Submitted by Hiolanda Rêgo (hiolandarego@gmail.com) on 2016-04-04T16:30:34Z No. of bitstreams: 1 Tese_Med_Guilherme Teixeira Valença.pdf: 1840128 bytes, checksum: 121ea4642b179f76915bacfa9b2f6d14 (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2016-04-12T16:06:03Z (GMT) No. of bitstreams: 1 Tese_Med_Guilherme Teixeira Valença.pdf: 1840128 bytes, checksum: 121ea4642b179f76915bacfa9b2f6d14 (MD5) / Made available in DSpace on 2016-04-12T16:06:03Z (GMT). No. of bitstreams: 1 Tese_Med_Guilherme Teixeira Valença.pdf: 1840128 bytes, checksum: 121ea4642b179f76915bacfa9b2f6d14 (MD5) / CAPES / Recentemente, foi demonstrado que o lócus MAPT relacionado com a Doença de Parkinson (DP) está também associado com sinais parkinsonianos em idosos sem diagnóstico clínico de DP. Entretanto, a biologia subjacente a esta associação é desconhecida. Vários estudos têm demonstrado que o haplótipo H1 e subhaplótipo H1c MAPT estão associados com doenças neurodegenerativas como a DP, doença de Alzheimer (DA), paralisia supranuclear progressiva (PSP) e degeneração córticobasal (DCB). Aqui, vamos refinar a análise desta importante região e avaliar o efeito dos haplótipos MAPT no parkinsonismo em idosos, na expressão total e no splicing alternativo deste gene. Foram utilizados dados ante-mortem e post-mortem dos participantes em dois estudos clínico-patológicos sobre o envelhecimento, Religious Orders Study e Memory and Aging Project. Os escores de parkinsonismo global e seus quatro principais componentes, bradicinesia, rigidez, tremor, e alteração da marcha foram coletados prospectivamente a cada ano. Nas análises de regressão, controladas para idade, sexo, índices post-mortem de PD, AD, outros taupatias, macroinfartos e microinfartos, o haplótipo MAPT H2 estava associado com parkinsonismo global (p = 0,049), bradicinesia (p = 0,008) e menor expressão total de MAPT (p = 1,72-14). Quando analisamos o splicing alternativo MAPT, parkinsonismo global e bradicinesia também estavam inversamente associados com a expressão de apenas uma isoforma (1N/4R, p=0,01 e p = 0,008, respectivamente); ao explorar cada característica clínica separadamente, descobrimos que haplótipo H2 é associado com bradicinesia e marcha, mas não tremor ou rigidez. Não houve associação entre essas características motoras e subhaplótipo H1c. No geral, estes resultados sugerem que ambos, menor expressão total de MAPT associado ao haplótipo H2 em comparação com H1 e splicing alternativo MAPT, podem parcialmente explicar a associação do gene MAPT com o parkinsonismo em idosos e, em certa medida, a associação deste último com doenças neurodegenerativas relacionadas à idade.

The microtubule-associated protein tau forms pathological neuronal filaments in Alzheimer's disease (AD) and other neurodegenerative disorders, known collectively as tauopathies. Previous studies in transgenic mouse models of AD suggest that reducing tau expression may be safe and beneficial for the prevention or treatment of AD and possibly other tauopathies. As a first step toward identifying novel therapeutic strategies to reduce tau levels, the studies presented in this dissertation aim to investigate the role of the human tau 3'-untranslated region (3'-UTR) in regulating tau expression. Tau expresses two 3'-UTR isoforms, long and short, as a result of alternative polyadenylation. The exact sequence of these two 3'-UTR isoforms was determined by rapid amplification of cDNA 3'-ends (3'-RACE), and the two 3'-UTR isoforms were cloned into a luciferase reporter vector. Using these reporter constructs, the expression of these isoforms was found to be differentially controlled in human neuroblastoma cell lines M17D and SH-SY5Y by luciferase assays and quantitative PCR (qPCR). Through an unbiased screen of tau 3'-UTR deletions and fragments using luciferase reporter constructs, several regions in the long tau 3'-UTR isoform that contain regulatory cis-elements were identified. Additionally, several microRNAs were computationally identified as candidates that might bind the long tau 3'-UTR and thereby differentially control the expression of long versus short tau 3'-UTR isoforms. Screening these candidate microRNAs via luciferase reporter assay identified miR-34a, which was subsequently shown to repress the expression of endogenous tau protein and mRNA in M17D cells using Western blot and qPCR, respectively. Conversely, inhibition of endogenously expressed miR-34 family members leads to increased endogenous tau expression. Taken together, these studies suggest that the expression of the two tau 3'-UTR isoforms is differentially regulated and that this differential regulation is due to the presence of regulatory cis-elements found only in the long tau 3'-UTR isoform, including a binding site for miR-34 family members. Improved understanding of the regulation of tau expression by its 3'-UTR may ultimately lead to the development of novel therapeutic strategies for the treatment of Alzheimer's disease and other tauopathies.

