Dissertations / Theses on the topic 'Glia Progenitors'

Neurogenesis occurs in exclusive regions in the adult nervous system, the subventricular zone and dentate gyrus in the brain, and olfactory epithelium (OE) in the periphery. Cell replacement after death or injury, occurs to varying degrees in neural tissue, and is thought to be dependent upon the biological responses of stem and/or progenitor cells. Despite the progress made to identify adult OE and central nervous system (CNS) progenitors and lineage trace their progeny, our spatial and temporal understanding of embryonic OE neuroglial progenitors has been stalled by the paucity of identifiable genes able to distinguish individual candidate progenitors. In the developing CNS, radial glia serve as both neural progenitors and scaffolding for migrating neuroblasts and are identified by the expression of a select group of antigens, including nestin. Here, I show that the embryonic OE contains a novel radial glial-like progenitor (RGLP) that is not detected in adult OE. RGLPs express the radial glial antigens nestin, GLAST and RC2, but not brain lipid binding protein (BLBP), which, distinct from CNS radial glia, is instead found in olfactory ensheathing cells, a result confirmed using lineage tracing with BLBP-cre mice. Nestin-cre-mediated lineage tracing with three different reporters reveals that only a subpopulation of nestin-expressing RGLPs activate the “CNS-specific” nestin regulatory elements, and produce spatially restricted neurons in the OE and vomeronasal organ. The dorsal-medial restriction of transgene-activating cells is also seen in the embryonic OE of Nestin-GFP transgenic mice, where GFP is found in a subpopulation of GFP+ Mash1+ neuronal progenitors, despite the fact that endogenous nestin expression is found in RGLPs throughout the OE. In vitro, embryonic OE progenitors produce three biologically distinct colony subtypes, that when generated from Nestin-cre/ZEG mice, produce GFP+ neurons, recapitulating their in vivo phenotype, and are enriched for the most neurogenic colony subtype. Neurogenesis in vitro is driven by the proliferation of nestin+ progenitors in response to FGF2. I thus provide evidence for a novel neurogenic precursor, the RGLP of the OE, that can be regulated by FGF2, and provide the first evidence for intrinsic differences in the origin and spatiotemporal potential of distinct progenitors during OE development.

Precise regulation of neurogenesis is achieved by differentially allocating the neurogenic competence along the tissue. In the hindbrain proneural gene expression is stereotypically confined in segment boundary-adjacent regions, hence, being absent in segment centers. This segregation of proneural gene expression therefore hints rhombomere centers as a putative non-neurogenic population. In this work, we unveil their spatiotemporal molecular profile as well as one of the mechanisms involved in their maintenance as non-committed progenitors. By 4D imaging we shed light for the first time into the in vivo cell behavior this population displays. We propose this population in rhombomere centers is indeed heterogeneous as it harbors cells with different proliferative capacity.
La regulació precisa de la neurogènesi s’aconsegueix localitzant la competència neurogènica de manera diferencial al llarg del territori. Al cervell posterior, l’expressió de gens proneurals es restringeix a les zones adjacents a les cèl·lules de les fronteres, i per tant és absent als centres així doncs assenyalant els centres dels rombòmers com una població no neurogènica. En aquest treball, hem revelat el seu perfil molecular espai-temporal així com un dels mecanismes que manté aquestes cèl·lules com a no neurogèniques. Mitjançant imatges 4D hem aportat llum per primera vegada a l’enteniment del seu comportament cel·lular en viu, i proposem que aquesta població dels centres dels rombòmers és de fet heterogènia ja que conté cèl·lules amb diferent capacitat proliferativa.

Glial cells play important roles in the development and homeostasis of metazoan nervous systems. However, while their involvement in the development and function in the central nervous system (CNS) of vertebrates is increasingly well understood, much less is known about invertebrate glia and the evolutionary history of glial cells more generally. An investigation into amphioxus glia is therefore timely, as this organism is the best living proxy for the last common ancestor of all chordates, and hence provides a window on the role of glial cells development and function at the transition between invertebrates and vertebrates. We report here our findings on amphioxus glia as characterized by molecular probes correlated with anatomical data at the TEM level. The results show amphioxus glial lineages express genes typical of vertebrate astroglia and radial glia and segregate early in development, forming what appears to be a spatially separated cell proliferation zone positioned laterally, between the dorsal and ventral zones of neural cell proliferation. Our study provides strong evidence for the presence of vertebrate-type glial cells in amphioxus while highlighting the role played by segregated progenitor cell pools in CNS development. There are implications also for our understanding of glial cells in a broader evolutionary context and insights into patterns of precursor cell deployment in the chordate nerve cord.

