abstract: Alzheimer’s Disease (AD) and Frontotemporal Dementia (FTD) are the leading causes of early onset dementia. There are currently no ways to slow down progression, to prevent or cure AD and FTD. Both AD and FTD share a lot of the symptoms and pathology. Initial symptoms such as confusion, memory loss, mood swings and behavioral changes are common in both these dementia subtypes. Neurofibrillary tau tangles and intraneuronal aggregates of TAR DNA Binding Protein 43 (TDP-43) are also observed in both AD and FTD. Hence, FTD cases are often misdiagnosed as AD due to a lack of accurate diagnostics. Prior to the formation of tau tangles and TDP-43 aggregates, tau and TDP-43 exist as intermediate protein variants which correlate with cognitive decline and progression of these neurodegenerative diseases. Effective diagnostic and therapeutic agents would selectively recognize these toxic, disease-specific variants. Antibodies or antibody fragments such as single chain antibody variable domain fragments (scFvs), with their diverse binding capabilities, can aid in developing reagents that can selectively bind these protein variants. A combination of phage display library and Atomic Force Microscopy (AFM)-based panning was employed to identify antibody fragments against immunoprecipitated tau and immunoprecipitated TDP-43 from human postmortem AD and FTD brain tissue respectively. Five anti-TDP scFvs and five anti-tau scFvs were selected for characterization by Enzyme Linked Immunosorbent Assays (ELISAs) and Immunohistochemistry (IHC). The panel of scFvs, together, were able to identify distinct protein variants present in AD but not in FTD, and vice versa. Generating protein variant profiles for individuals, using the panel of scFvs, aids in developing targeted diagnostic and therapeutic plans, gearing towards personalized medicine. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2018

The discovery that the (GGGCC)n>30 repeat expansion in the non-coding region of C9orf72 (C9) is the most prevalent mutation in familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) has led to a massive effort to discover the mechanism by which the expansion causes ALS. One effect of the repeat expansion is the reduction in C9 mRNA and protein levels. Therefore, we chose to concentrate on studying the function of C9 and how its reduction contributes to ALS progression. First, we show that C9 short (C9S) and long (C9L) isoforms have different cellular localization, and that C9 knockdown in several cell lines, results in significant changes in gene expression. Specifically, we show that differentially expressed genes were enriched in immune system activation pathway. Additionally, we observed gene expression changes in important glutamate-glutamine cycling genes and show that C9 knockdown results in accumulation of intracellular glutamate. We also show that C9S isoform may regulate gene expression as it interacts with chromatin, and can be ChIPed on the promoter of endothelin-1 (EDN1). C9 knockdown also leads to significant morphological changes that include increased cell sizes and nucleus, massive vacuolization, and results in reduced cell viability. Investigation into the vacuoles revealed that they originate from hyperactivation of macropinocytosis. The hyperactivation of macropinocytosis results in a caspase-independent cell death known as methuosis. We show that vacuolization is a p53-dependent process, and we present evidence that p53 mediates vacuolization via the repression of the mevalonate pathway. Furthermore, we found that inhibition of isoprenylation, a process depending on mevalonate pathway products (geranylgeranyl pyrophosphate and farnesyl pyrophosphate), participates in the induction of vacuoles. Importantly, we also reveal that C9 knockdown leads to mitochondrial dysfunction, increased ROS, increased DNA-damage and p53 activation, all of which are seen in C9 ALS patient samples or in C9 patient derived motor neurons (C9 iMNs). Our results reveal several previously unknown pathways which are affected by C9 knockdown , which have potential therapeutic implications, that include endothelin signaling and macropinocytosis, both of which can be blocked with FDA approved drugs.

Patients with Amyotrophic lateral sclerosis (ALS) often experience cognitive impairment that accompanies degeneration of the motor system. A valuable tool for assessing cognitive control over behaviour is the antisaccade task which requires: 1) inhibition of the automatic response to look towards an eccentric visual stimulus (prosaccade) to instead 2) redirect gaze in the opposite direction of the stimulus (antisaccade). Psychometric tests were used to quantify the degree of impairment, while eye tracking, functional magnetic resonance imaging (fMRI) and structural MRI were combined to identify the neural correlates of cognitive impairment in ALS. We predict ALS patients will have executive dysfunction and grey matter loss in executive and oculomotor control areas that will affect antisaccade performance and will alter the corresponding brain activation. ALS patients and age-matched controls participated in a rapid-event-related fMRI design with interleaved pro- and antisaccade trials. Catch trials (no stimulus presented after instructional cue to prepare pro- or antisaccade) allowed us to discern the preparatory period from the execution period. ALS patients were biased towards automatic saccade responses, and had greater difficulty with antisaccades relative to controls in terms of correct and timely responses. We found that worsened antisaccade performance in ALS correlated with the degree of cognitive impairment. Generally, we found trends of increased brain activation during the preparatory period of antisaccades in ALS patients compared to controls in most oculomotor areas; meanwhile few differences were seen during execution. Structural analyses revealed ALS patients had decreased grey matter thickness in frontotemporal and oculomotor regions such as the frontal and supplementary eye fields (FEF, SEF) and the dorsolateral prefrontal cortex (DLPFC). These findings suggest that loss of structural integrity and executive dysfunction may elicit compensation mechanisms to improve functional and behavioural performance. Despite this compensation, ALS patients still performed worse on antisaccades than controls. Further investigation to expand the current data set should improve our ability to assuredly identify the neural correlates of cognitive decline in ALS, and may provide a model system to use for critical evaluation of future therapies and interventions for ALS. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2011-09-22 14:20:39.704

