<div>Vertebrate neurogenesis is a multistep process that coordinates complex signaling pathways and chromatin-based regulatory machinery to generate highly specialized cells (Hsieh and Zhao 2016; Urban and Guillemot 2014; Alunni and Bally-Cuif 2016; Yao and Jin 2014; Schmidt, Strahle, and Scholpp 2013). Epigenetic factors play a fundamental role in underwriting neurogenesis in part by contributing to control of gene expression in differentiating neurons. A mechanistic understanding of the epigenetic machinery underlying neurogenesis in vertebrates is necessary both to fully understand biogenesis of neural tissue in this subphylum as well as to develop effective therapeutic strategies to treat diseased or damaged neural tissue. </div><div>An example of an epigenetic factor that is important for both neuronal differentiation and disease states is CHD5, a vertebrate-specific member of the CHD family of ATP-dependent chromatin remodeling proteins. Chromodomain / Helicase / DNA-binding (CHD) proteins play a variety of roles in vertebrate development, and misregulation or loss of CHD proteins has been linked to numerous diseases (Mayes et al. 2014; Marfella and Imbalzano 2007; Bartholomew 2014). CHD5 is expressed primarily in neural tissue, where it is thought to contribute to neurogenesis, and has been strongly linked to tumor suppression (Thompson et al. 2003; Vestin and Mills 2013). Loss of CHD5 plays a significant role in development of neuroblastoma, a devastating tumor that is a leading cause of cancer-related death in children (Jiang, Stanke, and Lahti 2011; Maris and Matthay 1999). Consistent with the disease phenotype associated with loss of CHD5, reduced expression of CHD5 impairs differentiation of neuronal cells (Egan et al. 2013b). However, ablation of CHD5 in mice surprisingly resulted in no detectable defects in brain development (Li et al. 2014; Zhuang et al. 2014). A subsequent report revealed that mice conditionally ablated for CHD5 in neural tissue exhibit symptoms consistent with an autism spectrum disorder (Pisansky et al. 2017). Much remains to be learned about the role of CHD5 in these processes to clarify these observations.</div><div>Further, Chd5 is unique among the family of Chd remodelers in that it provides a biochemical basis for crosstalk between the critical epigenetic marks H3K27me3 and DNA methylation. Chd5 and the closely related remodelers Chd3 and Chd4 are all components of the Mi-2/NuRD histone deacetylase complex that plays a critical role in mediating transcriptional repression in response to DNA methylation in mammals (Allen, Wade, and Kutateladze 2013). Only CHD5 is preferentially expressed in neural tissue, however, and only Chd5 remodelers have biochemical evidence of direct interaction with H3K27me3, which plays a critical role in enabling proper expression of transcriptional programs during neurogenesis (Egan et al. 2013b). Chd5 is thus unique among CHD remodelers in that it is biochemically linked to both DNA methylation and H3K27me3 in addition to being preferentially expressed in neural tissue.</div><div>With regards to mechanism, much remains to be learned regarding how Chd5 remodelers contribute to gene expression and tumor suppression. However, the data to date do not show extensive transcript phenotypes and it is not clear how the biochemical action of CHD5 contributes to the neurological phenotypes ascribed to altered expression of CHD5. Therefore, it is critical to determine a suitable context to study the role of CHD5 in these processes. Identification of CHD5-dependent genes in neurons is likely to generate insight into how loss of CHD5 contributes to tumorigenesis, in particular with regards to development of neuroblastoma. Regulatory pathways that drive neurogenesis have been found to be extensively conserved between humans and zebrafish. Therefore, we have turned to the power of the zebrafish model system to characterize how loss of Chd5 alters brain development during embryogenesis.</div><div>Importantly zebrafish development, and neurogenesis in particular, occurs largely over the first 5-days of development. Zebrafish are born outside of the mother, which can produce large clutches of several hundred embryos per week, providing us with an accessible context to study the role of chd5, the zebrafish homolog of human CHD5. The central nervous system of the zebrafish develops rapidly, and shares many of the organization features of the mammalian brain (Kalueff, Stewart, and Gerlai 2014). In particular, neuroblastoma arises from a population of cells known as sympathetic ganglion cells that are derived from the neural crest (Pei et al. 2013). These cells are conserved in vertebrates, and several models to study how these cells transform into neuroblastoma exist in zebrafish (Zhu et al. 2017; Morrison et al. 2016; Zhu and Thomas Look 2016). However, our understanding of the mechanisms controlling ganglion cell differentiation is incomplete and requires further investigation to understand how epigenetic and transcriptional mechanisms contribute to development of these cells and how failure of these processes leads to cancer. The neural crest undergoes a series of differentiation steps to form mature sympathetic neurons that are guided by bone morphogenic protein signaling, and transcription changes (Ernsberger and Rohrer 2018). These cells express key enzymes for synthesizing dopamine and norephinephrine to control the sympathetic system throughout the central nervous system (Ernsberger and Rohrer 2018).