With 9.9 million new dementia cases each year, Alzheimer’s and Parkinson’s disease (AD and PD) are the most prevalent form of neurodegenerative disorder (NDG) affecting the aging population. Despite years of pharmaceutical research, no cure is yet available. Most neuropathological aspects of these diseases are extremely complex but the study of the rare genetic cases allowed to model these diseases in animals and uncover key pathophysiological processes. Transgenic Drosophila NDG models have been used for in vivo studies for many years with a range of relevant phenotypes. The cellular and molecular biology of the Central Nervous System, as well as the mechanisms underlying neurodegeneration, are well conserved between Drosophila and Humans (with a 75% of human disease-related genes having homologs in flies). Most NDG studies are performed in the aging flies. However, there are reports of measurable phenotypes for a variety of AD and PD models in juvenile Drosophila melanogaster (larval stage) with an unexploited considerable potential for drug discovery and screening for this outstanding model. Here I sought to develop a new assay for research into NDGs that focus on the earliest phenotypes. During this Ph.D. project a customized crawling assay apparatus was developed, for the assessment of locomotor ability in humanised larval Drosophila (overexpressing human proteins/peptides linked to AD and PD). A locomotor phenotype was identified in larvae overexpressing different variation of Amyloid-β42, tau and α-Synuclein pan neutrally: these animals crawl on agarose surface at a reduced mean speed when compared to controls. The defect was proven partially rescuable by administration of Tacrine and Methylene Blue, renewing the importance of such models for future applications in drug discovery and screening. The motor impairment supports the hypothesis of a neurotoxic effect of the protein/peptide. Thus, to test this further, the overexpression of the human transgenes was restricted to neurons involved in larval olfaction (olfactory impairment is often the earliest symptom in PD and AD) and odour associated learning tasks (both PD and AD are characterized by severe cognitive dysfunction). Interestingly, larvae overexpressing the Amyloid-β42 ARC peptide in the Olfactory Sensory Neurons showed a subtle navigation defect during chemotaxis (in 1-Hexanol odour gradient) that could possibly be addressed to premature neural habituation to the olfactory stimulus. Furthermore, the overexpression of the peptide in the larval Mushroom Bodies influenced the performances of the animals in associative learning tasks. Lastly, using immunohistochemistry and confocal imaging techniques I showed that the gross morphology of neurons is not altered by the targeted overexpression of the Amyloid-β42 ARC. Even though physiological studies are required to characterize the chemosensory/learning defect shown by the Amyloid-β42 ARC larvae, this Ph.D. work further confirms that the effects of the overexpression of the human transgenes are robust and measurable already at larval stage. These findings may also be relevant to the development of new, fast, and cost-effective compound screening procedures, for applications in early stages of the drug discovery process.

Neurodegenerative diseases are associated with extensive physical and mental debilitation, significant costs to the healthcare system, as well as great emotional and financial burden to the patients, their families and care providers. Despite progress in our understanding of the mechanisms behind neurodegenerative diseases, the vast majority are still currently untreatable. Synapses are important pathological targets in a range of disorders, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and lysosomal storage disorders, such as Batten disease. Loss of synaptic connections and impairments in synaptic function are present in the initial stages of neurodegenerative conditions and throughout the course of disease progression. Therefore, synaptoprotective strategies are regarded as a potentially key factor in the development of effective therapies aimed at preventing or halting neurodegeneration. Despite the continuously growing body of research elucidating the molecular mechanisms that modulate synaptic function and vulnerability, the contribution of these pathways to neurodegenerative diseases is far from fully characterized. In addition, there are frequent issues regarding the applicability of the research performed using in vitro and small animal models of disease to develop therapeutic strategies for use in human patients. In the work described in this thesis, we initially validated the involvement of a selection of key synaptic targets, previously identified as regulators of synaptic degeneration in lower animal models, including mice and Drosophila, in a large animal model of neurodegenerative disease: CLN5 Batten sheep. Subsequently, we explored two of these individual synaptic protein targets in more detail (calretinin and α-synuclein), to further investigate their contribution to synaptic function and stability. Calretinin is a poorly characterized protein, primarily known for its calcium buffering capacities and high levels of expression in a subpopulation of interneurons. In this work, we show calretinin is expressed in previously unreported cell populations, including motor axons and synapses from the peripheral nervous system, and that it is enriched in synapses in vitro. Furthermore, we show calretinin responds dynamically to synaptic activity and is directly involved in neurodegenerative pathways, as demonstrated by its ability to influence the course of Wallerian degeneration and apoptotic cell death. α-synuclein plays a central role in the pathophysiology of Parkinson’s disease and contributes to the maintenance of synaptic transmission and mitochondrial function. However, questions still remain about how to effectively manipulate α- synuclein to obtain therapeutic benefits. Therefore, we sought to explore downstream targets of α-synuclein in order to uncover new pathways through which this protein may influence synaptic stability. Using proteomics on mice lacking α-synuclein and in vitro cell systems we identified sideroflexin 3 (sfxn3). We show sfxn3 is localized at the inner mitochondrial membrane and that it functions outside the main canonical pathways of mitochondria energy production. In addition, overexpression of sfxn3 in Drosophila led to a significant loss of synaptic boutons at the level of the neuromuscular junction, suggesting regulated levels of sfxn3 are important for the maintenance of synaptic connections. Altogether, the work developed in this thesis provides novel insights into pathways regulating synaptic stability and function. We not only provide evidence that the molecular targets studied are affected in a large animal model of neurodegenerative disease, and are therefore likely to be relevant to studies in human conditions, but we also uncover two new molecular targets capable of independently regulating synaptic form and function.