Este trabajo se centra en el estudio de la implicación de los genes MAPT y PGRN en la degeneración lobar frontotemporal (DLFT). Se describen 2 nuevas mutaciones en estos genes (P301T en el gen MAPT y la A303AfsX57 en el gen PGRN) y no se encontraron variaciones en el número de copias del gen MAPT en las muestras estudiadas, apoyando que la existencia de alteraciones en la dosis génica de MAPT no seria una causa de DLFT. El mecanismo patogénico de la mutación P301T es potencialmente múltiple e incluye la reducción de la capacidad de promover el ensamblaje entre tau y los microtúbulos, la estimulación del ensamblaje de filamentos anómalos de tau y la creación de un nuevo sitio de fosforilación en la proteína tau. El mecanismo patogénico de la mutación A303AfsX57 es la haploinsuficiencia. Respeto al fenotipo clínico los pacientes con mutaciones en el gen MAPT presentan una historia familiar con patrón autosómico dominante de inicio precoz, siendo el diagnóstico clínico más frecuente el de demencia frontotemporal, si bien en ocasiones pueden presentar sintomatología similar a la degeneración corticobasal o la parálisis supranuclerar progresiva. El examen neuropatológico muestra una DLFT con presencia de depósitos de filamentos de proteína tau hiperfosforilada. Los pacientes con mutaciones en el gen PGRN suelen presentar una edad de inicio más tardía que los pacientes con mutaciones en el gen MAPT. El fenotipo clínico de estos pacientes suele ser también similar a la demencia frontotemporal, si bien la importante alteración del lenguaje puede conducir a un diagnóstico de afasia progresiva no fluente. El examen neuropatológico muestra una DLFT con inclusiones ubiquitin positivas (DLFT-U) con presencia de inclusiones neuronales intranucleares. Estas inclusiones, sin embargo no son patognomónicas de la presencia de mutaciones en PGRN. La proteína depositada en las inclusiones neuronales ubiquitinadas presentes en los pacientes con mutaciones en PGRN es la TDP-43. Por otro lado se describe que la frecuencia del genotipo H1/H1 de MAPT en pacientes con DLFT no es significativamente diferente de la frecuencia en controles sanos en nuestro estudio. Tampoco encontramos diferencias en la ratio cerebral 4R/3R de RNAm de MAPT en pacientes con DLFT. Estos hallazgos sugieren que los eventos post-traduccionales podrían ser el principal factor que causa el depósito de isoformas específicas de tau en algunas taupatías, como algunos subtipos de la DLFT. Sin embargo, el incremento de la ratio 4R/3R de RNAm de MAPT en los portadores del genotipo H1/H1 sugiere que esta alteración podría ser el mecanismo a través del cual este genotipo incrementa el riesgo para desarrollar una taupatía. Finalmente se realizó un estudio de correlación clínico-genético-patológica en 32 casos con DLFT confirmada patológicamente. En este estudio se hallaron un amplio espectro de entidades clínicas y neuropatológicas, si bien se pudo establecer alguna asociación clínico-genética-patológica: el sustrato patológico de la demencia frontotemporal es impredecible, las mutaciones en el gen MAPT y los fenotipos clínicos de afasia progresiva no fluente y degeneración corticobasal se asocia a DLFT tau-positiva, mientras que la presencia de signos de motoneurona, la demencia semántica o las mutaciones en PGRN se asocian a DLFT-U. / In this work we describe two new mutations. The first is a mutation (P301T) in the MAPT gene in a patient with familial frontotemporal dementia and parkinsonism, and