Thèses sur le sujet « Caenorhabditis elegans – Système nerveux »

Cette étude sur la locomotion de C. Elegans vise à mieux comprendre le fonctionnement de son système nerveux et à apporter des éléments nouveaux de réflexion pour la conception de modèles ou d'objet biomimétiques. Ce travail débute par une description du ver, de sa physiologie ainsi que des principaux modes de locomotion connus : la nage (en milieu liquide) et la reptation (sur gel aqueux). Puis dans le cas de la nage, nous mettons en évidence une dissymétrie du mouvement, nécessaire pour la progression en milieu visqueux. L'analyse des vitesses des déplacements locaux permet de faire un bilan des forces exercées sur le ver, en admettant que celles-ci sont visqueuses. On montre ainsi que les coefficients de friction transverse et longitudinale peuvent être assimilés à ceux d'un ellipsoïde allongé. Dans le cas du mode reptation, on observe une diminution de l'amplitude de la tête vers la queue. L'interaction ver-substrat est abordée d'abord théoriquement (hypothèse de lubrification). Il en résulte des prédictions pour les coefficients de friction en désaccord avec les résultats expérimentaux. Ce désaccord est expliqué par la mise en évidence de seuils de friction statique. On mesure aussi la rigidité passive d'un ver. Un confinement vertical du ver en milieu liquide permet d'observer une transition continue de la nage vers la reptation. On montre que la période ainsi que le déphasage entre les mouvements de la tête et la queue augmentent avec le placage. Un confinement horizontal du ver sur substrat permet de contraindre l'amplitude. On montre que la longueur d'onde diminue avec l'amplitude
This study on the locomotion of C. Elegans aims at a better understanding of its nervous system and at giving birth to news ideas concerning the conception of new biometics models or objects. We first gave a description of the worm, of its physiology, and of its main modes of locomotion, that is to say the swimming - in liquid medium, and the crawling - on gel substract. When swimming, we analyzed how the dissymmetry pf the movement is necessary for the worm to move on when in viscous medium. Thanks to the analysis of the velocity of the local displacements and by supposing that the forces are viscous, we balanced the forces. We thus demonstrated that transversal and longitudinal friction coefficients could be compared to the coefficients obtained theoretically from an oblong ellipsoïd. When crawling, we were able to observe a diminution of the amplitude from the head to the tail. We first studied the worm-substract interaction theorically - lubrification hypothesis, but the friction coefficients predicted were in contradiction with experimental results. This difference, according to our experiments, was due to static friction. We also measured the rigidity of the worm. By confining the worm vertically in liquid medium, we observed a continuous transition from swimming to — crawling. We proved that the movement of the tail, in comparison with the movement of the head, was more and more delayed as the confinement increased. In these conditions, the global movement of the worm got slower. On substract, we were able to constrain the amplitude thanks to a horizontal confinement; we observed that wavelength decreased with amplitude

L‟épissage alternatif est un mécanisme de régulation de l‟expression des gènes ayant pris une importance croissante dans l‟étude du vivant. Si des méthodes existent pour déterminer les gènes qui y sont soumis, peu d‟outils sont disponibles pour suivre ces événements d‟épissage in vivo au cours du développement. Pourtant, la caractérisation des régulations sous-jacentes à ces évènements et la détermination des facteurs impliqués sont dépendantes de stratégies fiables pour les visualiser dans des conditions physiologiques.Nous avons développé un système adapté à l‟étude d‟événements d‟épissage basé sur un rapporteur fluorescent bicolore. Nous l‟avons appliqué à cinq gènes de l‟organisme modèle Caenorhabditis elegans et avons suivi leur épissage in vivo.Parmi les différents gènes suivis, deux d‟entre eux suivaient un modèle d‟épissage potentiellement stochastique, un autre une absence d‟épissage alternatif détectable. Les deux derniers gènes présentent un profil d‟épissage spécifique à certain types cellulaires mais ont un effet toxique sur l‟organisme lorsque nous les avons exprimés à partir de concatémères extrachromosomiques. Pour remédier à cela, nous avons choisi de mettre en place une méthode simplifiée d‟insertion en simple copie des rapporteurs utilisant le CRISPR-Cas.Nos résultats indiquent que le système rapporteur fonctionne avec succès. Cependant, il peut encore être amélioré pour se rapprocher des taux physiologiques de transcription grâce à une insertion en simple copie dans le génome de l‟organisme. Nous avons également révélé un événement sous le contrôle de régulations spatiales, temporelles et conditionnelles. De plus, nous avons créé une série de constructions capables de déterminer les éléments en cis impliqués dans la régulation du gène top-1
Alternative splicing is a regulatory mechanism of gene expression which is increasingly studied in Life Science. Methods exist to study this mechanism but specific tools to follow each alternative splicing event in a spatio-temporal manner are lacking. Yet, the characterization of the regulation and the elements that determines them depends on valide strategies for visualising them in physiological conditions.We have developped a dual-fluorescent reporter-based system in order to follow alternative splicing event regulation in vivo. It has been applied to five different genes in the model organism Caenorhabditis elegans. Among the genes followed, two follow a potentially stochastic scheme, one show no visible sign of alternative splicing. The last display tissue specific splicing patterns but developed a toxic effect in the animal when expressed from a multicopy extrachromosomal array. To remediate this problem, we decided to develop a method that allows for simpler single copy insertion of fluorescent reporter using CRISPR-Cas.Our results indicates that the dual-fluorescent reporter works well. However, this system can be upgraded by getting close to physiological rates of transcription allowed by single-copy insertion in the genome of C.elegans. We also discovered an alternatiove splicing event which follows a spatial, temporal and conditionnal regulation. Moreover, we constructed a set of different reporter to unravel the regulation observed in the gene top-1