Les avancées récentes dans la technique de transgénèse utilisant l’approche AAV ont permis de générer de nouveaux modèles animaux. Depuis quelques années, le développement des modèles de la maladie de Parkinson (MP) a amélioré la compréhension des mécanismes physiopathologiques de ce trouble dégénératif. Cependant, aucun modèle mammifère ne reproduit à ce jour la neurodégénérescence liée à l’âge associée à la pathologie synucléine et la symptomatologie motrice et non motrice. L’objectif de mon travail de thèse fut de développer de nouvelles stratégies de transgénèse chez le rat en utilisant ces vecteurs viraux pour la modélisation de la MP. Le challenge est de parvenir à une infection virale la plus précoce possible afin de transduire un maximum de neurones dopaminergiques. Pour cela, différentes voies d’approche ont été testées pour améliorer le processus de transgénèse : 1) l’injection de vecteurs viraux dans le rete testis de jeunes mâles pour transduire les cellules de la lignée germinale, 2) l’injection dans les embryons pré-implantatoires, 3) l’injection in utero intracérébroventriculaire et enfin 4) l’injection intracardiaque au premier jour de vie des animaux. Parmi elles, les injections in utero et intracardiaques ont permis d’aboutir à une transduction importante d’un grand nombre de structures cérébrales avec un tropisme neuronal. Ces techniques ouvrent la voie à la génération de nouveaux modèles animaux de maladies à composante génétique et à la thérapie génique. / Recent advances in the transgenesis technique using the AAV approach have led to the generation of new animal models. In recent years, the development of models of Parkinson's disease (PD) has improved understanding of the pathophysiological mechanisms of this degenerative pathology. However, no mammalian model recapitulates the required age-dependant parkinsonian degeneration, the α-synuclein inclusion pathology and motor and non-motor symptoms. The objective of my Ph.D work was to develop new transgenesis strategies in rats using these viral vectors for modeling PD. The challenge is to achieve a viral infection as early as possible in order to transduce as many dopaminergic neurons as possible. To this end, different strategies have been tested to improve transgenesis efficacy : i) injection in rete testis in young male to transduce germinal cells, ii) injection into early stage embryos, iii) in utero intracerebroventricular injection and iv) intracardiac injection in one day-old animals. Among them, in utero and intracardiac injections led to neuronal transgene expression in most regions of the brain. These techniques pave the way for the generation of new animal models of genetic diseases and offer unique opportunities for gene therapy.

