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In patients with Parkinson’s disease (PD), the molecularly misfolded form of α-synuclein was recently identified in cutaneous autonomic nerve fibers which displayed increased accumulation even in early disease stages. However, the underlying mechanisms of synucleinopathic nerve damage and its implication for brain pathology in later life remain to be elucidated. To date, specific diagnostic tools to evaluate small fiber pathology and to discriminate neurodegenerative proteinopathies are rare. Recently, research has indicated that deposition of α-synuclein in cutaneous nerve fibers quantified via immunohistochemistry in superficial skin biopsies might be a valid marker of PD which could facilitate early diagnosis and monitoring of disease progression. However, lack of standardization of techniques to quantify neural α-synuclein deposition limits their utility in clinical practice. Additional challenges include the identification of potential distinct morphological patterns of intraneural α-synuclein deposition among synucleinopathies to facilitate diagnostic discrimination and determining the degree to which structural damage relates to dysfunction of nerve fibers targeted by α-synuclein. Answering these questions might improve our understanding of the pathophysiological role of small fiber neuropathy in Parkinson’s disease, help identify new treatment targets, and facilitate assessment of response to neuroprotective treatment.

In patients with Parkinson’s disease (PD), the molecularly misfolded form of α-synuclein was recently identified in cutaneous autonomic nerve fibers which displayed increased accumulation even in early disease stages. However, the underlying mechanisms of synucleinopathic nerve damage and its implication for brain pathology in later life remain to be elucidated. To date, specific diagnostic tools to evaluate small fiber pathology and to discriminate neurodegenerative proteinopathies are rare. Recently, research has indicated that deposition of α-synuclein in cutaneous nerve fibers quantified via immunohistochemistry in superficial skin biopsies might be a valid marker of PD which could facilitate early diagnosis and monitoring of disease progression. However, lack of standardization of techniques to quantify neural α-synuclein deposition limits their utility in clinical practice. Additional challenges include the identification of potential distinct morphological patterns of intraneural α-synuclein deposition among synucleinopathies to facilitate diagnostic discrimination and determining the degree to which structural damage relates to dysfunction of nerve fibers targeted by α-synuclein. Answering these questions might improve our understanding of the pathophysiological role of small fiber neuropathy in Parkinson’s disease, help identify new treatment targets, and facilitate assessment of response to neuroprotective treatment.

<p dir="ltr">My research projects focuses on the dual targeting of small molecules to abrogate aberrant α-syn, tau (2N4R), and p-tau (1N4R) aggregation and to reduce the spread of AD and related dementias. Not very many drug discovery programs focus on the specific isoforms of the tau protein. We established two series of compounds: aminoindole compounds connected by a carboxamide and triazine compounds connected by a triazine linker. Using biophysical methods we evaluated the effectiveness of both series of compounds in decreasing the amount of misfolded α-syn and tau protein in order to explore their anti-aggregation potential.</p><p><br></p>

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.

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are defined by neurodegeneration and accumulations of misfolded proteins that spread through the brain in a well characterized manner. In AD these accumulations consist mainly of β-amyloid (Aβ) and tau, while in PD, α-synuclein (α-syn) make up the characteristic lewy pathology.     The general aim of this thesis was to investigate mechanisms associated with neurotoxic peptide activity by Aβ, tau and α-syn in relation to cellular degradation and transfer with a cell-to-cell transfer model system.    We found that intercellular transfer of oligomeric Aβ occurs independently of isoform. However, the amount of transfer correlates with each isoforms ability to resist degradation or cellular clearance. The Aβ1-42 isoform showed particular resistance to clearance, which resulted in higher levels of cell-to-cell transfer of the isoform and lysosomal stress caused by accumulation.    As Aβ accumulations can inhibit the proteasomal degradation we investigated how reduced proteasomal degradation affected neuron-like cells. We found increased levels of phosphorylated tau protein, disturbed microtubule stability and impaired neuritic transport after reduced proteasomal activity. These changes was partly linked to c-Jun and ERK 1/2 kinase activity.    We could also show that α-syn transferred from cell-to-cell in our model system, with a higher degree of transfer for the larger oligomer and fibrillar species. Similar to Aβ, α-syn mainly colocalized with lysosomes, before and after transfer.     Lastly, we have developed our cell-to-cell transfer system into a model suitable for high throughput screening (HTS). The type of cells have been upgraded from SH-SY5Y cells to induced pluripotent stem cells (iPSCs), with a differentiation profile more similar to mature neurons. The next step will be screening a small molecular library for substances with inhibitory effect on cell-to-cell transfer of Aβ peptides.     The importance of the degradative systems in maintaining protein homeostasis and prevent toxic accumulations in general is well known. Our findings shows the importance of these systems for neurodegenerative diseases and also highlight the link between degradation and cell-to-cell transfer. To restore or enhance the degradative systems would be an interesting avenue to treat neurodegenerative diseases. Another way would be to inhibit the transfer of misfolded protein aggregates. By using the HTS model we developed, a candidate substance with good inhibitory effect on transfer can hopefully be found.

