Introdução. O transplante de células é possibilidade terapêutica promissora para muitas doenças neurológicas. Nos últimos anos, a possibilidade do isolamento de células-tronco dos tecidos adultos, por exemplo da medula-óssea, atrai a atenção da comunidade científica, estratégia que minimiza os problemas éticos relativos ao uso de tecido fetal para implantes visando ao tratamento de doenças neurológicas. Entretanto, a eficiência da transdiferenciação de células-tronco mesenquimais em neurônios, bem como os mecanismos envolvidos nesse processo, permanecem desconhecidos. A obtenção de neurônios maduros ocorreu somente em sistemas de co-cultura, o que induz a questão se a diferenciação representa um potencial das células per si, ou se é possível somente devido à fusão com neurônios maduros. Objetivos. No presente trabalho, pretendeu-se verificar o potencial de as células-tronco mesenquimais tornarem-se neurônios e esclarecer os possíveis mecanismos envolvidos nesse processo. Material e métodos. Células-tronco mesenquimais foram isoladas de 20 doadores voluntários normais e caracterizadas por análise de separação celular ativada por fluorescência. A multipotencialidade foi investigada ao se diferenciar as células em condrócitos e osteócitos. A capacidade de auto-renovação foi confirmada pelo ensaio de incorporação de BrdU. Ulteriormente, as células foram diferenciadas por uma semana em meio contendo AMPc, IBMX, ou combinação de ambos, e os resultados foram comparados com o cultivo em meio básico. Diferentes bloqueadores de Ca2+ ou inibidores de PKA foram usados como tentativa de se impedir a diferenciação, ocorrência que foi mensurada com imunocitoquímica para NF-200 (marcador de neurônios maduros). O registro eletrofisiológico por meio de patch clamp foi usado para se confirmar o fenótipo neuronal. As figuras foram configuradas em microscopia confocal. Para análise estatística foi utilizada ANOVA com teste post-hoc. Resultados. As células isoladas expressaram CD90, 105, 44 e 13 mas foram negativas para CD34 e 45. Isto significa que não são de origem hematopoiética; 98,74 ± 0,43% das células incorporaram BrdU em 24 horas. Após o isolamento, foi possível diferenciá-las em condrócitos ou osteócitos. Em situação controle, não foram evidenciadas células positivas para NF200. Por outro lado, ocorreu positividade em 10,75% ± 1,35 (p<0,0001) das células sob IBMX e, em 15,18% ± 1,12, sob a combinação cAMP e IBMX (p<0,0001). Foram registradas correntes de Na+ e K+ dependentes de voltagem, mas não potenciais de ação. A diferenciação foi inibida com PKAi (5,73% ± 0,42, p<0,0001), nifedipina (5,79% ± 0,98, p<0,0001), Ni2+ (7,06% ± 1,68, p<0,0001) e Cd2+ (0 ± 0, p<0,0001). Discussão. Isolou-se uma população de células-tronco estromais da medula-óssea de seres humanos que se mostrou multipotencial e auto-renovável. O aumento da concentração de AMPc no meio elevou a concentração de neurônios para 15%. A diferenciação parece depender da via PKA mas também envolve a concentração intracelular de Ca2+. Conclusão. O correto entendimento de como as células-tronco mesenquimais diferenciam-se pode contribuir para aumentar a eficácia do método e, talvez um dia, tornar possível o uso dessa ferramenta no campo clínico. / Introduction. Cell transplantation has been considered a promising therapeutic approach for many neurological diseases. The possibility of isolation of stem cells from adult tissues, i.e. bone marrow, has attracted the attention of the scientific community in the recent years. This strategy is interesting on avoiding the ethical issues regarding the use of fetal tissue for neural implants. Moreover, the efficiency of the transdifferentiation of