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Three-dimensional (3D) in vitro culture systems that provide controlled, biomimetic microenvironments would be a significant technological advance for both basic cell biology research and the development of clinical therapeutics (e.g., as in vivo cell delivery constructs). A variety of signals determine cell phenotype, including those from soluble factors, immobilized biomolecules, mechanical substrates, and culture geometry. My research seeks to create hydrogel culture systems that incorporate these signals in a defined, controllable manner. Specifically, I have focused on developing hydrogels based on the extracellular matrix (ECM) component hyaluronic acid (HA) with precisely specified mechanical, chemical and geometrical microenvironments. For example, the mechanical environment presented by HA hydrogels was tuned to span the threefold range measured for neonatal brain and adult spinal cord by modifying HA with varying numbers of photocrosslinkable methacrylate groups. These hydrogels were used to evaluate the effects of mechanical properties of a 3D culture paradigm on the differentiation of ventral midbrain-derived neural progenitor cells (NPCs) and results demonstrated that the mechanical properties of these scaffolds can assert a defining influence on differentiation. In addition, whole fibronectin was incorporated into HA hydrogels as an adhesive factor to encourage angiogenesis in 3D cultures, as interplay between endothelial cells and neurons is an important determining factor during NPC development and axonal regeneration after injury. To create spatially defined neuronal cultures in three-dimensions, multiphoton excitation (MPE) was used to photocrosslink protein microstructures within HA hydrogels. This method can be used to create complex, 3D architectures that provide both chemical and topographical cues to direct cell adhesion and guidance on size scales relevant to in vivo environments. Using this approach, both dorsal root ganglion cells (DRGs) and hippocampal NPCs could be guided along user-defined, 3D paths. In future studies, these strategies can be combined into a single hydrogel to create a culture microenvironment with multiple types of highly specified cues (i.e., chemical, topographical, and mechanical). / text

A first-of-its-kind, proof-of-concept drug screen with implications for two unmet medical needs. / The actin-bundling protein fascin is a key mediator of tumor invasion and metastasis and its activity drives filopodia formation, cell-shape changes and cell migration. Small-molecule inhibitors of fascin block tumor metastasis in animal models. Conversely, fascin deficiency might underlie the pathogenesis of some developmental brain disorders. To identify fascin-pathway modulators we devised a cell-based assay for fascin function and used it in a bidirectional drug screen. The screen utilized cultured fascin-deficient mutant Drosophila neurons, whose neurite arbors manifest the 'filagree' phenotype. Taking a repurposing approach, we screened a library of 1040 known compounds, many of them FDA-approved drugs, for filagree modifiers. Based on scaffold distribution, molecular-fingerprint similarities, and chemical-space distribution, this library has high structural diversity, supporting its utility as a screening tool. We identified 34 fascin-pathway blockers (with potential anti-metastasis activity) and 48 fascin-pathway enhancers (with potential cognitive-enhancer activity). The structural diversity of the active compounds suggests multiple molecular targets. Comparisons of active and inactive compounds provided preliminary structure-activity relationship information. The screen also revealed diverse neurotoxic effects of other drugs, notably the 'beads-on-a-string' defect, which is induced solely by statins. Statin-induced neurotoxicity is enhanced by fascin deficiency. In summary, we provide evidence that primary neuron culture using a genetic model organism can be valuable for early-stage drug discovery and developmental neurotoxicity testing. Furthermore, we propose that, given an appropriate assay for target-pathway function, bidirectional screening for brain-development disorders and invasive cancers represents an efficient, multipurpose strategy for drug discovery.