</div><div>To address these questions about Chd5, we have used CRISPR/Cas9 to generate chd5-/- zebrafish that are protein nulls as determined by western blot. These chd5-/- fish are phenotypically indistinguishable from wild-type fish under standard growth conditions as was previously observed for mice lacking CHD5 (Zhuang et al. 2014; Li et al. 2014). By using zebrafish, we are able to perform transcriptome analyses to identify Chd5 target genes at stages much earlier than has previously been performed in mice because we can harvest large amounts of the tissue of interest from the readily accessible embryos. We have therefore undertaken RNA-seq analysis of isolated brains from wild-type and chd5-/- fish to identify chd5-dependent genes in predominantly differentiating (2-day old) and substantially differentiated (5-day old) neural tissue. These data provide a substantively different perspective from previous studies that examine the role of CHD5 in gene expression of differentiated SY-SH5Y cells (Egan et al. 2013a) or in the forebrain of 8-week-old mice (Pisansky et al. 2017). (Jiang, Stanke, and Lahti 2011). One role we identified from this data, is the promotion of development of sympathetic ganglion cells (detailed below), illuminating for the first time a role for chd5 in promoting differentiation of cells directly involved in neuroblatoma.</div><div>We observe not only extensive changes in gene expression, but also identify a novel role for Chd5 in enabling proper splicing during this critical window of neurogenesis in the zebrafish brain. We are further exploring the role of CHD5 in these processes by creating comparable cell culture-based models of loss of CHD5 to determine the conservation of molecular phenotypes observed in zebrafish. Furthermore, this model enables us to leverage the extensive biochemical tools available in cell culture to examine alterations to the chromatin that are difficult to interpret from studies of complex tissues such as the brain. </div><div>Herein I will describe the research progress we have made to understand the role of Chd5 in gene expression and splicing in zebrafish, as well as ongoing work to engineer mouse embryonic stem cells as an additional model to study the transcriptional consequences of loss of CHD5. Critically, the addition of the cell culture model will greatly enable biochemical characterization of the changes that are leading in particular to the changes in gene expression and splicing and will provide us with a context to test for a direct role of CHD5 in these processes. In addition, this thesis will detail the results from ongoing projects using the zebrafish model system, including: development of models in zebrafish to study the tumor suppressive role of Chd5, phenotypes observed using a targeted chemical-genetic screen, and advancement in developing new tools in zebrafish to engineer specific genomic modifications that will greatly expand the power of this vertebrate model.</div><div><br></div>

Investigamos o papel de condutâncias iônicas lentas na transmissão/codificação de informação entre neurônios que disparam em rajadas ou bursts. Para isso, desenvolvemos um protocolo experimental no qual a interação em tempo real entre computador e neurônio biológico permite isolar o efeito da dinâmica de um determinado tipo de canal iônico e estudar sua inuência nos mecanismos de codificação de informação. Os experimentos foram realizados com neurônios do gânglio estomatogástrico do siri azul, Callinectes sapidus, que não possuem condutâncias lentas capazes de fazê-los apresentar rajadas de disparos quando in vitro, condição na qual apresentam comportamento quiescente ou disparam tonicamente. Durante os experimentos, alteramos artificialmente o comportamento de um destes neurônios, conectando-o a um computador que introduz uma corrente capaz de fazê-lo apresentar rajadas. Essa corrente possui uma componente senoidal (vinda de um gerador de funções) e uma componente devido a uma condutância iônica lenta modelada matematicamente. A condutância lenta pode ser escolhida entre duas versões: uma em que a condutância é calculada em tempo real, a partir do valor instantâneo do potencial de membrana do neurônio biológico, e outra em que o valor da condutância é oriundo de uma série temporal previamente gravada. A fonte de informação utilizada nos experimentos é um neurônio artificial pré-sináptico, que possui uma distribuição de potenciais de ação (spikes) escolhida pelo experimentador, e é conectado ao neurônio biológico modificado através de um modelo de sinapse química inibidora. A quantidade de informação do neurônio artificial (variabilidade dos padrões de disparo) codificada pelo neurônio biológico é inferida calculando-se a informação mútua média entre eles para as duas versões da condutância lenta (dinâmica ou previamente gravada). Nossos