L'étude des systèmes vivants est notoirement difficile. La complexité déconcertante des systèmes biologiques, souvent citée, est principalement due à la complexité de leurs interactions, à leurs multiples niveaux d'organisation et à leur nature dynamique. Dans la quête de compréhension de cette complexité, les parallèles établis avec la physique standard - en particulier la physique statistique - sont à la fois utiles et d'une utilité limitée. D'une part, ils fournissent un riche ensemble d'éléments théoriques et méthodologiques pour construire des théories et concevoir des expériences. D'autre part, la vie biologique se déroule aussi selon des principes qui sont sans équivalent dans la physique de la matière conventionnelle. Une différence cruciale réside dans la notion de fonction : les systèmes biologiques sont façonnés par la nécessité d'accomplir des tâches spécifiques. Un problème général pour les systèmes vivants est de trouver et de promouvoir les configurations qui produisent des fonctions améliorées ou optimales, ce que nous appelons le problème de l'exploration-exploitation (EE). Un exemple spécifique de ce problème se trouve dans la biologie évolutive. Dans ce cas, des mutations génétiques aléatoires soutiennent l'exploration de l'espace de configuration, celles qui correspondent à un succès reproductif plus élevé étant favorisées par la sélection naturelle. Inspirés par ce dernier cas, nous développons un nouveau formalisme qui encode une dynamique générale d'exploration-exploitation pour les réseaux biologiques, représentée comme une exploration d'un paysage fonctionnel. En particulier, notre dynamique d'EE consiste en des changements de configuration stochastiques combinés à l'optimisation dépendante de l'état d'une fonction objective (métrique F). Nous commençons par étudier ses principales caractéristiques à travers l'étude de paysages fonctionnels simples et analytiquement traitables. Nous déployons des simulations pour des applications plus générales et plus complexes. Nous nous penchons ensuite sur le problème du câblage du cerveau, c'est-à-dire le développement du système nerveux d'un individu tout au long de sa vie. Nous soutenons que ce dernier est un autre exemple spécifique du problème de l'EE et qu'il peut donc être traité à l'aide de notre cadre théorique. En particulier, nous nous concentrons sur la maturation du cerveau chez le nématode C. elegans, le seul organisme pour lequel un réseau complet de neurones et de connexions neuronales a été reconstruit, à plusieurs moments du développement. Nous fixons le réseau à la naissance et utilisons le stade adulte pour déduire (i) une description max.ent. parcimonieuse (ERG) de la métrique F pour le cerveau du ver et (ii) les deux paramètres de notre dynamique EE. Selon la topographie de son paysage fonctionnel, le cerveau adulte est caractérisé par une tendance à former des triades et des nœuds de degré supérieur. Nous montrons que notre dynamique d'EE dans un tel paysage est capable de retracer toute l'histoire du développement. En particulier, nous montrons que la trajectoire que nous obtenons reproduit étroitement les autres points temporels expérimentaux que nous n'avons pas utilisés pour l'inférence. Ceci est vrai à la fois dans l'espace des statistiques du modèle et pour un certain nombre d'autres propriétés du réseau. En outre, nous discutons d'une interprétation micro-niveau de la dynamique de l'EE en termes de processus sous-jacent de formation des synapses. Notre étude est un premier pas vers la compréhension au niveau du système du développement d'un cerveau naturel et peut être étendue (i) à des paysages fonctionnels plus complexes, (ii) à d'autres organismes que le C. elegans et (iii) à d'autres problèmes que le câblage du cerveau. En effet, nous pensons que le paradigme de l'exploration-exploitation fait partie de ces principes spécifiques à la vie que nous commençons à peine à découvrir
The study of living systems is notoriously challenging. The often-quoted daunting complexity of biological systems is primarily due to the intricacies of their interactions, their multiple organisation levels and their dynamic nature. In the quest to understand this complexity, parallels drawn with standard physics – in particular, statistical physics -- are both useful and of limited use. On the one hand, they provide a rich set of theoretical and methodological building blocks for constructing theories and designing experiments. On the other hand, life also unfolds according to principles that are unparalleled in the physics of conventional matter. A crucial difference lies in the notion of function: biological systems are shaped by the need to perform specific tasks. A general problem for living systems is to find and promote those configurations that yield improved or optimal functions, we call this the exploration-exploitation (EE) problem. One specific instance of the above is found in evolutionary biology. There, random genetic mutations sustain the exploration of the configuration space, with those leading to higher reproductive success being favoured by natural selection. Inspired by the latter, we develop a novel formalism that encodes a general exploration-exploitation dynamics for biological networks. In particular, our EE dynamics is represented as an exploration of a functional landscape and consists of stochastic configuration changes combined with the state-dependent optimisation of an objective function (F metric). We begin by investigating its main features through the study of simple, analytically tractable functional landscapes. We deploy simulations for more general and complex applications. We then turn to the brain wiring problem, i.e., the development of an individual's nervous system during its early life. We argue that this is another specific instance of the EE problem and therefore can be addressed by using our theoretical framework. In particular, we focus on brain maturation in the nematode C.elegans, the only organism for which a complete network of neurons and neuronal connections has been reconstructed, at multiple developmental time points (seven). We fix the network at birth and use the adult stage to infer (i) a parsimonious maxent (ERG) description of the F metric for the worm brain and (ii) the two parameters of our EE dynamics. According to the topography of its functional landscape, the adult brain is characterised by a tendency to form both triads and high degree nodes. We demonstrate that our EE dynamics in such landscape is capable of tracking down the entire developmental history. In particular, we show that the trajectory we obtain closely reproduces the other experimental time points that we did not use for inference. This is true both in the space of model statistics and for a number of other network properties. Additionally, we discuss a micro-level interpretation of the EE dynamics in terms of the underlying synapse formation process. Our study is a first step towards the system-level understanding of the development of a natural brain and can be extended (i) to encompass more complex functional landscapes, (ii) to different organisms than the C. elegans and (iii) to several different problems than the brain wiring. Indeed, we posit that the exploration-exploitation paradigm is among those life-specific principles that we are just beginning to uncover

TRPV channels play a role in both mammalian insulin signaling, with TRPV1 expression in pancreatic beta-cells, and in C. elegans insulin-like signaling through expression of OSM-9, OCR-1, and OCR-2 in stress response pathways. In response to a glucose-supplemented diet, C. elegans are know to have sensitivity to anoxic stress, exhibit chemotaxis attraction, and display reduced egg-laying rate. Transcriptome analysis reveals that glucose stimulates nervous system activity with increased transcript levels of genes regulating neurotransmitters. Ciliated sensory neurons are needed for a reduced egg-laying phenotype on a glucose-supplemented diet. Egg-laying rate is not affected when worms graze on glucose-supplemented Delta-PTS OP50 E. coli, which is defective in glucose uptake. This suggests a possible sensory neuron obstruction by exopolysaccharides produced by standard OP50 E. coli on glucose, eliciting a starvation response from the worm and causing reduced egg-laying rate. Glucose chemotaxis is affected in specific TRPV subunit allele mutants: ocr-2(vs29) and osm-9(yz6), serotonin receptor mutants: ser-1(ok345) and mod-1(ok103), and G-alpha protein mutant: gpa-10(pk362). TRPV deletion mutants had no effect on glucose chemotaxis, alluding to the modality role pf TRPV alleles in specific sensory neurons. The role of serotonin in a reduced egg-laying rate with glucose remains unclear.