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.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. Genetic testing for this pathogenic mutation is challenging due to its GC rich, repetitive nature. I developed PCR based assays to detect the presence of the pathogenic variant, which were used in screening an archival cohort of Scottish ALS patients, and have also been implemented within a diagnostic setting. These PCR assays allow amplification of larger repeat expansions than have previously been reported, and can determine whether a C9orf72 expansion of greater than 100 repeats is present or not. It is not well understood how the repeat expansion leads to disease, but several potential mechanisms have been hypothesised, including reduced expression, RNA toxicity and protein toxicity via dipeptide repeat proteins produced through repeat associated non-AUG translation. Motor neurons are an understandably well studied target in amyotrophic lateral sclerosis, however the role of glia, particularly oligodendrocytes, in the pathogenesis of the disease has recently been highlighted from studies on rodent models and post mortem tissue. To investigate the effect of the C9orf72 repeat expansion on oligodendrocytes, we have applied a differentiation protocol to hiPSCs with the expansion and controls, including an isogenic control which has been generated in the lab. There was no difference in the production of neuronal and glial cell types between these cell lines. I went on to look for evidence of the main proposed pathological mechanisms of C9orf72 repeat expansions: loss of function or gain of function through either RNA or protein toxicity. hiPSC derived oligodendrocytes from both carrier and control showed low expression of C9orf72 mRNA, and there was no difference due to the presence of a repeat expansion. Carrier hiPSC derived oligodendrocytes displayed sense RNA foci, which did not appear to have an effect on cellular morphology. The detection of dipeptide repeat proteins proved challenging, and the results were inconclusive as to their presence in hiPSC derived oligodendrocytes. I went on to show there was no evidence of mislocalisation of TDP-43 in C9orf72 carrier oligodendrocytes. Finally, the study showed similar levels of cell death in basal conditions in carrier and control cells, and no clear difference in the response to endoplasmic reticulum stress. Further research will be required to elucidate the role of oligodendrocytes in C9orf72 related amyotrophic lateral sclerosis.

La catégorisation est un ensemble d’opérations mentales qui permettent de classer les objets et les évènements. C’est un processus crucial pour de nombreuses situations, telles que la survie dans le monde animal, l’apprentissage chez l’enfant, ou encore le raisonnement abstrait et la résolution de problèmes. Les patients ayant des lésions du cortex préfrontal présentent des difficultés pour les tâches de catégorisation, et l’existence de ces difficultés est corrélée au handicap fonctionnel de ces patients. Dans une première partie, nous avons mis au point une tâche de catégorisation adaptée pour l’utilisation chez le patient, intitulée SimiCat. A l’aide de cette tâche, nous avons précisé les difficultés de catégorisation des patients et montré que les erreurs de type différenciations sont spécifiques des patients frontaux. La tâche SimiCat présente une très bonne valeur diagnostique pour distinguer les patients ayant une démence fronto-temporale de ceux ayant une maladie d’Alzheimer. Dans une deuxième partie, nous avons utilisé l’IRM fonctionnelle pour préciser les bases cérébrales de deux processus clés pour la catégorisation : l’abstraction et la détection de similitudes. Nous avons montré que l’abstraction repose sur le cortex préfrontal dorsolatéral gauche, alors que la détection de similitudes repose sur le cortex préfrontal ventrolatéral bilatéral. A l’aide de la morphométrie basée sur le voxel, nous avons montré que la variabilité des performances de catégorisation des sujets sains était corrélée au volume de la portion antérieure du gyrus temporal moyen et inférieur droit, avec un gradient postéro-antérieur selon le niveau d’abstraction de la catégorisation. Dans une troisième partie, nous avons mis au point une tâche de double amorçage sémantique que nous utiliserons pour étudier les processus de catégorisation automatique chez les patients ayant des lésions frontales et temporales. A partir de ces résultats, nous proposons un modèle d’organisation cérébrale pour la catégorisation, reposant sur les régions temporales antérieures, le cortex préfrontal ventrolatéral bilatéral et dorsolatéral gauche. Nos résultats permettent également de mieux comprendre les déficits de catégorisation des patients, ce qui permettra d’adapter leur prise en charge diagnostique et thérapeutique. / Categorization is a set of mental processes that allow classifying objects and events. It is crucial in various contexts such as survival in animals, concept learning in children, abstract reasoning and problem solving. Patients with brain lesions involving the prefrontal cortex are impaired in categorization tasks. Categorization impairment correlates with functional autonomy in dementia. In the first part, we have developed a task, named SimiCat that we designed in order to assess categorization abilities in patients. With the help of this task, we showed that differentiation errors are specific of frontal patients. The SimiCat task has a good diagnostic value to distinguish behavior variant Frontotemporal dementia from Alzheimer disease.In the second part, we assessed the brain correlates of categorization. With functional MRI, we showed that abstraction involves the left dorsolateral prefrontal cortex, while similarity detection involves bilateral ventrolateral prefrontal cortex. With voxel-based morphometry we showed that variability in categorization performances correlates with the volume of the right anterior temporal lobe, with a caudo-rostral gradient according to abstraction. In the third part, we developed a double priming task that we will use to assess automatic categorization processes in patients with temporal and frontal lesions. Based on these results, we propose a model of brain organization for categorization. This model involves both anterior temporal lobes, as well as bilateral ventrolateral and left dorsolateral prefrontal cortices. Our results also contribute to a better understanding and management of patients suffering from categorization deficits.