The pre-synaptic protein α-Synuclein (αS) is often linked to the pathology of Parkinson’s disease (PD), an age-related neurodegenerative disorder. Lewy bodies, the cytopathological hallmarks of PD, are found to be rich in aggregates of misfolded αS protein. Metal dyshomeostasis has also been linked to PD due to the accumulation of iron in the substantia nigra pars compacta, and diminished copper levels reported in this same region. Metal dyshomeostasis in the brain coupled with oxidative stress can enhance the aggregation of αS. Recently, it was confirmed that mammalian αS is universally acetylated at the N-terminus, a common post-translational modification in humans. The consequences of this modification have been understudied, and it is believed to impart a functional role under physiological conditions with respect to membrane-interactions and protein folding. In an attempt to elucidate the pathological mechanism behind PD with respect to the structural dynamics of the protein, our investigations were focused on physiologically prevalent, N-terminally acetylated αS (NAcαS) and its interaction with the most prevalent redox-active metal ions in the brain (iron and copper) under both aerobic and/or anaerobic conditions. The structural features associated with metal-bound NAcαS differed depending on the iron oxidation states, where under aerobic conditions Feᴵᴵ stabilized an oligomer-locked, anti-parallel right-twisted β-sheet conformation that could potentially impart toxicity to neurons. In contrast, Feᴵᴵᴵ promoted a fibrillar structure rich in parallel β-sheets. N-terminal capping also altered the Cuᴵᴵ coordination sphere and had a dramatic effect on protein aggregation. Parallel studies on NAcαS variants with different site mutations near the putative copper binding sites (ex: H50Q and F4W) indicated that preferential binding shifts upon changes in the side chain residues. In depth analysis of the electron structure of Cuᴵᴵ-bound NAcαS using electron paramagnetic resonance spectroscopy (EPR) revealed a coordination sphere of N3O1 that includes the H50 residue in the wild-type protein that shifts to an O4 coordination sphere at the C-terminus upon Cuᴵᴵ binding to the disease-relevant H50Q variant. Immunoblotting analyses revealed that copper-induced redox chemistry promoted O2-activation and the subsequent formation of dityrosine crosslinks, a post-translational modification identified as a biomarker of PD. EPR-detection of tyrosyl radical formation in the presence of Cuᴵ-bound NAcαS further supported this radical coupling mechanism. Intermolecular crosslinks within the fibrillar core of NAcαS as well as intramolecular crosslinks within the C-terminal region underpin the role of metal-dioxygen chemistry in PD-related pathology. The unique structural features resulting from iron vs copper coordination to NAcαS inspired studies directed at the synergistic effect of each individual metal species as revealed by photo-initiated crosslinking of NAcαS. C-terminal intramolecular tyrosine interactions were mainly impacted by the presence of both metals, which each have binding sites around the same region. These findings emphasize that protein dynamics, metal binding site conformational changes, as well as aggregation pathways can deviate drastically upon N-terminal acetylation of αS and that protein-metal interactions may play a vital role in PD etiology.

Partie 1 : De plus en plus de molécules fluorées sont utilisées dans bon nombre de domaines variés, allant des matériaux aux sciences de la vie. Ces dernières années, un intérêt croissant a émergé avec l'association du groupement CF3 avec un hétéroatome, comme OCF3 ou SCF3. Le groupement SCF3 est très intéressant à cause de son paramètre d'hydrophobie (π=1.44). Par conséquent, les composés portant ce groupement sont des cibles importantes pour de nombreuses applications, en particulier en chimie médicinale. Cependant, la majorité des précédentes méthodes décrites dans la littérature utilisent des réactifs toxiques dans des conditions drastiques. Les trifluorométhanesulfénamides (1ère et 2nde génération) ont démontré leur potentiel dans la trifluorométhylthiolation électrophile. En raison de leur réactivité intéressante, ces deux générations de réactifs stables sont maintenant dans la boîte à outils de la chimie organique pour la trifluorométhylthiolation de molécules. Partie 2 : Des aggrégats d'α-synucléine sont une caractéristique neuropathologique de nombreuses maladies neurodégénératives, notamment la maladie de Parkinson (MP) et la démence à corps de Lewy (DLB), collectivement appelés synucléinopathies. L'imagerie TEP pourrait révéler la quantité et la distribution des agrégats d'α-synucléine dans le cerveau et serait plus avantageuse à utiliser pour le diagnostic spécifique de synucléinopathies présymptomatiques à différents stades de lamaladie. Nous avons concentré nos efforts sur des dérivés de benzimidazole comme composés de petites tailles, plans et π-délocalisés pour concevoir des traceurs radioactifs des agrégats de la synucléine. Ainsi des structures assemblant des benzimidazoles, un espaceur rigide (alcyne et triazole) et enfin une autre partie aromatique ont été envisagées. Le radiomarquage pourra être effectué par une substitution nucléophile avec K18F au cours de la dernière étape. Avec cette stratégie convergente, nous pourrions avoir accès à une grande série de molécules à évaluer / Part 1 : More and more applications for fluorinated molecules