Though symptoms such as loss of vision, decline in cognition and memory are evident during aging, the underlying processes that affect neuronal function during aging are not well understood. Unlike changes in other tissues and organs, age-related changes in the nervous system affect the overall physical, mental as well as social state of human beings. To start elucidating the molecular mechanisms underlying normal age-dependent brain decline, we have characterized structural neuronal changes occurring during Caenorhabditis elegans aging. Our analysis reveals distinct neuronal alterations that arise with age and that the types of changes and their age of onset are neuronal-type specific, highlighting the differential susceptibility of neurons to the stresses of life. We also find that these age-dependent neuronal changes are largely uncoupled from lifespan. As a first step towards understanding the neuropathological conditions manifested during senescence, we have characterized the role of the neuronal maintenance gene sax-7/L1CAM in normal C. elegans aging. Our comparison of age-related structural changes in the wild-type nervous system with that of sax-7 mutants, indicates that loss of function of sax-7 results in accelerated neuronal deterioration that mimics alterations occurring during normal aging. Conversely, overexpressing wild-type copies of SAX-7 delays some of the neuronal changes that accompany normal aging, indicating that SAX-7 plays a neuroprotective role. Additionally we find that x mechanical stress from body movements impacts the neuronal changes during adulthood. Taken together, our results give an entry point into the mechanisms of age-related neuroanatomical changes and neuronal protection.

Though symptoms such as loss of vision, decline in cognition and memory are evident during aging, the underlying processes that affect neuronal function during aging are not well understood. Unlike changes in other tissues and organs, age-related changes in the nervous system affect the overall physical, mental as well as social state of human beings. To start elucidating the molecular mechanisms underlying normal age-dependent brain decline, we have characterized structural neuronal changes occurring during Caenorhabditis elegans aging. Our analysis reveals distinct neuronal alterations that arise with age and that the types of changes and their age of onset are neuronal-type specific, highlighting the differential susceptibility of neurons to the stresses of life. We also find that these age-dependent neuronal changes are largely uncoupled from lifespan. As a first step towards understanding the neuropathological conditions manifested during senescence, we have characterized the role of the neuronal maintenance gene sax-7/L1CAM in normal C. elegans aging. Our comparison of age-related structural changes in the wild-type nervous system with that of sax-7 mutants, indicates that loss of function of sax-7 results in accelerated neuronal deterioration that mimics alterations occurring during normal aging. Conversely, overexpressing wild-type copies of SAX-7 delays some of the neuronal changes that accompany normal aging, indicating that SAX-7 plays a neuroprotective role. Additionally we find that x mechanical stress from body movements impacts the neuronal changes during adulthood. Taken together, our results give an entry point into the mechanisms of age-related neuroanatomical changes and neuronal protection.

Caenorhabditis elegans fait partie des modèles animaux les plus étudiés en laboratoire. La découverte des premiers virus infectant naturellement des Caenorhabditis apporte de nouveaux modèles animal-virus très prometteurs.Le virus d'Orsay, infectant spécifiquement C. elegans, et le virus de Santeuil, infectant spécifiquement C. briggsae, sont des virus à ARN positif simple brins (Hépatites A, C et E, Chikungunya, Coronavirus etc¿) qui provoquent une désorganisation de la structure des cellules intestinales de leur hôte. Par ailleurs, une variation dans la sensibilité à ce virus au sein des espèces est observée. Grâce à une étude statistique d'association pangénomique portant sur la sensibilité au virus d'Orsay de 97 isolats naturels de C. elegans, nous avons pu mettre en évidence une région chromosomique qui contient un polymorphisme responsable de cette sensibilité. Il s'agit une délétion dans le gène drh-1 qui code une protéine similaire à la protéine RIG-I, connue pour initier une réponse antivirale chez les vertébrés. Cependant, C. elegans est incapable de produire la même réponse antivirale que ces derniers. Ainsi, DRH-1 est impliquée dans la reconnaissance des ARN viraux et dans la production d'une réponse spécifique à l'infection virale par les petits ARN. Les gènes impliqués dans l'immunité sont soumis à une forte pression de sélection. Pourtant, de manière surprenante, la délétion du gène drh-1 est présente dans 23% des isolats étudiés. Cette délétion est génétiquement liée à une région plus large de 3 Mb. Cependant, au laboratoire, cette région n’apporte pas d’avantage sélectif qui expliquerait comment cette délétion peut se propager dans les populations
Caenorhabditis elegans is a commonly studied animal model in laboratories. The discovery ofthe first natural viral infections of Caenorhabditis brings new models to study animal-virusinteractions.The Orsay virus, specifically infecting C. elegans, and the Santeuil virus, specificallyinfecting C. briggsae, are positive single strand RNA viruses (Hepatites, Chikungunya,Coronavirus etc…) disrupting the structure of intestinal cells of their host. However, weobserved a strong variability in the sensitivity to those viruses at the intraspecific level.To identify the genetic basis of the sensitivity, we performed a genome wide association studyon 97 wild isolates of C. elegans. We were able to identify the center of chromosome IV as aregion containing the locus responsible for this sensitivity. A deletion in the drh-1 gene,coding for a RIG-I-Like protein, confers sensitivity to their carrier. RIG-I is known torecognize viral RNA and to trigger an antiviral response through the production of interferonsin vertebrates. However, C. elegans is not able to produce interferons but it appears thatDRH-1 initiates a viral specific siRNA pathway.Immunity genes are under strong selective pressure. Thus, it is surprising that such animportant protein for the antiviral pathway appears to be disrupted in 23% of the wild isolates.This deletion shows high linkage disequilibrium with a broader region of 3Mb, suggestingthat the deletion propagates with this region. However, this region does not seem to provideany advantage to their owner under laboratory conditions

How does a nervous system orchestrate compound behaviors? Finding the neural basis of behavior requires knowing which neurons control the behavior and how they are connected. To accomplish this we measured and manipulated neural activity in a live, behaving animal with a completely defined connectome. The C. elegans escape response is a compound behavior consisting of a sequence of behavioral motifs. Gentle touch induces a reversal and suppression of head movements, followed by a deep turn allowing the animal to navigate away from the stimulus. The connectome provides a framework for the neural circuit that controls this behavior. We used optical physiology to determine the activity patterns of individual neurons during the behavior. Calcium imaging of locomotion interneurons and motor neurons reveal unique activity profiles during different motifs of the escape response. Furthermore, we used optogenetics and laser ablations to determine the contribution of individual neurons to each motif. We show these that the suppression of head movements and turning motifs are distinct motor programs and can be uncoupled from the reversal. The molecular mechanisms that regulate these motifs involve from signaling with the neurotransmitter tyramine. Tyramine signaling and gap junctions between locomotion interneurons and motor neurons regulate the temporal orchestration of the turning motif with the reversal. Additionally, tyramine signaling through a GPCR in GABAergic neurons facilitates the asymmetric turning during forward viii locomotion. The combination of optical tools and genetics allows us to dissect a how a neural circuit converts sensory information into a compound behavior.