Neurons, the building blocks of the brain, are an astonishingly capable type of cell. Collectively they can store, manipulate and retrieve biologically important information, allowing animals to learn and adapt to environmental changes. This universal adaptability is widely believed to be due to plasticity: the readiness of neurons to manipulate and adjust their intrinsic properties and strengths of connections to other cells. It is through such modifications that associations between neurons can be made, giving rise to memory representations; for example, linking a neuron responding to the smell of pancakes with neurons encoding sweet taste and general gustatory pleasure. However, this malleability inherent to neuronal cells poses a dilemma from the point of view of stability: how is the brain able to maintain stable operation while in the state of constant flux? First of all, won’t there occur purely technical problems akin to short-circuiting or runaway activity? And second of all, if the neurons are so easily plastic and changeable, how can they provide a reliable description of the environment? Of course, evidence abounds to testify to the robustness of brains, both from everyday experience and scientific experiments. How does this robustness come about? Firstly, many control feedback mechanisms are in place to ensure that neurons do not enter wild regimes of behaviour. These mechanisms are collectively known as homeostatic plasticity, since they ensure functional homeostasis through plastic changes. One well-known example is synaptic scaling, a type of plasticity ensuring that a single neuron does not get overexcited by its inputs: whenever learning occurs and connections between cells get strengthened, subsequently all the neurons’ inputs get downscaled to maintain a stable level of net incoming signals. And secondly, as hinted by other researchers and directly explored in this work, networks of neurons exhibit a property present in many complex systems called sloppiness. That is, they produce very similar behaviour under a wide range of parameters. This principle appears to operate on many scales and is highly useful (perhaps even unavoidable), as it permits for variation between individuals and for robustness to mutations and developmental perturbations: since there are many combinations of parameters resulting in similar operational behaviour, a disturbance of a single, or even several, parameters does not need to lead to dysfunction. It is also that same property that permits networks of neurons to flexibly reorganize and learn without becoming unstable. As an illustrative example, consider encountering maple syrup for the first time and associating it with pancakes; thanks to sloppiness, this new link can be added without causing the network to fire excessively. As has been found in previous experimental studies, consistent multi-neuron activity patterns arise across organisms, despite the interindividual differences in firing profiles of single cells and precise values of connection strengths. Such activity patterns, as has been furthermore shown, can be maintained despite pharmacological perturbation, as neurons compensate for the perturbed parameters by adjusting others; however, not all pharmacological perturbations can be thus amended. In the present work, it is for the first time directly demonstrated that groups of neurons are by rule sloppy; their collective parameter space is mapped to reveal which are the sensitive and insensitive parameter combinations; and it is shown that the majority of spontaneous fluctuations over time primarily affect the insensitive parameters. In order to demonstrate the above, hippocampal neurons of the rat were grown in culture over multi-electrode arrays and recorded from for several days. Subsequently, statistical models were fit to the activity patterns of groups of neurons to obtain a mathematically tractable description of their collective behaviour at each time point. These models provide robust fits to the data and allow for a principled sensitivity analysis with the use of information-theoretic tools. This analysis has revealed that groups of neurons tend to be governed by a few leader units. Furthermore, it appears that it was the stability of these key neurons and their connections that ensured the stability of collective firing patterns across time. The remaining units, in turn, were free to undergo plastic changes without risking destabilizing the collective behaviour. Together with what has been observed by other researchers, the findings of the present work suggest that the impressively adaptable yet robust functioning of the brain is made possible by the interplay of feedback control of few crucial properties of neurons and the general sloppy design of networks. It has, in fact, been hypothesised that any complex system subject to evolution is bound to rely on such design: in order to cope with natural selection under changing environmental circumstances, it would be difficult for a system to rely on tightly controlled parameters. It might be, therefore, that all life is just, by nature, sloppy.

Yes / Monolayers of neurons and glia have been employed for decades as tools for the study of cellular physiology and as the basis for a variety of standard toxicological assays. A variety of three dimensional (3D) culture techniques have been developed with the aim to produce cultures that recapitulate desirable features of intact. In this study, we investigated the effect of preparing primary mouse mixed neuron and glial cultures in the inert 3D scaffold, Alvetex. Using planar multielectrode arrays, we compared the spontaneous bioelectrical activity exhibited by neuroglial networks grown in the scaffold with that seen in the same cells prepared as conventional monolayer cultures. Two dimensional (monolayer; 2D) cultures exhibited a significantly higher spike firing rate than that seen in 3D cultures although no difference was seen in total signal power (<50 Hz) while pharmacological responsiveness of each culture type to antagonism of GABAAR, NMDAR and AMPAR was highly comparable. Interestingly, correlation of burst events, spike firing and total signal power (<50 Hz) revealed that local field potential events were associated with action potential driven bursts as was the case for 2D cultures. Moreover, glial morphology was more physiologically normal in 3D cultures. These results show that 3D culture in inert scaffolds represents a more physiologically normal preparation which has advantages for physiological, pharmacological, toxicological and drug development studies, particularly given the extensive use of such preparations in high throughput and high content systems.