mesenchymal stem cells (MSCs) into neurons, and the mechanisms involved in this process remain largely unknown. The obtention of mature neurons was described only in coculture systems, what raised the question if the differentiation is a potential of the cells itself, or if it is possible only due to fusion with mature neurons. Objectives. In the present investigation, we aimed to verify the potential of MSCs to differentiate into neurons, and also to clarify the possible mechanisms involved on it. Material and methods. MSCs were isolated from 20 healthy human subjects and characterized by FACS-analysis. Multipotentiality was addressed by differentiating them into chondrocytes and osteocytes. The self-renewal capacity was confirmed with BrdU-incorporation assay. Afterwards, cells were differentiated for 1 week in a medium containing cAMP, IBMX, or a combination of both, and the results were compared with cells treated in basal-medium condition. Different Ca2+-blockers and PKA-inhibitor peptide were used on an attempt to impair differentiation, which was quantified with NF-200 immunostaining (a marker of mature neurons). Patch-clamp recording was used to confirm neuronal phenotype. Pictures were taken in confocal microscope. For statistical analysis ANOVA with a post-hoc test was used. Results. The isolated cells expressed CD90, 105, 44, and 13, but were negative for CD34 and 45, meaning that they were non-hematopoiethic; 98.74 ± 0.43 % of them incorporated BrdU in 6hs. After isolation, they differentiated into chondrocytes and osteocytes. In a control situation, no NF200 positive cell was seen. On the other hand, 10.75% ± 1.35 (p<.0001) of positivity was seen under IBMX and 15.18% ± 1.12 in the combination of cAMP with IBMX (p<.0001). Na+ and K+-voltage gated currents were recorded. Differentiation was impaired with PKAi (5.73% ± 0.42, p<.0001), nifedipin (5.79% ± 0.98, p<.0001), Ni2+ (7.06% ± 1.68, p<.0001), and Cd2+ (0 ± 0, p<.0001). Discussion. We were able to isolate a population of stromal stem cells from the bone marrow of human subjects, since they were multipotential and self-renewable. Increasing the concentration of cAMP raised the percentage of neurons up to 15%. The differentiation seems to be dependent on the PKA pathway, but also involved the intracellular concentration of Ca2+. Conclusions. The complete understanding of how MSC differentiate can contribute to increase the efficiency of the method and thus make possible to use this powerful tool in the clinical practice.

No description available.

Beta-N-methylamino-L-alanine (BMAA) is a non-protein amino acid produced by many cyanobacteria, and thought to induce neurotoxic effects through excitotoxicity, contributing to neurodegenerative diseases such as Amyotrophic Lateral Sclerosis/Parkinsonism-dementia complex (ALS-PDC) and Alzheimer’s. The ubiquitous nature of cyanobacteria, and evidence of biomagnification through our food web, creates a dire need for the development of an analytical platform that will provide accurate identification and quantification of BMAA amounts in our ecosystem and potential food supply. The present study evaluated the ability of a MALDI-ToF-MS method to detect and quantify BMAA in a variety of biological matrices. Through validation procedures, it was demonstrated that this MALDI-ToF-MS method provided comparable data to currently accepted analytical methods, specifically LC-MS/MS. Further, the development of said method reduced sample preparation and data acquisition time (1-2 seconds per sample), while providing high throughput analysis and eliminating the need for derivatization, chromatographic separation, and modification of amino acids.

