Ces dernières années, un nouveau composant de l'environnement des tumeurs (ET) a été mis en évidence: les projections des neurones du système nerveux. En effet, les tumeurs sont infiltrées par des axones, ce qui pourrait réguler la progression du cancer.Le cancer du pancréas fait partie des cancers les plus mortels. Les traitements thérapeutiques actuels qui ciblent ce cancer ne sont pas efficaces. Il est donc important de mieux comprendre les différentes composantes de l'ET de ce cancer afin d’identifier de nouvelles cibles thérapeutiques. Nous proposons de décrire l’innervation des tumeurs pancréatiques ce qui est le point de départ pour mieux comprendre l’importance de cette composante de l'ET. Les objectifs ont été d’analyser en 3D les réseaux d'axones qui innervent le pancréas sain et cancéreux, ainsi que leurs relations avec d'autres types cellulaires de l'ET (vaisseaux sanguins (VS)).Pour cela, nous avons utilisé une méthode d'imagerie 3D de pancréas entiers, rendus transparents, qui proviennent de modèles génétiques de souris qui développent des cancers du pancréas similaires à ceux de l'homme. Nous avons observé que les réseaux d'axones sont plus denses et plus complexes dans les régions cancéreuses du pancréas par rapport aux régions saines. Alors que dans les tissus sains les axones sont associés aux VS, ils ne le sont plus dans les régions cancéreuses. Nous avons de plus identifié des groupes morphologiques de réseaux d'axones qui permettent de discriminer une région saine d'une région cancéreuse.L’analyse de la structure en 3D de ces réseaux d'axones pourrait donc représenter une donnée prédictive et pronostique de l'état d'avancé clinique de la maladie. / Cancers are diseases in which cancer cells interact with a complex tumor environment (TE). In recent years, a new component of TE has been highlighted: neuronal projections of the nervous system. Indeed, the axons of neurons innervate the tumors, which could regulate cancer progression.Pancreatic cancer is among the most deadly cancers. Indeed, the current therapeutic treatments that target this cancer are not effective. It is therefore important to better understand the different components of the TE of this cancer in order to identify new potential therapeutic targets.In this thesis, we propose to describe the innervation of pancreatic tumors which is the starting point to better understand the importance of this component of the TE. The objectives were to visualize and analyze in 3D the networks of axons that innervate the healthy and cancerous pancreas, as well as their relations with other cell types of the TE (blood vessels (BV)).For this, we used a method of 3D imaging of whole pancreas, made transparent, which come from genetic models of mice that develop pancreatic cancer similar to that of humans.We observed that axon networks are denser and more complex in cancerous regions of the pancreas compared to healthy regions. Moreover, while in healthy tissue, axons are associated with BV, they are no longer in cancerous areas.We also identified morphological groups of axon networks that discriminate a healthy region from a cancerous region.The analysis of the 3D structure of these axon networks could thus represent a predictive and prognostic value for the progression of the disease.

Lesioned axons do not regenerate in the adult mammalian central nervous system, owing to the overexpression of inhibitory molecules such as myelin-derived proteins or chondroitin sulphate proteoglycans. In order to overcome axon inhibition, strategies based on extrinsic and intrinsic treatments have been developed. For myelin-associated inhibition, blockage with NEP1-40, receptor bodies or IN-1 antibodies has been used. In addition, endogenous blockage of cell signalling mechanisms induced by myelin-associated proteins is a potential tool for overcoming axon inhibitory signals. We examined the participation of glycogen synthase kinase 3 (GSK3beta) and ERK1/2 in axon regeneration failure in lesioned cortical neurons. We also investigated whether pharmacological blockage of GSK3beta and ERK1/2 activities facilitates regeneration after myelin-directed inhibition in two models: i) cerebellar granule cells and ii) lesioned entorhino-hippocampal pathway in slice cultures, and whether the regenerative effects are mediated by Nogo Receptor 1 (NgR1). We demonstrate that, in contrast to ERK1/2 inhibition, the pharmacological treatment of GSK3beta inhibition strongly facilitated regrowth of cerebellar granule neurons over myelin independently of NgR1. Lastly these regenerative effects were corroborated in the lesioned EHP in NgR1 -/- mutant mice. These results provide new findings for the development of new assays and strategies to enhance axon regeneration in injured cortical connections. On the other hand, and focused in the OMgp, by using recording electrophysiological nano-devices we found that, OMgp has a role in synaptic transmission, since it can induce excitatory postsynaptic potentials (EPSPs) in cultured hippocampal neurons.