The neocortex is the most recently evolved part of the mammalian brain which is involved in a repertoire of higher order brain functions, including those that separate humans from other animals. Humans have evolved an expanded neocortex over the course of evolution through a massive increase in neuron number (compared to our close relatives-­‐‑ the chimpanzees) in spite of sharing similar gestation time frames. So what do humans do differently compared to chimpanzees within the same time frame during their development? This dissertation addresses this question by comparing the developmental progression of neurogenesis between humans and chimpanzees using cerebral organoids as the model system. The usage of cerebral organoids, has enabled us to compare the development of both the human neocortex, and the chimpanzee neocortex from the very initiation of the neural phase of embryogenesis until very long periods of time. The results obtained so far suggest that the genetic programs underlying the development of the chimpanzee neocortex and the human neocortex are not very different, but rather the difference lies in the timing of the developmental progression. These results show that the chimpanzee neocortex spends lesser time in its proliferation phase, and allots lesser time to the generation of its neurons than the human neocortex. In more scientific terms, the neurogenic phase of the neocortex is shorter in chimpanzees than it is in humans. This conclusion is supported by (1) an earlier onset of gliogenesis in chimpanzees compared to humans which is indicative of a declining neurogenic phase, (2) an earlier increase in the chimpanzee neurogenic progenitors during development, compared to humans, (3) a higher number of stem cell– like progenitors in human cortices compared to chimpanzees, (4) a decline in neurogenic areas within the chimpanzee cerebral organoids over time compared to human cerebral organoids.

In this work we have assessed the possible contribution of the human chromosome-21 gene DYRK1A in the developmental cortical alterations associated with Down Syndrome using the mBACTgDyrk1a mouse, which carries 3 copies of Dyrk1a, and a trisomic model of the syndrome, the Ts65Dn mouse. We show that trisomy of Dyrk1a changes the cell cycle parameters of dorsal telencephalic radial glial (RG) progenitors and the division mode of these progenitors leading to a deficit in glutamatergic neurons that persist until the adulthood. We demonstrate that Dyrk1a is the triplicated gene that causes the deficit in early-born cortical glutamatergic neurons in Ts65Dn mice. Moreover, we provide evidences indicating that DYRK1A-mediated degradation of Cyclin D1 is the underlying mechanism of the cell cycle defects in both, mBACTgDyrk1a and Ts65Dn dorsal RG progenitors. Finally, we show that early neurogenesis is enhanced in the medial ganglionic eminence of mBACTgDyrk1a embryos resulting in an altered proportion of particular cortical GABAergic neuron types. These results indicate that the overexpression of DYRK1A contributes significantly to the formation of the cortical circuitry in Down syndrome.
En aquest treball s'ha avaluat la possible contribució del gen DYRK1A, localitzat en el cromosoma humà 21, en les alteracions del desenvolupament de l’escorça cerebral associades a la Síndrome de down (SD) mitjançant l’estudi de dos models murins: el ratolí mBACTgDyrk1a, el qual conté 3 còpies de Dyrk1a, i el ratolí Ts65Dn, un dels models trisòmics de la SD més ben caracteritzats. Els nostres resultats mostren que la trisomia de Dyrk1A altera alguns paràmetres del cicle cel•lular i el tipus de divisió dels progenitors neurals del telencèfal dorsal, donant lloc a un dèficit de neurones glutamatèrgiques que persisteix fins l’edat adulta. Hem demostrat que Dyrk1a és el gen triplicat responsable del dèficit inicial en la generació de neurones glutamatèrgiques corticals observat en el ratolí Ts65Dn. A més a més, hem proporcionat evidències de que la degradació de Ciclina D1 induïda per DYRK1A és el mecanisme molecular subjacent a les alteracions de cicle cel•lular observades en els progenitors neuronals dels embrions mBACTgDyrk1a i Ts65Dn. Per altra banda, hem demostrat que la neurogènesis inicial està incrementada en l’eminència ganglionar medial dels embrions mBACTgDyrk1a, fet que altera la proporció de subtipus específics d’interneurones GABAèrgiques en l’escorça cerebral adulta. En conclusió, els nostres resultats indiquen que la sobreexpressió de DYRK1A contribueix significativament a la formació dels circuits cortical en la SD.