La communication entre les différentes organelles se fait par l’intermédiaire du trafic vésiculaire, un processus qui nécessite un remodelage continu des membranes. Les vésicules fortement courbées bourgeonnent d'un compartiment donneur et fusionnent avec un compartiment accepteur. Les protéines impliquées dans le bourgeonnement et fusion des vésicules ont été largement étudiées. Récemment, la découverte de détecteurs de courbure membranaire a révélé que le trafic membranaire pourrait être régulé à un niveau supplémentaire, par la détection de la forme de la membrane. Le premier détecteur de courbure membranaire identifié était le motif ALPS (Amphipathic Lipid Packing Sensor), qui a été trouvé dans un certain nombre de protéines de la voie sécrétoire précoce et l'enveloppe nucléaire. La protéine d’arrimage GMAP-210 localisé au niveau du cis-Golgi, est composée d’une longue superhélice (coiled-coil) et d’un motif ALPS à l'extrême N-terminale. Il a été démontré in vitro, que ce motif se replie et forme une hélice amphipathique capable de se fixer sur des petits liposomes. Toutefois, l'identité des vésicules, reconnues par ce détecteur de courbure dans la cellule, reste inconnue. α-Synucléine est une autre protéine qui se lie préférentiellement à des membranes très courbées. Cette protéine localisée sur les vésicules synaptiques, est impliquée dans la régulation du taux de vésicules au niveau des terminaisons nerveuses pré-synaptiques. Connue pour son rôle central dans le développement de la maladie de Parkinson, α-synucléine contient une région non structurée en solution, mais qui forme une hélice amphipathique au contact de petits liposomes in vitro. Les hélices amphipathiques formées par le motif ALPS et α-synucléine sont très différentes aussi bien sur le plan chimique que sur le plan conformationel. Le motif ALPS possède une face hydrophobe bien développée, mais un coté polair pauvre avec très peu de résidus chargés. α-Synucléine, en revanche, a un côté hydrophobe modéré, et une face polaire zwitterionique riche en résidus chargés. L'objectif principal du projet était de comparer les propriétés de liaison aux membranaires in vivo et in vitro de ces deux hélices amphipathiques de structure opposée. L’expression de ces deux sondes chez la levure, favorise l'accumulation de structures vésiculaires de propriétés différentes. L'extrémité N-terminale de la protéine GMAP-210 contenant son motif ALPS (GMAPN) co-localisé spécifiquement avec des marqueurs de la voie sécrétoire précoce, alors une sonde contenant une portion de l’hélice amphipathique d’α-synucléine co-localise avec des marqueurs endocytiques et post-Golgiens. La mutagenèse du motif ALPS et l'inversion de la séquence de ALPS dans GMAPN confirment que ce détecteur de courbure membranaire se fixe spécifiquement aux vésicules via des interactions directes protéines-lipides, plutôt que les interactions protéines-protéines. Notre analyse a montré que ces détecteurs de courbure mammifères, exprimés dans la levure préservent leur capacité à cibler des vésicules spécifiques, vésicules de la voie sécrétoire précoce pour les motifs ALPS, et vésicules d’endocytose/post-Golgi pour α-synucléine. La composition membranaire de ces vésicules correspond à la composition des liposomes fixés par le motif ALPS et α-synucléine in vitro. Les propriétés biochimiques opposées du motif ALPS et α-synucléine, sont parfaitement adaptés à chacun de ces deux environnements membranaires dans la cellule. Le programme HeliQuest est conçu pour identifier des hélices amphipathiques capables de se lier sur les membranes, y compris les motifs ALPS. Un nouveau module conçu pour identifier les hélices amphipathiques avec des propriétés similaires à α-synucléine a été récemment élaboré. Les recherches effectuées dans les bases de données de protéines de levure et humaines ont permis d’identifier des hélices amphipathiques candidats qui ont des propriétés similaires à α-synucléine, dans de nombreuses protéines. Nous avons préparé un ensemble de sondes, dans lequel ces hélices sont insérées à la fin de la superhélice de GMAPN. Une première étude de leur co-localisation dans les cellules de levure avec un ensemble de marqueurs démontre une localisation spécifique, ce qui suggère que ces hélices peuvent avoir la capacité de cibler des membranes de manière