No description available.

Durant le développement du système visuel, les cellules ganglionnaires de la rétine (CGRs) envoient des axones qui seront influencés par divers signaux guidant leur cône de croissance, permettant ainsi la navigation des axones vers leurs cibles terminales. Les endocannabinoïdes, des dérivés lipidiques activant les récepteurs aux cannabinoides (CB1 et CB2), sont présents de manière importante au cours du développement. Nous avons démontré que le récepteur CB2 est exprimé à différents points du tractus visuel durant le développement du hamster. L’injection d’agonistes et d’agonistes inverses pour le récepteur CB2 a modifié l’aire du cône de croissance et le nombre de filopodes présents à sa surface. De plus, l’injection d’un gradient d’agoniste du récepteur CB2 produit la répulsion du cône de croissance tandis qu’un analogue de l’AMPc (db-AMPc) produit son attraction. Les effets du récepteur CB2 sur le cône de croissance sont produits en modulant l’activité de la protéine kinase A(PKA), influençant la présence à la membrane cellulaire d’un récepteur à la nétrine-1 nommé Deleted in Colorectal Cancer (DCC). Notamment, pour que le récepteur CB2 puisse moduler le guidage du cône de croissance, la présence fonctionnelle du récepteur DCC est essentielle.. Suite à une injection intra-occulaire d’un agoniste inverse du récepteur CB2, nous avons remarqué une augmentation de la longueur des branches collatérales des axones rétiniens au niveau du LTN (noyau lateral terminal). Nous avons également remarqué une diminution de la ségrégation des projections ganglionnaires au niveau du dLGN, le noyau genouillé lateral dorsal, chez les animaux transgéniques cnr2-/-, ayant le gène codant pour le récepteur CB2 inactif. Nos données suggèrent l’implication des endocannabinoïdes et de leur récepteur CB2 dans la modulation des processus de navigation axonale et de ségrégation lors du développement du système visuel. / Retinal projection navigation towards their visual targets is regulated by guidance molecules and growth cone transduction mechanisms. Here, we show that cannabinoid receptor 2 (CB2R) is widely expressed throughout the visual pathway during development and in cultured retinal ganglion cells (RGCs) and cortical neurons. Both the number of filopodia and the surface area of the growth cone (GC) are modulated by CB2R activity in a PKA-dependant manner. Furthermore, DCC is required for CB2R induced morphological changes of the GC. CB2R agonists induce GC chemorepulsion. Treatments altering CB2R activity change retinal explants projections length. These effects are specific to CB2R as no changes were observed in cnr2 knockout mouse. A single intraocular injection of AM630 increases retinal projection branch length and aberrant projections were also observed. Moreover, we report defect in eye-specific segregation of retinal projections in the dLGN in cnr2-/- mice. These findings highlight the modulatory role of endocannabinoids and their CB2R during axon guidance.

Introdução: Ativação exacerbada de processos excitatórios mediados por NMDA e excesso de inibição mediada por GABA são descritos, respectivamente, nas fases agudas e subagudas após o traumatismo cranioencefálico (TCE). No entanto, existem poucos estudos a respeito do funcionamento desses circuitos na fase crônica do TCE. Objetivo: Avaliar a excitabilidade cortical (EC) de pacientes em fase crônica que sofreram TCE, especificamente diagnosticados com lesão axonial difusa (LAD). Métodos: Todos os 31 pacientes adultos foram avaliados após 1 ano, pelo menos, do TCE moderado ou grave. Inicialmente, os pacientes foram submetidos à avaliação de funções executivas - atenção, memória, fluência verbal e velocidade de processamento de informação - por meio de bateria neuropsicológica. Em seguida, a avaliação da EC foi realizada utilizando-se uma bobina circular para aplicar pulsos simples e pareados de estimulação magnética transcraniana na região cortical representativa do abdutor