutants did not remain the ability to promote tubulin assembly that suggested both functional domains, N-terminus and C-terminus of Tau, are necessary and essential for the biological function of Tau. On the other hand, we used of phosphorylation assay and site directed mutagenesis to demonstrate that T231 of Tau is one of important phosphorylation sites of cdc2 and GSK3£]. Finally, we used tubulin assembly assay to show that phosphorylated Tau by GSK3£] can negatively regulate the ability of Tau to promote tubulin assembly that indicated that the phosphorylation at T231 may play a role in regulating Tau.