experimentos reproduziram qualitativamente as observações feitas por nosso grupo no circuito pilórico intacto do siri, que possui neurônios conectados por mutua inibição que naturalmente apresentam bursts. Além disso, observamos que vários picos de informação mútua média, presentes quando a condutância é dinâmica, desaparecem quando esta é substituída pela série temporal previamente gravada da condutância. Assim, pudemos confirmar os resultados previamente obtidos com simulações computacionais em que foram utilizados apenas modelos matemáticos e na ausência de ruído e demonstramos que as condutâncias iônicas lentas constituem um mecanismo biofísico que permite a codificação de estímulos sinápticos em neurônios que apresentam rajadas. / We investigated the role of slow ionic conductances on information processing by bursting neurons. A real time experimental protocol was developed to allow interacting computer models and biological neurons to address the effect of dynamical details of a single type of ion channel in information coding mechanisms. We experimented on Callinectes sapidus (blue crab) stomatogastric ganglion neurons. Such neurons were chosen because they do not present the slow conductances that can led to bursting activity in vitro (in such conditions they can be found either in a quiescent or in a tonic firing state). The experiments consisted in artificially changing the behavior of one of these neurons by injecting a computer generated current to achieve bursting. Such current has a sinusoidal component (from a function generator) and a component due to mathematical model of a slow ionic conductance. The slow conductance was implemented in two versions: in one of them the instantaneous value of the conductance is computed in real time and according to the membrane potential of the biological neuron, in another version the value of the conductance simply comes from a time series previously stored in the computer. A pre-synaptic artificial neuron, with a spike distribution chosen by the experimenter, provided input for the biological neuron through an artificial chemical inhibitory synapse. The amount of information (variability of spike patterns from the artificial neuron) coded by the biological neuron was inferred by calculating the average mutual information along stimulus and response bursts for the two conditions of the slow conductance (dynamically calculated or from file previously stored). Our experiments reproduced the results found in intact pyloric central pattern generator bursting neurons connected by mutual inhibition. Moreover, we show that the average mutual information peaks, found when the conductance is dynamically calculated, disappear when we use the previously recorded time series of the conductance. Such results validate those only found previously in numerical simulations in the absence of noise and point the role of the slow ionic conductances in a biophysical mechanism that allow bursting motor neurons to encode in a nontrivial fashion the information they receive through a single synapse.

A doença de Huntington (DH) é um distúrbio neurodegenerativo hereditário e autossômico dominante e tem como características alterações motoras e mentais progressivas. Recentemente, além das alterações verificadas no sistema nervoso central, também têm sido descritas alterações em órgãos periféricos, tais como osteoporose, atrofia muscular, problemas intestinais, alterações cardíacas e, sobretudo, alterações no sistema nervoso autônomo. São evidentes as alterações autonômicas do coração nos portadores da DH, as quais, são, sobretudo, um risco potencial, tornando os pacientes suscetíveis a problemas cardiovasculares. No entanto, os mecanismos pelos quais a doença afeta os componentes autonômicos do coração não são totalmente conhecidos, por isso a importância de se estudar os componentes da inervação cardíaca, sobretudo o gânglio estrelado (GE). A DH pode ser induzida através do ácido 3-nitropropiônico (3-NP), pois essa substância produz efeitos neurotóxicos inibindo a succinato desidrogenase. Esta pesquisa objetiva analisar, por meio da indução através do 3-NP, os efeitos da DH no GE, identificando possíveis alterações morfoquantitativas dos neurônios ganglionares, com uso de técnicas baseadas em delineamento estereológico 3D e de bioimagem associadas à teste comportamental e perfil hemodinâmico, a fim de contribuir para o entendimento de como a doença age na inervação do coração. Para isso foram utilizados 14 camundongos C57BL-6 machos que foram alocados em dois grupos: Grupo Controle com 7 animais induzidos com solução salina (0,9%); Grupo 3NP com 7 animais induzidos com doses subagudas de 60 mg.kg-1dia-1 de 3-NP. Foram realizados o teste comportamental, a avaliação cardíaca e a análise estereológica. Os principais achados dessa pesquisa foram: (I) diminuição da atividade exploratória dos animais; (II) prejuízo da função sistólica; (III) aumento de 76% no volume ganglionar; (IV) aumento de 70% no volume médio dos neurônios, concluindo-se que o 3-NP produz efeitos na função cardíaca, ocasionando hipertrofia do gânglio / Huntington\'s disease (HD) is a hereditary and autosomal dominant