In der vorliegenden Dissertation wurde die subzelluläre Lokalisation der Rezeptoren für die neurotrophen Faktoren BDNF, CNTF und GDNF in primären embryonalen und adulten Motoneuronen erstmalig genau charakterisiert. Die Rezeptoruntereinheiten des BDNF und CNTF Rezeptors, TrkB, p-TrkB, gp130 und p-Stat3, sind im Perikaryon, in Dendriten, im Axon und an den Axonterminalen bzw. Wachstumskegeln von Motoneuronen lokalisiert. Dabei sind die nativen Formen (TrkB, gp130) im Axon überwiegend membranständig, die aktivierten Formen (p-TrkB, p-Stat3) überwiegend im Inneren des Axons lokalisiert. Demgegenüber sind die Rezeptoruntereinheiten des GDNF Rezeptors, Ret und p-Ret, besonders stark in den Dendriten exprimiert. Auch im Perikaryon und an der neuromuskulären Endplatte sind Ret und p-Ret lokalisiert, nicht jedoch im Axon. Im zweiten Teil der Arbeit wurde das durch neurotrophe Faktoren bedingte Neuritenwachstum genau quantifiziert. Dabei wurde zwischen einer Stimulation des Axon- bzw. des Dendritenwachstums differenziert. Die mit GDNF behandelten Dendriten werden etwa doppelt so lang wie die Dendriten, der mit BDNF oder CNTF behandelten Motoneurone. GDNF ist somit ein potenter Stimulator des Dendritenwachstums bei isolierten primären Motoneuronen. Dieser Befund korreliert gut mit der starken Expression von Ret und p-Ret in den Dendriten. Des Weiteren wurde eine Analyse der Interaktion der neurotrophen Faktoren mit dem glutamatergen AMPA Rezeptor in Hinblick auf das Neuritenwachstum durchgeführt. Dabei zeigte sich, dass die Interaktion zwischen neurotrophen Faktoren und dem AMPA Rezeptor besonders für das Dendritenwachstum von Bedeutung ist. Die klinische Bedeutung neurotropher Faktoren und deren Rezeptoren wird im dritten Teil der Arbeit dargestellt. Die pmn Maus ist ein Mausmodell für humane degenerative Erkrankungen des Motoneurons, wie der ALS und der SMA. Pmn Motoneurone, die mit BDNF oder GDNF kultiviert werden, weisen den charakteristischen axonalen Wachstumsdefekt der pmn Motoneurone auf und werden nur etwa halb so lang wie gesunde Kontrollmotoneurone. Bemerkenswerterweise führt die Behandlung der pmn Motoneurone mit CNTF zu einer kompletten Remission des axonalen Wachstumsdefekts, so dass die Axone eine normale Axonlänge erreichen. Auch die Anzahl der pathologischen axonalen Schwellungen werden in vitro durch CNTF stark reduziert. CNTF scheint demnach der interessanteste neurotrophe Faktor für eine Behandlung degenerativer Motoneuronerkrankungen zu sein. / Neurotrophins are important factors for many different functions of motoneurons, such as survival, neurite growth, as well as neuromuscular signalling. Neurotrophin receptors are therefore thought to be differently distributed in dendrites and axons. However, their precise localization and regulation in motoneurons were not well defined. This thesis characterized the exact subcellular localisation of the BDNF, CNTF and GDNF receptor subunits on adult and embryonic motoneurons. The BDNF und CNTF receptor subunits, gp130 and p-Stat3, are located in the perikaryon, in dendrites, in the axon as well as the growth cones and neuromuscular junctions of motoneurons. Immunofluorescent staining for the native forms (TrkB, gp130) is mainly found close to the membrane of the axon. In contrast, the activated forms (p-TrkB, p-Stat3) are mainly located inside the axon. GDNF receptor subunits Ret and p-Ret are highly expressed in the dendrites of motoneurons. In addition, Ret and p-Ret are also located in the perikaryon as well as the neuromuscular junction. Moreover, neurite outgrowth stimulated by neurotrophic factors was analyzed, differentiating axonal and dendritic growth. Primary motoneurons treated with GDNF grew dendrites which were twice as long as dendrites treated with BDNF or CNTF. Thus, GDNF is an important and potent stimulator of dendrite outgrowth in isolated primary motoneurons. This finding correlates well with the high expression of Ret and p-Ret in dendrites. On the other hand BDNF, CNTF and GDNF had equally potent effects on stimulating axonal growth. This thesis also characterized the interactions of neurotrophic factors with AMPA receptors regarding effects on neurite outgrowth. Interestingly, this interaction seems to be of greater importance for dendritic growth rather than axonal growth. The pmn mouse is a mouse model for neurodegenerative diseases of motoneurons, such as amyotrophic lateral sclerosis and spinal muscular atrophy. Pmn Motoneurons, which were cultured in presence of BDNF or GDNF, displayed the characteristic axonal growth deficiency as well as typical axonal swellings. The axon of these motoneurons reached only half the length of healthy control motoneurons. Surprisingly, treatment with CNTF rescued the pmn phenotype as the axons grew to the lengths of healthy control motoneurons. CNTF treatment also significantly reduced the number of pathological axonal swellings in vitro. Therefore CNTF seems to be the most promising therapeutic neurotrophic factor for treatment of neurodegenerative diseases of the motoneuron.

