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.

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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.

Dans la maladie de Parkinson (MP), la perte progressive des neurones dopaminergiques (DA) touche principalement la substantia nigra pars compacta (SNc). Les symptômes moteurs sont classiquement gérés par une thérapie dopaminergique de remplacement (TDR). Conjointement à la levodopa, l’utilisation d’agonistes dopaminergiques permet de prévenir les complications motrices mais peut être associée à des troubles du système de récompense. Jusqu’à 14% des patients parkinsoniens sous TRD peuvent souffrir de comportement « addiction-like » tels que le pari pathologique, l’hypersexualité ou une prise compulsive de la médication DA. A ce jour la seule solution thérapeutique consiste à diminuer la TRD ce qui détériore les symptômes moteurs. Les neuroadaptations conduisant à ces troubles du système de récompense demeurent mal comprises. Nous proposons un travail dans lequel nous avons évalué les propriétés appétitives de l’agoniste D2/D3 pramipexole (ppx) après une exposition chronique à la L-dopa dans un modèle de rat parkinsonien alpha-synucléine. Dans une première étude, nous avons évalué l’effet d’une stimulation répétée des récepteurs DA sur la sensibilisation du système de récompense en contexte parkinsonien. Nos résultats montrent un effet récompensant du ppx après administrations chronique de L-dopa et perte DA nigrostriatal induite par surexpression de l’alpha-synucléine. Aucune modification transcriptionnelle n’a été observée pour les récepteurs DA. Cependant, nous avons identifié une association entre lésion/traitement pharmacologique et des changements transcriptionnels potentiellement liés à un contexte d’addiction aux psychostimulants. Cette étude fournit des preuves suggérant fortement la lésion parkinsonienne et la thérapie L-dopa comme des facteurs conjointement impliqués dans le remodelage cérébral sous-tendant une préférence de place conditionnée pour le ppx. Les données moléculaires et pharmacologiques générées ont suggéré un rôle clé de la voie glutamatergique dans cette réponse comportementale. Ce résultat est cohérent avec la littérature décrivant un déséquilibre glutamatergique striatal dans les contextes d’addiction aux psychostimulants et de complications motrices associées à la MP. Ainsi, nous avons conçu une deuxième étude visant à investiguer plus avant le potentiel thérapeutique d’une inhibition des récepteurs glutamatergiques. Une lésion bilatérale de la SNc a été réalisée par surexpression de la protéine alpha-synucléine au moyen d’un vecteur AAV. Suite à cette lésion, un traitement chronique à la L-dopa a été réalisé. L’effet de l’antagoniste des récepteurs mGluR5 (metabotropic glutamate receptor 5) MPEP sur les propriétés renforçatrices du ppx a été évalué dans un paradigme de préférence de place conditionnée. Enfin, une analyse des changements d’expression de protéines d’intérêt a été réalisé afin d’associer changements comportementaux drogue/lésion induits et paramètres moléculaires. L’acquisition et l’expression de la préférence de place ppx-induite a été abolie par le MPEP. De plus, nous avons identifié des réseaux neuraux et des modifications d’expression protéiques sous-tendant les plasticités striatales associées à la réponse comportementale. L’ensemble de ces travaux apporte de nouvelles idées sur le contexte physiopathologique associé aux troubles du système de récompense dans la MP. Des données moléculaires et pharmacologiques convergentes suggèrent fortement le mGluR5 comme une cible thérapeutique prometteuse. / In Parkinson’s Disease (PD), the progressive dopaminergic (DA) cell loss mainly affects the substantia nigra pars compacta (SNc). The motor symptoms are classically managed by DA replacement therapies (DRT). Although adding DA agonists to levodopa treatment may contribute to prevent motor complications, it may be associated with drug‑induced changes in reward related pathways. Up to 14% of PD patients under DRT may suffer from ‘addiction‑like’ behavior such as pathological gambling, hypersexuality or DA medication‑induced substance abuse. To date, the only therapeutic answer consists in lowering the DA medications which deteriorates the motor symptoms. Neuroadaptations leading to reward bias in PD patients under DRT are still poorly understood. To address this challenge, we propose a work in which we have assessed the rewarding effect of the D2/D3 agonist pramipexole (ppx) after chronic exposure to L‑dopa in an alpha-synuclein PD rat model. In a first study, we assessed the effect of repeated DA receptors stimulations on sensitization of the reward system in a parkinsonian context. Our findings demonstrated that ppx had a rewarding effect after chronic L-dopa administrations and alpha-synuclein-mediated nigral loss. No transcriptional changes within DA receptors were highlighted. However, we identified an association between the main drug or lesion and transcriptional changes which were potentially related to the context of psychostimulant addiction. This study provides evidences strongly suggesting that PD-like lesion and L-dopa therapy were concomitant factors involved in striatal remodeling underlying the ppx-induced place preference. Molecular and pharmacological data suggested a key involvement ofthe glutamatergic pathway in this behavioral outcome. These data were consistent with literature describing major striatal glutamate imbalance as a common feature of drug addiction and Parkinson’s disease physiopathological contexts. Hence, we designed a second study aiming to further investigate the therapeutic potential of glutamatergic receptors inhibition. A bilateral lesion of the SNc was performed in the rat using AAV-mediated overexpression of the alpha-synuclein. This lesion was followed by chronic L-dopa administrations. Then, the effect of the metabotropic glutamate receptor 5 (mGluR5) antagonist MPEP on ppx reinforcing properties was assessed in a place conditioning paradigm. Finally, analysis at the protein level was conducted to associate drug and lesion induced behavioral changes to molecular endpoints. Acquisition and expression of the ppx-induced place preference was abolished by the MPEP. Furthermore, we identified neural networks and protein changes underlying the striatal remodeling associated with the behavioral outcome. All this work provides new insights into the physiopathological context associated to the PD/DRT related reward bias. Convergent molecular and pharmacological data strongly suggest mGluR5 as a promising therapeutic target.

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