No description available.

La dégénérescence frontotemporale est une pathologie rare, plaçant au premier plan les désordres comportementaux. Après analyse des troubles psycho-comportementaux et de leurs méthodes actuelles d’évaluation, nous avons élaboré et validé un nouvel outil, appelé DAPHNE (pour Desinhibition-Apathie-Persévérations-Hyeroralité-Négligence-Empathie) spécialement conçu pour dépister et quantifier la sévérité et la progression des troubles du comportement dans la variante frontale de dégénérescence frontotemporale (vfDFT), en pratique clinique courante. Cette validation a été effectuée de manière prospective, auprès d’une population de patients présentant une vfDFT suivis pendant 2 ans et également d’une population contrôle afin d’établir sa spécificité. DAPHNE, adaptée des critères révisés de vfDFT, présente d’excellentes caractéristiques psychométriques. Explorant six domaines et dix symptômes comportementaux, avec un système de cotation innovant en 5 points, elle permet à la fois le screening mais également une appréciation quantitative et une aide au diagnostic. Parallèlement à cela, nous avons étudié les liens entre les différents troubles psycho-comportementaux, les fonctions cognitives proprement dites et la cognition sociale chez les patients vfDFT. Plus particulièrement, nous nous sommes intéressés à l’évaluation des troubles émotionnels des patients DFT. À l’aide d’une tâche originale TABEAU, portant sur l’étude de la sensibilité esthétique des patients DFT, nous avons observé des liens entre émotion et troubles du comportement (trouble de l’identification de certaines émotions et émoussement affectif etc.). Si le jugement esthétique est préservé en apparence, le patient est en difficulté sur le plan des processus cognitifs du fait de difficultés d’abstraction et en difficulté sur le plan des processus émotionnels. Ce travail montre quel spectateur est le patient DFT vis à vis de l’art et vient compléter les données de la littérature qui portaient jusque-là sur l’expression artistique. Ce type d’approche cognitive permet donc d’avancer dans la compréhension des interactions entre émotions et troubles du comportement, qui font toute la singularité de cette maladie. Pris dans leur ensemble, nos résultats soulignent l’intérêt majeur de l’étude fine du comportement en pratique quotidienne. À l’ère des biomarqueurs, la clinique garde une place de choix pour le diagnostic et la compréhension des maladies neurodégénératives. / Frontotemporal degeneration is a rare disease with early decline in social behavior and personal conduct. We have first presented behavioral disorders and their current evaluation methods. We developed and validated then a new tool, named DAPHNE (Disinhibition-Apathy-Perseverations-Hyerorality-Neglect-Empathy) specially designed to detect and quantify the severity and the progression of behavioral disorders in the behavioral variant of frontotemporal dementia (bvFTD), in clinical practice. This validation was performed prospectively, in patients with bvFTD with a follow-up of 2 years and also in AD, progressive supranuclear palsy and bipolar patients with cognitive disorders, in order to establish the specificity of this scale. DAPHNE, adapted from revised criteria of bvFTD, has excellent psychometric characteristics. Exploring six areas and ten behavioral symptoms, with an innovative scoring system in 5 points, it enables both the screening but also a quantitative assessment and a diagnostic support.As a second part of our works, we studied the links between the various psycho-behavioral disorders, cognitive functions and social cognition in bvFTD patients. Specifically, we focused on emotion assessment of FTD patients. Using an original task entitled TABEAU, based on the study of aesthetic sensibility of FTD patients, we observed the links between emotions and behavior disorders (such as trouble identifying certain emotions or emotional blunting). The aesthetic judgment seemed preserved. But the patient is in trouble in terms of cognitive processes due to abstraction problems and difficulties with emotional processes. This work shows what type of viewer is the FTD patient in front of a piece of art. It adds to the literature data that were previously based only on artistic expression. This cognitive approach improves the understanding of interactions between emotions and behavior disorders which make the singularity of this disease.Taken together, our results highlight the major interest of the detailed assessment of behavior in daily practice. In biomarkers’ era, clinical practice remains central in diagnosing and understanding of neurodegenerative diseases.