Les maladies d’Alzheimer et de Parkinson (MA et MP) sont des maladies neurodégénératives entrainant une diminution des fonctions cognitives et locomotrices et toujours dépourvues de traitements curatifs. La consommation d’huile d’olive et des polyphénols qu’elle contient atténuerait la survenue des démences telles qu’observées dans la MA ou la MP. À ce jour, ces composés phénoliques ont été étudiés de manière isolée afin de caractériser leur réactivité vis à vis des radicaux et leurs effets biologiques, mais les concentrations utilisées (5–100 µM) sont supérieures aux concentrations retrouvées dans le plasma (0.5-2,8 ng/ml). Jusqu’à présent, aucune étude n’a observé les effets de ces composés combinés, alors que l’oleuropéine et le tyrosol, des composés phénoliques parmi les plus abondants dans l’huile d’olive et que l’acide p-coumarique, un dérivé d’acide hydroxycinnamique, sont connus pour leurs activités antioxydantes et leur biodisponibilité. Dans un contexte cellulaire, les composés phénoliques agissent à travers leurs capacités de donneurs d’atomes d’hydrogène et/ou d’électrons, et impactent la régulation des défenses antioxydante. En conditions oxydatives, ils peuvent subir des modifications structurelles et mener à de nouveaux composés. L’impact cellulaire de ces derniers est encore peu étudié, car leur devenir dans le contexte d’un métabolisme oxydatif reste obscur. L’étude des métabolites issus de l’oxydation des composés phénoliques est donc d’une réelle importance afin d’approfondir les connaissances sur leurs mécanismes d’actions au niveau biologique. Nous avons émis l’hypothèse que la combinaison de l’oleuropéine, du tyrosol et de l’acide p-coumarique potentialise leurs effets neuroprotecteurs à travers leurs capacités de donneurs d’électrons et en lien avec la production des métabolites associés. Ce propose une approche pluridisciplinaire intégrant des composantes biologiques et des questions relevant de la physico-chimie, en s’appuyant sur des outils électrochimiques. En premier lieu, nous avons démontré que l’association de ces trois composés (Mix 1) permettait de potentialiser leurs effets neuroprotecteurs avec concentrations efficaces à 0.1 et 1µM, contrairement aux concentrations neuroprotectrices lorsque ces composés sont utilisés individuellement (5 à 10 fois plus élevées). Le Mix1 augmente la réactivité des composés phénoliques vis-à-vis d’espèces réactives de l’oxygène et permet une diminution de leur génération intracellulaire. Cette neuroprotection semble en partie médiée par une régulation des facteurs de régulation rédox endogènes. Dans un second temps, nous avons comparé le comportement en conditions oxydatives acellulaires de l’oleuropéine, du tyrosol et de l’acide p-coumarique individuellement et dans le Mix1. L’analyse en LC-MS2 des composés stables issus de l’oxydation du Mix 1 a montré une diminution des taux d’oxydation relatifs de chacun des composés, ainsi que la détection de trois néocomposés spécifiques de l’oxydation du Mix1. De plus, un effet neuroprotecteur significatif du Mix1 oxydé a été observé dès 1 et 5 nM, ce qui soutient l’hypothèse que les produits issus de l’oxydation des composés phénoliques peuvent participer aux effets biologiques des composés natifs. Notre troisième objectif était d’évaluer si les composés issus de l’oxydation des composés phénoliques pouvaient être générés dans un système biologique. L’analyse en LC-HRMS des extraits de cellules neuronales traitées avec le Mix1 en conditions oxydatives a révélé que ces composés étaient toujours présents sous leurs formes natives dans les cellules après plusieurs heures de traitement. Ce projet multidisciplinaire a permis d’observer les effets des composés phénoliques sous plusieurs angles. Il confirme qu’un effet neuroprotecteur de composés issus de l’oxydation est possible et ouvre la voie à de nouvelles approches pour le développement de formulations efficaces pour limiter le stress oxydatif au niveau cérébral. / Alzheimer and Parkinson diseases (AD and PD) are neurodegenerative disorders leading to cognitive function decline and curative treatments are still missing. Olive oil and related phenolic compounds consumption could prevent or slow down cognitive decline through neuronal preservation. Indeed, they have direct antioxidant properties as electrons or H-atom donors, which are largely suspected of being responsible for their biological effects, but also of some indirect properties as modulators of endogenous antioxidant defenses. The secoiridoid oleuropein and the monophenols tyrosol and p-coumaric acid have shown abilities to counteract oxidative injuries and associated neuronal death in several cellular and in vivo models. However, the basic mechanisms of action of phenolic compounds remain to be elucidated. In fact, phenolic compounds are highly sensitive to oxidative modifications and could be transformed into new compounds in an oxidative context. Cellular