Post-traumatic stress disorder (PTSD) is characterized by fear extinction deficit; chronic stress worsens this deficit. Using a Chronic Unpredictable Stress (CUS) model, we previously found that CUS increased fear extinction deficit in female mice and knockout of Ciliary Neurotrophic Factor (CNTF) attenuated it. The amygdala, specifically the medial amygdala, is strongly associated with fear conditioning and extinction. CUS increased CNTF and reduced Urocortin 3 (UCN3) in the medial amygdala, suggesting CNTF-mediated UCN3 inhibition may be involved in CUS-induced deficit of fear extinction. The medial amygdala connects to the hypothalamic paraventricular nucleus (PVN) via posterior bed nucleus of stria terminalis (pBNST) and mediates the stress response (Fig. 1). The objective of this project is to determine whether CUS affects CNTF, UCN3, and CNTF-related cytokine leukemia inhibitory factor (LIF) and interleukin-6 (IL-6) in the pBNST and hypothalamic PVN. Hippocampal CNTF expression was also examined as a brain region outside of the medial amygdala-pBNST-hypothalamic PVN circuitry. 4 groups (5 mice/group) of CNTF+/+ and CNTF-/- mice were treated with 4 weeks of CUS or control handling. At the end, fresh brain samples were collected. The hypothalamic PVN, pBNST and hippocampus were punched out from 600-700 um cryostat frozen sections. CUS was applied for 4 weeks. The control mice were handled daily for 4 weeks. RNA was extracted from tissue using QIANGEN Rneasy mini kit. BCA assay was performed to analyze protein concentration, then 10% SDS gel was used to run the protein samples. Statistical analysis included one-way ANOVA followed by Bonferroni multiple comparison or 2-tailed T test. p <0.05 was defined as significant difference. In the pBNST, CUS did not affect CNTF and UCN3 mRNA expression. However, UCN3 protein was upregulated by CUS in CNTF+/+ but not CNTF-/- mice, suggesting CNTF inhibits UCN3 expression, possibly through post-transcriptional mechanism. CUS did not alter LIF and IL-3 in the pBNST. CUS did not alter CNTF mRNA expression in the PVN and further study will measure UCN3 mRNA and protein in the PVN. Finally, there was no CUS effect on CNTF, LIF and IL-6 mRNA in the hippocampus. These results and further studies are useful in development of therapeutic medications and drug targets in the case of chronic stress.

