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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.

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.

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Les protéines alpha-synucléine (αsyn) et « Leucine-Rich Repeat Kinase 2 » (LRRK2), jouent toutes deux un rôle majeur dans la physiopathologie des formes sporadiques et génétiques de la maladie de Parkinson (MP). En particulier, la mutation G2019S de LRRK2, située dans son domaine kinase, est la cause la plus fréquente de formes génétiques de la MP. Il a été suggéré que l’αsyn et LRRK2 agiraient de concert pour induire la neurodégénérescence des neurones dopaminergiques de la substance noire pars compacta (SNpc) dans cette maladie. Dans notre laboratoire, il a été montré qu’en effet LRRK2 G2019S pouvait potentialiser la mort des neurones dopaminergiques induite par l’αsyn dans la SNpc de rats, confirmant l’existence d’une interaction fonctionnelle entre les deux protéines. De plus, il est connu depuis plusieurs années que les dysfonctionnements mitochondriaux joueraient un rôle central dans la MP. De nombreuses études ont montré que les deux protéines individuellement pouvaient entraîner des dysfonctionnements de cet organite. Notre hypothèse est donc que l’interaction fonctionnelle entre l’αsyn et LRRK2 pourrait passer par une action commune sur la mitochondrie. Nous avons ainsi pu montrer in vitro, dans des cultures primaires de neurones de rat surexprimant l’αsyn et LRRK2, que LRRK2 G2019S, mais pas sa forme sauvage (WT) ni sa forme sans activité kinase (DK, Dead Kinase) augmentait significativement le nombre de neurones présentant un marquage pathologique de l’αsyn (phospho-S129), sans induire de mort cellulaire. Au niveau cellulaire et moléculaire, une diminution significative du taux de production d’ATP mitochondrial a été mise en évidence dans les cellules co-exprimant LRRK2 (WT, G2019S, et encore plus DK) avec l’αsyn par rapport à celles exprimant l’αsyn seule, ceci sans différence dans la quantité totale d’ATP. Les mitochondries des neurones co-exprimant LRRK2 et l’αsyn parcouraient également de plus longues distances le long des neurites que celles des neurones exprimant uniquement l’αsyn. Pour résumer, dans ce modèle in vitro, LRRK2 augmente donc l’accumulation somatique d’une forme pathologique de l’αsyn, d’une manière dépendante de son activité kinase. Dans ces conditions, les mitochondries sont capables de maintenir leur homéostasie, notamment en adaptant leur production d’ATP. Cela semble indiquer l’existence d’un stress mitochondrial modéré, induit par la co-expression de l’αsyn et de LRRK2. / The proteins alpha-synuclein (αsyn) and Leucine-Rich Repeat Kinase 2 (LRRK2) both play major roles in the physiopathology of sporadic and genetic forms of Parkinson’s Disease (PD). In particular, the G2019S mutation of LRRK2, located in its kinase domain, is the most prevalent cause of genetic forms of PD. It has been suggested that αsyn and LRRK2 could act together to induce the selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) in the pathogenesis of this disease. In our laboratory, it has been shown that G2019S LRRK2 could increase the dopaminergic cell loss induced by αsyn in the SNpc of rats, confirming the existence of a functional interaction between the two proteins. Moreover, it has been known for years that mitochondrial dysfunction played a major role in PD. Many studies showed that both LRRK2 and αsyn induced mitochondrial dysfunction. Therefore, we hypothesized that the functional interaction between αsyn and LRRK2 could take place through a common effect on mitochondria. We showed in vitro, in primary rat neurons, that G2019S LRRK2, but not the wild type (WT) form nor the dead kinase mutant (DK), significantly increased the number of neurons expressing a pathological form of αsyn (phospho-S129). This was not associated with any cell loss. At the cellular and molecular levels, there was a significant decrease in the mitochondrial ATP production rate in cells co-expressing LRRK2 (WT, G2019S and even more pronounced with DK) with αsyn, without any change in total ATP levels. The mean distance travelled by mitochondria along neurites was higher in neurons co-expressing αsyn and LRRK2 than in neurons only expressing αsyn. To summarize, in this in vitro model LRRK2 increases the somatic accumulation of a pathologic form of αsyn, in a kinase-dependent manner. In these conditions, mitochondria are able to maintain their homeostasis, in particular by adapting their ATP production rate. This seems to indicate a moderate mitochondrial stress induced by the co-expression of αsyn and LRRK2.