a pattern of autosomal dominant inheritance. The fact that this new mutation is located in the same codon that other missense mutations (P301L and P301S) associated with tau pathology, highlights the importance of this site for the physiological function of tau protein. The second is a mutation in the PGRN gene (A303AfsX57) associated with late-onset frontotemporal dementia and with "cat's eye" shaped intranuclear and cytoplasmatic ubiquitin immunoreactive inclusions in the neuropathological exam. The A303AfsX57 mutation is consistent with a nucleotide deletion in exon 8 (c908delC). This deletion causes a frameshift at codon 303 that introduces a premature termination codon (A303AfsX57). Furthermore we determine that MAPT gene copy number variation is not involved in 70 patients with clinical diagnosis of FTLD.On the other hand we evaluated the 4R/3R tau mRNA ratio in 18 patients with frontotemporal lobar degeneration (FTLD), and the effect of the H1/H1 genotype on this ratio. The 4R/3R mRNA ratio in frontal cortex was similar in FTLD patients and controls. The H1/H1 genotype carriers showed a significant increase in 4R/3R mRNA ratio, suggesting that this genotype could modulate the tau mRNA splicing.Finally we performed a clinicopathological correlation and genetic analysis in 32 consecutive patients with neuropathological diagnosis of FTLD or CBD. According to previous studies, we described a broad spectrum of clinical and pathological features in these patients. However, we found certain degree of association of some clinical subtypes to specific pathological substrates: pathology underlying sporadic FTD is heterogeneous and not predictable. MAPT mutations and clinical diagnosis of PNFA and CBD were associated to tau-positive pathology. The presence of signs of lower MND and SD correlated with FTLD-U.

Parkinson's disease (PD) primarily manifests as loss of motor control through the degeneration of nigrostriatal dopaminergic neurons. The microtubule-associated protein tau (MAPT) locus is highly genetically associated with PD, wherein the H1 haplotype confers disease risk and the H2 haplotype is protective. As this haplotype variation does not alter the amino acid sequence, disease risk may be conferred by altered gene expression, either of total MAPT or of specific isoforms, of which there are six in adult human brain. To investigate haplotype-specific control of MAPT expression in the neurons that die in PD, induced pluripotent stem cells (iPSCs) from H1/H2 heterozygous individuals were differentiated into dopaminergic neuronal cultures that expressed all six mature isoforms of MAPT after six months' maturation. A reporter construct using the human tyrosine hydroxylase locus was also generated to identify human dopaminergic neurons in mixed cultures. Haplotype-specific differences in the inclusion of exon 3 and total MAPT were observed in iPSC-derived dopaminergic neuronal cultures and a novel variant in MAPT intron 10 increased the inclusion of exon 10 by two-fold. RNA interference tools were generated to knockdown total MAPT or specific isoforms, wherein knockdown of the 4-repeat isoform of tau protein increased the velocity of axonal transport in iPSC-derived neurons. MAPT knockdown also reduced p62 levels, suggesting an impact of tau on macroautophagy, likely through altered axonal transport. These results demonstrate how variation at a disease susceptibility locus can alter gene expression, thereby impacting on neuronal function.