How does a nervous system orchestrate compound behaviors? Finding the neural basis of behavior requires knowing which neurons control the behavior and how they are connected. To accomplish this we measured and manipulated neural activity in a live, behaving animal with a completely defined connectome. The C. elegans escape response is a compound behavior consisting of a sequence of behavioral motifs. Gentle touch induces a reversal and suppression of head movements, followed by a deep turn allowing the animal to navigate away from the stimulus. The connectome provides a framework for the neural circuit that controls this behavior. We used optical physiology to determine the activity patterns of individual neurons during the behavior. Calcium imaging of locomotion interneurons and motor neurons reveal unique activity profiles during different motifs of the escape response. Furthermore, we used optogenetics and laser ablations to determine the contribution of individual neurons to each motif. We show these that the suppression of head movements and turning motifs are distinct motor programs and can be uncoupled from the reversal. The molecular mechanisms that regulate these motifs involve from signaling with the neurotransmitter tyramine. Tyramine signaling and gap junctions between locomotion interneurons and motor neurons regulate the temporal orchestration of the turning motif with the reversal. Additionally, tyramine signaling through a GPCR in GABAergic neurons facilitates the asymmetric turning during forward viii locomotion. The combination of optical tools and genetics allows us to dissect a how a neural circuit converts sensory information into a compound behavior.

Le développement de Caenorhabditis elegans est finement régulé et aboutit au nombre constant de 959 cellules par individu. Une partie de ces régulations repose sur les microARN, ARN simple brin d’environ 22 nucléotides. Ils peuvent diminuer l'expression des gènes en ciblant des séquences homologues dans les régions 3' non transcrites (3'UTR) des ARN messagers. Pour déterminer l'impact de cette régulation, nous utilisons une méthode permettant de visualiser in vivo une différence d'expression induite post-transcriptionnellement. Cette méthode se base sur l'utilisation de deux protéines fluorescentes : la GFP (verte) et la mCherry (rouge) exprimées sous le contrôle du promoteur du gène d'intérêt. La mCherry est suivie par un 3'UTR contrôle et reflète l'activité du promoteur. La GFP est suivie par le 3'UTR du gène d'intérêt et subit les mêmes régulations que le gène endogène. La différence d'expression entre les deux protéines devrait donc refléter la régulation s'opérant sur le 3'UTR du gène. La transparence de C. elegans permet de localiser les protéines rapportrices par microscopie en fluorescence à tous les stades du développement dans l'organisme entier. Initialement, le 3'UTR du gène unc-54 (myosine de type II) a été choisi comme contrôle. Le rapporteur bicolore a révélé une régulation s’opérant sur unc-54 3’UTR jusqu’ici considérée comme permissif. La régulation observée s’opère dans les muscles et les neurones ADF. La caractérisation de cette régulation a permis de mettre en évidence le rôle potentiel de mir-1820. Le profil d’expression de mir-1820 a pu être établi grâce à une fusion avec la GFP et correspond au profil des régulations observées sur unc-54 3’UTR
Caenorhabditis elegans development is very tightly regulated, leading to the same number of cells in each individual. Part of this regulation network relies on small single strand RNAs (miRNAs), which can target homologous sequences in the 3’ untranslated regions (3’UTR) of messenger RNAs. We want to investigate the contribution of miRNAs during neurons differentiation. In order to study the miRNA contribution to gene regulation we use double fluorescent reporters that allow us to visualize the posttranscriptional contribution to regulation throughout development. The GFP and the mCherry are expressed under the control of the gene promoter, but followed by either the 3’UTR of interest, or a control 3’UTR. We first chose as a control 3’UTR the unc-54 3’UTR (myosin class II). The gene unc-54 is expressed through all larval stages and in the adult worms. The two colors reporter system showed that unc-54 3’UTR undergoes a regulation in the ADF pair of neurons and partially in the body wall muscle. The characterization of this regulation pointed out a potential role for mir-1820. The GFP was cloned between mir1820 5’ and 3’ sequences and the construction displayed an expression profile overlapping with the regulation pattern observed on unc-54 3’UTR

Alzheimer’s disease (AD) is a neurodegenerative disease and the most common form of dementia associated with amyloid-beta peptide deposition and loss of mitochondrial function and regulation. Currently, there is no cure for AD, thus, there is a need to continuously develop therapeutic strategies that could address the complex multifactorial causes of AD development. Due to this necessity, this study has investigated the role of vitamin B2 as a disease modifying drug for AD by employingamyloid-beta and mitochondrial based AD therapeutic strategies. Using a transgenic C. elegans AD worm model expressing amyloid-beta (Aβ1-42) in muscle cells at temperature upshift to 25°C, we screened for protective effect of dose-dependent concentrations of active forms of vitamin B2, FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide), against amyloid-beta mediated paralysis. Protective concentrations were then assayed for improvement of mitochondrial metabolic functions by performing ATP, oxygen consumption and reactive oxygen species (ROS) production assays. Consequently, we investigated for drug protective mechanisms of FMN and FAD using RNAi genetic screening technique. FMN and FAD significantly delayed amyloid-beta mediated paralysis and improved mitochondrial metabolic functions at final concentrations of 0.74mM and 0.74µM respectively. More so, both compounds induced activation of stress response FOXO transcription factor, daf-16. Specifically, FMN treatment induced mitochondrial unfolded protein response (UPRmt) pathway through ubiquitin-like protein (ubl-5) activation as well as other stress response pathway signature such as Activating Transcription Factor Associated with Stress (atfs-1). This study will be useful in understanding the importance of micronutrients such as vitamin B2 in normal cellular function as related to neurodegenerativediseases and aging. Therefore, vitamin B2 supplementation could be an important source of Alzheimer’s disease therapeutic strategy.