Tetrabromobisphenol-A (TBBPA) is the most widely used flame retardant. Flame retardants are sprayed on furniture, mattress beds, children’s pajamas, car seats, upholstery, carpets, and rugs in the United States. Chemical immune reactivity may play a role in the epidemic of autoimmune disease. The goal of this research is to investigate whether any correlation exists between immunological reactivity to TBBPA, a key chemical used in most flame retardants, and neurological autoimmune target sites that are associated with neurological autoimmune diseases with a diverse and specific list of antibodies that include myelin basic protein, myelin-associated glycoprotein, alpha-synuclein, aquaporin receptors, and S100B antibodies with human serum samples. The outcomes of this research can be used to support the development of safety regulations and for identifying potential health concerns for current mandatory flame-retardant legislation. Additionally, this research may support the decisions made in respect of those suffering from neurological autoimmune diseases, as to whether removing flame retardant chemicals is a factor for consideration.

The pesticide/neurotoxin, rotenone, has been shown to cause systemic inhibition of mitochondrial complex I activity, with consequent degeneration of the nigrostriatal pathway, as observed in Parkinson’s disease. A novel intrastriatal rotenone model of Parkinson’s disease was used to examine the neuroprotective effects of valproic acid (VPA) and melatonin, both of which are known to induce neurotrophic gene expression in the central nervous system via mechanisms which may involve epigenetic modulation. In these studies, sham or lesioned rats were treated with either vehicle, VPA (4mg/mL), or melatonin (4µg/mL) in drinking water. Results from a forelimb asymmetry test indicated a significant decrease in use of the contralateral forelimb in rotenone-infused animals, in the third week post-surgery, which was abolished by VPA treatment. Apomorphine administration resulted in significantly higher ipsilateral rotation in rotenone-lesioned (12µg) animals, as compared to controls, which was attenuated by melatonin treatment. Subsequent immunohistochemical examination revealed a decrease in tyrosine hydroxylase immunoreactivity within the striatum and substantia nigra of rotenone-infused animals. VPA or melatonin treatment prevented this decrease in tyrosine hydroxylase in the striatum and substantia nigra. Stereological cell counting indicated a significant decrease in dopamine neurons within the substantia nigra of rotenone-treated animals. Importantly, this loss of dopamine neurons in rotenone-infused animals was blocked by chronic VPA or melatonin treatment. A third study explored whether rotenone infusion into the medial forebrain bundle and substantia nigra in mice could provide a model of Parkinson's disease. Densitometric analysis revealed a significant depletion of tyrosine hydroxylase immunofluorescence within the ipsilateral striatum and substantia nigra of lesioned animals, and a significant bilateral overexpression of α-synuclein in the substantia nigra, as compared to control animals. These novel findings support the use of intracranial rotenone as a Parkinsonian model, and provide a solid platform for future combinatorial therapeutic approaches with VPA and melatonin. / Dissertation / Doctor of Philosophy (PhD)

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Parkinson’s disease (PD) is a common neurodegenerative movement disorder, characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of aggregated alpha synuclein (aSyn). The disease often presents with early prodromal non-motor symptoms and later motor symptoms. Diagnosing PD based purely on motor symptoms is often too late for successful intervention, as a significant neuronal loss has already occurred. Furthermore, the lower prevalence of PD in females is not well understood, highlighting the need for a better understanding of the interaction between sex and aSyn, the crucial protein for PD pathogenesis. Here, we conducted a comprehensive phenotyping study in 1- to 5-month-old mice over- expressing human aSyn gene (SNCA) in a bacterial artificial chromosome (BAC-SNCA). We demonstrate a SNCA gene-dose-dependent increase of human aSyn and phosphorylated aSyn, as well as a decrease in tyrosine hy- droxylase expression in BAC-SNCA mice, with more pronounced effects in male mice. Phosphorylated aSyn was already found in the dorsal motor nucleus of the vagus nerve of 2-month-old mice. This was time-wise associated with significant gait altrations in BAC-SNCA mice as early as 1 and 3 months of age using CatWalk gait analysis. Furthermore, anxiety-related behavioral tests revealed an increase in anxiety levels in male BAC-SNCA mice. Finally, 5-month-old male BAC-SNCA mice exhibited a SNCA gene-dose-dependent elevation in energy expen- diture in automated home-cage monitoring. For the first time, these findings describe early-onset, sex- and gene- dose-dependent, aSyn-mediated disturbances in BAC-SNCA mice, providing a model for sex-differences, early- onset neuropathology, and prodromal symptoms of PD.