impacts of these products are rarely considered, probably because their intracellular detection is a great challenge and remain an important field to explore. Moreover, oleuropein, tyrosol and p-coumaric were individually evaluated in order to characterize their own reactivity towards ROS and their biological properties. However, the concentration ranges used to observe neuroprotection in cellular models (5 – 100 µM) were far from the possibly recovered phenolic concentration in plasma (0.5 - 2,8 ng /ml of plasma). To observe an effect with concentration range closer to the physiological ones, several antioxidant combinations could be proposed. Nevertheless, despite the biological promises of phenolic compounds from olive oil, studies which combined some of the most abundant phenolic compounds in the neuroprotection field is still missing. We thus hypothesized that the combination of oleuropein, tyrosol and p-coumaric acid in equimolar proportion (Mix 1) could potentiated their ROS scavenging and neuroprotective properties to counteract oxidative stress-induced neuronal death. These effects could be in part due to their oxidized metabolites. First, we demonstrated that the association of these three phenolic compounds potentiated their neuroprotective effects with efficient concentrations at 0.1 and 1µM, contrary to concentration ranges when there are used individually (5 to 10 higher). The Mix1 increase the reactivity of phenolic compounds towards ROS and decreases their intracellular production. These neuroprotective properties seems, at least in part, lead by a regulation of the endogenous redox factors. Then, we compared the behavior of Oleuropein, tyrosol and p-coumaric acid individually and in the Mix1 under acellular oxidative conditions. The oxidized Mix1 LC-ESI-MS2 profiles showed a regeneration of oleuropein through a decrease of the phenolic compounds oxidation rate and highlight the presence of specific oxidized products in Mix 1. This study also showed a significant neuroprotection of oxidized Ole and oxidized Mix1 with very low concentrations (1 and 5 nM), suggesting the putative relevant role of oxidized Ole products to protect or delay neuronal death. Our third objective was to evaluate if these oxidized metabolites could be produced in a biological system. Even under oxidative conditions, we recovered these compounds in cellular extracts under their native forms after few hours of treatments, attesting their accumulation in neuronal cells. Taking together, our results pointed out the relevance to study the phenolic compounds from a chemical and a biological point of view, which should always be as cellular redox reactions are between biological and chemical fields. Lastly, we confirmed the significance to take into account the oxidized metabolites from phenolic compounds to deeper understand their biological properties.

Les synucléinopathies regroupent les maladies neurodégénératives de Parkinson, les démences à corps de Lewy et l'atrophie multi-systématisée. Des études suggèrent que les synucléinopathies seraient des maladies à prion. Aujourd'hui, certains aspects manquent pour que l'α-synucléine soit reconnue comme un prion. Par exemple, il est à démontrer que chaque synucléinopathie est causée par une souche précise d'α-synucléine. Durant ma thèse j’ai mis au point une méthode d'amplification fiable des dépôts présents dans le cerveau des patients atteints de synucléinopathies. J’ai aussi documenté les procédures de nettoyage à adopter envers des matériels souillés, par diverses fibres amyloïdes, afin de réduire le risque de contamination. Finalement, j’ai été associé à une étude montrant les capacités de propagation d'assemblages d'α synucléine, dans un réseau de neurones humains en culture. Ces résultats permettront des études structurales, et fonctionnelles, des souches d’α-synucléine dans les synucléinopathies. / The aggregation of α-synuclein protein has been shown to be associated with Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, called synucleinopathies. Increasing amount of evidences suggest that synucleinopathies are prion diseases. Some aspects are missing for α-synuclein to be recognized as a prion, such as the existence of strains associated to synucleinopathies. During my thesis I set up a reliable method to amplify α-synuclein-rich deposits from patients tissues. I validated the method using all synucleinopathies tissues. This should allow the identification of α-synuclein strain related to each synucleinopathy. In addition, I also documented cleaning procedures for materials soiled with various amyloid fibers, in order to reduce the risk of contamination. Finally, I was associated to a study that shows the propagation abilities of different α-synuclein assemblies in a neuronal network mimicking human cortico-cortical connections. These results open the way to structural and functional studies of the amplified deposits.