are being found in various fields, from materials to life sciences. In recent years, a growing interest has emerged in the association of the trifluoromethyl group with heteroatoms such as CF3O or CF3S. The CF3S moiety is of particular interest, because of its high hydrophobicity parameter (π=1.44). Consequently compounds bearing this group are important targets for various applications, in particular in medicinal chemistry and agrochemistry. However, the majority of previous methods described in the literature use toxic reagents under harsh conditions. Trifluoromethanesulfenamides (1st and 2nd generation) have demonstrated their potential in the electrophilic trifluoromethylthiolations. Because of their interesting reactivity, these two generations of shelf-stable reagents are now in the toolbox of organic chemists for the trifluoromethylthiolation of molecules, providing a convenient method to pursue less toxic pathways. Part 2 : α-synuclein aggregation is a neuropathological hallmark of many neurodegenerative diseases including Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), collectively termed synucleinopathies. PET imaging can reflect the amount and distribution of alpha-synuclein aggregates in the brain and would be advantageous to use for specific diagnosis of synucleinopathies in presymptomatic stages of disease. We focused our interest onto benzimidazole derivatives as small, planar and -delocalized compounds to design radiotracers of synuclein aggregates. Compounds based on the association of benzimidazole moiety, rigid linker (alkyne and triazole) and another aromatic part have been designed. The radiolabeling could be performed by nucleophilic substitution with K18F during the last step. With this convergent strategy, we could have acces to a large series of molecules to be evaluated

The field of proteomics studies the structure and function of proteins in a large scale and high throughput manner. My work in the field of proteomics focuses on identifying interactions between proteins and discovering novel interactions. The identification of these interactions provides new information on metabolic and disease pathways and the working proteome of a cell. Cells are lysed and purified using antibody based affinity purification followed by digestion and identification using an HPLC coupled to a mass spectrometer. In my studies, I looked at the interaction networks of several AD related genes (Apolipoprotein E, Clusterin variant 1 and 2, Low-density lipoprotein receptor, Phosphatidylinositol binding clathrin assembly protein, Alpha-synuclein and Platelet-activating factor receptor) and an endosomal recycling pathway involved in cholesterol metabolism (Eps15 homology domain 1,2 and 4, Proprotein convertase subtilisin/kexin type 9 and Low-density lipoprotein receptor). Several novel and existing interactors were identified and these interactions were validated using co-immunopurification, which could be the basis for future research.

Synucleins represent a conserved family of small proteins that include α-, β-, and γ- isoforms, which are highly expressed in neurons of the vertebrate nervous system. The normal function of these proteins is not well understood. However, in humans α- synuclein dysfunction is causatively linked to Parkinson’s Disease (PD), where it abnormally accumulates in neuronal cell bodies as protein aggregates that are associated with neuronal death. Although the associations between synuclein accumulation and cellular death are established in PD, the extent to which this occurs in other contexts, such as neuronal injury, is unknown. Furthermore, the effects of synuclein aggregation on the function of synapses, where synuclein is normally localized, are not well understood. To address these questions I took advantage of the experimentally accessible nervous system of the sea lamprey (Petromyzon marinus). I used molecular cloning and phylogenetic analyses to characterize three lamprey synuclein orthologues, one of which is highly expressed within a class of neurons called the giant reticulospinal (RS) neurons. Spinal cord injury induces the accumulation of synuclein protein only within a population of poor surviving RS neurons, and this accumulation is correlated with cellular death. Thus, similar to PD, the abundance of synuclein protein is associated with neuronal toxicity. In a related project, I demonstrated that elevating synuclein levels at synapses, such as occurs in PD, is deleterious to synaptic function through an inhibition of synaptic vesicle (SV) recycling. By injecting excess synuclein protein directly into the axons of giant RS neurons, and analyzing the ultrastructural morphology of synapses, I have shown that clathrin-mediated synaptic vesicle endocytosis was greatly inhibited. The conserved N-terminal domain was sufficient to inhibit vesicle recycling, and injecting synuclein mutants with disrupted N-terminal α-helices caused reduced defects in SV recycling. Therefore the α-helical structure of the N-terminus is necessary to inhibit SV recycling at early stages of clathrin-mediated endocytosis. Binding interactions with clathrin-mediated endocytosis components, such as the phosphoinositide lipid PI(4)P support this hypothesis. These studies provide a better understanding of the mechanisms by which synuclein dysfunction leads to neuronal death after injury and synaptic dysfunction in PD and other synuclein-associated diseases. / text