Le remplacement des oligodendrocytes myélinisants par des progéniteurs endogènes a été observé dans le contexte des pathologies démyélinisantes via le recrutement de deux populations distinctes de progéniteurs : les progéniteurs dérivant des cellules souches adultes de la zone sous ventriculaire (SVZ) et les précurseurs d'oligodendrocytes (OPCs) du parenchyme. Réactivés par des facteurs présents dans le cerveau après lésion, ces progéniteurs acquièrent de nouvelles propriétés migratoires et sont recrutés au niveau du site lésionnel. Toutefois, ces tentatives d'autoréparation endogènes sont limitées et ne permettent pas une récupération fonctionnelle, dans la grande majorité des cas. Cet échec dans la régénération peut être imputé en parti à des défauts de migration de ces cellules vers les zones lésées. Divers signaux sont impliqués dans le recrutement des cellules progénitrices allant de signaux développementaux ré&#8208;exprimés au niveau de la lésion à l'expression de cytokines induite par la neuroinflammation. Nos données dévoilent de nouvelles fonctions pour Reelin et CNTF dans le contrôle de la migration des progéniteurs neuraux pendant la phase de remyélinisation chez la souris. Alors que Reelin induit la dispersion des cellules dérivant de la SVZ hors de leur niche rendant plus efficace les processus de recrutement spontané vers la lésion démyélinisée, CNTF participe au contrôle directionnel de leur migration vers de la zone endommagée in vivo. L'utilisation de tests in vitro nous a permis de montrer que Reelin, en plus de son effet décrit sur le détachement des neuroblastes de la SVZ migrant en chaîne, exerce un effet chimiocinétique. / Replacement of myelinating oligodendrocytes by endogenous progenitors has been demonstrated to occur in demyelinating diseases via the recruitment of two distinct pools of progenitors: Subventricular zone (SVZ)&#8208;derived progenitors and parenchymal oligodendrocyte precursors (OPCs). Becoming re&#8208;activated by factors present in the brain after injury, these progenitors acquire new migration properties and are recruited to the lesion sites. However these spontaneous repair attempts are limited and do not permit efficient functional recovery. This failure can be due in part to an inefficient migration of these cells to the lesion site. Numerous signals are involved in the migration of precursor cells into the area of brain damage ranging from developmental signals re&#8208;expressed at the lesion site to neuroinflammation&#8208;induced cytokines. Our data uncover new functions for Reelin and CNTF in the control of neural progenitor's recruitment during the remyelination phase in mouse model of demyelination. While Reelin induces SVZ&#8208;derived cells dispersion form their niche which potentiates the spontaneous recruitment processes to the demyelinated lesion, CNTF participates in the control of their migration toward the damaged site in vivo. Using in vitro assays we show that Reelin, in addition to its already described detachment effect on SVZ neuroblasts chain migration, is chemokinetic. CNTF is acting on SVZ derived progenitors and parenchymal OPCs as a chemoattractant, which cells respond to it in gradient sensing manner. All together, these two studies reveal key function for Reelin and CNTF in the post&#8208;lesional migration.

Les cytokines de la famille de l’IL-6 possèdent comme caractéristique commune la capacité à recruter une chaine réceptrice membranaire nommée gp130. Au début de notre étude, cette famille comptait 8 membres : IL-6 et son homologue viral (vIL-6), IL-11, LIF, OSM, CNTF, CT-1 et CLC. Depuis, cette famille s’est enrichie de trois nouveaux membres, NP, IL-27 et IL-31. Ces cytokines sont essentielles au développement (LIF et CT-1) ou sont impliquées dans l’hématopoïèse, les réponses immunitaires et l’inflammation (IL-6, IL-11, IL-27, IL-31 et OSM) alors que d’autres agissent plus particulièrement au niveau du système nerveux, comme NP, CLC et CNTF. Le premier axe de recherche de ma thèse a porté sur l’identification d’un nouveau membre de cette famille,la neuropoïétine (NP), et plus précisément sur la caractérisation de son profil d’expression tissulaire et l’identification de son récepteur, des principales voies de signalisation intracellulaire induites ainsi que ses fonctions biologiques. Le deuxième axe de recherche a consisté à étudier la capacité de CLC à se comporter comme un ligand alternatif du récepteur tripartite au CNTF, impliqué dans le développement neuronal. Pour cela, nous avons mis en évidence la présence de la cytokine CLC et de ses partenaires de sécrétion, dans l’environnement des motoneurones lors du développement embryonnaire chez la souris. Enfin, le dernier axe de recherche s’est focalisé sur le rôle des états de glycosylation de CLC sur la sécrétion des cytokines composites CLC/CLF et CLC/solCNTFRα. / Members of the interleukin 6 (IL-6) cytokine family share a common characteristic, the capacity to signal via thegp130 subunit receptor. This family contains 8 members : IL-6 and its viral counterpart (vIL-6), IL-11, LIF, OSM, CNTF, CT-1 and CLC. This family has been more recently enriched with three new members, neuropoietin (NP), IL-27 and IL-31. These proteins are involved in development (LIF and CT-1), in hematopoiesis, immune responses and inflammation, such as IL-6, IL -11, IL-27, IL-31 and OSMor act in the nervous system (NP, CLC and CNTF). My first research work was focused on the identification of a new member of this family, neuropoietin. More specifically, this study allowed determining its tissue expression profile, its complex receptor and the signaling pathways induced, and its biological functions. A second research project was focused on determining whether CLC may constitute an alternative ligand for the CNTF receptor, involved in the neuronal development. We have evidenced the presence of CLC and its secretory partners, in the environment of motoneurons during embryonic development in mice. Finally, I have evaluated the role of the glycosylationstates of CLC on the secretion of the composite cytokines CLC/CLF and CLC/solCNTFR.