Mutant mRNA and protein both contribute to the clinical manifestation of many repeat-associated neurodegenerative and neuromuscular disorders. The presence of nuclear RNA clusters is a feature shared amongst these diseases, such as C9ORF72/ALS and myotonic dystrophy 1/2 (DM1/2); however, this pathological hallmark has not been conclusively demonstrated in Huntington’s disease (HD) in vivo. Investigations into HD – caused by a CAG repeat expansion in exon 1 of the huntingtin (HTT) gene – have largely focused on toxic protein gain-of-function as a disease-causing feature, with fewer studies investigating the role of mutant HTT mRNA in pathology or pathogenesis. Here we report that in two HD mouse models, YAC128 and BACHD-97Q-ΔN17, mutant HTT mRNA is preferentially retained in the nucleus in vivo. Furthermore, we observed the early, widespread formation of large mutant HTT mRNA clusters (approximately 0.6 to 5 µm3 in size) present in over 50-75% of striatal and cortical neurons. Affected cells were limited to one cluster at most. Endogenous wild-type mouse Htt or human HTT mRNA containing 31 or fewer repeats did not form clusters. Additionally, the aberrantly spliced N-terminal exon 1-intron 1 RNA fragment, HTT1a, also formed clusters that fully co-localized with the mutant HTT mRNA clusters. These results suggest that multiple repeat-containing transcripts can coalesce to form a single cluster in a given cell. Treating YAC128 mice with antisense oligonucleotides efficiently silenced individual HTT mRNA foci but had limited impact on clusters. Our findings identify mutant HTT mRNA clustering as an early, robust molecular signature of HD, further supporting HD as a repeat expansion disease with suspected mRNA involvement.

SAMMANFATTNING Bakgrund: Vid hjärnundersökningar finns även andra strukturer utanför hjärnan som är innerverade av nervsystemet. Det är möjligt att sjukdomen i hjärnan avspeglas där också. Det finns många sjukdomar och tillstånd med liknande symptom och att ställa rätt diagnos kan vara svårt. Många sjukdomar påverkar det autonoma nervsystemet och ett sätt att hitta rätt diagnos kan vara att undersöka hur det fungerar.  Syfte: Att se vilka variationer av radioaktivitetsupptag som fanns i parotiskörtlarna mellan patienterna som avbildades med 11C-PE2I PET/DT. Samt att kunna se om denna information kan utnyttjas till att identifiera dysfunktion av autonoma nervsystemet (MRT-bilder är även en tillhjälps verktyg). Metod: Studiedesignen var en retrospektiv kvantitativ studie. Hundra (konsekutiva) patienter indelades i grupper så som: normal, Parkinsons, Parkinsons sjukdom med kombination av vaskulära förändringar och atypisk Parkinson sjukdom. Isotopupptag i basala ganglier och parotiskörtlar har mätts. Några patienters MRT- bilder var tillgängligt till kvantifiering.   Resultat: kombinerade gruppen med Parkinsons sjukdom, atypisk Parkinsons sjukdom och vaskulära förändringar har en uppreglering av antalet fria dopaminreceptorer i parotis jämfört med friska (även i bara PSP samt bara vaskulära grupper) med (p &lt;0.05).   Slutsats: Det finns variation av upptaget mellan en del av grupper därmed kan sjukdomen i hjärnan avspeglas i körtlar också.   Nyckelord: Neurodegenerativa sjukdomar, Autonom dysfunktion, 11CPE2I-PET/DT, parotiskörtlar, basala ganglier / ABSTRACT Background: At brain examination, there are also other structures outside the brain that are innervated by the nervous system. It is possible that the disease of the brain is reflected there as well. There are many diseases and conditions with similar symptoms and making the right diagnosis can be difficult. Many diseases affect the autonomic nervous system and one way to find the right diagnosis may be to investigate how it works.     Purpose: to see what variations in radioactivity uptake were found in the parotic glands between the patients depicted with the 11C-PE2I PET/CT. As well as being able to see how valuable this information is in being used to identify dysfunction of the autonomic nervous system (MRT- images are also an aids tool).   Method: The study design was a retrospective quantitative study. One hundred (consecutive) patients were divided in groups such as normal, Parkinson's, Parkinson's disease with combination of vascular changes and atypical Parkinson's disease. Isotope uptake in basal ganglia and parotic glands has been measured. Some patients' MRT images were available for quantification.     Result: the results show that the combined group of Parkinson's disease, atypical Parkinson's disease and vascular changes has an upregulation of the number of free dopamine receptors in parotid compared to healthy ones (also in PSP only and only vascular groups) with (p &lt;0.05).    Conclusion: There is variation of uptake between some of the groups thus the disease in the brain can be reflected in glands as well.     Keywords: Neurodegenerative Diseases, Autonomic Dysfunction, 11CPE2I-PET/CT, Parotid Glands, Basal Ganglia