Alzheimer’s disease (AD) is associated with amyloid-beta peptide deposition and loss of mitochondrial function. Using a transgenic C. elegans AD worm model expressing amyloid-beta in body wall muscle, we determined that supplementation with either of the forms of vitamin B2, flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) protected against amyloid-beta mediated paralysis. FMN and FAD were then assayed to determine effects on ATP, oxygen consumption, and reactive oxygen species (ROS) with these compounds not significantly improving any of these mitochondrial bioenergetic functions. Knockdown of the daf-16/FOXO transcriptional regulator or the FAD synthase enzyme completely abrogated the protective effects of FMN and FAD, while knockdown of the mitochondrial unfolded protein response factors ubl-5 or atfs-1 also blocked the protective effects. Therefore, vitamin B2 supplementation could lead to the activation of conserved signaling pathways in humans to delay the onset and progression of neurodegenerative diseases such as AD.

Neuronal migration is an essential aspect of nervous system development; improper or incomplete neuronal migration can lead to debilitating disorders. The model organism Caenorhabditis elegans has 302 neurons and is ideal for studying nervous system development. The cytoplasmic adaptor protein, MIG-10, is necessary for the long range anteroposterior migration during embryogenesis of the neurons CAN, ALM, and HSN. Mutations in the mig-10 gene result in incomplete migrations of all three neurons. MIG-10 is a homologue of the vertebrate proteins lamellipodin and RIAM-1, which are involved in directing actin polymerization during axon outgrowth and guidance. RIAM-1 is known to interact with proteins from the Ras GTPase family. The MIG-10 protein has a pleckstrin homology (PH) domain, a Ras-associating (RA) domain, and a proline-rich region. We used a yeast two-hybrid system to investigate which Ras family proteins MIG-10 interacts with. Three isoforms of MIG-10, MIG-10A, MIG-10B, and MIG-10C, as well as the RAPH domain alone, were used as baits. No evidence of interaction was observed for any of the baits used. These results do not reject our hypothesis as the constitutively active Ras clones may need to be used or there may not be a direct interaction between MIG-10 and the Ras family members. We are currently screening a C. elegans cDNA library for interactions with all three isoforms of MIG-10. In the future we plan to investigate how MIG-10 may be involved in the WAVE/SCAR actin nucleation pathway.

Caenorhabditis elegans fait partie des modèles animaux les plus étudiés en laboratoire. La découverte des premiers virus infectant naturellement des Caenorhabditis apporte de nouveaux modèles animal-virus très prometteurs.Le virus d'Orsay, infectant spécifiquement C. elegans, et le virus de Santeuil, infectant spécifiquement C. briggsae, sont des virus à ARN positif simple brins (Hépatites A, C et E, Chikungunya, Coronavirus etc¿) qui provoquent une désorganisation de la structure des cellules intestinales de leur hôte. Par ailleurs, une variation dans la sensibilité à ce virus au sein des espèces est observée. Grâce à une étude statistique d'association pangénomique portant sur la sensibilité au virus d'Orsay de 97 isolats naturels de C. elegans, nous avons pu mettre en évidence une région chromosomique qui contient un polymorphisme responsable de cette sensibilité. Il s'agit une délétion dans le gène drh-1 qui code une protéine similaire à la protéine RIG-I, connue pour initier une réponse antivirale chez les vertébrés. Cependant, C. elegans est incapable de produire la même réponse antivirale que ces derniers. Ainsi, DRH-1 est impliquée dans la reconnaissance des ARN viraux et dans la production d'une réponse spécifique à l'infection virale par les petits ARN. Les gènes impliqués dans l'immunité sont soumis à une forte pression de sélection. Pourtant, de manière surprenante, la délétion du gène drh-1 est présente dans 23% des isolats étudiés. Cette délétion est génétiquement liée à une région plus large de 3 Mb. Cependant, au laboratoire, cette région n’apporte pas d’avantage sélectif qui expliquerait comment cette délétion peut se propager dans les populations
Caenorhabditis elegans is a commonly studied animal model in laboratories. The discovery ofthe first natural viral infections of Caenorhabditis brings new models to study animal-virusinteractions.The Orsay virus, specifically infecting C. elegans, and the Santeuil virus, specificallyinfecting C. briggsae, are positive single strand RNA viruses (Hepatites, Chikungunya,Coronavirus etc…) disrupting the structure of intestinal cells of their host. However, weobserved a strong variability in the sensitivity to those viruses at the intraspecific level.To identify the genetic basis of the sensitivity, we performed a genome wide association studyon 97 wild isolates of C. elegans. We were able to identify the center of chromosome IV as aregion containing the locus responsible for this sensitivity. A deletion in the drh-1 gene,coding for a RIG-I-Like protein, confers sensitivity to their carrier. RIG-I is known torecognize viral RNA and to trigger an antiviral response through the production of interferonsin vertebrates. However, C. elegans is not able to produce interferons but it appears thatDRH-1 initiates a viral specific siRNA pathway.Immunity genes are under strong selective pressure. Thus, it is surprising that such animportant protein for the antiviral pathway appears to be disrupted in 23% of the wild isolates.This deletion shows high linkage disequilibrium with a broader region of 3Mb, suggestingthat the deletion propagates with this region. However, this region does not seem to provideany advantage to their owner under laboratory conditions

Alzheimer’s disease (AD) is symptomized by amyloid-beta plaques in the brain and accounts for more than 65 percent of dementia cases. Vitamin B12 (cobalamin) deficiency can result in similar cognitive impairment and roughly 15% of the elderly are vitamin B12 deficient. Vitamin B12 deficiency results in the accumulation of toxic methylmalonic acid and homocysteine. Hyperhomocysteinemia is a strong risk factor for AD. To test if vitamin B12 deficiency stimulates amyloid-beta toxicity, Caenorhabditis elegans expressing amyloid-beta in muscle were fed either vitamin B12-deficient OP50-1 or vitamin B12-rich HT115(DE3) E. coli bacteria. Increased amyloid-beta toxicity was found in worms fed the 0P50-1 diet. Supplementation of the OP50-1 diet with vitamin B12 did not rescue the increased C. elegans toxicity. Knockdown of either of the only two C. elegans vitamin B12-dependent enzymes metr-1 or mmmc-1 protected against toxicity. Therefore, vitamin B12 deficiency does not stimulate Alzheimer’s amyloid-beta-mediated toxicity in C. elegans.