neurodegenerative disorder and is characterized by progressive motor and mental changes. Recently, in addition to changes in the central nervous system, alterations in peripheral organs such as osteoporosis, muscular atrophy, intestinal problems, cardiac alterations and, above all, changes in the autonomic nervous system have also been described. Autonomic heart alterations in DH patients are evident, which are a potential risk, making patients susceptible to cardiovascular problems. However, the mechanisms by which the disease affects the autonomic components of the heart are not fully understood, therefore, the importance of studying the components of cardiac innervation, especially the stellate ganglion (SG). HD can be induced through 3-nitropropionic acid (3-NP), as this substance produces neurotoxic effects inhibiting succinate dehydrogenase. The aim of this research was to analyze the effects of HD on the SG by means of 3-NP induction, identifying possible morpho-quantitative changes in ganglion neurons, using techniques based on 3D stereological and bioimaging techniques associated with behavioral and hemodynamic profile, In order to contribute to the understanding of how the disease acts in the heart innervation. For this, 14 male C57BL-6 mice were used, which were allocated in two groups: Control Group with 7 animals induced with saline solution (0.9%); Group 3NP with 7 animals induced with subacute doses of 60 mg.kg-1day-1 of 3-NP. Behavioral test, cardiac evaluation and stereological analysis were performed. The main findings of this research were: (I) decrease in the exploratory activity of the animals; (II) impairment of systolic function; (III) 76% increase in ganglion volume; (IV) increase of 70% in the mean volume of the neurons, concluding that 3-NP produces effects on cardiac function, causing hypertrophy of the ganglion

O gânglio estrelado (GE) é o principal componente da inervação cardíaca extrínseca e está envolvido na gênese de diversas cardiomiopatias. Durante o envelhecimento, o controle neural do coração dos mamíferos é alterado de forma complexa e não clara, geralmente ocasionando decremento da função cardíaca e maior propensão a doenças degenerativas. A ocorrência de resultados dissonantes quanto aos parâmetros morfoquantitativos durante o envelhecimento, como o aumento ou diminuição do número total de neurônios simpáticos, é assunto para discussões interessantes. Esta pesquisa foi conduzida em preás machos (Galea spixii), um pequeno roedor da fauna brasileira. Desta forma, estudou-se o efeito do desenvolvimento pós-natal (maturação e envelhecimento) na macro e microestrutura do gânglio estrelado esquerdo (GEe) de preás, por meio de microscopia quantitativa tridimensional (Estereologia) associada a técnicas de imuno-histoquímica. De acordo com a fase específica do desenvolvimento pós-natal, os animais foram alocados nos seguintes grupos etários: Neonatos, Jovens, Adultos e Senis. Inicialmente, os animais foram submetidos à eutanásia e seus gânglios estrelados esquerdos coletados e fixados em solução de formaldeído (4%) em PBS. Foi realizada amostragem sistemática e uniformemente aleatória (SURS), estimando-se: volume do GEe, volume neuronal e número total de neurônios do GEe. Os principais achados deste estudo foram: i) aumento do comprimento do gânglio - 42% entre Neonato e Senil; 34% entre Jovem e Senil e 35% entre Adulto e Senil; ii) hipertrofia do GEe - 171% entre os grupos Neonato e Adulto; iii) aumento do tecido não neuronal - 268% entre os grupos Neonato e Adulto; iv) estabilidade no número total de neurônios uninucleados, binucleados e total (uni+bi); v) estabilidade no tamanho (volume) dos neurônios uninucleados e binucleados; e vi) estabilidade no número total de neurônios imunorreativos ao Ki-67 (uni+bi). Espera-se que os resultados gerados por esta pesquisa possam esclarecer alguns aspectos estruturais da plasticidade neural durante o desenvolvimento pós-natal de preás, avançando assim o conhecimento acerca da inervação cardíaca extrínseca / The stellate ganglion (SG) is a main component of extrinsic cardiac innervation and is involved in the genesis of various cardiomyopathies. During ageing, the neural control of heart in mammals is altered in the complex shape and unclear, generally cause decrement in the cardiac function and a greater propensity to degenerative diseases. The occurrence of discordant results regarding the morphoquantitative parameters during ageing, such as increase or decrease in the total number of sympathetic neurons, is a subject for interesting discussions. This research was conducted in males preas (Galea spixii), a small rodent of the Brazilian fauna. Therefore, this work aimed to study the effect of postnatal development (maturation and ageing) in the macro and microstructure of the left stellate ganglion (LSG) in preas by dimensional quantitative microscopy (Stereology) associated to immunohistochemistry techniques. According to a specific stage of postnatal development, the animals were allocated into the following age groups: Newborn, Young, Adult and Elderly. The animals were euthanised and the left stellate ganglia were collected and fixed in 4% formaldehyde