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.

Behavioral characterization of animal models for Alzheimer's Disease is critical for the development of potential therapeutics and treatments against the disease. While there are several known animal models of AD, three current models--APPsw, P301L, and APPsw+P301L--have not been well characterized, if at all. This study, therefore, aimed to perform a full behavioral characterization of these three models in order to better understand the impairments associated with each one. Between 5 and 8.5 months of age, animals were behaviorally tested in a variety of sensorimotor, anxiety, and cognitive tasks. The number of tau+ neurons in the forebrains of P301L mice was then compared to their behavioral performance. Results of this study indicate that retinal degeneration (rd) seriously impairs the performance of mice in behavioral tasks. Animals that carry the homozygous allele of this mutation must, therefore, be eliminated from any such study requiring visual acuity. After this elimination, my findings indicate that APP mice are impaired in several cognitive tasks (including platform recognition, Morris maze, Y-maze, and radial-arm water maze) at a young early age (5 to 8.5 months of age). These mice have fairly normal sensorimotor function, showing significant impairment only in balance beam performance starting at 5 months. Although P301L mutant Tau mice, as a group, did not have significant impairments in any sensorimotor or cognitive task, correlation analysis revealed that higher numbers of tau+ neurons in cortex and hippocampus were associated with poorer cognitive performance. Finally, discriminant function analysis (DFA) appears able to accurately discriminate between the three transgenic groups of mice using only an 8-measure data set. In conclusion, this study provides the first comprehensive, multiple task behavioral assessment of the APPsw and P301L animal models of AD, indicating that APPsw mice are cognitively impaired at an early age while P301L mice are largely unimpaired through 8.5 months. Nonetheless, correlational analysis implicates the formation of neurofibrillary tangles in the onset of cognitive impairments. Finally, my findings recommend the continued use of DFA to determine if groups of animals, based on different transgenicity or therapeutic treatment, could be discriminated between from their behavior alone.

Fluorodeoxyglucose Positron Emission Tomography (FDG PET) and Magnetic Resonance Imaging (MRI) are commonly used in a clinical setting as an examination in Alzheimer’s Disease (AD) patients. FDG PET is an imaging tool to evaluate hypometabolism; meanwhile, the MRI observes the brain volume. However, it is still unclear which examination better reflects the tau tangles, which have been known as the hallmark’s pathology of AD. Therefore, this study was conducted to compare FDG PET and MRI in assessing tau pathology in AD, by utilizing a database from the Alzheimer’s Disease Neuroimaging Initiative (ADNI). The presence of tau tangles was confirmed by using the Tau-PET images. In total, 275 cognitively impaired subjects were included as well as a subgroup of 147 subjects with positive amyloid status. Based on the analysis, it was observed that higher Tau-PET is significantly associated with FDG PET hypometabolism and MRI atrophy. A similar result was also seen in the amyloid positive subgroups. By comparing the spearman’s correlation coefficients, it was found that that the correlation was stronger between Tau PET and FDG PET (r=-0.414, p<0.001, and r=-0.475, p<0.001 in the positive amyloid subgroup) compared to Tau-PET and MRI (r=-0.331, p<0.001 and r=-0.440, p<0.001 in the positive amyloid subgroup). Inconclusion, FDG PET better reflects the tau pathology compared to MRI in AD.

Tau mutations promote the formation of tau oligomers and filaments, which are neuropathological signs of several tau-associated dementias. Types of neurons in the CNS are spared of tau pathology and are surrounded by a specialized form of extracellular matrix; called perineuronal nets (PNs). Aggrecan, the major PN proteoglycans, is suggested to mediate PNs neuroprotective function by forming an external shield preventing the internalization of misfolded tau. We recently demonstrated a correlation between aggrecan amount and the expression and phosphorylation of tau in a TauP310L-acan mouse model, generated by crossbreeding heterozygous aggrecan mice with a significant reduction of aggrecan and homozygous TauP301L mice. Neurodegenerative processes have been associated with changes of PN structure and protein signature. In this study, we hypothesized that the structure and protein expression of PNs in this TauP310L-acan mouse is regulated by tau. Immunohistochemical and biochemical analyses demonstrate that protein levels of PN components differ between TauP301LHET-acanWT and TauP301LHET-acanHET mice, accompanied by changes in the expression of protein phosphatase 2 A. In addition, tau can modulate PN components such as brevican. Co-immunoprecipitation experiments revealed a physical connection between PN components and tau. These data demonstrate a complex, mutual interrelation of tau and the proteoglycans of the PN.