Orientador: Antonio Carlos Boschero / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T00:36:09Z (GMT). No. of bitstreams: 1 Rezende_LuizFernandode_D.pdf: 9509933 bytes, checksum: fc8e235ac3d4242d8e48c28911285e25 (MD5) Previous issue date: 2009 / Resumo: Introdução: O CNTF pertence à família da IL-6, e sendo assim, sinaliza pelo complexo receptor gp130, ativando diversas vias de sinalização dependendo do tipo celular, principalmente as vias STAT3, MAPK e PI3K. Seus efeitos incluem diferenciação e/ou sobrevivência neuronal, e é diferencialmente expresso ao longo da vida do animal. As ilhotas pancreáticas, por sua vez, são ricamente enervadas e expressam receptores para NGFs, podem apresentar respostas neurotípicas e expressam o CNTF. O objetivo desse estudo foi investigar os possíveis efeitos do CNTF sobre a diferenciação e/ou sobrevivência de ilhotas pancreáticas de ratos neonatos, qual(is) via(s) de sinalização ele ativa nessas ilhotas e como é expresso nelas ao longo da vida dos animais. Material e Métodos: Ilhotas pancreáticas de ratos neonatos (1-2 dias) foram isoladas pelo método de colagenase e cultivadas por 3 dias em meio RPMI com (CNTF) ou sem (CTL) 1nM de CNTF. Após isso, foram analisados a secreção de insulina estimulada por glicose (RIE), metabolismo (MTS, produção de NADPH), metabolismo de glicose (produção de 14CO2), expressão gênica (RTPCR), protéica (Western-Blot), atividade de caspase-3 (DEVD) e apoptose (fragmentação de DNA). Resultados: O CNTF reduziu a secreção de insulina estimulada por glicose e o metabolismo de ilhotas pancreáticas, não alterando o metabolismo de glicose e expressão de proteínas cruciais para a função das ilhotas. Por outro lado, o CNTF aumentou a expressão de proteínas relacionadas à sobrevivência das ilhotas pancreáticas, como Cx36, PAX4, e BCL-2, reduziu a atividade da caspase-3 e a apoptose das ilhotas. O CNTF também aumentou a fosforilação de STAT3, sua translocação ao núcleo e expressão de genes-alvo, como a SOCS-3, levando à redução da GSIS e sobrevivências observadas, apesar de não ativar as vias da MAPK e PI3K. Mais ainda, a expressão do CNTF é aumentada em ilhotas pancreáticas de ratos de 2 meses de idade, e assim permanecendo até os 20 meses de idade. Conclusão: O CNTF não promove a maturação de ilhotas pancreáticas, mas sim sua sobrevivência, e esses efeitos são mediados através da via JAK/STAT3, sem ativar as vias MAPK ou PI3K. Finalmente, o CNTF possui expressão diferenciada ao longo da vida do animal / Abstract: Introduction: CNTF belongs to the IL-6 cytokine family and as such, it signals through gp130 receptor complex, activating many pathways depending on the cell-type, mainly STAT3, MAPK and PI3K. Its effects include increased neuron differentiation and/or survival, and are differentially expressed throughout the animal life. Meanwhile, pancreatic islets are richly innervated and express receptors for NGFs, may undergo neurotypic responses, and express CNTF. The aim of this study was to investigate possible effects of CNTF on neonatal rat pancreatic islet differentiation and/or survival, which signalling pathway (s) it activates on pancreatic islets and how it is expressed in the pancreatic islets throughout the animal life. Methods: Pancreatic islets from neonatal rats (1-2 d old) were isolated by the collagenase method and cultured for 3 days in RPMI medium with (CNTF) or without (CTL) 1nM of CNTF. Thereafter, glucose-stimulated insulin secretion (RIA), general metabolism by (NAD(P)H production) (MTS), glucose metabolism (14CO2 production), gene (RT-PCR), protein expression (Western-Blot), caspase-3 activity (DEVD), and apoptosis (DNA fragmentation) were analysed. Results: CNTF reduced pancreatic islets GSIS and metabolism, whereas not affecting glucose metabolism and the expression of proteins crucial for the islets function. Conversely, CNTF significantly expression of proteins related pancreatic islets survival, such as Cx36, PAX4, and BCL-2, reduced caspase-3 activity and islet-cells apoptosis. CNTF also increases STAT3 fosforilation, translocation to the nuclei and expression of target genes, resulting in the reduced GSIS and survival observed, although not affecting MAPK and PI3K pathways. Moreover, CNTF expression is increased in rats pancreatic islets after 2 months of age, and it remains so until 20 months of age. Conclusion: CNTF has no effect over maturation of pancreatic islets function, whereas it improves pancreatic islets survival, and also that these effects are mediated through JAK/STAT3 but not through MAPK or PI3K pathways. Finally, CNTF is differentially expressed in rat pancreatic islets throughout the animal life / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