spécifique. D'autres travaux seraient nécessaires pour confirmer ou pas si ces hélices amphipathiques font partie d'une nouvelle classe de détecteurs de courbure ayant les mêmes propriétés que α-synucléine. / Communication between membrane-bound organelles is mediated by vesicular trafficking, a process which requires continual membrane remodeling. Highly curved vesicles bud from a donor compartment through functioning of different coat protein complexes, and fuse with an acceptor compartment thanks to proteins of the membrane fusion machinery. The proteins involved in vesicle budding and fusion have been extensively studied. Recently, the discovery of membrane curvature sensors revealed that membrane trafficking could be regulated at an additional level, through detection of the shape of a membrane. The first membrane curvature sensor identified was the ALPS (Amphipathic Lipid Packing Sensor) motif, which has been found in a number of proteins that function in the early secretory pathway and nuclear envelope. One example is GMAP-210, a long coiled-coil tether localizing to cis-Golgi membranes, which has an ALPS motif at its extreme N-terminus. This ALPS motif was found to fold into an amphipathic helix and bind to small liposomes in vitro. However, the identity of the vesicles that this curvature sensor binds to in cells is not known. Another protein - α-synuclein - has also been reported to bind preferentially to highly curved membranes. This neuronal protein localizes to synaptic vesicles and is involved in maintaining the reserve pool of vesicles in pre-synaptic nerve terminals. α-Synuclein, known for its central role in the development of Parkinson’s disease, contains a region that is unstructured in solution, but forms an amphipathic helix upon binding to small liposomes in vitro. The chemistry and geometry of the amphipathic helices formed by ALPS motifs and α-synuclein are very different. The ALPS motif has a well-developed hydrophobic face but a poor polar side with few charged residues. α-Synuclein, in contrast, has a restrained hydrophobic side, and a zwitterionic polar face rich in charged residues. The main goal of the project was to compare the in vivo and in vitro membrane binding properties of these two amphipathic helices of opposite structure. When expressed in yeast cells, these two curvature sensors promoted the accumulation of vesicular structures possessing different characteristics. The N-terminus of GMAP-210 containing its ALPS motif (GMAPN) co-localized specifically with early secretory pathway markers, whereas a probe containing a portion of the amphipathic membrane-binding helix of α-synuclein co-localized with endocytic and post-Golgi markers. Mutagenesis of the ALPS motif and the inversion of the ALPS sequence in GMAPN support the conclusion that this membrane curvature sensor is targeted to specific vesicles in cells through direct protein-lipid, rather than protein-protein interactions. Our analysis has shown, remarkably, that mammalian curvature sensors expressed in yeast cells preserve their capacity to target specific vesicles, those of the early secretory pathway for ALPS motifs, and endocytic/post-Golgi vesicles for α-synuclein. The membrane composition of these vesicles corresponds to the preferred in vitro liposome binding properties of these membrane curvature sensors. The contrasting chemistries of ALPS motifs and α-synuclein are well adapted to each of these two major membrane environments in the cell. The HeliQuest algorithm is designed to search databases for membrane-binding amphipathic helices, including ALPS motifs. A new module designed to identify amphipathic helices with properties similar to α-synuclein has recently been developed. Searches of both yeast and human protein databases has identified candidate α-synuclein-like amphipathic helices in numerous proteins. We prepared a set of probes, in which these helices are displayed at the end of the GMAPN coiled-coil. An initial study of their co-localization in yeast cells with a set of organelle markers demonstrates specific localization patterns, suggesting that these helices may have specific membrane targeting capacities. Further work will explore the question of whether these amphipathic helices are part of a novel class of α-synuclein-like curvature sensors.