Un certain nombre de régions du cerveau des vertébrés, y compris chez l’homme, continuent d’être le siège de l’ajout de nouveaux neurones à l’âge adulte. Ces nouveaux neurones sont produits à partir de cellules spécialisées, appelées cellules souches neurales (CSN). Celles-ci sont capables de s’auto-renouveler et sont principalement trouvées dans un état d’arrêt transitoire du cycle cellulaire que l’on appelle quiescence. A l’heure actuelle, les mécanismes cellulaires et moléculaires permettant aux CSN de trouver un équilibre entre maintien et différentiation, ainsi que les règles générales gouvernant l’évolution de leur population, ne sont que partiellement compris. A l’échelle moléculaire, plusieurs facteurs et voies de signalisation apparaissent déterminants pour l’homéostasie des CSN. Notamment, la voie de signalisation du récepteur Notch s’avère essentielle pour maintenir à la fois l’état de quiescence et le caractère souche des CSN. Il demeure néanmoins inconnu si la signalisation Notch affecte ces deux propriétés de manière indépendante ou non. A l’échelle cellulaire, la plupart des modèles actuels suggèrent que les CSN se divisent rarement et principalement de manière asymétrique. Cette dernière propriété permettrait aux CSN de se perpétuer tout en donnant naissance à des cellules filles déterminées à se différencier en neurones. Le pallium du poisson-zèbre abrite une population particulièrement importante de CSN, que l’on appelle glies radiaires (GR), et qui possèdent les mêmes caractéristiques fondamentales que leurs homologues chez les mammifères. Notre laboratoire avait précédemment démontré que le récepteur Notch3 était nécessaire au maintien de la quiescence des GR. Le travail présenté dans ce manuscrit se décompose en deux études complémentaires dont les objectifs respectifs étaient: (1) d’améliorer notre compréhension du rôle de la voie de signalisation Notch3 dans l’homéostasie des GR et (2) d’étudier les schémas de divisions adoptés par les GR afin de maintenir leur nombre sur une longue durée. Dans la première étude, nous démontrons que le rôle de la signalisation Notch3 s’étend au-delà du simple contrôle de la quiescence des GR en contribuant également au maintien de leur caractère souche par l’intermédiaire de son gène cible hey1. Un point important de cette découverte est que l’action du facteur Hey1 sur le caractère souche des GR apparaît indépendante du rôle de Notch3 dans le maintien de leur quiescence. Dans la seconde étude, nous avons réalisé une analyse clonale du devenir des GR exprimant le gène her4.1. Ceci nous a permis de mettre en évidence que leurs choix entre différentiation, amplification et auto-renouvellement apparaissent stochastiques, mais équilibrés, ce qui leur permet de maintenir leur population dans le temps. De façon très intéressante, nous avons aussi observé que le nombre total de GR du pallium augmente au cours de la vie, ce qui, au regard du comportement homéostatique de la population de GR exprimant her4.1, nous amène à proposer que la zone neurogénique du pallium est organisée selon une hiérarchie dans laquelle une population inconnue de progéniteurs produit continuellement de nouvelles GR, qui ensuite se maintiennent grâce à un équilibre probabiliste entre leurs différents lignages
New neurons continue to be added into discrete brain regions of most adult vertebrate species, including humans. Adult born neurons arise from precursor cells, called neural stem cells (NSCs), endowed with self-renewal potential and mostly found in a state of reversible cell cycle arrest, named quiescence. Currently, the molecular, cellular and population rules allowing NSC to balance maintenance and differentiation remain incompletely understood. At the single cell level, several factors and signalling pathways were demonstrated to be essential for NSC homeostasis. Among them, the Notch signalling pathway is critically involved in the control of NSC quiescence and stemness. However, whether these two properties represent molecularly distinct or overlapping outputs of the Notch signalling pathway remains unknown. At the cellular level, current models state that NSCs divide rarely and mostly asymmetrically, allowing both self-renewal and the generation of a more committed progeny that ultimately exits the cell cycle and fulfils neuronal differentiation. The adult zebrafish pallium harbours NSCs, called radial glia (RG), which share with their mammalian counterparts the same basic properties. Previously, our laboratory demonstrated that Notch3 was necessary to maintain RG quiescence. Here, in two different and complementary works, we took advantage of the widespread neurogenic ventricular zone (VZ) of the adult zebrafish pallium to (1) explore further the role of Notch3 signalling in RG homeostasis and (2) investigate the division pattern and dynamics allowing the RG population to be maintained on the long run. In the first study, we demonstrate that the role of Notch3 signalling extends beyond the simple maintenance of RG quiescence and that Notch3 also contributes to RG stemness. By overlapping the transcriptomic profiles of both notch3 mutant RG and adult pallial VZ progenitors, we identified different sets of Notch3 target genes potentially responsible for its pleitropic effect in RG. Notably, we show that the Notch3 signalling contribution to RG stemness critically relies on the transcriptional activation of its canonical target gene hey1 and this, independently of Notch3 action on RG quiescence. In the second study, we performed a quantitative analysis of the fates of individual her4.1(Hes5)-expressing RG. We demonstrate that these cells adopt balanced stochastic fates, which allows their population to reach homeostasis. We also report that the overall RG population of the zebrafish pallium continues to grow during adulthood and that this expansion is very likely driven by a yet undefined upstream population of progenitors. As a consequence, we propose that the adult zebrafish is organised into a hierarchy of progenitors dominated by an unknown population that fuels the ongoing production of an intrinsically homeostatic population of RG which, itself, follows neutral drift dynamics