La production physiologique continue de cellules myélinisantes dans le système nerveux (SN) de mammifère offre de nouvelles perspectives thérapeutiques. Lors d’une atteinte de la myéline, une régénération endogène impliquant la génération d’oligodendrocytes s’engage. Ce processus repose sur la mobilisation de progéniteurs oligodendrocytaires parenchymateux et de progéniteurs de la zone sous-ventriculaire (SVZ). Cette réparation ne permet cependant pas une récupération fonctionnelle systématique. Nos travaux ont pour but d’identifier les facteurs qui contrôlent les différentes étapes de régénération. Ils révèlent une réexpression du CNTF et une surexpression des héparans sulfates protéoglycanes (HSPGs) suite à une démyélinisation du corps calleux. Ces changements de l’environnement péri-lésionnel régulent positivement le processus de remyélinisation. Nous avons montré un impact direct de l’expression post-lésionnelle du CNTF sur la mobilisation des deux sources cellulaires. Différents tests in vitro ont identifié le CNTF comme facteur chémoattractant pour ces cellules. Nos données montrent également que des modifications de sulfatation des héparans sulfates (HS) protéoglycanes contrôlées par la N-désacétylase-Sulfotransférase 1 des cellules du lignage oligodendrocytaire s’établissent en bordure de lésion et créent un microenvironnement favorable à la régénération. Divers test fonctionnels in vivo et in vitro révèlent le rôle clef des HSPGs dans la cinétique de démyélinisation et de remyélinisation en régulant la mobilisation des cellules du lignage oligodendrocytaire et l’activation microgliale. / In the mammal’s nervous system, the ongoing production of new myelinating cells on has open news therapeutic perspectives for demyelinating diseases. An endogenous regeneration process involving the generation of oligodendrocytes can occur following demyelination. This process relies on the mobilization of an endogenous reservoir of progenitor cells located in the adult brain: The parenchymal oligodendrocyte precursors and the subventricular zone derived neural progenitors. However, these endogenous repair attempts do not permit an efficient functional recovery. These failures are mainly due to mobilization, differentiation or to the generation of a hostile environment for the repair process. Our work is focusing on the identification of factors regulating those events. Our data show the reexpression of CNTF and overexpression of heparan sulphate proteoglycans (HSPGs) following a focal demyelination of the corpus callosum in adult mice. These environmental changes favor myelin repair. We show a direct impact of the post-lesional expression of CNTF on the mobilization of both cellular sources. Using various in vitro assays, we showed that CNTF is acting on the two cellular sources as a chemoattractant factor. Our data also show that sulfation modifications of HSPGs performed by the deacetylase-N-sulfotransferase 1 (Ndst1) on oligodendrocyte lineage cells occurred around the lesion and created a permissive microenvironment for the regenerative process. Various in vitro and in vivo functional assays demonstrated the key role of HSPGs in demyelination and remyelination dynamic by controlling mobilization of the oligodendrocyte lineage cells and microglial activation.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

One of the major limitations in drug development and gene therapy for brain diseases is the natural defensive structure called the blood brain barrier (BBB), which prevents therapeutic polypeptide drugs and viral vectors from entering the brain. Intranasal delivery of therapeutic gene products into the brain offers a non-invasive alternative towards a feasible gene and protein therapy for neurological diseases. From recent studies involving axonal transport, it is tempting to speculate that therapeutic macromolecules including neurotrophic factors and viral vectors can be delivered into the brain by peripheral neurons, such as olfactory receptor neurons (ORNs), which span the BBB. It is thought that the nasal pathway into the brain involves two general mechanisms; intracellular (intraneuronal) or extracellular routes of transport. However