solution in PBS. A systematic uniformly random sampling (SURS) was performed to estimate: the volume of LSG, neuron volume and the total number of LSG neurons. The main findings of this study were: i) increase in length ganglia - 42% between Newborn and Elderly; 34% between Young and Elderly and 35% between Adult and Elderly; ii) hypertrophy of LSG - 171% between the groups Newborn and Adult; iii) increase of non-neuronal tissue - 268% between the groups Newborn and Adult; iv) stability for the total number of uninucleate neurons, binucleate neurons and total (uni+bi); v) stability in the size (volume) of uninucleate and binucleate neurons; and, vi) stability for the total number of neurons immunorreactive to Ki-67 (uni+bi). It is expected that the results generated for this research may clarify structural aspects of neural plasticity during the postnatal development of preas, thus advancing the knowledge about the extrinsic cardiac innervation

O presente trabalho tem por objetivo analisar o efeito do metilmercúrio na retina de peixe tropical Hoplias malabaricus (Traíra) através de baixas doses agudas. As intoxicações foram realizadas, por meio de injeção intraperitoneal, nas doses de 0,01, 0,05, 0,1 e 1,0 g/g, com um período de quinze dias de depuração do MeHg. Após o término do período de depuração, os olhos foram enucleados e as retinas isoladas foram fixadas em PFA 4% por 3 horas. As retinas foram conservadas, até o momento do uso (ou por no mínimo 9 horas), em tampão PB 0,1M a 4ºC. Após os procedimentos imunohistoquímicos para marcação de células bipolares do tipo ON com estratificação na sublâmina b da CPI, as retinas foram aplanadas para confecção de montagens planas para a análise quantitativa de células bipolares ON imunorreativas a proteína cinase C _. A análise quantitativa das células da camada de células ganglionares (CCG) também foi realizada. Células da CCG foram coradas pela técnica de Nissl, as retinas foram aplanadas em lâminas gelatinizadas e submetidas a uma bateria de desidratação (com diferentes concentrações alcoólicas) e coloração, utilizando cresil violeta como corante. Estas análises foram realizadas em 3 ou 4 retinas para cada dose testada. Análises idênticas foram realizadas nas retinas controle. Todas as retinas foram dividas nos quadrantes dorsal, ventral, nasal, temporal e em centro e periferia. Campos foram fotografados por toda a retina com intervalos de 1 mm, com auxilio do programa Axio Vision por meio de uma câmera digital e um microscópio acoplados a um computador. Os campos amostrados foram contados com o auxilio do programa NIH Scion Imagem 2.0. A densidade média de células foi estimada para cada retina e os grupos intoxicados foram comparados com o grupo controle (Teste T-student). A partir dos dados de densidade celular, mapas de isodensidade foram confeccionados, além de permitir estimar o poder de resolução teórico da acuidade visual de cada um dos animais experimentais utilizados para análise de células da CCG a partir da densidade máxima de células. Evidenciamos que as baixas doses agudas testadas não causam diminuição na densidade célular de células bipolares ON e células da CCG, comparado ao grupo controle. Não houve reduções significativas na densidade de células para ambos os tipos celulares analizados em nenhuma das regiões retinianas nas doses de MeHg testadas. Assim, a intoxicação de MeHg por baixas doses agudas não alterou o poder de resolução teorio da acuiade visual dos animais testados / This study aims to examine the effects of low acute doses of methylmercury (MeHg) on the retina of the tropical fish Hoplias malabaricus (Thraira). Four levels of MeHg intoxication were induced by intraperitoneal injection of doses of either 0.01, 0.05, 0.1 or 1.0 g MeHg/g of body weight, followed by a fifteen day period of depuration of MeHg. After the depuration period, the eyes were harvested, and the retinas were isolated and fixed in 4% paraformaldehyde for 3 hours. The retinas were then stored (for at least for 9 hours) in 0.1 M sodium phosphate PB buffer at 4°C until the time of analysis. ON bipolar cells in sublamina b of the inner plexiform layer immunoreactive to protein Kinase C_ were immunohistochemically labeled, and the retinas were flattened to make whole mounts for quantitative analysis of ON bipolar cell densities. Quantitative analysis of cells in the retinal ganglion cell layer (GCL) was also performed. GCL cells were Nissl stained, and the retinas were flattened on gelatinized slides and subjected to another battery of dehydration (with different alcohol concentrations) and staining using cresyl violet. These analyses were carried out in 3 or 4 retinas for each dose tested. Identical analyses were performed on the control retinas. All retinas were divided into regions: dorsal, ventral, nasal, temporal, center and periphery. Sample retinal fields were photographed throughout the retina at intervals of 1 mm, with a digital camera attached to a microscope using Axio Vision software coupled to a computer. ON bipolar and GCL cells within the fields were counted with the help of the NIH Scion Image 2.0 software. The average density (mm2) of both types of cells