curto do polegar (pollicis brevis) na área M1 de ambos hemisférios. Os parâmetros de EC medidos foram: Limiar Motor de Repouso (LMR), Potenciais Evocados Motores (PEM), Inibição Intracortical de Intervalo Curto (IICIC) e Facilitação Intracortical (FIC). Todos os dados foram comparados aos dados normativos de EC já descritos na literatura e também aos de um grupo controle de pessoas saudáveis. Resultados: Não houve diferença significativa entre os hemisférios direto e esquerdo. Desta forma, os dados foram analisados de forma agrupada (\"pooled data\"). Os valores de LMR e FIC dos pacientes com LAD estavam dentro dos valores de normalidade. No entanto, os valores de PEMs a 120% do LMR, a 140% do LMR e IICIC estavam aumentados (respectivamente p=0,013; p=0,012; p < 0,001): PEM-120% LAD 524,95 [365,42 ; 616,66] versus Controles 303,50 [241,49 ; 399,19]; PEM-140% LAD 1150,00 [960,56 ; 1700,00] vs Controles 670,5 [575,43 ; 1122,78] e IICIC LAD 1,09 [0,82 ; 1,35] vs Controles 0,34 [0,28 ; 0,51]; pp02-Rel LAD 0,85 [0,64 ; 1,36] vs Controles 0,28 [0,20 ; 0,37]; pp04-Rel LAD 1,03 [0,88 ; 1,34] vs Controles 0,38 [0,29 ; 0,62] - sugerindo um possível desarranjo no sistema inibitório (p < 0.001). Os achados neuropsicológicos mostraram alterações na memória, atenção e velocidade de processamento de informação, mas possuíam correlação fraca com os dados de EC. Conclusão: Como os processos inibitórios envolvem circuitos mediados por GABA, além de outros, existe a possível inferência de que a própria fisiopatologia do LAD (rompimento de axônios) possa depletar GABA contribuindo com a desinibição do sistema neural na fase crônica do LAD / Background: Overactivation of NMDA-mediated excitatory processes and excess of GABA-mediated inhibition are described after a brain injury on the acute and subacute phases, respectively. Nevertheless, there are few studies regarding the circuitry on the chronic phase of brain injury. Objective: To evaluate the cortical excitability (CE) on the chronic phase of Traumatic Brain Injury (TBI) victims, specifically diagnosed with Diffuse Axonal Injury (DAI). Method: All 31 adult patients were evaluated after one year, at least, from the moderate and severe TBI. First, all patients underwent a broad neuropsychological assessment to evaluate executive functions - attention, memory, verbal fluency and information processing speed. Then, subsequently, the CE assessment was performed with a circular coil applying single-pulse and paired-pulse transcranial magnetic stimulation over the cortical representation of the abductor pollicis brevis muscle on M1 of both hemispheres. The CE parameters measured were: Resting Motor Threshold (RMT), Motor-Evoked Potentials (MEP), Short Interval Intracortical Inhibition (SIICI), and Intracortical Facilitation (ICF). All data were compared to normative data previously described on literature and to a control group that consisted of healthy subjects. Results: No significant difference between Left and Right hemispheres were found on these DAI patients. Therefore, parameters were analyzed as pooled data. Values of RMT and ICF from DAI patients were found within the normality. However, MEPs and SIICI values were higher on DAI patients (respectively p=0,013; p=0,012; p < 0,001): MEP-120% DAI 524,95 [365,42 ; 616,66] versus Control 303,50 [241,49 ; 399,19]; MEP-140% DAI 1150,00 [960,56 ; 1700,00] vs Control 670,5 [575,43 ; 1122,78] and SIICI DAI 1,09 [0,82 ; 1,35] vs Control 0,34 [0,28 ; 0,51]; pp02-Rel DAI 0,85 [0,64 ; 1,36] vs Control 0,28 [0,20 ; 0,37]; pp04-Rel DAI 1,03 [0,88 ; 1,34] vs Control 0,38 [0,29 ; 0,62] - suggesting a disarranged inhibitory system (p < 0.001). The neuropsychological findings had weak correlation with CE data. Conclusion: As inhibition processes also involve GABA-mediated circuitry, it is likely to infer that DAI pathophysiology itself (disruption of axons) may deplete GABA contributing to a disinhibition of the neural system on the chronic phase of DAI

No description available.