Les Tunneling nanotubes (TNT) sont des protrusions cellulaires à base d'actine qui médient la communication cellulaire en transférant des cargos cellulaires. Les différents types de communication intercellulaires sont de plus en plus considérés comme des cibles potentielles pour le traitement de différentes maladies, telles que les maladies infectieuses liées aux virus et bactéries, les cancers ou les maladies neurodégénératives. Des études récentes ont mis en évidence un mécanisme de propagation d'agrégats protéiques ressemblant à la propagation du prion dans diverses maladies neurodégénératives non infectieuses telles que la maladie d'Alzheimer (AD), la démence frontotemporelle (FTD), la maladie de Parkinson (PD) et la maladie de Huntington. Ces maladies se caractérisent par l'accumulation de protéines mal repliées dans le cerveau des patients. Ainsi, on peut envisager de nouvelles stratégies thérapeutiques pour bloquer la propagation des protéines anormales dans tout le cerveau. Il a été démontré que les TNT pourraient jouer un rôle essentiel dans la propagation des agrégats de prions au sein du système nerveux central (SNC) et périphérique. Par conséquent, l'étude du mécanisme de la formation de TNT pourrait fournir de nouvelles idées sur le mécanisme de propagation de la maladie et de nouvelles cibles thérapeutiques. L'objectif de ma thèse était d'étudier le rôle du transfert des agrégats de protéines par les TNT entre les cellules et d'étudier le mécanisme de formation des TNT. Dans notre laboratoire, nous avons déjà montré que les TNT permettent le transfert de prions entre les cellules. Dans la première partie de mon doctorat, j'ai confirmé que les transferts d'agrégats de prions entre les cellules de CAD neuronales se faisaient par les TNT à l'intérieur de vésicules endocytiques (Zhu et al., 2015). De plus, en collaboration avec un collègue, nous avons fourni des preuves que les agrégats de prions pourraient être transférés entre des astrocytes primaires et des neurones et que ce transfert était médié par un contact cellulaire (Victoria et al., 2016). J'ai également collaboré à une autre étude où nous avons montré que les agrégats d'α-synucléine (caractéristiques de la maladie de Parkinson) peuvent être transférés entre les cellules à l'intérieur des lysosomes, et que ce transfert intercellulaire est médié par les TNT (Abounit et al., 2016). Dans mon deuxième projet, afin d'étudier le mécanisme de la formation de TNT, j'ai effectué un crible à haut débit pour les Rab GTPase. J'ai trouvé que Rab8 et Rab11 peuvent favoriser la formation des TNT, et que les cascades Rab8-VAMP3, Rab11-ERM et Rab8-Rab11 sont impliquées dans la formation des TNT. Mes données suggèrent que la polymérisation de l'actine et le trafic de membranes sont impliqués dans la formation des TNT. Ces résultats permettent d'éclairer le mécanisme de la formation des TNT et de fournir des preuves moléculaires que les Rab GTPases régulent ce processus. / Tunneling nanotubes are actin-based cell protrusions that mediate cell-to-cell communication by transferring cellular cargos. The different types of intercellular communication are increasing by being considered as potential targets for the treatment of various diseases, such as infectious diseases linked to viruses and bacteria, cancers or neurodegenerative diseases. Recent studies have highlighted a prion-like mechanism of propagation of protein misfolding in a variety of common, non-infectious, neurodegenerative diseases such as Alzheimer’s disease (AD), Frontotemporal dementia (FTD), Parkinson’s disease (PD), and Polyglutamine (PolyQ) diseases, which are characterized by the accumulation of misfolded proteins in the brain of patients. Thus, new therapeutic strategies to block propagation of protein misfolding throughout the brain can be envisaged. It has been shown that TNTs might play a critical role in spreading of prion aggregates within the CNS and from the periphery. Therefore, the study of mechanism of TNT formation could provide new insights on the mechanism of disease propagation and novel therapeutic targets. The aim of my thesis was to study the role of TNT-mediate protein aggregates transfer between cells and to investigate the mechanism of TNT formation. In our lab, we already reported TNT