was estimated for each retina and the data from each of the four MeHgintoxicated groups were compared with the control group values (Student t-test). From the density data we derived isodensity maps, permitting us to estimate the theoretical resolving power (maximum visual acuity) of each of the experimental animals used from the maximum density of cells in the ganglion cell layer. We showed that low acute doses of MeHg/g do not decrease cell densities of either ON bipolar cells or cells in the GCL, compared to controls. There were no significant decreases in cell density (counts) for either cell type in any of the retinal regions, for any of the MeHg doses tested. Thus, acute low-dose MeHg intoxication did not degrade the estimates of the animals theoretical resolving power

Les dystrophies rétiniennes héréditaires (DRH) sont un ensemble de pathologies rétiniennes incurable provoquant la cécité. Les DRH sont caractérisées par le dysfonctionnement/dégénérescence des photorécepteurs et le remodelage de la structure de la rétine. Une des approches thérapeutiques envisagées pour traiter les DRH est la thérapie génique spécifique, c’est à dire le remplacement du gène défectueux par un gène sain. Cependant, bien qu’efficace, la thérapie génique spécifique n’est pas toujours applicable, en particulier quand la dégénérescence est trop avancée ou quand le gène muté n’est pas connu. Afin de traiter tous les cas de DRH quelle que soit leur origine génétique et leur stade de progression, une approche de thérapie génique d’addition est envisagée : Le transfert d’optogène. Cela consiste à convertir les cellules encore présentes dans la rétine malgré la dégénérescence, en cellule photosensible suite à l’expression d’un optogène (protéine photosensible). Mon projet de thèse a consisté dans un premier temps à évaluer le transfert de gène avec un vecteur AAV2/2 dans les cellules ganglionnaires rétiniennes de chien et de primate non-humain. Cette première partie a permis d’initier un second projet qui a eu pour objectif d’évaluer l’efficacité du transfert d’optogène (Channelrhodopsin-2) pour la restauration de la fonction visuelle dans un modèle canin de dystrophie rétinienne (le chien Rpe65- /-). / Inherited retinal dystrophies (IRD), a group of incurable retinal pathologies, are associated with visual impairments due to a malfunction and/or degeneration of photoreceptors and/or retinal pigment epithelium (RPE). Significant progress in the field of gene therapy has allowed the development and the characterization of an innovative tool to treat IRD patients: recombinant adeno-associated viral vectors (AAV) that carry and deliver therapeutic nucleic acids. However, due to the heterogenic nature of IRD, gene supplementation will not allow to treat all forms of IRD because: (i) the numbers of mutated genes are unknown according to the state of art; (ii) the dominant forms of IRD in which mutations lead to negative effects are not eligible; (iii) the limit of AAV packaging excludes large-sized mutated genes and (iv) this approach is only applicable when photoreceptors are still alive. To treat all IRD patients, a novel therapeutic approach, independently of the mutated gene and the disease kinetic is suitable: the optogene transfer (light-sensitive protein) to restore photosensitivity in neurodegenerative retina by converting surviving retinal cells into photosensors. The primary goal of my research was to promote and characterize adeno-associated virus type 2-(AAV2) transduction in retinal ganglion cells of dog and non-human primate. A second aim was to investigate the feasibility of AAV2-mediated optogenes transfer in retinal ganglion cells as a therapeutic approach to restore visual function in RPE65 deficient dog, a canine model of IRDs.

Le Syndrome de Wolfram (SW; OMIM #222300, prévalence 1-9 / 1000 000) est une maladie neurodégénérative, qui se présente avec un début juvénile, intégrant le diabète insipide, diabète sucré, l’atrophie optique (AO), et la surdité. AO est généralement son premier symptôme neurologique, commençant à l’âge de 11 ans et se terminant par la cécité 8 ans plus tard. Malheureusement, un modèle murin du SW approprié aux symptômes ophtalmologiques n'a pas encore été trouvé, donc la recherche de la thérapie pour sauver la vision en est à ces débuts. Dans cette thèse j’ai étudié l’atteinte visuelle de deux modèles de souris mutantes pour le SW et succès d’une approche de thérapie génique (TG) avec le gène humain WFS1.Premièrement, les souris Wfs1exon8del sont été examinées à 3 et 6 mois pour l’acuité visuelle (AV) et la sensibilité aux contrastes (SC) via changements dans le reflexe optomoteur (ROM), la fonction rétinienne neurale par électrorétinogramme (ERG), ainsi que la physiologie de l’œil par la fondoscopie et tomographie par cohérence optique (TCO). De plus, la proportion des cellules ganglionnaire de la rétine (CGRs) et la perte axonale dans le nerf optique (NO) à 7 mois ont été examinés avec marquage anti-Brn3a et microscopie électronique, respectivement. Il y avait une perte progressive de l’AV et la SC chez les souris KO à partir du 1 mois. Elle était accompagnée d'une pâleur du disque optique (DO), d'amincissement