In translational research, disease models in preclinical studies are used as media for discovery of drugs or novel therapeutics. Development of in vitro models for various neurological diseases that enable efficient pharmacological or toxicological screening has been ongoing but challenging. Recognizing the potential benefit of in vitro disease models, dysfunctions in the cortical neuronal networks were induced to mimic the functional pathology of neurological symptoms using microelectrode arrays. Two different disease states – tinnitusand excitotoxicity – were investigated and discussed. In this model, pentylenetetrazol-induced increase in spontaneous firing rate and synchrony in the auditory cortical networks was used as correlate of tinnitus. Potential tinnitus treatment drugs from several different classes – including the novel class of potassium channel openers – were screened and quantified. The potentialtherapeutic values of these drugs were also discussed as the basis for drug repurposing. Functional excitotoxicity was induced by cisplatin (a cancer drug that causes neurological sideeffects) and glutamate (the major excitatory neurotransmitter). As proof-of-principle that the model may contribute to expediting the development of therapeutics, cisplatin excitotoxicity wasprevented by the antioxidant D-methionine, while glutamate excitotoxicity was prevented by ceftriaxone (a modulator of a glutamate reuptake transporter). In the latter part of the study, with results linking two of the screened drugs L-carnitine and D-methionine to GABAA receptor activation, it was demonstrated that this model not only served as an efficient drug-screening platform, but can be utilized to functionally investigate the underlying mechanism of drugs. Inaddition, several practical or conceptual directions for future studies to improve on this in vitro disease model are suggested.

Status epilepticus (SE) is a major clinical emergency resulting in continuous seizure activity that can cause brain injury and many molecular and pathophysiologic changes leading to neuronal plasticity. The neuronal plasticity following SE-induced brain injury can initiate epileptogenesis and lead to the ultimate expression of acquired epilepsy (AE), characterized clinically by spontaneous, recurrent seizures. Epileptogenesis is the process wherein healthy brain tissue is transformed into hyperexcitable neuronal networks that produce AE. Understanding these alterations induced by brain injury is an important clinical challenge and can lend insight into possible new therapeutic targets to halt the development of AE. Currently there are no means to prevent epileptogenesis following brain injury; thus, the elucidation of mechanisms of epileptogenesis will be useful in preventing the long-term clinical sequela. It has been demonstrated in vivo that calcium (Ca2+) dynamics are severely altered during SE and that elevations in intracellular Ca2+ ([Ca2+]i) in hippocampal neurons are maintained well past the duration of the injury itself (Ca2+ plateau). Here we report that similar changes in [Ca2+]i are observed in the hippocampal neuronal culture model of SE-induced AE. As an important second messenger, the maintenance of a Ca2+ plateau following injury can lead to several changes in gene expression, neurotransmitter release, and overall, neuronal plasticity. Thus, changes in post-SE [Ca2+]i and Ca2+ homeostasis may be important in understanding epileptogenesis and eventually preventing the progression to chronic epilepsy. This dissertation examines the development and maintenance of the Ca2+ plateau after SE and demonstrates the novel finding that pharmacological modulation of [Ca2+]i following SE may inhibit epileptogenesis in vitro.