[ES] La esquizofrenia (SC) es un trastorno del neurodesarrollo que afecta los procesos cognitivos y el comportamiento social. A diferencia de otras neuropatologías, los cerebros de los pacientes con SC no muestran características histológicas evidentes y los mecanismos moleculares subyacentes a la enfermedad siguen siendo desconocidos, lo que dificulta mucho su prevención y tratamiento eficaz. Los endofenotipos más consistentes en la SC incluyen la reducción del neuropilo, la conectividad funcional deteriorada entre las áreas corticales y cambios específicos en las conexiones sinápticas. El cuerpo calloso (CC) es el haz más grande de fibras nerviosas cortico-corticales, conectando ambos hemisferios cerebrales, y su desarrollo es un proceso complejo crucial para la formación adecuada de los circuitos corticales. Numerosa evidencia convergente apoya la hipótesis de que el CC se encuentra hipoconectado en pacientes con SC. Está bien establecido que la SC tiene un fuerte componente genético. Un gen clave implicado en el riesgo de SC es neuregulina 1 (NRG1), controlando varios aspectos del desarrollo neuronal. Estudios previos se han centrado principalmente en la señalización de Nrg1 en las interneuronas inhibitorias, descuidando su papel en las neuronas excitatorias. Para comprender el papel de Nrg1 en los circuitos corticales, en este trabajo estudiamos el papel de Nrg1 en el desarrollo de los axones del cuerpo calloso y específicamente, su función celular autónoma en las neuronas excitatorias. Para este objetivo quisimos, 1) evaluar la participación de la Nrg1 en el desarrollo de las neuronas de proyección callosa; 2) investigar el efecto autónomo de Nrg1 en el desarrollo de las neuritas; 3) estudiar los mecanismos moleculares subyacentes al papel de Nrg1 en el desarrollo neuronal; 4) explorar el potencial de Nrg1 en la reprogramación de astrocitos a neuronas, como un nuevo posible enfoque terapéutico después de una lesión cerebral. Para ello, primero desarrollamos un modelo in vivo de pérdida de función para determinar el papel de Nrg1 en el desarrollo de las proyecciones callosas.Descubrimos que la eliminación de Nrg1 en el modelo de ratón condicional impedía el desarrollo de axones callosos in vivo. A nivel mecanístico, encontramos que la señalización intracelular de Nrg1 es tanto necesaria como suficiente para promover el desarrollo axonal en las neuronas corticales in vivo. En segundo lugar, para determinar específicamente el papel de Nrg1 en el desarrollo axonal de las neuronas excitatorias, empleamos un modelo in vitro con un enfoque más reduccionista. Realizamos cultivos primarios de neuronas corticales. En este modelo, llevamos a cabo experimentos de ganancia y pérdida de función para investigar específicamente el efecto autónomo celular de Nrg1 sobre el desarrollo de dendritas y axones. Nuestros experimentos con cultivos primarios de neuronas confirmaron que la señalización intracelular de Nrg1 es necesaria y suficiente para promover el crecimiento axonal in vitro. En tercer lugar, estudiamos los mecanismos moleculares subyacentes al papel de Nrg1 en el desarrollo neuronal. Descubrimos mediante Western blot e inmunofluorescencia que la expresión de la proteína GAP43 está altamente disminuida en neuronas knockout para Nrg1. Además, observamos que disminución del desarrollo axonal en neuronas knockout para Nrg1 es parcialmente rescatado al sobreexpresar la proteína GAP43. Estos resultados sugieren que la señalización a través de GAP43 podría ser uno de los mecanismos involucrados en el papel de Nrg1 en el crecimiento axonal. En conjunto, nuestro estudio indica un papel crucial para la señalización intracelular de Nrg1 en el desarrollo de las conexiones cortico-corticales que conectan ambos hemisferios cerebrales. Nuestros resultados sugieren que la disfunción de Nrg1 en las neuronas excitatorias puede contribuir a la hipoconectividad asociada a la SC y las alteraciones del desarrollo neurológico. / [CA] L'esquizofrènia (SC) és un trastorn del neurodesenvolupament que afecta els processos cognitius i el comportament social. A diferència d'altres neuropatologies, els cervells dels pacients amb SC no mostren característiques histològiques evidents i els mecanismes moleculars subjacents a la malaltia continuen sent desconeguts, la qual cosa dificulta molt la seua prevenció i tractament eficaç. Els endofenotipus més consistents en la SC inclouen la reducció del neuropil, la connectivitat funcional deteriorada entre les àrees corticals i els canvis específics en les connexions sinàptiques. El cos callós (CC) és el feix més gran de fibres