de la rétine ainsi que des lésions axonales. Par contre, il n’avait pas de perte des CGRs ni stress du réticulum endoplasmique dans la rétine. Brièvement, les souris KO présentent un phénotype ophtalmique du SW significatif et peuvent servir comme modèle.Deuxièmement, à la recherche d'un autre modèle du SW, les fonctions visuelles de la lignée Wfs1E864K de la souris ont été étudiées. Déjà à 1 mois, les souris Wfs1E864K/E864K avait une perte drastique de la fonction des CGRs, mais en gardant le nombre de cellules à un niveau normal. Ceci a été accompagné par un amincissement de la rétine et d’un sévère dommage du NO, comme montré par le TCO et la fondoscopie, respectivement. En conséquence, les souris Wfs1E864K/E864K, avec leur fort phénotype ophtalmique, pourraient servir comme modèle du SW classique.Enfin, pour enquêter sur les futures options de traitement contre le SW, les souris de la lignée Wfs1exon8del à 1 mois ont subi une TG intravitréenne avec AAV-2/2-CMV-WFS1. Les examens à 3 et 6 mois ont montré une amélioration de l’AV, ainsi que le sauvetage de la pâleur du DO et réduction des lésions axonales chez les souris KO. En outre, aucun effet indésirable lié à des injections TG n’ont été noté. Suivant cette idée, les souris Wfs1E864K/E864K ont également été soumis à la TG intravitréenne, délivrée à P14, mais sans succès.En conclusion, la lignée Wfs1exon8del de la souris est un modèle fiable du SW, y compris les aspects visuels. Je propose le modèle Wfs1E864K/E864K comme une alternative, en particulier pour enquêter sur la fonction de Wfs1 dans l'œil. Enfin, la GT intravitréenne avec WFS1 a un potentiel pour sauver partiellement le phénotype ophtalmique, ouvrant la voie vers le traitement pour les patients du SW / Wolfram Syndrome (WS; OMIM #222300, prevalence 1-9 / 1 000 000) has a juvenile onset and incorporates diabetes insipidus, diabetes mellitus, optic atrophy (OA), and deafness; leading to death in middle age. OA is its first neurological symptom, starting in adolescence and ending with blindness within 8 years. Unfortunately, a suitable WS mouse model comprising ophthalmologic symptoms has not yet been found, therefore the search for its treatment is delayed. In this thesis, I studied visual impairment in two WS mouse models along with a success of a gene therapy (GT) approach with the human WFS1 gene.Firstly, 3 and 6 months old Wfs1exon8del mice were examined for the visual acuity (VA) and contrast sensitivity via changes in the opto-motor reflex (OMR), the neural retinal function via electroretinogram (ERG), as well as the eye physiology via fundoscopy and optic coher-ence tomography (OCT). Also, the proportion of retinal ganglion cells (RGC) and the axonal loss at the age of 7 months were determined with anti-Brn3a immuno-labeling of retinal sections and electron microscopy of optic nerve (ON) sections, respectively. There was a progressive loss of VA and contrast sensitivity in Wfs1exon8del-/- mice, starting already at 1 month of age. It was accompanied by optic disc pallor, retinal thinning as well as axonal damage. However, there was no RGC loss and the endoplasmic reticulum (ER) stress in the retina was at a normal level. It suggested a presence of another cause for the reported degeneration in KO mice; in opposition to what was proposed in the literature. I brief, KO mice exhibit significant WS ophthalmic phenotype.Secondly, in search for another model, visual functions of Wfs1E864K mouse line were investigated. This line was originally a model of Wolfram-like Syndrome, characterized by dominant mutations in WFS1 leading to congenital progressive hearing impairment, diabetes mellitus and OA. Only homozygous mutants, however, showed expected visual impairment. Already at 1 month of age, Wfs1E864K/E864K mice had drastic loss of RGC function, albeit keeping the cell number at a normal level. This was accompanied by retinal thinning and a severe ON damage, as shown with OCT and fundoscopy, respectively. In contrast, the RGC function in Wfs1E864K/+ mice dropped slightly only at the age of 7 and 12 months, showing that the pathology of the E864K mutation-driven disease in mice is different than in humans. Therefore, Wfs1E864K/E864K mice, with their strong ophthalmic phenotype, could potentially serve as a model of the classical WS.Finally, to investigate future treatment options, 1 month old Wfs1exon8del+/+ (WT) and Wfs1exon8del-/- (KO) mice underwent a uni- and bi-lateral intravitreal gene therapy (GT) with AAV-2/2-CMV-WFS1. Exams at 3 and 6 months of age showed improved VA, as well as optic pallor and axonal damage rescue in KO mice. Also, no adverse effects related to either GT or sham injections were noted. Following this idea, the Wfs1E864K/E864K mice were also subjected to intravitreal GT, delivered at P14, but without success.In conclusion, Wfs1exon8del mouse line is a reliable model of WS, including the visual aspects. I propose the Wfs1E864K/E864K model as an alternative, especially to investigate Wfs1 function in the eye. Finally, the intravitreal AAV-driven GT with WFS1 has a potential to partially rescue the ophthalmic phenotype, paving the wave towards the treatment for WS patients.