Made available in DSpace on 2014-12-17T15:28:52Z (GMT). No. of bitstreams: 1 HermanyMV_DISSERT.pdf: 1765987 bytes, checksum: dedd84315ca1c69652d27407ffd67d85 (MD5) Previous issue date: 2013-03-04 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Recently, genetically encoded optical indicators have emerged as noninvasive tools of high spatial and temporal resolution utilized to monitor the activity of individual neurons and specific neuronal populations. The increasing number of new optogenetic indicators, together with the absence of comparisons under identical conditions, has generated difficulty in choosing the most appropriate protein, depending on the experimental design. Therefore, the purpose of our study was to compare three recently developed reporter proteins: the calcium indicators GCaMP3 and R-GECO1, and the voltage indicator VSFP butterfly1.2. These probes were expressed in hippocampal neurons in culture, which were subjected to patchclamp recordings and optical imaging. The three groups (each one expressing a protein) exhibited similar values of membrane potential (in mV, GCaMP3: -56 ?8.0, R-GECO1: -57 ?2.5; VSFP: -60 ?3.9, p = 0.86); however, the group of neurons expressing VSFP showed a lower average of input resistance than the other groups (in Mohms, GCaMP3: 161 ?18.3; GECO1-R: 128 ?15.3; VSFP: 94 ?14.0, p = 0.02). Each neuron was submitted to current injections at different frequencies (10 Hz, 5 Hz, 3 Hz, 1.5 Hz, and 0.7 Hz) and their fluorescence responses were recorded in time. In our study, only 26.7% (4/15) of the neurons expressing VSFP showed detectable fluorescence signal in response to action potentials (APs). The average signal-to-noise ratio (SNR) obtained in response to five spikes (at 10 Hz) was small (1.3 ? 0.21), however the rapid kinetics of the VSFP allowed discrimination of APs as individual peaks, with detection of 53% of the evoked APs. Frequencies below 5 Hz and subthreshold signals were undetectable due to high noise. On the other hand, calcium indicators showed the greatest change in fluorescence following the same protocol (five APs at 10 Hz). Among the GCaMP3 expressing neurons, 80% (8/10) exhibited signal, with an average SNR value of 21 ?6.69 (soma), while for the R-GECO1 neurons, 50% (2/4) of the neurons had signal, with a mean SNR value of 52 ?19.7 (soma). For protocols at 10 Hz, 54% of the evoked APs were detected with GCaMP3 and 85% with R-GECO1. APs were detectable in all the analyzed frequencies and fluorescence signals were detected from subthreshold depolarizations as well. Because GCaMP3 is the most likely to yield fluorescence signal and with high SNR, some experiments were performed only with this probe. We demonstrate that GCaMP3 is effective in detecting synaptic inputs (involving Ca2+ influx), with high spatial and temporal resolution. Differences were also observed between the SNR values resulting from evoked APs, compared to spontaneous APs. In recordings of groups of cells, GCaMP3 showed clear discrimination between activated and silent cells, and reveals itself as a potential tool in studies of neuronal synchronization. Thus, our results indicate that the presently available calcium indicators allow detailed studies on neuronal communication, ranging from individual dendritic spines to the investigation of events of synchrony in neuronal networks genetically defined. In contrast, studies employing VSFPs represent a promising technology for monitoring neural activity and, although still to be improved, they may become more appropriate than calcium indicators, since neurons work on a time scale faster than events of calcium may foresee / Neur?nios se comunicam por meio de sinapses, trocando mensagens capazes de modificar o potencial de membrana de outros neur?nios. Demonstrar o papel desses sinais e decodificar essa linguagem el?trica representa o grande objetivo da neuroci?ncia moderna. Atualmente, a eletrofisiologia ? o ramo da neuroci?ncia capaz de investigar esses recursos el?tricos de neur?nios - que v?o desde registros de condut?ncia e comportamento cin?tico de canais i?nicos individuais at? a demonstra??o de neur?nios individuais implicados em comportamentos complexos. Nesse sentido, diferentes estados cerebrais e comportamentos implicam o recrutamento de grandes conjuntos de neur?nios se comunicando em um estado coerente, din?mico. Al?m disso, essas grandes popula??es s?o formadas por diversos subtipos neuronais cuja an?lise requer t?nicas que possibilitem uma resolu??o temporal e espacial de c?lulas individuais e, prefencialmente, de subtipos espec?ficos. Apenas recentemente, indicadores ?pticos geneticamente codificados surgiram como ferramentas n?o invasivas de alta resolu??o espacial e temporal utilizados para monitorar a atividade de neur?nios individuais e popula??es neuronais espec?ficas. O n?mero crescente de novos indicadores optogen?ticos, juntamente com a aus?ncia de compara??es em condi??es id?nticas, gerou dificuldade em escolher a mais adequada das prote?nas, dependendo do desenho experimental. Portanto, o objetivo deste estudo foi comparar tr?s prote?nas rep?rter recentemente desenvolvidas: os indicadores de c?lcio GCaMP3 e R-GECO1, e o indicador de voltagem VSFP butterfly1.2. Foram expressos em neur?nios do hipocampo em cultura, os quais foram submetidos a registros de patch-clamp e de imageamento ?ptico. Os tr?s grupos (cada um expressando uma prote?na) exibiram valores semelhantes de potencial de membrana (em mV, GCaMP3: -56 ? 8,0; R-GECO1: -57 ? 2,5; VSFP: -60 ? 3,9; p = 0,86), no entanto, o grupo de neur?nios que expressam VSFP mostrou uma m?dia mais baixa de resist?ncia de entrada do que os outros grupos (em Mohms, GCaMP3: 161 ? 18,3; GECO1-R: 128 ? 15,3; VSFP: 94 ? 14,0; p = 0,02). Cada neur?nio foi submetido a inje??es de correntes com frequ?ncias diferentes (10 Hz, 5 Hz, 3 Hz, 1,5 Hz, e 0,7 Hz) e as suas respostas de fluoresc?ncia foram registradas. Em nosso estudo, apenas 26,7% (4/15) dos neur?nios que expressam VSFP mostraram sinal de fluoresc?ncia detect?vel em resposta a potenciais de a??o. O valor m?dio de sinal-para-ru?do (SNR), obtido em resposta a cinco potenciais de a&#1195;?o (a 10 Hz) foi pequeno (1,3 ? 0,21), no entanto a cin?tica r?pida do VSFP permite a discrimina??o de disparos, como picos individuais, com detec??o de 53% dos APs evocados. Freq??ncias abaixo de 5 Hz, assim como varia&#1195;?es no potencial de membrana subliminares, foram indetect?veis devido ao alto ru?do do sinal de fluoresc?ncia. Por outro lado, os indicadores de c?lcio mostraram maior altera??o na fluoresc?ncia, seguindo o mesmo protocolo (cinco potenciais de a&#1195;?o a 10 Hz). Entre os neur?nios expressando GCaMP3, 80% (8/10) exibiram sinal, com um valor m?dio de SNR de 21 ? 6,69 (soma), enquanto que para os neur?nios expressando R-GECO1, 50% (2/4) dos neur?nios demonstraram sinal com um valor m?dio SNR de 52 ? 19,7 (soma). Para protocolos de 10 Hz, 54% dos disparos foram detectados com evocado GCaMP3 e 85% com o R-GECO1. Disparos foram detectados em todas as frequ?ncias e os sinais de fluoresc?ncia foram tamb?m detectados a partir de despolariza??es subliminares. Sendo GCaMP3 o indicador mais prov?vel de produzir sinal de fluoresc?ncia e com alto SNR, alguns experimentos foram realizados somente com essa prote?na. Observamos que GCaMP3 ? eficaz na detec??o de inputs sin?pticas (envolvendo influxo de Ca2+), com alta resolu??o espacial e temporal. Tamb?m foram observadas diferen?as entre os valores de SNR resultantes dos disparos evocados, em compara??o com os disparos espont?neos. Em registros de grupos de c?lulas, GCaMP3 mostrou clara discrimina??o entre c?lulas ativadas e sil?ncio, revelando-se como uma ferramenta potencial em estudos de sincroniza??o neuronal. Assim, nossos resultados sugerem que os indicadores de c?lcio dispon?veis atualmente permitem estudos detalhados sobre a comunica??o neuronal, que v?o desde dendritos individuais at? a investiga??o de eventos de sincronia em redes neuronais geneticamente definidas. Em contraste, VSFPs representam uma tecnologia promissora para monitorar a atividade neural e, apesar de ainda requererem melhoramentos, podem se tornar mais apropriados do que os indicadores de c?lcio, uma vez que os neur?nios trabalham em uma escala de tempo mais r?pida do que eventos de c?lcio podem prever

Le récepteur cannabinoique de type 1 (CB1R) est l’un des récepteurs couplés aux protéines G les plus abondants du cerveau mammifère. CB1R a longtemps été décrit comme un récepteur présynaptique régulant de manière rétrograde la transmission synaptique. Cependant, depuis les vingt dernières années, de nouveaux rôles ont été découverts et il est maintenant clairement admis que l’action des endocannabinoides (eCBs) ne se limite pas à la régulationde la neurotransmission au niveau de synapses adultes déjà établies. En effet, les eCBs et le CB1R sont des acteurs majeurs de l’ensemble des phases du développement cérébral. Cependant, les mécanismes moléculaires impliqués n’ont toujours pas été identifiés. Les mécanismes cellulaires auxquels nous nous intéressons ne dépendant pas de l’environnement cellulaire, nous proposons donc de combiner la puissance génétique du modèle drosophile à l’accessibilité et la haute résolution offerte par la culture primaire de neurones. De plus, le récepteur CB1 ne possédant pas d’orthologue parmi les invertébrés, ce système offre la possibilité d’étudier la biologie du récepteur en s’affranchissant de la machinerie endocannabinoide. Cependant, actuellement, aucun protocole de culture primaire de neurones de drosophile ne permet d’obtenir des cellules hautement différenciées et polarisées à basse densité. Ainsi, nous avons tout d’abord développé, optimisé et validé un nouveau protocole permettant de d’obtenir des neurones fonctionnels, hautement différenciés et polarisés en culture de basse densité. Dans un second temps, nous avons démontré que l’activation durécepteur CB1, exprimé ectopiquement dans les neurones de drosophile, entrainait son internalisation, de manière identique à ce qui avait déjà été observé chez les mammifères. Puis, nous avons étudié l’effet de l’expression et de l’activation ectopique de CB1R sur le développement neuronal chez la drosophile. Ainsi, nous avons démontré que l’activation du récepteur module directement la dendritogénèse. Afin de compléter la caractérisation de notremodèle, nous avons démontré que l’activation transitoire du récepteur dans les corps pédonculés (le centre de la mémoire olfactive chez la drosophile) altérait spécifiquement la formation d’une forme consolidée de mémoire après un conditionnement aversif. En conclusion, la validation du modèle drosophile dans l’étude de la biologie cellulaire durécepteur CB1 ouvre de nouvelles perspectives quant à la détermination des mécanismes moléculaires régissant l’action du récepteur sur le fonctionnement neuronal. / The type-1 cannabinoid receptor (CB1R), the neuronal receptor for the major psychoactive substance of marijuana, is one, of the most abundant G-protein coupled receptors in the mammalian central nervous system. CB1R is traditionally described as a presynaptic receptor that retrogradely regulates synaptic transmission. In addition to this now relatively wellcharacterized function, in the last two decades it has become widely recognized that endocannabinoid (eCB) actions in the brain are not limited to the regulation of neurotransmission at established adult synapses. Indeed, eCB and CB1R are now recognized to be involved in brain development at the synaptic, neuronal and network levels. However, precise mechanisms underlying these processes remain poorly described. Since cellular mechanisms that mediate CB1R-activition dependent neuronal remodeling and subneuronal targeting have been demonstrated to be cell-autonomous, we aimed to combine the power of Drosophila genetics with the experimental accessibility and single-cell resolution of lowdensity primary neuronal cultures, a tool currently lacking in Drosophila. Moreover, becauseDrosophila does not have a CB1R ortholog, CB1R cell biology may be observed independently from eCB machinery. Thus, we first developed and validated an in vitro culture protocol that yields mature and fully differentiated Drosophila neurons. Secondly, we showed that activation-dependent endocytosis of ectopically expressed CB1R is conserved in Drosophila neurons. Next, we investigated whether ectopic expression and activation of CB1R in Drosophila modulate neuronal development. As observed in mammals, we observed that activation of CB1R impairs dendritogenesis in a cell-autonomous manner. For further characterization of our model, we showed that, as with mammals, transient ectopic CB1R expression and activation in mushroom body neurons (the center of olfactory memory in Drosophila) modulate the formation of a consolidated form of aversive memory. In conclusion, the validation of this new animal model opens new perspectives to better characterize mechanisms underlying modulation of neuronal functions induced by CB1Ractivity

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.