The generation of complex behaviors often requires the coordinated activity of diverse sets of neural circuits in the brain. Activation of neuronal circuits drives behavior. Inappropriate signaling can contribute to cognitive disorders such as epilepsy, Parkinson’s, and addiction (Nordberg et al., 1992; Quik and McIntosh, 2006; Steinlein et al., 2012). The molecular mechanisms by which the activity of neural circuits is coordinated remain unclear. What are the molecules that regulate the timing of neural circuit activation and how is signaling between various neural circuits achieved? While much work has attempted to address these points, answers to these questions have been difficult to ascertain, in part owing to the diversity of molecules involved and the complex connectivity patterns of neural circuits in the mammalian brain. My thesis work addresses these questions in the context of the nervous system of an invertebrate model organism, the nematode Caenorhabditis elegans. The locomotory circuit contains two subsets of motor neurons, excitatory and inhibitory, and the body wall muscle. Dyadic synapses from excitatory neurons coordinate the simultaneous activation of inhibitory neurons and body wall muscle. Here I identify a distinct class of ionotropic acetylcholine receptors (ACR-12R) that are expressed in GABA neurons and contain the subunit ACR-12. ACR-12R localize to synapses of GABA neurons and facilitate consistent body bend amplitude across consecutive body bends. ACR-12Rs regulate GABA neuron activity under conditions of elevated ACh release. This is in contrast to the diffuse and modulatory role of ACR-12 containing receptors expressed in cholinergic motor neurons (ACR-2R) (Barbagallo et al., 2010; Jospin et al., 2009). Additionally, I show transgenic animals expressing ACR-12 with a mutation in the second transmembrane domain [ACR-12(V/S)] results in spontaneous contractions. Unexpectedly, I found expression of ACR-12 (V/S) results in the preferential toxicity of GABA neurons. Interestingly loss of presynaptic GABA neurons did not have any obvious effects on inhibitory NMJ receptor localization. Together, my thesis work demonstrates the diverse roles of nicotinic acetylcholine receptors (nAChRs) in the regulation of neuronal activity that underlies nematode movement. The findings presented here are broadly applicable to the mechanisms of cholinergic signaling in vertebrate models.

Le système nerveux est un réseau complexe qui détecte et analyse les informations. Ces informations sont transmises entre cellules grâce à des connections synaptiques et des jonctions communicantes. Ce réseau n’est pas statique et évolue au cours du développement, de l’apprentissage mais aussi durant le processus de vieillissement – naturels ou pathologiques. Comprendre le système nerveux et son fonctionnement requiert une analyse des connections synaptiques in vivo chez un animal model tel que Caenorhabditis elegans. Cependant les techniques actuellement disponibles pour C. elegans sont laborieuses, ne dépendent pas forcément d’une interaction trans-synaptique ou fixent la synapse. Par conséquent, ces approches ne permettent pas de réaliser des études de populations et dynamiques des modifications synaptiques. Dans ce manuscrit, je décris tout d’abord une nouvelle technique pour visualiser les synapses in vivo chez le vers C. elegans. Cette technique appelé iBLINC (in vivo Biotin Labeling of INtercellular Contacts) qui consiste en la biotinylation d’un peptide par une ligase d’Escherichia coli, BirA. Ces deux molécules sont fusionnées à des protéines trans-membranaires qui forment un complexe à la synapse. La biotinylation est détectée grâce à une streptavidin monomérique taguée avec un fluorophore qui est secrétée dans l’espace extracellulaire. J’ai démontré que cette technique est directionnelle et dynamique. En utilisant iBLINC pour visualiser des synapses faisant partie du circuit sensoriel de C. elegans, une évolution du nombre et de la taille des synapses a pu être observée avec l’âge. Il semblerait que ce changement soit dépendant du segment de la zone synaptique observée. Ces résultats ont été corroborés par l’observation d’une diminution du nombre de vésicules pendant le vieillissement grâce à un marqueur pré-synaptique des synapses GABAergique de la jonction neuromusculaire. Pour conclure, ce manuscrit décrit une nouvelle technique permettant d’observer les synapses chez le vers vivant et démontre une évolution naturelle du nombre de synapses et du nombre de vésicules pré-synaptiques avec l’âge
The nervous system is a complex network that senses and processes information and is essential for the survival of both vertebrates and invertebrates as it is involved in behavior responses. Information within the network is transmitted through specialized cell-cell contacts, including synaptic connections. Importantly, the network is not static and is believed to change during development and learning, as well as during pathological or normal age-related decline. Studying the nervous system in vivo requires the use of animal models such as Caenorhabditis elegans. Understanding of behavior and development requires visualization and analysis of synaptic connectivity. However, existing methods are laborious and may not depend on trans-synaptic interactions, or otherwise ‘trap’ the synapses by fixing the connections, thus precluding dynamic studies. In order to study synaptic modifications, we developed a new transgenic approach for in vivo labeling of specific connections in C. elegans, called iBLINC (in vivo Biotin Labeling of INtercellular Contacts). iBLINC involves the biotinylation of an acceptor peptide (AP) by the Escherichia coli biotin ligase BirA. Both are fused to two interacting post- and pre-synaptic proteins, respectively. The biotinylated acceptor peptide fusion is detected by a monomer streptavidin fused to a fluorescent protein that is transgenically expressed and secreted into the extracellular space. The method is directional, bright and dynamic. Moreover it correlates well with electron micrograph reconstruction. Using this new technique to observe synapses, which are part of the thermosensory circuitry of C. elegans, during aging, we could conclude that the connection pattern varies with age and within a population. Changes of the number and size of the synapses were observed during aging. Some segments of the synaptic region seem to be more affected than others by the aging process. Those results were corroborated by using a GABAergic pre-synaptic marker which allowed us to visualize a decline of the vesicle number with aging. In summary, in this thesis I explained a new in vivo trans-synaptic method to visualize synapses in C. elegans. Then I demonstrated that a natural decline in the number of synapses as well as the number of vesicles occurs during aging