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Septinas são proteínas ligantes a GTP encontradas desde fungos até metazoários. A primeira função identificada para septinas foi o seu papel central na organização e dinâmica do septo de divisão de leveduras. Uma das características marcantes é que septinas se organizam em heterofilamentos de 7 a 9 nm de espessura que foram purificados de diversos organismos tais como Saccharomyces cerevisiae, Drosophila e cérebro de camundongos. Hoje se sabe que septinas não estão envolvidas apenas nos processos de divisão celular, mas em uma variedade de processos como tráfico de vesículas, exocitose, interação com proteínas do citoesqueleto e com a membrana plasmática, o que resulta em alterações da morfologia celular. Neste trabalho foram desenvolvidos estudos da septina 4 humana (SEPT4) nos quais foi realizado a expressão e purificação da SEPT4 pelo uso do sistema de expressão heteróloga em E. coli e em células de insetos (Sf-9) via baculovírus. A tentativa de expressão usando o vetor pETTEV em E.coli não obteve sucesso, pois a proteína não foi expressa na forma solúvel. A construção do baculovírus recombinante AcSept4 e expressão da SEPT4 nas células de insetos foi realizada com êxito, mas o processo de purificação não foi satisfatório. Com o intuito de obter informações sobre possíveis proteínas que interagem com a SEPT4 e conseqüentemente sobre as funções desempenhadas por ela na célula, a SEPT4 foi utilizada como isca para ensaios de interação proteína-proteína pela técnica de duplo híbrido. Para isso, o gene da SEPT4 foi clonado fusionado ao domínio de ligação ao DNA Lex-A. A realização do ensaio de duplo híbrido com a proteína completa não foi possível, pois a mesma provocou a auto ativação do sistema, por isso uma nova construção foi realizada com a região GTPase e C-terminal SEPT4GC (124-478) como isca. Dentre as interações identificadas, foram encontradas apenas septinas do grupo II (SEPT6, SEPT8, SEPT10 e SEPT11) e quatro novas interações, que ainda precisam ser confirmadas. Por outro lado, uma interação já descrita na literatura envolve a proteína &#945-sinucleína, que é uma proteína abundantemente expressa no cérebro e associada à doença de Parkinson. O foco do estudo dessa interação foi realizar ensaios com os diferentes domínios da SEPT4 para comprovar uma interação direta e com isso tentar mapear o sítio de interação com a &#945-sinucleína. Os resultados obtidos pela ressonância plasmônica de superfície (SPR) indicam que o domínio C-terminal participa da interação com baixa afinidade (K,D=390 &#181M) e sugerem que o domínio GTPase também pode estar envolvido. Já os dados obtidos com os experimentos de RMN e anisotropia de fluorescência mostram indícios que a interação é dependente da conformação da &#945-sinucleína por que a interação aconteceria com maior afinidade quando a &#945-sinucleína está na presença de SDS. / Septins are a family of GTP binding proteins found in a great diversity of organisms. These proteins have been identified as having a central role in septum organization during yeast division. Septins are organized into heterofilaments which are 7 to 9 nm wide and these have been purified from yeast, Drosophila and mice brain. Septins are not only required for cell division, but seem to play a role also in vesicle trafficking and in the formation of diffusion barriers within cells, since they interact with cytoskeleton proteins and the plasma membrane causing changes in cell morphology. In the present work, the aim was investigate human Septin 4 (SEPT4), a septin highly expressed in the brain. One objective of this work was to find a suitable expression system and purification method for SEPT4. The protein was expressed in both E.coli and insect cells (Sf-9). Expression in E. coli with the vector pETTEV was unsuccessful because the protein was insoluble. Expression in insect cells using the recombinant baculovirus AcSept4, was obtained successfully, but the purification was difficult. Important information concerning SEPT4 function might be acquired, if interactions partners involved in cellular process were identified. With this goal in mind, a yeast two hybrid assays were performed. The sept4 gene was fused to the Lex-A DNA binding domain and used as bait in the yeast two hybrid essays. However, full length SEPT4 showed autonomous activation of reporter genes. A second construct was prepared including only GTPase domain and the carboxy terminus domain, (residues 124 to 478) and the screen of interactions were carried out only with SEPT4GC. All of the group II septins (SEPT6, SEPT8, SEPT10 and SEPT11) were identified together with four new interactions. The latter still need be confirmed. In addition, another interaction already described in the literature is between SEPT4 and &#945-synuclein, which is a protein highly expressed in brain and related to Parkinson\'s disease. Different spectroscopic methods and SPR were used to identify which domain of SEPT4 interacts directly with &#945-synuclein and in which region. The surface plasmon resonance (SPR) results indicate that the carboxy terminus participates in the interaction with low affinity (KD = 390 &#181M) and suggests that the GTPase domain may also be involved. The results obtained by fluorescence anisotropy and NMR studies provide evidence that the interaction is dependent on the &#945-synuclein conformation, because the affinity of SEPT4 and &#945-synuclein seemed to be higher in the presence of SDS.