nervioses cortico-corticals, connecta tots dos hemisferis cerebrals, i el seu desenvolupament és un procés complex crucial per a la formació adequada dels circuits corticals. Nombrosa evidència convergent dona suport a la hipòtesi que el CC es troba hipoconnectat en pacients amb SC. Està ben establit que la SC té un fort component genètic. Un gen clau implicat en el risc de SC és neuregulina 1 (NRG1) que controla diversos aspectes del desenvolupament neuronal. Estudis previs s'han centrat principalment en la senyalització de Nrg1 en les interneurones inhibitòries, descurant el seu paper en les neurones excitatòries. Per a comprendre el paper de Nrg1 en els circuits corticals, en este treball estudiem el paper de Nrg1 en el desenvolupament dels axons del cos callós i específicament, la seua funció cel·lular autònoma en les neurones excitatòries. Per a este objectiu, 1) avaluem la participació de Nrg1 en el desenvolupament de les neurones de projecció callosa; 2) investiguem l'efecte autònom de Nrg1 en el desenvolupament de les neurites; 3) estudiem els mecanismes moleculars subjacents al paper de Nrg1 en el desenvolupament neuronal; 4) explorem el potencial de Nrg1 en la reprogramació d'astròcits a neurones, com un nou possible enfocament terapèutic després d'una lesió cerebral. Per això, emprem ratolins knockout nounats per a Nrg1 i realitzem un rastreig neuronal de les projeccions calloses, així com també rastregem estes projeccions en ratolins wild-type mitjançant la tècnica d'electroporació in utero. Descobrim que l'eliminació de Nrg1 en el model de ratolí condicional impedia el desenvolupament d'axons callosos in vivo. A nivell mecanístic, trobem que la senyalització intracel·lular de Nrg1 era suficient per a promoure el desenvolupament axonal en les neurones corticals in vivo. En segon lloc, per a determinar específicament el paper de Nrg1 en el desenvolupament axonal de les neurones excitatòries, emprem cultius primaris de neurones corticals. En este model, duem a terme experiments de guany i pèrdua de funció per a investigar específicament l'efecte autònom cel·lular de Nrg1 sobre el desenvolupament de dendrites i axons. Els nostres experiments amb cultius primaris de neurones van mostrar que la senyalització intracel·lular de Nrg1 és necessària i suficient per a promoure el creixement axonal in vitro. En tercer lloc, estudiem els mecanismes moleculars subjacents al paper de Nrg1 en el desenvolupament neuronal. Descobrim que l'expressió de la proteïna GAP43 està altament disminuïda en neurones knockout per a Nrg1. A més, observem que la disminució del desenvolupament axonal en neurones knockout per a Nrg1 és parcialment rescatat al sobreexpresar la proteïna GAP43. Estos resultats suggerixen que la senyalització a través de GAP43 podria ser un dels mecanismes involucrats en el paper de Nrg1 en el creixement axonal. En conjunt, el nostre estudi indica un paper crucial per a la senyalització intracellular de Nrg1 en el desenvolupament de les connexions cortico-corticals que connecten tots dos hemisferis cerebrals. Els nostres resultats assenyalen que la disfunció de Nrg1 en les neurones excitatòries pot contribuir a la hipoconnectivitat associada a la SC i a les alteracions del desenvolupament neurològic. / [EN] Schizophrenia (SZ) is a neurodevelopmental disorder that affects cognitive processes and social behavior, impacts approximately 1% of the population but presents a major socio-economic impact. Unlike other neuropathologies, the brains of SZ patients do not display obvious histological hallmarks and their molecular mechanisms remain unknown, making it very difficult to prevent and treat effectively. The most consistent endophenotypes in SZ include reduced neuropil, impaired functional connectivity between cortical areas and specific changes in synaptic connections. Therefore, SZ is a pathology based on abnormal cortical neuronal connectivity. The corpus callosum (CC), connecting the brain's hemispheres, is the largest bundle of cortico-cortical nerve fibers and its development is a complex process crucial for proper cortical circuitry formation. Converging evidence supports the hypothesis that the CC is hypoconnected in SZ patients. While the developmental etiology of SZ remains largely unresolved, it is well established that SZ has a strong genetic component. A key gene implicated in SZ risk is neuregulin 1 (NRG1), which controls several aspects of neuronal development. Prior research has primarily focused on Nrg1 signaling in inhibitory interneurons, neglecting its role in excitatory neurons. To understand the function of