Afin de caractériser les voies de signalisation du système immunitaire inné, nous étudions l'interaction entre le ver C. elegans et le champignon Drechmeria coniospora. Une des réponses du ver à l'infection consiste en une augmentation de la production de peptides antimicrobiens (PAM) dans l'épiderme. Des vers transgéniques exprimant le gène rapporteur de la GFP sous le contrôle du promoteur d'un PAM, fluorescent vert après infection. Si un gène nécessaire à l'expression des PAM est inactivé, alors les vers transgéniques ne fluorescent plus après infection. Nous avons effectué un crible pour identifier les molécules de signalisation nécessaires à l'expression des PAM en utilisant une approche quantitative et semi-automatique par ARN interference (ARNi). Deux banques d'ARNi couvrant 95% du génome, soit 20 000 gènes, ont été criblées et 360 candidats bloquant l'induction de la GFP après infection ont été obtenus, correspondant à 343 gènes. Une caractérisation phénotypique a permis de placer les candidats dans différentes catégories fonctionnelles et permis d'identifier d'une part un récepteur agissant en amont de la voie de signalisation p38 nécessaire à l'activation des gènes PAM, d'autre part une implication des granules de stress lors de l'infection. Ces analyses sont le fondement pour l'établissement d'une description compréhensive du réseau génétique régulant le système immunitaire inné du ver et permettront de révéler les interactions complexes entre l'immunité et les processus physiologiques au niveau moléculaire, cellulaire et au niveau de l'organisme
To investigate innate immune signaling, we study the interaction of C. elegans with the fungus Drechmeria coniospora. One of the responses of the worm to this infection is the up-regulation of a variety of antimicrobial peptide (AMP) genes in the epidermis. Transgenic worms carrying a GFP reporter gene under the control of an AMP promoter fluoresce green after infection by D. coniospora. If a gene required for AMP gene expression is inactivated, the reporter strain will not turn green upon infection. Using this fluorescent read-out, we have been able to screen for signaling molecules required for AMP gene expression using a quantitative semi-automated RNAi approach. We have screened two RNAi libraries that together cover 95% of the ca. 20,000 genes in the C. elegans genome and we obtained 360 high-confidence candidates that reduced the level of induction of green fluorescence after infection, and correspond to 343 genes. A further phenotypic characterization allowed the candidates to be grouped into distinct functional categories and allowed the identification of both a receptor acting upstream the p38 MAPK pathway necessary for the activation of the AMPs, and the implication of stress granules during infection. Altogether, the screen data and its analysis represent the foundation for the establishment of a comprehensive description of the signaling network regulating the innate immune system of the worm and will shed light on the complex interactions between immunity and other physiological processes at the molecular, cellular and organismal level

Spinal muscular atrophy (SMA) and Charcot-Marie-Tooth disease type 2D (CMT2D)/distal SMA type V (dSMAV) are two incurable neuromuscular disorders that predominantly manifest during childhood and adolescence. Both conditions are caused by mutations in widely and constitutively expressed genes that encode proteins with essential housekeeping functions, yet display specific lower motor neuron pathology. SMA results from recessive inactivating mutations in the survival motor neuron 1 (SMN1) gene, while CMT2D/dSMAV manifests due to dominant point mutations in the glycyl-tRNA synthetase (GlyRS) gene, GARS. Using a number of different model systems, ranging from Caenorhabditis elegans to the mouse, this thesis aimed to identify potential novel therapeutic compounds for SMA, and to increase our understanding of the mechanisms underlying both diseases. I characterised a novel C. elegans allele, which possesses a point mutation in the worm SMN1 orthologue, smn-1, and showed its potential for large-scale screening by highlighting 4-aminopyridine in a screen for compounds able to improve the mutant motility defect. Previously, the gene encoding three isoforms of chondrolectin (Chodl) was shown to be alternatively spliced in the spinal cord of SMA mice before disease onset. I performed functional analyses of the three isoforms in neuronal cells with experimentally reduced Smn levels, and determined that the dysregulation of Chodl likely reflects a combination of compensatory mechanism and contributor to pathology, rather than mis-splicing. Finally, working with two Gars mutant mice and a new Drosophila model, I have implicated semaphorin-plexin pathways and axonal guidance in the GlyRS toxic gain-of-function disease mechanism of CMT2D/dSMAV.

Many neuronal patterning genes are expressed in distinct populations of cells in the nervous system, leading researchers to analyze their function in specific isolated cellular contexts that often obscure broader, themes of gene function. In this thesis, I aim to make clearer those overlooked common functional themes. I show that the C. elegans homeobox gene unc-42 is expressed in 15 out of a total of 118 distinct sensory, inter, and motor neuron classes throughout the C. elegans nervous system. Of these 15 unc-42(+) synaptically interconnected neuron classes, I show the extent to which unc-42 controls their identities and assembly into functional circuitry. I find that unc-42 defines the routes of communication between these interconnected neurons by controlling the expression of neurotransmitter pathway genes, neurotransmitter receptors, neuropeptides and neuropeptide receptors. I also show that unc-42 controls the expression of molecules involved in axon pathfinding and cell-cell recognition. Consequently, I show how the loss of unc-42 has effects on axon pathfinding and chemical synaptic connectivity, as determined by electron microscopical reconstruction of serial sections of unc-42 mutants. I conclude that unc-42 plays a critical role in establishing functional circuitry by acting as a terminal selector of functionally connected neuron types. I speculate that in other parts of the nervous system “circuit transcription factors” may also control assembly of functional circuitry and propose that such organizational properties of transcription factors may be reflective of not only an ontogenetic, but perhaps also phylogenetic trajectory of neuronal circuit establishment.

Sexual reproduction is an evolutionary innovation that arose 1.2 billion years ago, and in that time, has allowed a rapid diversification of species outpacing that of asexually reproducing organisms. Successful sexual reproduction in animals requires the incredible coordination of complex genetic and behavioral factors; from the most fundamental levels of ensuring correct chromosome segregation and ploidy to the most complex of behavioral mating rituals, any failure can result in a complete loss of evolutionary fitness. In this thesis, I have explored the developmental programs that function to ensure somatic sex determination, sexual differentiation, and mating behaviors in C. elegans. C. elegans is an androdiecious nematode species that has been extensively characterized in regard to the sexual dimorphism of its development, nervous system, and behavioral outputs. Sex determination pathways are widely diverged across phyla, and C. elegans has coopted a Gli family transcription factor to serve as a cell autonomous global regulator of somatic sex determination. I investigated the expression of this transcription factor, tra-1, with cellular, subcellular, sex-specific, and temporal resolution in both sexes of C. elegans and found that it is dynamically regulated to control sex determination. In contrast to the upstream sex determination pathway, genes that control downstream sexual differentiation in animals display much higher functional conservation, and many of the regulators of sexual differentiation belong to a family of transcription factors known as the DMRT family. Downstream of the tra-1 global regulator, I found that the highly conserved DMRT family gene dmd-4 acts much more specifically in adult hermaphrodites to generate sexual dimorphism at the level of the phasmid sensory neurons PHA and PHB. Furthermore, the sexual dimorphism of DMD-4 is regulated post-translationally by a ubiquitin-binding domain that I also found to be functionally conserved in the human ortholog, Dmrt3. Although these transcription factors both demonstrate the high degree of genetic control that contributes to sex determination and sexual differentiation, I also described male-specific effects of early life stress on sexual dimorphic synaptic connectivity and behavior generated by the phasmid sensory neurons, indicating that sexual differentiation is also plastic to environmental cues encountered during the life of an organism. This thesis provides insight into how genetic pathways function at multiple levels to give rise to extensive sexual dimorphism in the soma of an animal, both globally and in regard to the development on individual cells, in addition to the ways in which these genetic pathways can be modified by environmental factors and organismal life history.