No description available.

<p>Tauopathies are neurodegenerative diseases defined by the accumulation of pathological tau protein in neurons and glia. Alzheimer’s disease (AD), the most common tauopathy, is characterized by the presence of neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein aggregates in neurons. Emerging evidence suggests that the NFT burden correlates with neuron death and cognitive decline, contributing to disease progression. Besides AD, a similar deposition of tau inclusions is found to be associated with neurodegeneration in the brains of patients with other tauopathies including progressive supranuclear palsy, corticobasal degeneration, and Pick’s disease. These diseases display clinical, biochemical, and neuropathological heterogeneity. Little is known about how tau aggregation can lead to varied phenotypes in tauopathies, and there is no disease-modifying treatment. Thus, it is necessary to understand the role of diverse tau proteoforms in tauopathies for the development of new therapeutics to treat tauopathies, including AD.</p> <p>In the studies summarized in Chapter 2, we investigated how the molecular diversity of tau proteoforms could impact antibody-based assays of a phospho-tau variant serving as an AD biomarker. A tau variant phosphorylated on threonine 181 (pT181-tau) has been widely investigated as a potential AD biomarker in cerebrospinal fluid (CSF) and blood. pT181-tau is present in NFTs of AD brains, and CSF levels of pT181-tau correlate with overall NFT burden. Various immuno-based analytical methods, including Western blotting and ELISA, have been used to quantify pT181-tau in human biofluids. The reliability of these methods depends on the affinity and binding specificity of the antibodies used to measure pT181-tau levels. Although both of these properties could in principle be affected by phosphorylation within or near the antibody’s cognate antigen, such effects have not been extensively studied. Here, we developed a bio-layer interferometry (BLI)-based analytical assay to assess the degree to which the affinity of pT181-tau antibodies is altered by the phosphorylation of serine or threonine residues near the target epitope. Our results revealed that phosphorylation near T181 negatively affected the binding of pT181-tau antibodies to their cognate antigen to varying degrees. In particular, two of three antibodies tested showed a complete loss of affinity for the pT181 target when S184 or S185 was phosphorylated.</p> <p>In the studies outlined in Chapter 3, we examined the relative abilities of different tau proteoforms to induce seeded tau aggregation and to themselves undergo seeded aggregation in cultured cells. Accumulating evidence suggests that tau aggregates, including NFTs, spread in a stereotypical pattern across neuroanatomically connected brain regions. This spreading phenomenon is thought to occur via a prion-like mechanism involving the release of tau aggregates from a diseased neuron into the extracellular space, aggregate uptake by neighboring healthy neurons, and the formation of new aggregates in the cytosol of the recipient cells via a seeding process. Although research over the past decade has revealed key molecular events involved in the cell-to-cell transmission of tau aggregates, the impact of the protein’s domain structure and phosphorylation profile on the efficiency of prion-like propagation remains poorly defined. Here, we compared three tau variants – K18, 0N4R, and 2N4R – in terms of their aggregation and seeding efficiencies in recombinant protein solutions and in cell culture models. Our results revealed that K18 had the highest fibrillization rate and yield among the three tau variants. Recombinant preformed fibrils (PFFs) derived from all three variants had similar seeding efficiencies. Additionally, we investigated the relationship between tau phosphorylation and aggregation. We found that hyperphosphorylated tau did not undergo self-assembly in the absence of heparin, whereas it formed fibrils at low yield in the presence of the cofactor. Moreover, hyperphosphorylated tau PFFs produced under these conditions induced seeded tau aggregation in cell culture.</p> <p>Taken together, these results point to critical roles of tau proteoforms as both AD biomarkers and drivers of disease progression. Our results indicate that the presence of different combinations of phosphorylated residues near a target phospho-tau antigen can affect the accuracy of antibody-based biomarker assays. In addition, the domain structure and phosphorylation profiles of tau proteoforms associated with AD and other tauopathies likely have a profound influence on the evolution of tau pathology in these disorders. Our findings highlight the importance of accounting for the molecular diversity of tau proteoforms in tauopathies and provide valuable insights into molecular determinants influencing tau aggregation and propagation in the brains of patients.</p>