Nrg1 signaling in the development of cortical circuits, we studied the Nrg1's role in the development of callosal axons and specifically, the cell autonomous function in the excitatory neurons. To this aim, we sought to 1) evaluate the Nrg1's involvement in the development of callosal projecting neurons; 2) investigate the Nrg1's cell autonomous effect on neurites outgrowth; 3) study the molecular mechanisms underlying Nrg1's role in neuron development; 4) explore the Nrg1's potential in reprogramming astrocytes to neurons, as a novel therapeutic approach following brain injury. First, we developed an in vivo loss-of-function model to determine the role of Nrg1 in the development of callosal projections. We employed newborn Nrg1 knockout mice and performed neuronal tracing of the callosal projections, as well as we traced those projections in wild type mice by in utero electroporation. We found that the deletion of Nrg1 in a conditional mouse model impaired the development of callosal axons in vivo. On a mechanistic level, we found that the intracellular signaling of Nrg1 was sufficient to promote axonal development in cortical neurons in vivo. Second, to specifically determine the role of Nrg1 in axonal development of excitatory neurons, we employed a suitable in vitro model with a more reductionist approach. We performed primary cultures of cortical neurons. Using transfection by electroporation, we achieved sparse labeling and obtained internal controls. In this model, we carried out gain- and loss-of-function approaches to investigate specifically the Nrg1's cell autonomous effect on dendrites and axonal development. Our single-cell experiments in primary cultures showed that Nrg1 is cell-autonomously required and sufficient to promote axonal outgrowth in vitro. Third, we studied the molecular mechanisms underlying Nrg1's role in neuron development. We found by Western blot and immunofluorescence that the GAP43 protein expression is impaired in Nrg1 knockout neurons. Additionally, we observed that decreased axonal development in Nrg1 knockout neurons was rescued by overexpressing GAP43 protein. These results suggest that signaling through GAP43 may be one of the mechanisms involved in the role of Nrg1 in axonal growth. In conclusion, our study indicates a crucial role for Nrg1 intracellular signaling in the development of long-range cortico-cortical connections between brain hemispheres. It indicates that Nrg1 dysfunction in excitatory neurons may contribute to SZ-associated hypoconnectivity and neurodevelopmental alterations, providing new insights into the role of Nrg1 in the etiology of SZ. / Rodríguez Prieto, Á. (2024). Nrg1 Signaling in the Development of Cortical Circuits: Molecular Basis of Schizophrenia [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/212464

Im Gegensatz zum peripheren Nervensystem (PNS) ist die Regenerationsfähigkeit im adulten zentralen Nervensystem (ZNS) von Vertebraten sehr eingeschränkt. Diese eingeschrängte Regenerationsfähigkeit wird im Wesentlichen durch das Vorhandensein von Myelin im adulten ZNS determiniert. Einerseits ist dieses Lipid für die Stabilisierung und Ernährung von Axonen sowie für die schnelle Reizweiterleitung unbedingt notwendig, andererseits stellt es den größten Inhibitor axonaler Regeneration dar. Myelin ist außerdem Zielstruktur diverser ZNS Pathologien, wie z.B. der Multiplen Sklerose. Für das Verständnis dieser Pathologien sowie der auswachsinhibitorischen Wirkung von Myelin wurde der Hippocampus als eine der plastischten ZNS Regionen gewählt. Dazu waren genaue Kenntnisse der Myeloarchitektur dieses Gebietes notwendig. Nach Etablierung einer zuverlässigen Detektierung für Myelin konnten in der vorliegenden Arbeit detailliert Myelinisierungsvorgänge im sich entwickelnden, im adulten und im deafferenzierten Hippocampus der Ratte analysiert werden. Während der Entwicklung erreichen die ersten entorhinale Axone den Hippocampus bereits am embryonal Tag 17 (E17); Myelin kann jedoch erst am postnatal Tag 17 (P17) lichtmikrokopisch nachgewiesen werden. Die Anzahl myelinisierter Fasern erreicht um den P25 ein Verteilungsmuster, welches dem von adulten Tieren gleicht. Nach Entorhinaler Cortex Läsion (ECL), bei der die Durchtrennung des Tractus perforans (PP) eine Denervation des Hippocampus bewirkt, kommt es zu einem langanhaltenden Verlust von Myelin. Zehn Tage nach Läsion (10 dal), also zum Zeitpunkt maximaler Aussprossung (Sprouting), kommt es zu einem Wiederkehren myelinisierter Fasern. Mehrere myelin-assoziierte Proteine, mit wachstumshemmenden Eigenschaften sind bekannt, wie