mediate prion transfer between cells. In the first part of my PhD, I further confirmed that prion aggregates transfer between neuronal CAD cells through TNT inside endocytic vesicles (Zhu et al., 2015). Furthermore in collaboration with a colleague, we provided evidences that prion aggregates could transfer between primary astrocytes and neurons and the transfer was mediated by cell-to-cell contact (Victoria et al., 2016). I also collaborated to another study where we showed that α-synuclein aggregates (Parkinson’s disease) can transfer between cells inside lysosomes, and the intercellular transfer is mediated by TNTs (Abounit et al., 2016).In my second project, in order to investigate the mechanism of TNT formation, I performed a High-content screening of Rab GTPase. I found that Rab8 and Rab11 can promote TNT formation, that Rab8-VAMP3, Rab11-ERM and Rab8-Rab11 cascades are involved in TNT formation. My data suggests that both actin polymerization and membrane trafficking are involved in TNT formation. These results help to shed light on the mechanism of TNT formation, and provide molecular evidences that Rab GTPases regulate this process.

Glia are the understudied brain cells that perform many functions essential to maintain nervous system homeostasis and protect the brain from injury. If brain damage occurs, glia rapidly adopt the reactive state and elicit a series of cellular and molecular events known as reactive gliosis, the hallmark of many neurodegenerative diseases. However, the molecular pathways that trigger and regulate this process remain poorly defined. The fruit fly Drosophila melanogaster has glial cells that are strikingly similar to mammalian glia, and which also exhibit reactive responses after neuronal injury. By exploiting its powerful genetic toolbox, we are uniquely positioned to identify the genes that activate and execute glial responses to neuronal injury in vivo. In this dissertation, I use Wallerian degeneration in Drosophila as a model to characterize molecular pathways responsible for glia to recognize neural injury, become activated, and ultimately engulf and degrade axonal debris. I demonstrate a novel role for the GEF (guanine nucleotide exchange factors) complex DRK/DOS/SOS upstream of small GTPase Rac1 in glial engulfment activity and show that it acts redundantly with previously discovered Crk/Mbc/dCed-12 to execute glial activation after axotomy. In addition, I discovered an exciting new role for the TNF receptor associated factor 4 (TRAF4) in glial response to axon injury. I find that interfering with TRAF4 and the downstream kinase misshapen (msn) function results in impaired glial activation and engulfment of axonal debris. Unexpectedly, I find that TRAF4 physically associates with engulfment receptor Draper – making TRAF4 only second factor to bind directly to Draper – and show it is essential for Draper-dependent activation of downstream engulfment signaling, including transcriptional activation of engulfment genes via the JNK and STAT transcriptional cascades. All of these pathways are highly conserved from Drosophila to mammals and most are known to be expressed in mouse brain glia, suggesting functional conservation. My work should therefore serve as an excellent starting point for future investigations regarding their roles in glial activation/reactive gliosis in various pathological conditions of the mammalian central nervous system.

Neurons comprise the main information processing cells of the nervous system. To integrate and transmit information, neurons elaborate dendritic structures to receive input and axons to relay that information to other cells. Due to their intricate structures, dendrites and axons are susceptible to damage whether by physical means or via disease mechanisms. Studying responses to axon injury, called Wallerian degeneration, in the neuronal processes of Drosophila melanogaster has allowed the identification of genes that are required for injury responses. Screens in Drosophila have identified dsarm and highwire as two genes required for axon degeneration; when these genes are mutated axons fail to degenerate after injury, even when completely cut off from the neuronal cell body. We found that these genes are also required for dendrite degeneration after injury in vivo. Further, we reveal differences between axon and dendrite injury responses using in vivo timelapse recordings and GCaMP indicators of intracellular and mitochondrial calcium transients. These data provide insights into the neuronal responses to injury, and better define novel targets for the treatment of neurodegenerative diseases.