Résumé : La répétition de l’hexanucléotide GGGGCC est le facteur génétique le plus présent chez les patients atteints de démence fronto-temporale (DFT) et de sclérose latérale amyotrophique (SLA). Elle apparait au niveau du cadre de lecture ouvert 72 du chromosome 9 (C9orf72). D’une part, la DFT se caractérise par des troubles de la personnalité, du langage et du comportement causés principalement par une dégénérescence de neurones corticaux, thalamiques, hippocampiques et cérébelleux. D’autre part, les patients SLA sont atteints de paralysie des muscles squelettiques causée par la mort de motoneurones au niveau de la moelle épinière et du système moteur central. Ces deux pathologies partagent de nombreuses caractéristiques cliniques et génétiques, suggérant qu’il existe des mécanismes communs entre elles.D’abondantes inclusions cytoplasmiques de protéines issues de la traduction des ARN d’hexanucléotides ont été retrouvées dans le cerveau de patients décédés de SLA et de DFT. On appelle ces polypeptides : Protéines à Répétitions de Dipeptides (PRD), car ils ne sont composés que d’une suite de deux résidus : glycine-alanine (GA), glycine-proline (GP), glycine-arginine (GR), proline-arginine (PR) et proline-alanine (PA). La toxicité de ces PRD a été mise en évidence dans des modèles cellulaires, chez la drosophile mais aussi la souris. Cependant, peu d’études se sont concentrées sur la caractérisation biochimique de ces PRD.Le travail de cette thèse a consisté en la production et la caractérisation in vitro de PRD. J’ai réalisé la caractérisation biochimique de ces protéines et étudié leur capacité à s’auto-assembler au sein de l’équipe « Repliement des protéines in vitro et maladies conformationnelles » dirigée par Ronald Melki. Les PRD recombinants poly-GA, poly-PA et poly-GP ont été produits. Nous avons déterminé les conditions in vitro dans lesquelles ces protéines s’assemblaient. La morphologie des agrégats a été étudiée par microscopie électronique, tandis que la structure des protéines a été déterminée par spectromètre de dichroïsme circulaire pour la forme monomérique et par spectroscopie à infrarouge pour les assemblages.La toxicité, la propagation et la capacité de ces PRD à être internalisé sont testées dans des modèles cellulaires en collaboration avec l’équipe du Pr. Mimoun Azzouz de l’Université de Sheffield. / Abstract : The most common genetic factor between familial Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) is the repetition of (GGGGCC)n in the open reading frame 72 of chromosome 9 (C9orf72). FTD is characterized by personality, behavior and language disorders due to neuronal degeneration in the cortex, thalamus, hippocampus and cerebellum. ALS patients, on the other hand, exhibit motor symptoms primarily consisting of paralysis of the skeletal muscles caused by motoneuronal loss in the spinal cord and the central motor system. These pathologies have significant genetic and clinic overlaps suggesting common mechanisms.Large amounts of cytoplasmic inclusions of the Dipeptide Repeat Proteins (DRP), translated from C9orf72 RNA, were found in brains of patients who died from ALS or FTD.These proteins are constituted of repetitions of Dipeptides : glycine-alanine (poly-GA), glycine-proline (poly-GP), glycine-arginine (poly-GR), proline-arginine (poly-PR) and proline-alanine (poly-PA). The toxicity of the aggregated DRP has been shown in various cellular models as well as in drosophila and mice. Nevertheless, few studies have described the biochemistry of the DRP. The aim of my thesis is the in vitro caracterization of the DRP. I have characterized the biochemistry of these proteins and in particular their self-assembly abilities in Ronald Melki’s team « Proteins folding and conformational diseases ».Recombinant DRP of different lengths and nature have been produced to determined the in vitro conditions in which they assemble. The morphology of the related aggregates has been studied by transmission electron microscopy. Structure informations of DPR were obtained with Circular Dichroïsm spectrometry for monomers and Infrared spectroscopy for assemblies. Lastly, the toxicity and internalization abilities of these proteins and their assemblies are tested on culture cells in collaboration with Pr. Mimoun Azzouz of the University of Sheffield.