The effect of interleukin-1 beta (IL-1β) on the electrical properties of sensory neurons was assessed at comparable levels and exposure times to those found in animal models of neuropathic pain. Experiments involved whole cell current- or voltage-clamp recordings from rat dorsal root ganglion (DRG) neurons in defined medium, neuron enriched cultures. 5-6 days exposure to 100 pM IL-1β produced neuron specific effects. These included an increase in the excitability of medium diameter and small diameter isolectin B4 (IB4)-positive neurons that was comparable to that found after peripheral nerve injury. By contrast, a reduction in excitability was observed in large diameter neurons, while no effect was found in small diameter IB4-negative neurons. Further characterization of changes in medium and small IB4-positive neurons revealed that some, but not all, effects of IL-1β were mediated through its receptor, IL-1RI. Using appropriate voltage protocols and/or ion substitutions, it was found that neuron specific changes in several ionic currents, including alterations in hyperpolarization activated inward current (IH) and decreases in various K+ currents contribute to the increased excitability produced by IL-1β. Overall, these studies revealed that: 1. The effects of long-term exposure of DRG neurons to IL-1β are reflective of the enduring increase in primary afferent excitability reported after peripheral nerve injury. This expands the recognized role of IL-1β in acute inflammatory pain to neuropathic pain. 2. Hyperexcitability in medium neurons exposed to IL-1β likely includes mixed populations of neurons corresponding to nociceptive and non-nociceptive primary afferent fibres and, therefore, has relevance to hyperalgesia and allodynia, respectively. 3. The responsiveness of small IB4-positive neurons, but not IB4-negative, to prolonged IL-1β exposure is consistent with the suggestion that small IB4-negative afferents are involved in inflammatory pain, while small IB4-positive afferents are involved neuropathic pain. 4. The identification of receptor mediated effects and several contributing ionic mechanisms, may have relevance to the development of new therapeutic approaches to neuropathic pain. 5. IL-1β can contribute to increased neuronal excitability by mechanisms that are independent of IL-1RI signalling. This should be taken into account when targeting IL-1β, or more specifically IL-1RI, in the management of neuropathic pain.

En esta tesis estudiamos la población total de las células ganglionares de la retina (CGR) en rata y ratón, y desarrollamos un modelo experimental de hipertensión ocular mediante fotocoagulación láser. La población de CGR proyecta masivamente a los colículos superiores. Se observa una estría visual en la retina dorsal donde se encuentra las densidades más altas de CGR. La pequeña población de CGR ipsilateral se distribuye mayoritariamente en la periferia de la retina temporal. El aumento de la presión intraocular induce una compresión de los axones en la cabeza del nervio óptico que provoca una alteración del transporte axonal retrógrado, que induce una degeneración sectorial localizada y difusa de las CGR, preferentemente en la retina dorsal, así como de sus axones. La pérdida selectiva de las CGR en la capa de CGR, sugiere que la causa de la muerte de las CGR no se debe a una isquemia retiniana. / In this thesis we have studied the total population of retinal ganglion cells (RGCs) in rat and mouse, and developed an experimental model of ocular hypertension by laser photocoagulation. The RGC population projects massively to the superior colliculi. There is a visual streak in the dorsal retina where the highest densities of RGCs are found. The small population of ipsilateral RGCs is distributed mainly in the periphery of the temporal retina. The increase of intraocular pressure induces a compression of the axons at the optic nerve head that causes a disturbed retrograde axonal transport, inducing a localized, diffuse and sectorial degeneration of RGCs and their axons, preferably in the dorsal retina. The selective loss of RGCs in the RGC layer, suggests that the cause of the RGC loss is not due to retinal ischemia.

Neuronal networks produce reliable functional output throughout the lifespan of an animal despite ceaseless molecular turnover and a constantly changing environment. The cellular and molecular mechanisms underlying the ability of these networks to maintain functional stability remain poorly understood. Central pattern generating circuits produce a stable, predictable rhythm, making them ideal candidates for studying mechanisms of activity maintenance. By identifying and characterizing the regulators of activity in small neuronal circuits, we not only obtain a clearer understanding of how neural activity is generated, but also arm ourselves with knowledge that may eventually be used to improve medical care for patients whose normal nervous system activity has been disrupted through trauma or disease. We utilize the pattern-generating pyloric circuit in the crustacean stomatogastric nervous system to investigate the general scientific question: How are specific aspects of rhythmic activity regulated in a small neuronal network? The first aim of this thesis poses this question in the context of a single neuron. We used a single-compartment model neuron database to investigate whether co-regulation of ionic conductances supports the maintenance of spike phase in rhythmically bursting “pacemaker” neurons. The second aim of the project extends the question to a network context. Through a combination of computational and electrophysiology studies, we investigated how the intrinsic membrane conductances of the pacemaker neuron influence its response to synaptic input within the framework of the Phase Resetting Curve (PRC). The third aim of the project further extends the question to a systems-level context. We examined how ambient temperatures affect the stability of the pyloric rhythm in the intact, behaving animal. The results of this work have furthered our understanding of the principles underlying the long-term stability of neuronal network function.