The MAPT gene has been shown to be associated with several neurodegenerative disorders, including forms of parkinsonism and Parkinson disease (PD), but the results reveal population differences. We investigated the association of 10 single-nucleotide polymorphisms (SNPs) in the region of MAPT on chromosome 17q21 with PD and age at onset, by using 443 discordant sib pairs in PD from a public dataset (Mayo-Perlegen LEAPS Collaboration). Association with PD was assessed by the FBAT using generalized estimating equations (FBAT-GEE), while the association with age at onset as a quantitative trait was evaluated using the FBAT-logrank statistic. Five SNPs were significantly associated with PD (P < 0.05) in an additive model, and 9 SNPs were associated with PD (P < 0.05) in dominant and recessive models. Interestingly, 8 PD-associated SNPs were also associated with age at onset of PD (P < 0.05) in dominant and recessive models. The SNP most significantly associated with PD and age at onset was rs17649641 (P = 0.015 and 0.021, respectively). Two-SNP haplotypes inferred from rs17563965 and rs17649641 also showed association with PD (P = 0.018) and age at onset (P = 0.026). These results provide further support for the role of MAPT in development of PD.

L’avènement du séquençage haut débit permet actuellement d’étudier et d’analyser la part de lacomposante génétique des maladies complexes médiée par les variants rares. Cependant, leurinterprétation représente un défi majeur. En effet, le séquençage de milliers d’exomes et degénomes a révélé la complexité du polymorphisme humain et notamment la surreprésentation devariants rares. Et malgré le développement de logiciels d’analyse ainsi que de différentes bases dedonnées, la priorisation des variants rares reste difficile. Dans le cadre de cette thèse, nous avons focalisé nos analyses sur les variations génétiques rares impliquées dans la maladie d’Alzheimer (MA). D’un point de vue génétique, la MA répond dans une majorité des cas à un déterminisme multifactoriel mais une minorité des cas sont des formes précoces à transmission autosomique dominante. La caractérisation des gènes PSEN1, PSEN2 et APP responsables des formes mendéliennes de MA a permis de formuler l’hypothèse de la cascade amyloïde en plaçant le peptide amyloïde (Aβ) au centre du processus physiopathologique. Afin de détecter de nouveaux facteurs de risque génétique dans la survenue de la MA, nous avons réalisé une étude d’association à partir de données de séquençage d’exomes de 522 cas atteints de formes précoces de MA et 584 contrôles. Les premières analyses ont porté sur les variants mononucléotidiques ainsi que les courtes insertions/délétions et ont permis de mettre en évidence un enrichissement en variants rares prédits délétères dans le gène ABCA7 chez les individus malades. Notre attention s’est ensuite portée sur les variations du nombre de copies (CNVs). L’absence de récurrence à l’échelle d’un gène nous a amené à travailler sur une liste de gènes. En nous focalisant sur l’hypothèse amyloïdergique, nous avons construit une liste de 342 gènes impliqués dans le métabolisme et la toxicité du peptide Aβ. Grâce à cette stratégie, nous avons ainsi réussi à mettre en évidence un enrichissement de CNVs rares intersectant ce réseau centré sur le peptide Aβ. Le résultat majeur de cette étude de CNVs a été la mise en évidence d’une duplication du locus 17q21.31 chez 5 patients atteints d’une maladie neurodégénérative similaire à une maladie d’Alzheimer. Les patients porteurs présentent un diagnostic clinique de MA, des biomarqueurs et une imagerie métabolique en faveur d’une neurodégénérescence de type Alzheimer. En revanche, l’imagerie amyloïde et l’analyse neuropathologique n’ont pas révélé de pathologie amyloïde et sont donc en faveur d’une tauopathie pure. L’étude des CNVs a également révélé une délétion partielle du gène PSEN1, emportant les exons 9 et 10, pour laquelle nous avons pu réaliser des études fonctionnelles. Nous avons ainsi pu déterminer que la protéine mutante favorisait la production de peptides amyloïdes plus longs, ces derniers étant des médiateurs majeurs de la neurotoxicité d’Aβ. / Next-generation sequencing allows studying and analyzing the genetic component part of complexdiseases mediated by rare variants. However, their interpretation represents a major challenge.Indeed, the sequencing of thousands of exomes and genomes revealed the human polymorphismcomplexity