the pathways involved have not yet been fully characterized. In this study I firstly investigated the temporal and spatial localisation pattern of both biotinylated and I125 labelled ciliary neurotrophic factor (CNTF) following nasal delivery into Sprague-Dawley rats. Results showed that intranasal delivered CNTF was transported to several brain regions by both intracellular axonal pathway through ORNs and the extracellular trigeminal pathway. Excess unlabelled CNTF competed for receptor binding in the olfactory mucosa confirming receptor mediated intracellular transport to the olfactory bulb via ORNs. Denervation of the olfactory mucosa prior to CNTF delivery failed to prevent CNTF transport to trigeminal and hypothalamic brain regions. Intranasal delivered CNTF was biologically active, resulting in activation of the STAT3 signalling pathway in the thalamus and hypothalamus. To examine the functional activity of intranasal delivered CNTF, I conducted a weight loss trial using an obese Zucker rat (OZR) model to test whether CNTF treatment caused body weight loss. Intranasal administration of CNTF resulted in reduced body weight in the CNTF treated OZR group compared to the BSA control group during the 12 day trial and for 3 days after. Intranasal delivery of CNTF may be a valuable method for the treatment of obesity. In the second study, I investigated the temporal and spatial expression of Enhanced Green Fluorescent Protein (EGFP) transferred by a single nasal delivery of either a recombinant adenovirus vector (Ad5CMV-EGFP) or an adeno-associated virus vector (AAV2-EGFP) into Sprague-Dawley rats. Adenovirus mediated EGFP expression was localized in ORNs throughout the olfactory epithelium after 24 hours. EGFP in the ORNs appeared to be anterogradely transported along their axons to the olfactory bulb and transferred in glomeruli to second-order neurons. EGFP was transferred to several brain regions including the cortex, hippocampus, and brainstem after 7 days. EGFP expression co-localized with Olfactory Marker Protein and was confirmed with EGFP immunofluorescence labelling and western blotting. AAV expressed EGFP localized in similar olfactory and brain regions 6 weeks after delivery. mRNA levels suggested that the AAV-EGFP construct was only incorporated into olfactory mucosa cells and the viral vector was not present in olfactory bulb and brain regions. In conclusion, this simple and non-invasive polypeptide and gene delivery method provides ubiquitous macromolecule distribution throughout the rodent brain and may be useful for the treatment of neurological disorders.

Aucun traitement de la maladie d’Alzheimer (MA) n’existe, justifiant le développement de stratégies thérapeutiques. La toxicité des oligomères solubles du peptide amyloïde β (Aβ) est centrale dans les pertes synaptiques et cellulaires précoces dans la maladie. Dans le cadre de cette hypothèse, nous proposons qu’empêcher ces effets puisse prévenir le déclin cognitif dans la MA. Les facteurs neurotrophiques sont de bons candidats pour prévenir la mort cellulaire mais nécessitent une application ciblée et continue. Nous avons utilisé la technologie d’encapsulation cellulaire pour produire des bioréacteurs implantables contenant des cellules C2C12 sécrétant le facteur neurotrophique ciliaire (CNTF). Notre objectif était d’établir la preuve du concept que la production à long terme de CNTF in situ dans le cerveau puisse prévenir les déficits cognitifs liés à l’Aβ.Nos études prouvent que le CNTF produit par les bioréacteurs prévient la cytotoxicité et l’apoptose induites par le peptide Aβ in vitro. La neuroprotection dépend de l’activation de la PI3-Kinase et du facteur STAT3. In vivo, l’implantation des bioréacteurs dans le cerveau prévient les troubles cognitifs induits par l’injection icv d’Aβ ou retarde leur apparition chez la souris Tg2576. Dans nos deux modèles précliniques de la MA, la protection comportementale est associée au maintien des protéines synaptiques dans l’hippocampe. Aussi, la production in situ de CNTF est une approche thérapeutique préventive efficace contre la toxicité et les déficits cognitifs liés à l’Aβ. Ces résultats suggèrent également que l’implantation de cellules encapsulées est un bon procédé pour délivrer des molécules thérapeutiques au cerveau / No cure against Alzheimer’s Disease (AD) exists yet, justifying the development of therapeutic strategies. Toxicity of soluble amyloid β peptide is a key-player