z.B. die Familie der Nogo Gene (Nogo; englisch, kein Durchkommen). Diese werden ganz entschieden für den Verlust der Regenerationsfähigkeit des adulten ZNS verantwortlich gemacht. In der vorliegenden Arbeit wird die Expression der drei Nogo Gene (Nogo-A, -B, - C) und deren Rezeptor (Ng66R) während der postnatalen Entwicklung, im adulten ZNS sowie nach Läsion beschrieben. Ein erster überraschender Befund war die neuronale Expression der Nogos, die bisher nur in Oligodendrocyten nachgewiesen worden war. Zu einem Zeitpunkt, an dem entorhinale Fasern bereits in den Hippocampus eingewachsen, aber noch nicht myelinisiert sind (P0), wird Nogo-A, -B und Ng66R mRNA mit Ausnahme der Körnerzellschicht des Gyrus dentatus in allen Zellschichten des sich entwickelnden Hippocampus detektiert. Nogo-C und myelin basic protein (MBP) mRNA, werden erst am P15 expremiert, zu einem Zeitpunkt also, an dem myelinisierte Fasern erstmalig im Hippocampus auftreten. MBP wird ausschließlich in glialen, Nogo-C hingegen hauptsächlich in neuronalen Zellen exprimiert. Nach Deafferenzierung zeigt sich eine dynamische und Isoform- spezifische Regulation aller Nogo Transkripte. So zeigen die als erste von der Deafferenzierung betroffenen Körnerzellen zu Beginn der Waller`schen Degeneration sowie der neuronalen und glialen Antwort, eine starke Erhöhung aller Nogo Transkripte. Zum Zeitpunkt der maximalen Aussprossung kam es zu einem signifikanten Abfall der Nogo-C und Ng66R mRNA Expression, währendessen Nogo-A und Nogo-B bereits wieder das Kontrollniveau erreicht hatten. Vor allem im contralateralen Hippocampus, dem Hauptquellgebiet sproutender Fasern, imponierte die Runterregulation von Ng66R mRNA und zeigte erst nach Abschluß von axonalen Sproutingprozessen und der Synapsenformation wieder vergleichbare Werte mit den Kontrolltieren. Diese Korrelation der erniedrigten Ng66R Expression im contralateralen Hippocampus und dem axonalen Einwachsen in den deafferenzierten Hippocampus, läßt eine reduzierte axonale Ansprechbarkeit auf den Neuriten-Auswachshemmer Nogo-A vermuten, da bekannt ist, dass Axone, die kein Ng66R exprimieren, nicht durch die Nogo Gene im Wachstum gehemmt werden. Zusammenfassend kommt es während der Entwicklung und in der Reorganisationsphase zu einer spezifischen und geordneten Myelinisierung im Hippocampus. Die neuronale Expression von Nogo- A, -B und -C in einer so plastischen ZNS- Region unterstützt die Hypothese, dass den Nogo- Genen neben der reinen Hemmung von axonalen Auswachsen weitere Funktionen zuzuordnen sind. So scheinen sie vor allem während der Entwicklung und während der Stabilisierungsphase der hippocampalen Reorganisation eine wichtige Rolle einzunehmen. Die hier dargestellten Daten zeigen auf, dass vor einem therapeutischen Einsatz von Nogo- Antagonisten nach Schädigung deren Verträglichkeit bzw. unerwünschte Nebeneffekte ausgeschlossen werden müssen. / Compared to the peripheral neuronal system (PNS) the reorganisation capacity in the adult central neuronal system (CNS) is highly restricted. One important reason for the lack of reorganisation is the existence of myelin in the CNS. Myelin is crucial for the stabilization of axonal projections in the developing and adult mammalian brain. However, myelin components also act as a non-permissive and repellent substrate of outgrowing axons. In these thesis the appearance of mature, fully myelinated axons during hippocampal development and following entorhinal cortex lesion with the myelin-specific marker Black Gold is reported. Althrough entorhinal axons enter the hippocampal formation at the embryonic day 17, light and ultrastructural analysis revealed that mature myelinated fibres in the hippocampus occur in the second postnatal week. During postnatal development, increasing numbers of myelinated fibers appear and the distribution of myelinated fibers at postnatal day 25 was similar to that found in the adult. After entorhinal cortex lesion, a specific anterograde denervation in the hippocampus takes place, accompanied by a long- lasting loss of myelin. Quantitative analysis of myelin and myelin breakdown products at different time points after lesion revealed a temporally close correlation to the degeneration and reorganisation phases in the hippocampus. In conclusion, it could be shown that the appearance of mature axons in the hippocampus is temporally regulated during development. Reappearing mature axons were found in the hippocampus following axonal sprouting. Various myelin-associated proteins, with neurite inhibition properties are known. One is the family of Nogo genes (no go). They are