and in particular the overrepresentation of rare variants. Despite the development ofsoftwares and variant databases, the prioritization of rare variants remains arduous. My thesis subject was focused on the involvement of rare variants in Alzheimer's disease (AD). From a genetic point of view, AD is caused, in most cases, by a multifactorial determinism, but a minority of cases are autosomal dominant early-onset forms (ADEOAD). The characterization of mutations in the PSEN1, PSEN2 and APP genes as a cause of these Mendelian forms of AD led to the formulationof the amyloid cascade hypothesis, stating that the amyloid-β peptide (Aβ) is triggering the pathophysiological process. In order to detect new genetic risk factors involved in AD, we performed an association study using exome sequencing data from 522 cases with early-onset Alzheimer Disease and 584 controls. The first analyzes focused on single nucleotide variants and short insertions / deletions, and revealed an enrichment in cases of variants that are predicted to be deleterious in the ABCA7 genes. We then then focused on copy number variations (CNVs). The lack of recurrence at the gene-level incited us to work on a gene list. By focusing on the amyloidogenic hypothesis, we built a list of 342 genes involved in the metabolism and toxicity of the Aβ peptide. Thanks to this strategy, we found an enrichment of rare CNVs intersecting this Aβ network in cases.The main result of this CNV study was the identification of a duplication of the 17q21.31 locus in 5patients with a neurodegenerative disease similar to Alzheimer's disease. These patients have aclinical diagnosis of AD, as well as biomarkers and metabolic imaging consistent with an ADneurodegeneration. However, amyloid imaging and neuropathological analysis did not reveal anyamyloid pathology, and were therefore pointing to a pure tauopathy. This CNV study also revealed a partial deletion of the PSEN1 gene, overlapping exons 9 and 10, for which we performed functional studies. We demonstrated that the mutant protein enhanced the production of longer amyloid peptides, the latter being major mediators of Aβ neurotoxicity.

Alzheimer's disease (AD) is a large and growing problem and while we today lack a full understanding of this disease, we know that the protein tau and the amyloid fibrils it forms play a central role in its development. We also know that these fibrils can have different morphologies in different diseases and that fibrils produced in vitro not necessarily adopt any of the morphologies found in patients. This means there is a need for more pathologically relevant fibrils in vitro to be able to understand this disease better. One approach to satisfy this need is to use fibrils found in patients as seeds and thus transfer their morphology to recombinantly purified protein. To facilitate this process this study has attempted to develop a way to differentiate between different fibril morphologies using a FRET based system. This involves fluorescent fusion proteins (tau-EXFPs) and fluorescent amyloid probes as well as seeding experiments with pseudo wild type tau (PWT) and tau with the P301L mutation. Greater differences in terms of fibrillation rates and ThT fluorescence between PWT and P301L was shown than previously reported between WT and P301L. They were also shown to differ in fibril morphology in TEM. The ThT fluorescence intensity was to a certain degree transferable from PWT to P301L by seeding. Furthermore, this study confirms that the tau-EXFP fusion protein can be incorporated into amyloid fibrils and strongly suggests that a FRET effect between EXFP and BTD14 (as well as X34 and ThT) can be achieved. It also demonstrates differences in FRET efficiency between PWT and P301L fibrils using FLIM. These results indicate that a FRET based approach could be a useful method to discern different fibril morphologies from each other, but further measurements and optimization are needed before this method could be reliably applied. The fusion proteins could also be used to investigate tau spreading in vivo, e.g. in D. melanogaster. To find suitable FRET partners to the fusion proteins, a ligand screen was conducted. This could be used as an alternative to the FRET method. With the right selection of fluorescent amyloid probes, a unique fingerprint for each fibril morphology could maybe be generated and fulfill the same intended function as the FRET method.