in early synaptic and cellular loss in AD. According to this hypothesis, we propose that preventing Aβ peptide effects could prevent cogninitive decline in AD. Neurotrophic factors are good candidates to prevent cell death but require a targeted and continuous delivery. We used the cell encapsulation technology to produce graftable bioreactors that contain C2C12 cells secreting the Ciliary Neurotrophic factor (CNTF). Our goal was to realize the proof-of-concept that CNTF long term in situ delivery could prevent Aβ-induced cognitive decline.Our studies prove that bioreactor-produced CNTF prevents Aβ-induced cytotoxicity and apoptosis in vitro. Neuroprotection relies on PI3K and STAT3 activation. In vivo, bioreactor implantation in brain prevents cognitive impairment induced by Aβ icv injection or delays their appearance in Tg2576 mice. In both of our preclinical model of AD, behavioral protection was associated with synapse maintenance in hippocampus.Therefore, in situ long term CNTF delivery is an efficient preventive therapeutic strategy against toxicity and Aβ-linked cognitive disturbances. These results also suggest that encapsulated cells graft is a good way to deliver therapeutic molecules to the brain

Orientador: Francesco Langone / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T03:17:53Z (GMT). No. of bitstreams: 1 Rezende_AlexandreCesarSantosde_M.pdf: 2677724 bytes, checksum: 332acee58d6f9fc544b659f930833e41 (MD5) Previous issue date: 2005 / Resumo: A investigação de agentes terapêuticos para o tratamento de doenças neurodegenerativas e neurotraumas recebeu um grande impulso nas últimas décadas, em virtude do desenvolvimento de técnicas que permitiram a clonagem de moléculas com reconhecida ação neurotrófica. O emprego de modelos experimentais para o estudo de processos neurodegenerativos contribuiu significativamente ao conhecimento dos mecanismos de ação e especificidade dessas moléculas. O fator neurotrófico ciliar (CNTF) despertou grande interesse com a descoberta do seu efeito neuroprotetor sobre motoneurônios após secção de nervos periféricos em ratos neonatos e camundongos adultos. Contudo, os testes clínicos em pacientes com esclerose lateral amiotrófica revelaram efeitos colaterais importantes associados às diferentes doses e esquemas de tratamento com CNTF ecombinante humano. Estes resultados tiveram como conseqüência a interrupção dos testes clínicos e estimularam a busca de vias alternativas para a administração do CNTF com o objetivo de eliminar os efeitos colaterais indesejáveis. Uma via alternativa muito promissora parece ser a administração do CNTF conjugado com peptídeos que possuem domínio de transdução ou translocação de proteínas (PTDprotein transduction domain). Neste estudo investigamos o efeito do CNTF conjugado com um PTD derivado do vírus HIV-1 (Tat-CNTF) quando administrado a ratos neonatos (P2) após secção do nervo ciático. Um grupo de ratos (Wistar) teve o nervo ciático esquerdo seccionado e recebeu um fragmento de gelfoam embebido em CNTF (6µg), Tat-CNTF (6µg ou 3µg) ou PBS. Outro grupo sofreu o mesmo tipo de lesão e foi tratado diariamente, durante 5 dias, com doses subcutâneas de CNTF (1,2µg/g), Tat-CNTF (1,2, 0,6 ou 0,3µg/g) ou PBS. O peso corporal foi registrado diariamente entre P2 e P7. Na idade de P7 os animais foram perfundidos (paraformaldeído 4%) e a medula lombar foi embebida em parafina para obtenção de cortes seriais transversais. Os cortes foram corados com cresil violeta e os motoneurônios do grupamento ventrolateral de ambos os lados foram contados. A razão entre o número de motoneurônios do lado lesado e do lado íntegro correspondeu ao índice de sobrevivência neuronal (ISN). Uma série de 9 cortes de cada animal foi reagida para investigação imunoistoquímica da expressão de GFAP pelos astrócitos. Nossos resultados mostraram que o ISN de todos os grupos tratados com Tat-CNTF, local e subcutaneamente, foi similar aos dos grupos tratados da mesma forma com CNTF e maior que nos grupos tratados com PBS. O acompanhamento do ganho de peso dos animais entre P2 e P7 mostrou que aqueles que receberam tratamento local com CNTF ou Tat-CNTF apresentaram crescimento semelhante ao do observado nos animais do grupo controle. Por outro lado, a curva de crescimento dos animais tratados com doses subcutâneas diárias de CNTF (1,2µg/g), revelou que estes tiveram ganho de peso significativamente inferior ao dos animais tratados com a mesma dose, ou com as