distinctly responsible for the lack of reorganisation. In these thesis the expression pattern of Nogo-A, Nogo-B, Nogo-C and Nogo-66 receptor (Ng66R) mRNA during hippocampal development and lesion induced axonal sprouting is reported. The first surprising result was the neuronal expression of all Nogos, who were supposed to be only expressed by oligodendrocytes. Nogo-A, Nogo-B and Ng66R transcrips preceded the process of myelination and were highly expressed at postnatal day zero (P0) in all principal hippocampal cell layers, with the exception of dentate granule cells. Only a slight Nogo-C expression was found at P0 in the principal cell layers of the hippocampus. During adulthood, all Nogo splice variants and their receptor were expressed in the neuronal cell layers of the hippocampus, in contrast to the myelin basic protein mRNA expression pattern, which revealed a neuronal source of Nogo gene expression in addition to oligodendrocytes. After hippocampal denervation, the Nogo genes showed an isoform-specific temporal regulation. All Nogo genes were strongly regulated in the hippocampal cell layers, wheras the Ng66R transcrips showed a significant increase in the contralateral cortex. These data could be confirmed on protein levels. Futhermore, Nogo-A expression was up-regulated after kainat- induced seizure. These data show that neurons express Nogo genes with a clearly distinguishable pattern during development. This expression is further dynamically and isoform-specifically altered after lesioning during the early phase of structural rearrangements. Thus, these results indicate a role for Nogo-A, -B and –C during development and during stabilisation phase of hippocampal reorganization. Taken together with these data, the findings that neurons in a highly plastic brain region express Nogo genes supports the hypothesis that Nogo may function beyond its known neuronal growth inhibition activity in shaping neuronal circuits.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Sulfatide is a galactolipid and a major lipid component of the myelin sheath. Its production is catalyzed by the enzyme cerebroside sulfotransferase (CST). To determine the functions of sulfatide, the gene encoding CST was genetically disrupted resulting in mice incapable of sulfatide synthesis. Using these mice, it has been shown in the central nervous system (CNS) that sulfatide is essential for normal myelin synthesis and stability even though the onset of myelination is not impaired. Additionally, proper initial clustering of paranodal proteins and cluster maintenance of nodal proteins is impaired suggesting that paranodal domains are important for long-term node stability. In contrast to the CNS, a requirement for sulfatide in the initiation of myelination, and in initiation of paranodal and nodal clustering or in the long-term maintenance of these clusters in the peripheral nervous system (PNS) has not been analyzed. Therefore, we have employed a combination of electron microscopic, immunocytochemical, and confocal microscopic analyses of the CST KO mice to determine the role of sulfatide in PNS myelination and onset of protein domain formation and maintenance. For these studies we have quantified myelin thickness, paranodal structural integrity, and the number of paranodal and nodal protein clusters in the CST KO and wild type mice at 4 days, 7 days, and 10 months of age. Our findings indicate that myelination onset is not delayed in the absence of sulfatide and that both the node and paranode are grossly normal; however, closer analysis reveals that paranodal junctions are compromised, Schwann cell microvilli are disoriented and the myelin-axon interface along the internodal region is transiently disrupted. In addition, we report that the paranodal myelin protein neurofascin 155 (Nfasc155) shows a transient decrease in initial clustering in the CST null mice at 4 days of age that is restored to WT levels by 7 days of age that is also maintained in the adult mice. Whereas nodal clustering of neuronal voltage-gated sodium channels is initially normal, cluster number is significantly but also transiently reduced by 7 days of age. By 10 months of age, the number of sodium channel clusters is restored to normal levels. In contrast, clustering of neither the paranodal neuronal protein contactin nor the myelin nodal protein gliomedin is altered at any of the ages studied. Together our findings suggest that sulfatide is not essential for PNS myelination or for protein domain formation in contrast to its more vital role in the development and maintenance of the CNS.