Les tauopathies sont des maladies neurodégénératives caractérisées par l’agrégation intracérébrale de protéines tau anormales. Cependant ces maladies sont très hétérogènes sur le plan clinique, anatomopathologique mais aussi biochimique avec l'agrégation de différentes isoformes de protéines tau. De nombreux axes de recherche existent à ce jour afin de mieux comprendre ces maladies incurables. Au cours de cette thèse d'université, nous avons étudié les modifications de l’épigénome qui constituent une piste nouvelle et très prometteuse dans la recherche sur les maladies neurodégénératives. L'épigénétique est un processus dynamique et réversible qui peut être modifié par de nombreux facteurs génétiques ou environnementaux et qui joue un rôle très important dans la régulation des gènes. De nombreuses études rapportent une association entre certaines marques épigénétiques et les maladies neurodégénératives. Par exemple, dans la maladie d’Alzheimer, il a été observé une hyperméthylation de l'ADN, au niveau du promoteur du gène MAPT qui code les protéines tau.Dans ce contexte, nos objectifs étaient de déterminer si des variations de l'épigénome impliquant le gène MAPT contribuent à l'expression différentielle des protéines tau qui est observée dans les différentes classes de tauopathies. Nous avons donc constitué et caractérisé une banque de prélèvements cérébraux de témoins et de patients atteints de différentes tauopathies. Puis nous avons analysé la méthylation de l'ADN dans 3 tauopathies : la maladie d'Alzheimer, la paralysie supranucléaire progressive et la DCB. Notre étude a permis de mettre en évidence chez les patients atteints de PSP une hypométhylation dans l’inton 0 du gène MAPT. Cette hypométhylation ne concernait que le cortex frontal, affecté par la pathologie tau, mais pas le cortex occipital qui est épargné par la pathologie tau. De plus, nous avons également mis en évidence dans le tissu cérébral des patients atteints de PSP une hyperexpression des ARNm de MAPT par rapport aux témoins. Nous démontrons avec ce travail que l’hypométhylation de l'ADN de l’intron 0 de MAPT constitue une signature épigénétique spécifique de la PSP. Cette première étude nous a conduits à suspecter l'existence d'un promoteur alternatif du gène MAPT situé dans cette région de l'intron 0. Nous avons donc testé in vitro l'activité de ce promoteur et cloné des transcrits issu de ce promoteur alternatif. Nous avons ensuite confirmé ces analyses par la mesure de l'expression des ARNm par qPCR. Au total, ces expériences prouvent l'existence et la fonctionnalité de ce promoteur alternatif dans le cerveau humain. De plus, l'activation de ce promoteur alternatif aboutit à la transcription d'ARNm plus courts codant pour de nouvelles protéines tau qui pourraient être impliquées dans la survenue des tauopathies. / Tauopathies are neurodegenerative diseases characterized by intracerebral aggregation of abnormal tau proteins. However, these diseases are heterogeneous clinically, pathologically but also biochemically with the aggregation of different isoforms of tau protein. Many lines of research exist to date to better understand these incurable diseases. During this university thesis, we studied the changes in the epigenome that constitute a new and very promising approach in research on neurodegenerative diseases. Epigenetics is a dynamic and reversible process which can be modified by numerous genetic or environmental factors and plays a very important role in gene regulation. Many studies report an association between some epigenetic marks and neurodegenerative diseases. For example, in Alzheimer\'s disease, it has been observed hypermethylation of DNA in the promoter of the MAPT gene which encodes the tau protein.In this context, our objective was to determine if changes in epigenomic involving MAPT gene contribute to the differential expression of tau protein which is observed in the different classes of tauopathies. So we have established and characterized a human brainbank of controls and patients with different tauopathies. Then we analyzed the DNA methylation in 3 tauopathies: Alzheimer\'s disease, progressive supranuclear palsy, and CBD. Our study highlighted in PSP patients hypomethylation in intron 0 of MAPT gene. This hypomethylation concerned only the frontal cortex, affected by the tau-pathology but not the occipital cortex which is spared by tau-pathology. In addition, we also shown in the brain tissue of patients with PSP an overexpression of mRNA of MAPT compared to controls. We demonstrate in this work that hypomethylation of DNA in intron 0 of MAPT is a specific epigenetic signature of PSP. This first study has led us to suspect the existence of an alternative promoter of the MAPT gene located in this region of intron 0. We tested the in vitro activity of this promoter and cloned transcripts derived from this alternative promoter. We then confirmed this analysis by measuring mRNA expression by qPCR. In total, these experiments prove the existence and the functionality of this alternative promoter in the human brain. Furthermore, activation of the alternative promoter results in shorter mRNA transcripts encoding novel tau proteins that might be involved in the onset of the tauopathies.

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