Le gène GALIG produit deux protéines, la Mitogaligine et la Cytogaligine. Son expression conduit à la mort cellulaire par un processus encore mal défini. Dans ce cadre, nous nous sommes intéressés principalement à la Cytogaligine et avons précisé qu’elle est localisée dans le cytoplasme et le noyau mais également dans la mitochondrie au niveau de la membrane interne. Un test de complémentation montre que la Cytogaligine interagit avec la Mitogaligine. Cette interaction pourrait être un facteur important dans la mise en place de la voie d’apoptose médiée par l’expression de GALIG. D‘autres protéines interagissant avec la Cytogaligine ont été identifiées, notamment l’α-Synucléine, protéine centrale dans la maladie de Parkinson (MP), qui est connue pour s’agréger dans les cellules et induire la fragmentation des mitochondries. Dans la mesure où la surexpression de l’α-Synucléine conduit à des défauts de l’autophagie et du système Ubiquitine-protéasome dans la MP, nous avons recherché d’éventuels partenaires de la Cytogaligine associés à ces fonctions. De fait, la Cytogaligine interagit, en autre, avec les protéines de l’autophagie LC3B, GABARAP, p62/SQSTM1, la protéine chaperon Hsc70 ainsi que les protéines du système UPS, HUWE1 et UBQLN4. Ces résultats ouvrent de nouvelles pistes sur les conséquences fonctionnelles de l’expression de la Cytogaligine. Dans une deuxième partie, nous avons réalisé une étude clinique visant à évaluer le profil d’expression des gènes précédemment étudiés dans les cellules mononuclées du sang périphérique de patients atteints de la MP. Si l’expression du gène GALIG ne présente pas de variations entre les patients et les contrôles, une dérégulation de l’expression de différents gènes associés au processus de l’autophagie est mise en évidence. Parmi ces données, celles combinant l’expression du couple de gènes LC3B et GAPDH pourraient représenter un marqueur potentiel de la maladie dans le cadre d’un test diagnostic non invasif. / The GALIG gene produces two proteins, Mitogaligin and Cytogaligin. GALIG expression induces cell death by a still poorly defined process. In this context, we focused mainly on Cytogaligin and specified that it is localized in cytoplasm and nucleus but also in mitochondria close to the inner membrane. A functional complementation test showed that Cytogaligin interacted with Mitogaligin. This interaction could be an important factor in the establishment of the apoptosis pathway mediated by GALIG expression. Other proteins interacting with Cytogaligin have been identified, including α-Synuclein, a central protein in Parkinson's disease (PD), which is known to aggregate in cells and induce fragmentation of mitochondria. Since overexpression of α-synuclein leads to autophagy and Ubiquitin-proteasome system disruptions, we have looked for potential Cytogaligin partners associated with these functions. Cytogaligin interacted with the autophagy proteins LC3B, GABARAP, p62/SQSTM1, the chaperone protein Hsc70 as well as the UPS system proteins HUWE1 and UBQLN4. These results open perspectives regarding the functional consequences of the expression of Cytogaligin. In a second part, we carried out a clinical study aimed at evaluating the expression profile of the previously studied genes in the peripheral blood mononuclear cells of PD patients. If the expression of the GALIG gene does not show variations between PD patients and controls, deregulation of the expression of genes associated with autophagy was highlighted. Among these data, those combining the expression of the two genes LC3B and GAPDH could represent a potential marker of the disease as a non-invasive diagnostic test.

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.

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