The advent of sexual reproduction in early evolutionary history had profound effects on the evolution of animals. In most sexually reproducing species, males and females have distinct morphological and behavioral differences that are shaped by the evolutionary imperatives of each sex. Underlying the behavioral differences between males and females are distinct and measurable dimorphisms in the nervous system. These dimorphisms can arise in the form of connectivity, neurotransmitter usage, gene expression or combinations of all three. The androdioecious nematode Caenorhabditis elegans, with its stereotyped development and simple nervous system, offers a remarkably powerful system for studying the conserved mechanisms of sex determination that shape neural development. In this thesis, I present my work on the characterization of several genes that regulate the development of sexual dimorphisms in the nervous system. The first part of the thesis concerns the characterization of the gene ham-3, which codes for a subunit of the C. elegans ortholog of the SWI/SNF chromatin remodeling complex. ham-3 is required for the proper terminal differentiation of the HSN, a serotonergic neuron of the sex-specific nervous system, which it manages by regulating the expression of transcription factors required for crucial steps of migration, axon guidance and serotonergic fate adoption. The second part of the thesis concerns the investigation of sexually dimorphic pruning mechanisms. I show that unc-6/Netrin is subject to direct transcriptional repression in hermaphrodites by tra-1, the master transcriptional regulator of sexual fate determination in C. elegans. This regulation is required for the proper timing of the sexually dimorphic pruning of synapses in the tail region in hermaprhodites. In males, where unc-6 is not repressed by tra-1, unc-6 expression perdures into adulthood and the synapse is maintained. Together, these data provide insight into the ways in which conserved genetic and developmental mechanisms manage the generation differentiation, connectivity, and maintenance of sexually dimorphic nervous systems.

The ability to move is essential to an animal’s ability to interact with and respond to its changing environment. The nematode Caenorhabditis elegans is a commonly used organism in the study of the genetic and neural bases of behaviours, yet the mechanistic explanation for its ability to move in a smooth sinusoidal wave remains elusive. Here, I present studies of an uncharacterized gene, sto-6, encoding a stomatin protein that regulates C. elegans motor behaviour. I show that this gene plays a role in two unexplained and fundamental processes to C. elegans locomotion: wave initiation and wave propagation. Furthermore, I examine the genetic interaction between sto-6 and an innexin gene unc-7, providing support for the hypothesis that stomatins regulate gap junction proteins in C. elegans. Together, these studies push forward our understanding of the mechanistic basis of C. elegans locomotion, and open up avenues of further inquiry.

Indiana University-Purdue University Indianapolis (IUPUI)
Methylmercury (MeHg) exposure from occupational, environmental and food sources is a significant threat to public health. MeHg poisonings in adults may result in severe psychological and neurological deficits, and in utero exposures can confer significant damage to the developing brain and impair neurobehavioral and intellectual development. Recent epidemiological and vertebrate studies suggest that MeHg exposure may contribute to dopamine (DA) neuron vulnerability and the propensity to develop Parkinson’s disease (PD). I have developed a novel Caenorhabditis elegans (C. elegans) model of MeHg toxicity and have shown that low, chronic exposure confers embryonic defects, developmental delays, reduction in brood size, decreased animal viability and DA neuron degeneration. Toxicant exposure results in an increase in reactive oxygen species (ROS) and the robust induction of several glutathione-S-transferases (GSTs) that are largely dependent on the PD-associated phase II antioxidant transcription factor SKN-1/Nrf2. I have also shown that SKN-1 is expressed in the DA neurons, and a reduction in SKN-1 gene expression increases MeHg-induced animal vulnerability and DA neuron degeneration. Furthermore, I incorporated a novel genome wide reverse genetic screen that identified 92 genes involved in inhibiting MeHg-induced animal death. The putative multidrug resistance protein MRP-7 was identified in the screen. I have shown that this transporter is likely expressed in DA neurons, and reduced gene expression increases cellular Hg accumulation and MeHg-associated DA neurodegeneration. My studies indicate that C. elegans is a useful genetic model to explore the molecular basis of MeHg-associated DA neurodegeneration, and may identify novel therapeutic targets to address this highly relevant health issue.

Indiana University-Purdue University Indianapolis (IUPUI)
The etiology of many neurodegenerative diseases is unknown, but a number of studies indicate that a combination of both genetic and environmental factors contribute to the progression of disease. Exposure to environmental metals, such as Mn2+, Fe2+, Cu2+, and Al3+, has been shown to increase cell death that is characteristic of neurodegenerative disorders such as AD, PD, Wilson’s disease and Menkes disease. These metals are important in numerous biological processes in the brain and their homeostasis is regulated through multiple mechanisms of transport, storage, and secretion. The vertebrate divalent metal transporter-1 (DMT-1) has been implicated in transport and homeostasis of these divalent cations. In these studies I utilize Caenorhabditis elegans (C. elegans) to show that long term exposure to Mn2+ decreases animal viability in a dose-dependent manner, and I demonstrate that C. elegans homologues to DMT-1, SMF-1, SMF-2, and SMF-3, play specific roles in divalent metal ion-induced DA neurodegeneration. I show that SMF-1 contributes to Fe2+-induced DA neuron degeneration, SMF-3 contributes to Al3+-induced DA neuron degeneration, and both SMF-2 and DAT-1 contribute to Cu2+-induced DA neuron cell death. These studies utilize C. elegans as a powerful model to characterize molecules and pathways involved in metal toxicity and metal-induced DA neuron degeneration.