doses inferiores, de Tat-CNTF. O menor crescimento dos animais tratados com CNTF foi significativo a partir do segundo dia de tratamento. Na idade P7 o peso corporal desses animais foi 25% e 30% inferior aos dos animais tratados com Tat-CNTF e aos animais controle, respectivamente. Não houve diferença significativa de ganho de peso entre os animais tratados com as diferentes doses de Tat-CNTF e os animais tratados com PBS subcutaneamente. Além disso, a administração subcutânea de CNTF provocou intensa mobilização da gordura marrom interescapular, fenômeno não verificado nos animais tratados com Tat-CNTF. Os dados da análise imunoistoquímica para GFAP revelaram que a resposta astroglial nos animais tratados com Tat-CNTF foi menos intensa que a observada nos animais que receberam tratamento subcutâneo com CNTF. Nossos resultados revelaram que a Tat-CNTF além de possuir ação neuroprotetora não produziu os efeitos colaterais indesejáveis do CNTF, mesmo quando administrado em dose igual à deste último. Estes dados suportam a hipótese que a adição do domínio PTD a moléculas neurotróficas é uma estratégia de neuroproteção eficaz e abre perspectivas para possível emprego da Tat-CNTF em moléstias neurodegenerativas que afetam os motoneurônios espinhais, tais como a esclerose lateral amiotrófica / Abstract: The investigation of therapeutical agents for the treatment of neurodegenerative diseases and neurotrauma received a great attention in the last decades due to cloning techniques for neurotrophic molecules. Experimental models contributed to the knowledge of the mechanisms of action and specificity of such molecules. Ciliary neurotrophic factor (CNTF) is known as a neuroprotective agent on motoneurons after peripheral nerve section in neonatal rats and adult mice. However, clinical trials with human recombinat CNTF showed dose dependent-side effects. Consequently, new approaches for avoiding those side effects have been investigated, such as CNTF fused with domain transduction protein (PTD). In the present work, we studied the effects of CNTF fused with the PDT of the HIV-1 (Tat-CNTF) in neonatal rats (P2) after sciatic nerve transection. Wistar rats had their left sciatic nerve cut and embedded in gelfoam with either CNTF (6µg), Tat-CNTF (6µg or 3µg) or PBS. Other lesioned rats were treated subcutaneously with either CNTF (1.2µg/gr), Tat-CNTF (1.2, 0.6 or 0.3µg/gr) or PBS, once a day, for 5 days. The body weight was registered from P2 to P7. At P7 the rats were perfused (4% paraformaldehyde) and the lumbar spinal cord was infiltrated with paraffin. Serial transverse sections were stained with cresyl violet and used for ventrolateral motoneuron counting. The ratio between the number of motoneurons counted in the operated and control sides was defined as neuronal survival ratio (NSR). From each animal, a series of nine sections was used for immunohistochemical detection of glial fibrillary acidic protein (GFAP) expressed by astrocytes. The animals that received either local or subcutaneous Tat-CNTF showed NSR similar to those observed in CNTF groups and higher than the registered in the controls (PBS). Body weight of rats from P2 to P7 treated with a local dose of CNTF or Tat-CNTF was similar to that of the control groups. On the other hand, rats treated with subcutaneous daily doses of CNTF had lower body weight than the observed in the other groups. Animals treated with CNTF had lower weight gain from the second day on. At P7 the body weight of CNTF treated animals was 25% and 30% lower than those receiving Tat-CNTF and PBS, respectively. The animals treated with different subcutaneous doses of Tat-CNTF had similar body growth as the control groups. Moreover, only subcutaneous administration of CNTF led to fat mobilization from the intrascapular brown adipose tissue. Immunostaining for GFAP showed that astrocytic response was less intense after Tat-CNTF administration in comparison with subcutaneous treatment with CNTF. Our results showed that Tat-CNTFis a neuroprotective agent not associated with the side effects attributed to CNTF, even when administered in similar doses. These data support the hypothesis that the addition of PTD to neurotrophic molecules is an efficient neuroprotective strategy and opens up perspectives for using Tat-CNTF in neurodegenerative diseases that affect the spinal motoneurons, such as the amyotrofic lateral sclerosis / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural