Thesis
Reference
Analysis of LNP-1 involvement in synaptic vesicle trafficking and neurotransmission
GHILA, Luiza Mihaela
Abstract
Récemment, un nouveau gène de souris, Inp, a été identifié en amont de cluster HoxD. Chez C. elegans, Inp-1 code pour une protéine putative qui contient les domaines de séquences conservés parmi tous les orthologues actuellement identifiés. Inp-1 est principalement exprimé dans les cellules neuronales chez le ver et colocalise avec les marqueurs de vésicules synaptiques dans les cultures cellulaires de mammifères. Les animaux déficients pour Inp-1 présentent une augmentation significative de leur résistance à l'aldicarb comparé aux animaux de type sauvage. Encore, la mauvaise localisation des protéines presynaptiques, telles que synaptobrevin-1 ou RAB-3 chez les mutants Inp-1, suggère une fonction pour LNP-1 dans la neutrotransmission. Nous avons aussi identifié un partenaire putatif de LNP-1: SIAH-1, une ligase E3-ubiquitin. Elucider les mécanismes moléculaires par lesquels le système LNP-1-ubiquitine-protéasome régule la neurotransmission pourrait être un aspect essentiel pour comprendre les processus pathologiques qui sous-tendent certains des désordres du système nerveux central.
GHILA, Luiza Mihaela. Analysis of LNP-1 involvement in synaptic vesicle trafficking and neurotransmission . Thèse de doctorat : Univ. Genève, 2008, no. Sc. 4015
URN : urn:nbn:ch:unige-21624
DOI : 10.13097/archive-ouverte/unige:2162
Available at:
http://archive-ouverte.unige.ch/unige:2162
Disclaimer: layout of this document may differ from the published version.
UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Département de Zoologie et Biologie Animale Prof. Denis DUBOULE
NCCR Frontiers in Genetics Dr. Marie GOMEZ
_________________________________________________________________________________
Analysis of LNP-1 involvement in synaptic vesicle trafficking and neurotransmission
THÈSE
présentée à la Faculté des Sciences de l`Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie
par
Luiza Mihaela GHILA de
Bucarest (Roumanie)
Thèse N° 4015
GENÈVE
Atelier d'impression ReproMail
Abbreviations
5HT - serotonine
ACC - ACh-gated chloride channel Ach – acetylcholine
AChE – acetylcholinesterase AChR – acetylcholine receptor AZ – active zone
CAST- CAZ-associated structural protein CAZ - presynaptic cytomatrix at the active zone cha-1 - choline acetyltransferase gene
ChAT – choline acetyltransferase CNS – central nervous system-- DA – dopamine
DC – dorsal nerve cord
DCC - Deleted in Colorectal Cancer
DLK-1 - dual-leucine-zipper-bearing MAPKKK DOP – dopamine receptor
DYN-1 – dynamin
FIF - formaldehyde-induced fluorescence GABA - γ-aminobutyric acid
GAD - glutamic acid decarboxylase GFP – green fluorescen protein GLR – non-NMDA glutamate receptor GPCR - G-protein coupled receptors iGluR – ionotropic glutamate receptor KLC-2 – kinesin-1 light chain LDCV - large dense-core vesicle l-DOPA - 1-dihydroxypheny-l-alanine LNP – limb and neural pattern
NBRP – National BioResource Project NGF – nerve growth factor
PAR-1 – proteinase-activaed receptor-1 PSD – postsynaptic density
RAB-3 - Ras-related GTP-binding protein-3 RFP – red fluorescent protein
RIM – Rab3-interacting molecule SAD – PAR-1-like Ser/Thr kinase SCF - Skp1/Cullin/F-box ubiquitin ligase SIAH - seven in absentia homolog SINA - seven in absentia protein
SNARE - soluble N-ethylmaleimide-sensitive component attachment protein receptor SNB-1 – synaptobrevin-1
SNF - sodium-dependent neurotransmitter symporter family
SNT-1 – synaptotagmin
SV – synaptic vesicle
SYD-2 - α-Liprin/synapse defective-2 TH - tyrosine hydroxylase
Unc-104/KIF1A - kinesin-like motor protein homologous to human axonal transporter of synaptic vesicles UNC-116 - kinesin-1 heavy chain UNC-13 - Neurotransmitter release regulator
UNC-16 - JNK/SAPK-associated protein-1
UNC-17 - acetylcholine vesicle transporter
UNC-17 - synaptic vesicle acetylcholine transporter (VAChT)
UNC-18 - Vesicle trafficking protein Sec1
UNC-26 – synaptojanin
UNC-3 - HLH transcription factor EBF/Olf-1
UNC-30 - Transcription factor PTX1 UNC-5 – netrin
UNC-55 - nuclear receptor related to the vertebrate COUP (chicken ovalbumin upstream promoter) transcription factors
UNC-57 – endophilin
UPS - ubiquitin-proteasome system VAChT - vesicular acetylcholine transporter
VAMP - vesicle-associated membrane protein
VC – ventral nerve cord
VGAT - vesicular GABA transporter VGluT - vesicular glutamate transporter VMATs - vesicular monoamine
transporters
YAC – yeast artificial chromosome APC - Anaphase Promoting Complex RRF-3 - RNA-directed RNA polymerase QDE-1 required for posttranscriptional gene silencing and RNA interference N2 - C. elegans wild type, var. Bristol UNC-42 - calcium and calmodulin- dependent protein kinase II (CaMKII)
Table of contents
PREAMBLE 7
1. RESUME EN FRANÇAIS 8
2. SUMMARY 13
3. INTRODUCTION 17
3.1 CAENORHABDITIS ELEGANS AS MODEL SYSTEM 17
3.2. CAENORHABDITIS ELEGANS AS MODEL FOR STUDYING NERVOUS SYSTEM DEVELOPMENT
AND FUNCTION 21
3.2.1 BEHAVIOURAL TOOLS 22
3.2.2 PHARMACOLOGICAL TOOLS 24
3.3. MOLECULAR MECHANISMS INVOLVED IN THE ESTABLISHMENT AND MAINTENANCE OF
NEURONAL NETWORKS 25
3.3.1 THE STRUCTURE OF THE SYNAPSE IN C. ELEGANS 25
3.3.2 PROTEINS INVOLVED IN NEUROTRANSMISSION IN C. ELEGANS 26 3.4 STRATEGIES TO IDENTIFY AND STUDY PROTEINS INVOLVED IN NERVOUS SYSTEM
DEVELOPMENT AND FUNCTION 52
3.4.1 FORWARD GENETIC SCREENS 53
3.4.2 REVERSE GENETIC TECHNIQUES 53
3.4.3 LUNAPARK (LNP) A NOVEL MOUSE GENE THAT SHARES THE GLOBAL CONTROL REGION
(GCR) WITH EVX2 AND HOXD GENES 54
3.5 OBJECTIVES OF THIS WORK 58
3.5.1 ELUCIDATION OF THE ROLE OF LNP-1 IN C. ELEGANS 58
3.5.2 IDENTIFICATION OF LNP-1 PARTNERS 58
3.5.3 ANALYZING THE CONSERVATION OF LNP-1 THROUGH EVOLUTION 59
4. RESULTS 61
4.1. LNP-1 IS REQUIRED FOR SYNAPTIC VESICLE TRAFFICKING AND SYNAPTIC
TRANSMISSION IN C. ELEGANS 62
4.2. ADDITIONAL RESULTS 77
4.2.1 LNP-1 IS EXPRESSED VERY EARLY IN EMBRYOGENESIS 77 4.2.2 MUTATIONS IN LNP-1 INDUCE A REDUCTION OF THE C. ELEGANS LIFE SPAN AND EARLY
NEURODEGENERATION 79
4.2.3 SIAH-1: A PUTATIVE PARTNER FOR LNP-1 81
4.2.4 E3/UBIQUITIN LIGASE ACTIVITY OF SIAH-1 IS CONSERVED IN C. ELEGANS 83 4.2.5 SIAH-1 AND LNP-1 COLOCALIZE IN C. ELEGANS NERVOUS SYSTEM 85 4.2.6 E3/UBIQUITIN LIGASE SIAH-1 INVOLVEMENT IN SYNAPTIC FUNCTION 86 4.2.7 LNP-1 AND E3/UBIQUITIN LIGASE SIAH-1 REGULATE THE NORMAL DISTRIBUTION OF PRESYNAPTIC PROTEIN SNB-1 IN THE VENTRAL NERVE CORD 87
4.2.8 LNP-1 AND E3/UBIQUITIN LIGASE SIAH-1 REGULATE THE ABUNDANCE OF GLR-1 IN THE
VENTRAL NERVE CORD 88
4.2.9 PRESYNAPTIC PROTEIN SNB-1 ABUNDANCE IN VENTRAL CORD IS UBIQUITIN-DEPENDENT
90
4.3 LNP BIOINFORMATICAL ANALYSES 99
4.3.1 PREDICTED DOMAINS 99
4.3.2 PREDICTED POST-TRANSLATIONAL MODIFICATIONS 102
4.3.3 PREDICTED INTERACTIONS 104
5. DISCUSSIONS AND PERSPECTIVES 106
6. BIBLIOGRAPHY 112
ANNEX 1: MATERIALS AND METHODS 129
PREAMBLE
Every story has a start, and this one started with an interview for the Swiss NCCR Frontiers in Genetics Doctoral School in February 2004. For this reason, I would like to thank to the NCCR scientific committee who selected me as part of the 2004 student generation of their Doctoral School, especially to Prof. Ivan Rodriguez and Prof. Denis Duboule, whom supported me with their advices and guidance all these years. I would like also to thank to Bérénice Krebs, Stéphane Barges and Caroline Laemmli for their promptness and all the help regarding the administrative problems. My sincere gratitude is toward Dr. Brigitte Galliot for accepting me as a summer student before I started my pre-doctoral training. I am grateful for her guidance and training in molecular biology techniques, which helped the development of my scientific way of thinking.
I would like to thank to Dr. Marie Gomez, who accepted to guide the work presented here and to the past members of her laboratory, especially to Dr. Patrizia Latorre, which is a great person and a friend, but also to Patrick Tschopp and Ludovic Baillon for great discussions during their rotation in Gomez’s laboratory.
I am grateful for the numerous discussions and advices received during the Monday’s C. elegans lab Meetings to Dr. Marie Gomez, Dr. Vincent Menuz and Sébastien Gentina and during the Wednesday’s Developmental Biology Meetings to Dr. Brigitte Galliot, Dr. Roland Dosch, Dr. Simona Chera, Dr. Wanda Buzgariu, Dr Amandine Stein, Dr. Marijana Miljkovich Licina, Dr. Renaud de Rosa, Manon Quiquand, Franck Bontems, and Kevin Dobretz. In addition, special thanks to the members of NCCR Frontiers in Genetics Bioimaging Platform, especially to Dr.
Christoph Bauer for his assistance and advises over these years.
I also would like to thank to my great friend Simona, for her permanent support and good mood, and special thanks to Tatiana not only for her very good food and very pleasant home-vacations, but also for her great stories and interesting discussions. I am very grateful to Aleksei and Vladimir for making things seem a lot easier and a lot more fun. Thanks to all my friends from home and last, but not least, to my family.
Furthermore, I would like to thank to the members of my thesis committee: Prof.
Denis Duboule, Prof. Ivan Rodriguez and Dr. Jean-Louis Bessereau for their time and interest in my thesis.
Luiza
1. RESUME EN FRANÇAIS
Récemment, un gène de souris codant pour une protéine appelée LNP (Limb and Neural Pattern) a été identifié en amont des clusters Evx2 et HoxD. Lnp est un gène non-hox dont les éléments de régulation sont sous le contrôle d’une GCR (global control region – région de contrôle globale) commune. La GCR est essentielle à l’expression des gènes Lnp et Evx2 limitée au système nerveux central (SNC) ainsi qu’à l’expression séquentielle des gènes HoxD lors du développement des membres. Le patron d’expression de Lnp est corrélé, au moins partiellement, avec celui des gènes Evx2 et Hoxd13 à Hoxd10. Ces derniers codent pour des facteurs de transcription bien connus qui jouent un rôle crucial au cours de la morphogénèse et sont hautement conservés à travers le règne animal. Pendant l’embryogénèse de la souris, lnp est exprimé dans les membres, les yeux, le cœur, les organes génitaux ainsi que dans le système nerveux. De plus, l’expression de lnp dans les structures nerveuses comme le cervelet ou le tube neural coïncide avec l’expression des gènes evx. Des études génétiques chez C.
elegans, Drosophila et la souris ont montré que les homologues d’evx régulent la différentiation des neurones moteurs. Contrairement à evx et ses orthologues, la fonction de lnp chez les mammifères reste inconnue. Chez l’homme, des points d’interruption au voisinage du cluster lnp-evx2-hoxd, notamment une translocation spécifique (t(2;10)(q31.1;q26.5)) résulte, parmi d’autres effets, à une hypoplasie cérébelleuse sévère. Il a été proposé que les défauts cognitifs observés chez les patients porteurs de cette translocation pourraient provenir d’une altération de l’expression du gène lnp dans le SNC. Cette hypothèse est cohérente avec des études génétiques menées sur la souris, qui ont montré qu’une translocation similaire du cluster lnp- evx2-hoxd conduit à une diminution de l’expression de lnp. Du fait de sa localisation chromosomique et de sa co-expression avec les gènes Hoxd et Evx2, élucider le rôle de Lnp dans le système nerveux central présente un intérêt réel. Pour cette raison, nous nous sommes concentrés sur le développement d’un organisme modèle (C. elegans).
Chez C. elegans, Lnp-1 est localisé sur le chromosome X et cartographié sur le cosmide C05E11. Le gène lnp-1 code pour une protéine putative de 342 acides aminés, qui contient les domaines de séquences conservés parmi tous les orthologues actuellement identifiés. Aucun de ces domaines de séquences conservés ne montre de claire homologie avec des protéines connues, excepté deux domaines transmembranaires prédits qui se trouvent dans la partie N-terminale et un domaine en doigt de zinc atypique dans la partie C- terminale de la protéine. Nous avons utilisé des outils bioinformatiques pour une analyse détaillée de LNP chez l’humain, la souris et le ver (voir Résultats – Chapitre 4.3).
Plusieurs lignées de vers transgéniques exprimant le gène putatif lnp::gfp sous le contrôle d’une région de 3kb localisée en amont de la séquence codante ont été obtenus par microinjection. Une large expression, possiblement ubiquitaire, est d’abord visible pendant la
gastrulation. Après l’embryogenèse, le rapporteur Lnp est exprimé dans de nombreux corps cellulaires le long de la corde ventrale, autour du pharynx et de la queue. Cependant, en raison de la localisation spécifiquement sub-cellulaire de la protéine de fusion, il a été difficile de déterminer l’identité neuronale de certaines cellules. Pour résoudre ce problème, nous avons fusionné un promoteur non localisé avec les marqueurs GFP ou RFP. Nous avons injecté ces constructions dans des animaux de type sauvage exprimant une protéine pan-neuronal (un marqueur marquant toutes les cellules neuronales). Comme montré, les neurones moteurs dans la corde nerveuse ventrale, dans l’anneau nerveux et dans le queue sont marqués. Le long de la corde nerveuse ventrale, les neurones moteurs de type V- et D- sont marqués. De plus, une expression est observée dans les cellules musculaires et hypodermales. L’expression de lnp-1 est maintenue dans tous les tissus pendant l’âge adulte. Nous avons aussi montré que la localisation sub-cellulaire de LNP-1 est dépendante de la protéine UNC-104/kinesin (voir résultats – Chapitre 4.1).
Nous avons sollicité la génération d’une lignée déficiente pour lnp à “National Bioresource Project for the Experimental Animal Nematode C. elegans” (NBRP; Japan) et avons obtenu deux allèles différents : lnp-1(tm1247) dans lequel une partie des exons 2, 3, 4 et 5 ont été supprimés ainsi que lnp-1(tm733) dans lequel une partie des exons 3 et 4 ont été supprimés (figure 4A). Les deux lignées homozygotes sont viables et ne montrent pas de défaut évident de comportement ou de développement. Nous avons généré des anticorps dirigés contre la partie C-terminale de la protéine. Dans les analyses de western blots (immunotransfert) LNP-1 n’a pas été détecté dans les extraits protéiques totaux provenant des animaux lnp-1(tm733) et lnp-1(tm1247), suggérant que ces mutants sont des allèles nuls.
Puisque lnp-1 est principalement exprimé dans les cellules neuronales chez le ver et colocalise avec des marqueurs de vésicules synaptiques dans les cultures cellulaires de mammifères, nous avons d’abord analysé le comportement des mutants lnp-1(tm1247) et lnp-1(tm733) en utilisant des outils pharmacologiques comme la substance active aldicarb (2-methyl-2- (methylthio)propionaldehyde O-methylcarbamoyloxime). L’aldicarb est un inhibiteur de l’acétylcholinestérase qui provoque une accumulation du neurotransmetteur acétylcholine aux jonctions neuromusculaires. En présence d’aldicarb, les vers de type sauvage sont rapidement paralysés et une exposition prolongée induit la mort. Les animaux déficients pour lnp-1 présentent une augmentation significative de leur résistance à l’aldicarb comparé aux animaux de type sauvage. Nous avons ensuite examiné le comportement de ponte, qui est régulé par de multiples neurotransmetteurs incluant la sérotonine et l’acétylcholine (Weinshenker et al., 1995). Nous avons pu observer que le nombre moyen d’œufs pondus par animal était significativement réduit chez les mutants lnp-1(tm1247) comparé aux animaux de type sauvage et que la ponte en réponse à la sérotonine était réduite. La résistance à l’aldicarb est le résultat d’un défaut soit présynaptique soit postsynaptique. Le lévamisole, qui cause une hypercontraction des muscles du corps du nématode entraînant la paralysie et la mort, agit comme un agoniste sur les récepteur à l’acétylcholine (Jones and Sattelle, 2004).
Contrairement à l’aldicarb, le lévamisole affecte seulement les fonctions postsynaptiques.
Cependant, la sensitivité au lévamisole était similaire à celle des animaux sauvages, suggérant une fonction presynaptique de LNP-1 dans la neurotransmission. La mauvaise localisation des protéines presynaptiques, telles que synaptobrevin-1 ou RAB-3 chez les mutants lnp-1, étaye cette hypothèse. Ces résultats suggèrent que LNP-1 joue un rôle au cours de la synaptogénèse en régulant le transport vésiculaire et/ou la localisation des protéines synaptiques.
Nous avons examiné l’espérance de vie des mutants lnp-1 (voir Résultats – Chapitre 4.2.2). Nos résultats préliminaires suggèrent que LNP-1 pourrait être impliqué dans la régulation de l’espérance de vie. Nous avons observé une forte diminution de l’espérance de vie dans la lignée déficiente pour lnp-1 comparé à la lignée N2. 50% des animaux lnp-1(tm733) et lnp-1(tm1247) sont morts après 11 et 16 jours respectivement, alors que 50% des animaux N2 sont morts après seulement 23 jours, montrant une diminution de l’espérance de vie en moyenne de 60% et 30% chez les animaux lnp-1(tm733) et lnp-1(tm1247) comparé aux animaux N2. Il a été récemment proposé que l’activité neurale pourrait réguler l’espérance de vie chez C. elegans (Evason et al., 2005). Les vers traités avec de la Trimethadione, une drogue qui a une activité anticonvulsante, montrent une extension significative de leur espérance de vie. Ils montrent aussi une hypersensitivité à la paralysie entraînée par l’aldicarb, suggérant que cet anticonvulsant stimule la neurotransmission synaptique dans le système neuromusculaire. Ainsi, les résultats obtenus dans les tests à l’aldicarb et l’espérance de vie des animaux lnp-1(tm733) et lnp-1(tm1247) sont cohérents avec les phénotypes observés chez les vers traités avec des drogues anticonvulsantes (voir Résultats – Chapitre 4.1).
La seconde approche que nous avons utilisé pour comprendre la fonction de lnp-1 a été d’identifier les partenaires putatifs de LNP-1 (voir Résultats – Chapitre 4.2.3). Un Yeast two- hybrid screening en vu de l’identification des interactions protéine-protéine à petite et grande échelle chez C. elegans a été précédemment décrit (Crowe and Candido, 2004; Li et al., 2004;
Malone et al., 2003; Walhout et al., 2000). En utilisant cette approche, nous avons screené une banque d‘ADNc de C. elegans, en utilisant comme appât une forme de LNP-1 excluant les deux domaines transmembranaires prédits; l’expression de LNP-1 incluant ces domaines dans le système E. coli entraîne la production d’une protéine insoluble s’accumulant dans les corps d’inclusions. Dans deux yeast two-hybrid screens indépendants, nous avons obtenu huit clones positifs, qui ont été séquencés et analysés par la suite. Ils codent pour une ligase putative ring finger/E3-ubiquitin, probablement l’orthologue de la protéine Seven In Absentia (SINA), qui est une protéine hautement conservée au cours de l’évolution (voir Résultats – Chapitre 4.2.3). La protéine SINA contient plusieurs domaines conservés parmi tous les orthologues identifiés jusqu’à présent: un domaine N-terminal en doigt d’anneaux (C3HC4) impliqué dans l’ubiquitination comme l’ubiquitine ligase, suivi d’une région conservée cystéine/histidine (C2HC4H3) et une région C-terminale impliquée dans l’interaction avec des protéines cibles (Figure 6 ; (Hu and Fearon, 1999). Siah (l’homologue de sina) est localisé sur le chromosome IV de C. elegans et cartographié sur le yeast artificial chromosome (YAC) Y37E11AR. SINA a
été découvert à l’origine comme une protéine contenant un doigt en anneaux qui est indispensable au développement neuronal des cellules photoréceptrices R7 chez Drosophila (Carthew and Rubin, 1990). SINA, avec PHYLLOPOD, entraînent l’ubiquitination suivi de la destruction par le protéasome de TRAMTRACK, un régulateur de la différentiation neuronale (Tang et al., 1997). Chez les mammifères, il a été montré que les protéines Siah ciblent la dégradation de différentes protéines incluant Deleted in Colorectal Cancer (DCC), synaptophysin, synphylin-1, numb, glutamate receptors, etc. (Hu et al., 1997; Moriyoshi et al., 2004; Nagano et al., 2003; Susini et al., 2001; Wheeler et al., 2002). Ces études suggèrent que Siah pourrait agir dans la régulation du développement et de la fonction neuronale par le biais de la dégradation (via ubiquitination) d’un bon nombre de protéines neuronales cibles.
Nous nous sommes ensuite demandé si chez C-elegans, siah-1 et lnp-1 partagent des domaines d’expression (voir Résultats – Chapitre 4.2.5). Nous avons construit un gène de fusion entre le gène rapporteur gfp (ou rfp) et une région de 3 kb située en amont de la séquence codante de siah-1. Des vers transgéniques et doubles transgéniques exprimant soit siah-1 seul ou siah-1 et lnp-1 ont été générés par microinjection. Comme pour lnp-1, de nombreux corps cellulaires situés le long de la corde ventrale, autour du pharynx et de la queue exprimaient siah-1. De plus, les vers exprimant à la fois les transgènes lnp-1 et siah-1 montraient une bonne colocalisation des rapporteurs, particulièrement dans le système nerveux. Ces résultats ont été confirmés par des expériences immunohistochimiques sur des vers entiers, montrant une colocalistion de LNP-1 et SIAH-1 endogène.
Nous avons obtenu une lignée déficiente pour siah-1 : siah(tm1968) de NBRP consortium qui génère des lignées sur demande. La lignée homozygote est viable et ne montre aucun défaut de comportement et de développement évident. Nous avons généré des doubles mutants lnp-1(tm1247);siah-1(tm1968) and lnp-1(tm733);siah-1(tm1968) et réalisé les mêmes analyses comportementales et pharmacologiques décrites pour les simples mutants. Les analyses phénotypiques sur les doubles mutants et la comparaison avec les phénotypes des simples mutants ont confirmé que ces molécules agissent dans la même cascade (voir Résultats – Chapitre 4.2.6-8).
Nous avons étudié la sub-localisation de Lnp-1 dans des cellules en culture de mammifère (3T3 et C3H fibroblaste et lignées cellulaires PC12 traitées NGF) en utilisant des anticorps spécifiques que nous avons générés contre la protéine Lnp de souris. Nous avons fait des expériences de colocalisation avec des structures intracellulaires connues ou des marqueurs d’organelles comme la tubuline, l’actine, la vinculine, des marqueurs endosomales spécifiques des stades. Nous avons montré que à la fois dans les fibroblastes et les cellules PC12 différentiées NGF, Lnp révélait un marquage ponctué dans les corps cellulaires et les processus neuritiques. Une colocalisation partielle a été trouvée avec la synaptophysine, un marqueur de vésicule synaptique et la kinesine, une protéine motrice (voir Résultats – Chapitre 4.2.10). Il a été rapporté que Siah, comme Lnp, colocalise partiellement avec la synaptophysine dans les cellules PC12 différenciées NGF (Wheeler et al., 2002). Pour analyser d’avantage
l’interaction supposée entre ces deux protéines in vivo, nous avons réalisé des analyses immunohistochimiques sur des cultures de cellules de mammifères. Nous avons montré que dans les cellules PC12 différenciées NGF, Siah et Lnp colocalisaient dans les corps cellulaires et les processus neuronaux en des structures ponctuées, certaines d’entre elles étant positives pour le marqueur de vésicule synaptique synaptophysine (voir Résultats – Chapitre 4.2.10).
Élucider les mécanismes moléculaires par lesquels le système LNP-1/ubiquitine protéasome régule la synaptogénèse pourrait être un aspect essentiel pour comprendre les processus pathologiques qui sous-tendent certains des désordres du système nerveux central chez l’humain. Il est intéressant de noter que parkin – un gène muté responsable de la maladie de Parkinson, code pour une Ubiquitine E3 ligase. Il est très vraisemblable que dans un futur proche les défauts dans ce système seront liés à un nombre de maladies grandissantes.
2. SUMMARY
Recently, a mouse gene coding for a protein named LNP (limb and neural pattern) was identified upstream of the Evx2 and the HoxD cluster. Lnp is a non-hox gene having its regulatory elements under the control of a common GCR (global control region). The GCR is essential for the restricted expression of the Lnp and Evx2 genes in the CNS, as well as for the sequential expression of HoxD genes in the developing limbs. The expression pattern of Lnp is related, at least partially, to that of Evx2 and Hoxd13 to Hoxd10 genes, which encode for well- known transcription factors that are crucial players during morphogenesis and highly conserved throughout the animal kingdom. Mouse lnp is expressed in limbs, eyes, heart, genitalia and in the nervous system during embryogenesis. Furthermore, lnp expression in neuronal structures such as the cerebellum and the neural tube coincides with that of evx genes. Genetic studies in C. elegans, Drosophila, and mouse have shown that evx homologues distinguish alternative fates in motor neurons. In contrast to evx and its orthologs, the function of mammalian lnp remains unknown. In humans, breakpoints in the vicinity of the lnp-evx2-hoxd cluster, notably one specific translocation (t(2;10)(q31.1;q26.5)) results, among other defects, in severe cerebellar hypoplasia. It has been proposed that the cognitive defects observed in the patient carrying this translocation could be due to an alteration of lnp expression in the central nervous system. This hypothesis is consistent with genetic studies in mice, where a similar translocation of the lnp-evx2-hoxd cluster results in a down-regulation of the lnp gene expression. Due to these facts, its chromosomal location and co-expression with Hoxd and Evx2 genes, deciphering the Lnp role in the central nervous system is of real interest and for this reason we have concentrated on developing a C. elegans model system.
Lnp-1 in C. elegans is located on the chromosome X and maps to the C05E11 cosmid.
The lnp-1 gene encodes a putative 342 amino acid protein, which contains the sequence domains conserved among all orthologs identified so far. None of these conserved sequence domains show any clear homology with known proteins, except for two predicted transmembrane domains at the N-terminal part and an atypical zinc-finger domain at the C- terminal part of the protein. We used bio-informatics tools for a detailed analysis of the human, mouse and worm LNP (see Results – Chapiter 4.3).
Several transgenic worm lines expressing a putative lnp gene:gfp under the control of a 3kb region located upstream of the coding sequence were obtained by microinjection. Broad and possibly ubiquitous expression is first visible during gastrulation. Postembryonically, Lnp reporter is expressed in numerous cell bodies along the ventral cord, around the pharynx and tail. However, due to the specific sub-cellular localisation of the fusion protein, it was difficult to determine neuronal identity of some cells. To resolve this problem, we made a non-localized promoter fusion with GFP or RFP markers. We injected these constructs into wild-type animals and into animals expressing a pan-neuronal protein (a marker which labels all the neuronal
cells). As shown, motor neurons in the ventral nerve cord, in the nerve ring and in the tail are labelled. Along the ventral nerve cord, V- and D- types motor neurones are labelled. In addition, expression is observed in muscles and hypodermal cells. Expression of lnp-1 is maintained in all tissues throughout adulthood. We also showed that LNP-1 sub-cellular localization is dependent of the UNC-104/kinesin protein. (see Results – Chapiter 4.1).
We requested the generation of lnp-deleted strains from The “National Bioresource Project for the Experimental Animal Nematode C. elegans” (NBRP; Japan) and we obtained two different alleles lnp-1(tm1247) in which part of exon 2, exon 3, 4, 5 have been deleted, and lnp- 1(tm733) in which part of exons 3 and 4 have been deleted (Figure 4A). The two homozygous strains are viable and do not show any obvious behavioural or developmental defects. We generated antibodies raised against the C-terminal part of the protein. In western blots analyses LNP-1 was not detected in total protein extracts from lnp-1(tm733) and lnp-1(tm1247) animals, suggesting that these mutants are null alleles. Because lnp-1 is mainly expressed in neuronal cells in worms and colocalised with vesicle synaptic markers in mammalian cell cultures, we first analysed the behaviours of lnp-1(tm1247) and lnp-1(tm733) mutants using pharmacological tools such as the aldicarb drug. Aldicarb is an inhibitor of the acetylcholinesterase, leading to the accumulation of the acetylcholine neurotransmitter at the neuromuscular junction, causing paralysis and death. In the presence of aldicarb, wild type worms are rapidly paralysed and prolonged exposure induces death. lnp-1-deleted animals, on the other hand, showed a significant increase in aldicarb resistance compared to wild type animals. We examined the egg-laying behaviour, which is regulated by multiple neurotransmitters including serotonin and acetylcholine (Weinshenker et al., 1995). We could observe that the mean number of eggs laid/per animal was significantly reduced in lnp-1(tm1247) mutants compared with wild-type animals and that the egg laying response to serotonin was reduced. Resistance to aldicarb is the result of either presynaptic or postsynaptic defects. Levamisole, which causes hypercontraction of nematodes’ body-wall muscles leading to paralysis and death, acts as an agonist on acetylcholine receptors (Jones and Sattelle, 2004). Unlike aldicarb, levamisole only affects postsynaptic function. However, sensitivity to levamisole was similar to that of wild-type animals, suggesting a presynaptic function for LNP-1 in neurotransmission. The mislocalization of presynaptic proteins, such as synaptobrevin-1 or RAB-3, in lnp-1 mutants further supports this hypothesis. These results suggest that LNP-1 plays a role in synaptogenesis by regulating vesicular transport and/or localization of synaptic proteins.
We examined the life span of lnp-1 mutants (see Results – Chapiter 4.2.2). Our preliminary results suggest that LNP-1 might be involved in the regulation of life span. We could observe a strong decrease in life span in lnp-1-deleted strains compared with N2 strain. 50% of lnp-1(tm733) and lnp-1(tm1247) animals died after 11 and 16 days respectively, whereas 50%
of N2 animals died after only 23 days, meaning a decrease on the lifespan mean by 60% and 30% in lnp-1(tm733) and lnp-1(tm1247) animals compared with N2 animals. It has been
recently proposed that neural activity may regulate lifespan in C. elegans (Evason et al., 2005).
Worms treated with Trimethadione, a drug with anticonvulsant activity, display a significant extension in their lifespan. They also show hypersensitivity to aldicarb-mediated paralysis suggesting that this anticonvulsant stimulates synaptic neurotransmission in the neuromuscular system. Thus, the results obtained in aldicarb assays and lifespan in lnp-1(tm733) and lnp- 1(tm1247) animals are consistent with the phenotypes observed in worms treated with anticonvulsant drugs (see Results – Chapiter 4.1).
The second approach we used to understand the function of lnp-1 was to identify putative LNP-1 partners (see Results – Chapiter 4.2.3). Yeast two-hybrid screening for successful identification of protein-protein interactions at small and large scale in C. elegans has been previously described (Crowe and Candido, 2004; Li et al., 2004; Malone et al., 2003;
Walhout et al., 2000). Using this approach, we screened a C. elegans cDNA library, using as bait a deleted form of LNP-1 excluding the two predicted transmembranes domains; expression of LNP-1 including these domains in E. coli system leads to the production of an insoluble protein accumulating in inclusion bodies. In two independent yeast two-hybrid screens, we obtained eight positive clones, which were sequenced and further analysed. They all code for a putative ring finger/E3-ubiquitin ligase, probably the ortholog of the seven in absentia protein (SINA), which is a protein highly conserved during evolution (see Results – Chapiter 4.2.3).
SINA protein contains several domains conserved among all orthologs identified so far: an N- terminal RING finger domain (C3HC4) involved in ubiquitination as ubiquitin ligase, followed by a conserved cysteine/histidine region (C2HC4H3) and a C-terminal region involved in the interaction with protein targets (Figure 6; (Hu and Fearon, 1999). Siah (sina homolog) is located on chromosome IV of C. elegans and maps to the yeast artificial chromosome (YAC) Y37E11AR. SINA was originally discovered as a RING finger-containing protein that is critically involved in neuronal development of the R7 photoreceptor cell in Drosophila (Carthew and Rubin, 1990). SINA, together with PHYLLOPOD, promotes the ubiquitin proteasome-dependent degradation of TRAMTRACK, a negative regulator of neuronal differentiation (Tang et al., 1997). In mammals, Siah proteins have been shown to target to degradation different proteins including Deleted in Colorectal Cancer (DCC), synaptophysin, synphylin-1, numb, glutamate receptors, etc. (Hu et al., 1997; Moriyoshi et al., 2004; Nagano et al., 2003; Susini et al., 2001;
Wheeler et al., 2002). These studies suggest that Siah proteins may act to regulate neuronal development and function by mediating the ubiquitin-dependent degradation of a number of neuronal target proteins.
We then addressed the question whether in C. elegans siah-1 and lnp-1 share expression pattern domains (see Results – Chapiter 4.2.5). We constructed a gene fusion between the reporter gene gfp (or rfp) and a 3-kb region upstream the siah-1 coding sequence.
Transgenic and double transgenic worms expressing either siah-1 alone or siah-1 and lnp-1 were generated by microinjection. As for lnp-1, numerous cells bodies along the ventral cord,
around the pharynx and tail expressed siah-1. Furthermore, worms expressing both lnp-1 and siah-1 transgenes displayed good colocalization of the reporters, particularly in nervous system.
These results were confirmed by immunohistochemistry experimets on whole worm, showing colocalization of endogenous LNP-1 and SIAH-1.
We obtained a siah-1-deleted strain: siah(tm1968) from the NBRP consortium who generated the line upon our request. The homozygous strain is viable and does not show any obvious behavioural and developmental defects. We have generated double mutants lnp- 1(tm1247);siah-1(tm1968) and lnp-1(tm733);siah-1(tm1968) and performed the same behavioural and pharmacological analyses described for the single mutants. Analyses of the phenotypes resulting from double mutants and comparison with phenotypes from single mutants confirmed that these molecules are acting in the same pathway (see Results – Chapiters 4.2.6-8).
We studied the sublocalisation of Lnp-1 in mammalian cell cultures (3T3 and C3H fibroblast and NGF-treated PC12 cell lines) using specific antibodies we generated against the mouse Lnp. We performed colocalisation experiments with known intracellular structure or organelle markers like tubulin, actin, vinculin, stage-specific endosomal markers. We could show that in both fibroblast and NGF-differentiated PC12 cells, Lnp revealed punctuate staining pattern in cell bodies and neuritic processes. Partial colocalisation was found with synaptophysin, a synaptic vesicle marker and kinesin, a motor protein (see Results – Chapiter 4.2.10). It has been reported that Siah, like Lnp, partially colocalises with synaptophysin in NGF-differentiated PC12 cells (Wheeler et al., 2002). To further analyse the putative interaction between these two proteins in vivo, we performed immunohistochemistry in mammalian cell cultures. We showed that in NGF-differentiated PC12 cells, Siah and Lnp colocalised in the cell body and neuritic processes, to punctuate structures, some of which are positive for the synaptic vesicle marker synaptophysin (see Results – Chapiter 4.2.10).
Elucidating the molecular mechanisms by which LNP-1/ubiquitin proteaseome system regulates synaptogenesis could be an essential aspect of understanding the pathological processes that underlie some central nervous system disorders in humans. It is interesting to note that parkin – a mutated gene responsible for Parkinson’s disease, encodes an ubiquitin E3 ligase. It is very likely that in a near future defects in this system will be linked to a growing number of diseases.
3. INTRODUCTION
3.1 Caenorhabditis elegans as model system
Caenorhabditis elegans is a small, free-living, soil nematode found in temperate regions, in nutrient- and microorganism-rich habitats such as compost, mushroom beds and garden soil, feeding on bacteria and other microorganisms (Brenner, 1973). In the 1960s, Sydney Brenner began using this animal to study the genetics of development and neurobiology. C. elegans represents a very good model system for genetic analysis because of its rapid life cycle (Figure 1), small size (an adult has about 1.5 mm long and 75 µm cross- section) and easiness to cultivate it in laboratory. Despite the fact that C. elegans is a self- fertilising hermaphrodite, it is possible to set up genetic crosses because functional males are found, though rarely (about 0.05% of the wild-type population). However, after mating, male sperm out-competes hermaphrodite sperm (Ward and Carrel, 1979). Other key features are the nematode’s anatomical simplicity (less then 1000 cells), its small genome (100 Mbs) entirely mapped, its developmental lineage is known to the cellular level (Sulston et al., 1983), and the wiring diagram of its nervous system has been mapped out in detail (White et al., 1986).
Although some researchers are considering that this animal has few morphological and behavioural traits, the transparency of the body, the constancy of the cell number and the consistency of the cell position between individuals represent unique advantages of this model organism. First stage larvae can be frozen at -80°C in the presence of glycerol and other cryoprotectants and recovered after defrosting (years or decades later)(Brenner et al., 1974).
This possibility of long-term storage of mutant strains gave the opportunity to organise and maintain a comprehensive collection of genetic stocks containing all published strains, with
Figure1. The C. elegans life cycle at 22°C. 0 minutes is fertilization. Numbers in blue along the arrows indicate the length of time the animal spends at a certain stage. First cleavage occurs at about 40 minutes postfertilization.
Eggs are laid outside at about 150 minutes.
postfertilization and during the gastrula stage. The length of the animal at each stage is marked next to the stage name in micrometers.
(from WormAtlas)
open access for the entire research community – the Caenorhabditis Genetics Center (CGC - http://www.cbs.umn.edu/CGC/).
The early germ line in C. elegans is syncytial, which permits the uptake by multiple nuclei of any substance injected into the gonad (Figure 2). When DNA is injected into the gonad of a young hermaphrodite, the repair machinery catenates it into long extrachromosomal arrays (>100 kb), which can be stably maintained in the resulting offspring (Mello et al., 1991).
Transgenic extrachromosomal arrays can be stably integrated into the chromosomes by UV irradiation of transgenic lines or by co-injection of single stranded DNA (Fire, 1986). However, homologous recombination into endogenous loci can occur at low frequency suggesting that targeted gene replacement is possible (Broverman et al., 1993; Berezikov et al., 2004). The selection of transgenic nematodes can be performed by coinjecting a visible marker gene, for example a fluorescent-intestinal marker (elt-2::GFP) or by using a known dominant mutation in the cuticular collagen gene rol-6 (su1006) which causes the nematodes to develop a helically twisted cuticle and to roll longitudinally when they move forward (Kramer et al., 1990). In addition, genotyping can be performed on individual animals by “single-worm PCR”. The transparency of C. elegans allows in vivo microscopic analysis without animal dissection.
Transgenesis is a very useful research tool for:
•
proving that a DNA fragment contains the wild type copy of a mutated gene, by rescuing the mutant with the transgene (Fire and Waterston, 1989);•
generating "antisense knockout" strains. If a gene's open reading frame is hooked up "backwards" to its own promoter, antisense RNA will be made, and this will suppress expression (translation) of the wild type mRNA (Fire et al., 1991);Figure 2. Microinjection in the C. elegans gonad. The optimal position of the injection needle in the cytoplasmic core of the distal germ line is depicted. For DNA transformation, injection solution should flow in both directions through both the distal and proximal germ line (arrows)(from Evans, 2006 WormBook).
•
analysing promoter elements by hooking the 5' region of a gene up to enymatic (lacZ or !-galactosidase) (Fire et al., 1990) or fluorescent (GFP, RFP, Venus, Cherry, etc.) (Chalfie et al., 1994) markers/reporters;•
driving the overexpression of a given gene to look at gain-of-function mutations (Fire and Waterston, 1989);•
introducing in vitro mutated or foreign genes to examine structure-function relationships;•
constructing complexly mutated strains.An alternative method of C. elegans transformation named biolistic transformation, which relies on microparticle bombardment for DNA delivery into the gonad rather than on microinjections, has been developed (Wilm et al., 1999). In this method, transgene DNA is first coated on gold microparticles and then shot into the worms by means of helium pressure. The strong selection markers that allow easy identification of transformants surpasses the main disadvantage of this technique such as low absolute efficiency of biolistic transformation (i.e.
number of transformed progeny relative to the total number of bombarded worms). The bombardment procedure itself is a straightforward and already standardized technique. The main difference between conventional microinjection and biolistic transformation is that the latter can produce both extrachromosomal and low-copy-number integrated lines (Berezikov et al., 2004).
In-situ hybridization and immunofluorescence procedures for the examination of the distribution of mRNA (Seydoux and Fire, 1994) or protein (Strome and Wood, 1982) produced by the wild-type gene have also been developed. However, due to worms’ thick cuticle, the permeabilisation step, necessary for the probe/antibody penetration, is very difficult. Thus, the sensitivity and resolution may not be as good as that obtained with reporter gene fusion. The reporter gene-fusion approach remains the best technique to study genes expression pattern.
One of the main advantages of the nematode Caenorhabditis elegans model system is the easiness to conduct forward genetic screens and to isolate mutants with the phenotypes of interest. However, identifying the mutated genes requires positional cloning, which usually requires laborious work and a large amount of time. In the past few years, a new technique was adapted for C elegans: insertional mutagenesis with a heterologous transposon. The Drosophila mariner element Mos1 can be mobilized in the C. elegans germline to cause mutations (Bessereau et al., 2001). This technique bypasses the mapping steps and accelerates the process of identifying the mutated genes. Mutagenic insertions are subsequently localized within the genome using inverse PCR. As a disadvantage, the mutagenesis induced by using this technique is roughly one order of magnitude lower compared with chemical mutagenesis.
The main advantage is that the molecular identification of the mutated genes is extremely rapid.
Therefore, the decision of using Mos1-mediated mutagenesis or classical chemical mutagenesis must take into consideration the trade-off between the amount of time spent screening for mutants versus time spent mapping and rescuing a mutation (Bessereau, 2006;
Williams et al., 2005).
The suitability for genetic analysis is considered the principal advantage of this model system for biological investigation. On the other hand, some experimental procedures that are considered routine for other model systems may be difficult or impossible to use for C. elegans. There are no nematode cell lines available. The dissection of the specific tissues is unrealistic given the small size of the animal, although preparation of the pharynx can be used for the physiological studies (Pemberton et al., 2001) or careful dissection can be performed for patch-clamp recordings of individual neurons (Goodman et al., 1998). The small size of the embryo makes grafting experiments less feasible for studying development in C. elegans, although single-cell and/or early embryo manipulations are customary in the field.
For example, individual blastomeres can be isolated from the early embryo and are able to develop in culture. Moreover, applying pressure to the surface of the egg can change the position of the early blastomeres (Schierenberg and Wood, 1985). It is also possible, using a laser microbeam, to ablate specific cells in live animals at embryonic or post-embryonic stages (Sulston and White, 1980).
Most of the work with C. elegans involves either the whole animal or Figure 3. All C. elegans neurons are mapped and
graphically represented (from WormAtlas)
specific genes of interest. Preparing a time-scale schedule of the different experiments planned is very important for efficiently working with C. elegans. Most of the knowledge about C. elegans is gathered into an open-access virtual library – the C. elegans model organism database named WormBase (http://www.wormbase.org) containing the genomic, genetic, anatomy, people and literature information. Associated to this database, the WormBook contains original reviews on all aspects of C. elegans biology and up-to-date descriptions of technical procedures used to study this model system (http://www.wormbook.org). These very well organized informational resources ease the work with C. elegans.
3.2. Caenorhabditis elegans as model for studying nervous system development and function
The nervous system is the most complex tissue in most animals, including C. elegans, and genes involved in its development and function represent the main interest of many major researching projects. The C. elegans hermaphrodite nervous system comprises 302 neurons and 56 glial and support cells, all of which make up 37% of the somatic cells (Figure 3). These neurons are interconnected by circa 5000 chemical synapses, 700 gap junctions and 2000 neuromuscular junctions (White et al., 1986b). The structure of each neuron is nearly invariant from worm to worm and the cellular bodies are invariantly positioned allowing in vivo identification. Each of the 302 neurons has a unique combination of properties such as morphology, neurochemistry, and synaptic connectivity, which mediates a variety of behaviours:
locomotion, egg-laying, defecation, feeding, ability to respond to diverse environmental stimuli, etc. (Brenner, 1974). The majority of the neurons are located in the head surrounding the pharynx (Albertson and Thomson, 1976; Ward et al., 1975; Ware et al., 1975), along the ventral midline (White et al., 1976) and in the tail (Hall, 1977). Many neuronal processes form a ring around the pharynx (the nerve ring) or bundles along the body (the nerve cords)(Figure 4).
Figure 4. C. elegans nervous system. The 302 neurons of C. elegans are mostly located within the head and tail ganglia and along the ventral nerve cord. The nerve ring is the central region of neuropil in the animal. The ventral cord is the main process bundle that emanates from the nerve ring. It is composed of the processes of interneurons and motor neurons. Most of the processes that run in the dorsal cord are axons of motor neurons that originate in the ventral cord and enter in the dorsal cord via commisures.
Based on structure and functional information, about half of the neurons are inter-neurons, one- third are sensory and about one-quarter are motor neurons (Thomas and Lockery, 1999). The male has extra 79 nerve cells, totalling 381 neurons and 92 glial and support cells, which are probably related to differences in the sexual behaviour.
The simple organisation of the nervous system, along with its cellular complexity and knowledge base, makes the C. elegans nervous system a useful model to study the developmental and functional neurobiology. Hence, different assays for assessing the nervous system function, including electrophysiological, pharmacological and behavioural assays were developed to analyse evident or subtler phenotypes. Therefore the complete invariant cell lineages that reveal the developmental origin of every neuron (Sulston et al, 1983), the wiring diagram based on serial section electron micrographs that describe all the synapses between neurons (White et al., 1986a) or the possibility of ablating a specific neuronal cell (Bargmann and Avery, 1995; Bargmann and Kaplan, 1998; Sulston and White, 1980) represent unique advantages of this model system. Furthermore, using C. elegans as a model is relatively easy to genetically identify genes and molecularly confirm their role in specific processes (Schafer, 2005). A large number of proteins playing vital roles in nervous system development and function were first genetically identified in C. elegans, like, for example, the guidance molecule netrin (UNC-5) and its receptor UNC-6 (Hedgecock et al., 1990; Leung-Hagesteijn et al., 1992);
the acetylcholine vesicle transporter UNC-17 (Alfonso et al., 1993); the first olfactory receptor with defined odorant specificity ODR-10 (Sengupta et al., 1996).
One striking feature of C. elegans nervous system is that most of the neurons are non- essential for the life of the laboratory hermaphrodites. Only three neurons are required for normal development (two of them located in the body – CANL and CANR and one in the pharynx – M4)(Avery and Horvitz, 1989). Thus, many mutations leading to defects in the nervous system will not be lethal (Bargmann et al., 1993; Bargmann and Horvitz, 1991; Chalfie et al., 1985; Nonet et al., 1993).
3.2.1 BEHAVIOURAL TOOLS
Similarly to a human neurological exam, there are some standard behavioural tests used for the initial screen of new mutants (Brenner, 1974; de Bono and Maricq, 2005). The worms are able to move forward and backward by propagating sinusoidal waves along their bodies, to perform exploratory movements with their heads as they feed and to respond to a number of sensory stimuli, like mechanical stimulation, changes in chemical environment, osmolarity and temperature. The response to these sensory stimuli is reflected by specific movements, but can be also represented by feeding, egg laying or entry/escape from dauer stage. All these behaviours are scored and can be analyzed to determine the role of various genes in the nervous system. The most studied behaviours by mutational analyzes are
coordinated movement, chemotaxis, thermotaxis, osmotic avoidance, male mating, egg laying, mechanosensation. Some other behaviours are less studied or recently considered: pharyngeal pumping, response to light or “precipice response” (rapid backing when the animal comes to a sharply cut edge of the agar). Several of the most studied behaviours are presented below:
locomotion, egg-laying and mechanosensory responses.
3.2.1.1 Locomotion
Mutations in a large number of genes affect the normal movement of the worms. The uncoordinated mutants (unc) are among the first isolated locomotory mutations. This phenotype could be caused by defects in any of the 95 body-wall muscle cells or their attachments to the body wall, or from lesions that affect nervous system function/structure. Fairly different underlying lesions may produce similar behavioural phenotypes, which makes difficult to assess the site of action (neuron, muscle or hypodermis) for many of the unc genes. The alterations in the nervous system of the unc mutants can be connected to motor neurons, process placement, synaptic specificity or neurotransmitter function. Most of the ventral cord motor neurons arise after hatching, therefore mutations that affect these neurons result in worms that are initially coordinated but become uncoordinated in time. Several mutants have commissures that fail to reach the dorsal cord (unc-5, unc-6, unc-51), or highly disorganized processes for all classes of ventral motor neurons (unc-3), or restricted to a limited number of motor neurons classes (unc- 30). Only two mutants have been reported with changes in synaptic specificity: unc-4 and unc- 55. Assays for scoring locomotion are diverse: observation of the worm posture, movement response to tapping the Petri dish, measuring the rate or amplitude of sinusoidal oscillation and measuring the rate of movement. Because of the limited range of worm movements, even subtle defects in locomotion can be scored.
3.2.1.2 Egg laying
A hermaphrodite that has not being mated lays about 300 eggs during its reproductive life span. The mature adult is fertile for about four days. The vulval and uterine muscles control egg laying. Two types of neurons (two HSN cells and six VC motor neurons) make synapse with these muscles. Laser ablations showed that only the two HSN neurons are required for normal egg laying. The rate of egg laying is influenced by the presence of food as hermaphrodites are laying more eggs when fed. Three neurotransmitters, serotonin, acetylcholine and octopamine, are involved in the regulation of the egg laying. The first one, serotonin, stimulates the egg laying process. Furthermore, imipramine which prevents the serotonin reuptake into the pre- synaptic element, potentates the endogenous serotonin activity. Similarly, drugs which upregulates cholinergic activity stimulate the egg laying. In addition, octopamine inhibits egg
laying and phentolamine (octopaminergic antagonist) stimulates it. Most eggs are laid in short bursts of 3-4 eggs (active state) separated by 20 minutes breaks (inactive state). Serotonin seems to be responsible for the induction of active state while acetylcholine may trigger individual egg laying muscle contractions (Waggoner et al., 1998). The classical assay for scoring egg laying consists in moving the adults into microtitre wells filled with saline buffer with or without drugs/neurotransmitters and counting the eggs laid over time. Other assays measure the time from egg fertilisation to egg-laying by checking the stage of embryogenesis of newly laid egg, the stage of the oldest eggs in the uterus, or by counting the total number of eggs from uterus. Increased numbers of eggs inside the adult worm can suggest problems of the muscles or in the neuron’s function or structure. The modification concerning the stage of the laid eggs reflects, usually, developmental defects.
3.2.1.3 Mechanosensory responses
The nose-touch response represents the briefly backward movement which follows after the tip of the nose collides into an obstacle. There is also an independent response to touch by an eyelash to the side of the body, named Mec response: a touch in the anterior part makes the worm move vigorously back and a touch of the posterior part make it move forward. A reaction to a much harsher touch occurs even in the absence of the Mec response. Mainly, glutaminergic neurotransmission was associated with the nose-touch response (Hart et al., 1995; Maricq et al., 1995).
3.2.2 PHARMACOLOGICAL TOOLS
The exposure to exogenous neurotransmitters or other pharmacological agents represents one of the most intensively employed manipulations in worm neurological studies.
Most of these substances affect diverse aspects of neurotransmission, often at the neuromuscular junctions affecting locomotion. Consequently, scoring locomotion is an essential step in the behavioural assays. Some of the most used substances are presented in Table 1.
Neurotransmitter Substance Action Result
Acetylcholine Aldicarb Inhibitor of acetylcholine esterase
Levamisole Inotropic acetylcholine receptor agonist Body-wall muscle hypercontraction GABA Muscimol Inotropic GABA receptor agonist Flaccid paralysis of the body-wall
muscle
Dopamine Dopamine Flaccid paralysis of the body-wall
muscle
Glutamate Avermectine Glutamate-gated Cl- channel agonist Inhibit pharyngeal pumping (low conc.) Flaccid paralysis (high concentration) AMPA Glutamate-gated cation channel agonist Hyperactive foraging movements of
the nose Octopamine Octopamine invertebrate neurotransmitter Blocks egg-laying Serotonine Serotonine
Fluoxetine Serotonin reuptake inhibitor Imipramine Serotonin reuptake inhibitor
Increase egg-laying
Table 1. Some of the most used substances for studying the neurotransmission involvement in different behaviours (adapted from (Hope, 1999)
Therefore, these drugs provide exceptionally useful pharmacological tools to delineate fundamental aspects of cell signalling in C. elegans, primarily through their use in forward genetic screens, followed by the mapping and characterization of genes that confer altered receptivity to the drug.
3.3. Molecular mechanisms involved in the establishment and maintenance of neuronal networks
3.3.1 THE STRUCTURE OF THE SYNAPSE IN C. ELEGANS
One of the most important challenges in developmental neuroscience is to understand how neuronal cells can identify the correct partners, interact with them, and ultimately create a functional neuronal network. Synapses are critical structures mediating interactions between neurons; they are constituted of three major elements: the pre-synaptic element, the postsynaptic element and the synaptic cleft, which separates pre- and postsynaptic structures (Figure 5). The pre-synaptic terminals are locally enlarged and filled with synaptic vesicles.
Synaptic vesicles are docked at the pre-synaptic plasma membrane in regions called active zones (AZ). Vesicles close to the active zone are smaller and less electron-dense than transport vesicles, which travel along the microtubules from the soma to the synapse. Opposite is a region of the postsynaptic cell containing neurotransmitter receptors. The postsynaptic density (PSD) represents an elaborate complex of interlinked proteins, situated close to the Figure 5. Schematic drawing of a synapse. Synaptic vesicles (or vesicle precursors) are synthesized in the soma and transported to synaptic terminals by axonal transport and loaded with neurotransmitter. Loaded vesicles are translocated to active zone and docked to release sites. Calcium influx initiates a rapid fusion of SVs with
plasma membrane and
neurotransmitter is released in the synaptic cleft, where it will bind to neurotransmitter receptors from the postsynaptic cell. To terminate the neurotransmition signal in the synaptic celft, the trasmitter is either destroyed or transported back into pre-synaptic cell via transmitter re-uptake pumps.
Vesicle membranes are recycled by endocytosis..
postsynaptic membrane. PSD proteins are involved in anchoring and trafficking neurotransmitter receptors and also in the activity modulation of these receptors. The synaptic cleft is defined by circa 20 nm-wide gap between the pre- and postsynaptic cells. The cleft small volume allows neurotransmitter concentration to be raised and lowered rapidly. The membranes of the two adjacent cells are held together by cell adhesion proteins (Uchida et al., 1996). The main function of the synapse is to transmit information between neurons. When an electrical impulse propagates in the pre-synaptic neuron, small chemical molecules (neurotransmitters) are released into the synaptic cleft where they interact with specific receptors of the postsynaptic neuron, causing either the opening or closing of the ion channels, or the activation of signal transduction pathways through G-protein coupled receptors (GPCRs).
Most neurons in C. elegans are extremely simple in structure and contain one or few processes. The vast majority of the synapses are formed en passant (along the axonal shaft)(White et al., 1986a). The specificity of synaptic contacts made by individual neurons is relatively invariant from worm to worm. Individual synapses are varying widely in size and vesicle density. Most vesicles in C. elegans have circa 35-45 nm in diameter (SV), but large dense-core vesicles (LDCV) are 40-53 nm. The overall ultrastructural look of pre-synaptic terminal of worm synapses resembles a lot with those of vertebrate central nervous system depicted above, however the postsynaptic sites of worm synapses do not have visible density- like structures. Therefore, synaptic partners are allocated based on proximity (Jin, 2005). A key tool in analysing synapses in vivo is the synaptobrevin GFP fusion reporter (Nonet, 1999).
Synaptobrevin (SNB-1) is a small integral membrane protein of secretory vesicles, which are part of the vesicle-associated membrane protein (VAMP) family. Recently, a large number of fluorescent markers have been developed for the study of different aspects of synapse structure and/or function (Crump et al., 2001; Francis et al., 2005; Sieburth et al., 2005; Yeh et al., 2005;
Zhang et al., 2002; Zhen and Jin, 1999). Genetic screens for synaptogenesis mutants using the SNB-1::GFP pattern as a visual tracker helped identifying several proteins involved in the function of the synapse (Crump et al., 2001; Schaefer et al., 2000; Shen and Bargmann, 2003;
Sieburth et al., 2005; Zhen and Jin, 1999). Most of the mutations selected seem to have wide effects on many or all synapses, but cause modest behaviour abnormalities.
3.3.2 PROTEINS INVOLVED IN NEUROTRANSMISSION IN C. ELEGANS
Neurotransmission is the process by which neurons transfer information via chemical signalling at synaptic contacts with target cells, often on a rapid time scale. Consequently, synaptic transmission requires the coordinated activity of a wide array of proteins. These proteins regulate many aspects of the synaptic function including: synapse formation, neuron and muscle excitability, calcium signalling, synaptic vesicle cycling (exocytosis and endocytosis), the assembly, localization and function of the receptors. The electrical events that