Le développement du cortex cérébral résulte de processus de prolifération, neurogenèse, migration et différenciation cellulaire qui sont contrôlés génétiquement. Les malformations corticales qui résultent d'anomalies de ces processus sont associées à l'épilepsie et la déficience intellectuelle. Nous avons étudié la souris mutante HeCo (heterotopic cortex), qui présente une hétérotopie sous-cortical bilatérale (neurones présents dans la substance blanche) et nous avons identifié la présence d'une mutation sur le gène Eml1 (Echinoderm Microtubule-associated protein-Like 1). De plus, des mutations du gène EML1 ont été identifiées chez des patients atteints d'une forme sévère et rare d'hétérotopie. Dans le cerveau embryonnaire des souris HeCo, des progéniteurs ont été identifiés en dehors de la zone de prolifération, ce qui représente une nouvelle cause de cette malformation. Nous avons étudié la fonction d'Eml1 dans les progéniteurs de la glie radiaire, qui sont clés au cours de la corticogenèse. Nous avons montré qu'Eml1 se localise dans le fuseau mitotique où elle est susceptible de réguler la dynamique des microtubules. Nos données suggèrent qu'Eml1 peut jouer un rôle dans la régulation de la longueur du fuseau puisque celle-ci est perturbée dans les cellules de la glie radiaire chez la souris HeCo. Ceci pourrait représenter la cause primaire de leur ectopie. Nous avons analysé le nombre et la taille des cellules en métaphase dans la partie apicale de la zone ventriculaire où ont lieu les mitoses. Nous proposons ici de nouveaux mécanismes qui régissent l'organisation des progéniteurs dans la zone ventriculaire au cours du développement cortical normal et pathologique
The cerebral cortex develops through genetically regulated processes of cellular proliferation, neurogenesis, migration and differentiation. Cortical malformations represent a spectrum of heterogeneous disorders due to abnormalities in these steps, and associated with epilepsy and intellectual disability. We studied the HeCo (heterotopic cortex) mutant mouse, which exhibits bilateral subcortical band heterotopia (SBH), characterized by many aberrantly positioned neurons in the white matter. We found that Eml1 (Echinoderm Microtubule-associated protein-Like 1) is mutated in these mice. Screening of EML1 in heterotopia patients identified mutations giving rise to a severe and rare form of atypical heterotopia. In HeCo embryonic brains, progenitors were identified outside the normal proliferative ventricular zone (VZ), representing a novel cause of this disorder. We studied Eml1 function in radial glial progenitors (RGCs), which are important during corticogenesis generating other subtypes of progenitors and post-mitotic neurons, and serving as guides for migrating neurons. We showed that Eml1 localizes to the mitotic spindle where it might regulate microtubule dynamics. My data suggest a role in the establishment of the steady state metaphase spindle length. Indeed, HeCo RGCs in the VZ showed a perturbed spindle length during corticogenesis, and this may represent one of the primary mechanisms leading to abnormal progenitor behavior. I also analyzed cell number and metaphase cell size at the apical side of the VZ, where mitosis occurs. I thus propose new mechanisms governing normal and pathological VZ progenitor organization and function during cortical development

Le développement du cortex cérébral résulte de processus de prolifération, neurogenèse, migration et différenciation cellulaire qui sont contrôlés génétiquement. Les malformations corticales qui résultent d'anomalies de ces processus sont associées à l'épilepsie et la déficience intellectuelle. Nous avons étudié la souris mutante HeCo (heterotopic cortex), qui présente une hétérotopie sous-cortical bilatérale (neurones présents dans la substance blanche) et nous avons identifié la présence d'une mutation sur le gène Eml1 (Echinoderm Microtubule-associated protein-Like 1). De plus, des mutations du gène EML1 ont été identifiées chez des patients atteints d'une forme sévère et rare d'hétérotopie. Dans le cerveau embryonnaire des souris HeCo, des progéniteurs ont été identifiés en dehors de la zone de prolifération, ce qui représente une nouvelle cause de cette malformation. Nous avons étudié la fonction d'Eml1 dans les progéniteurs de la glie radiaire, qui sont clés au cours de la corticogenèse. Nous avons montré qu'Eml1 se localise dans le fuseau mitotique où elle est susceptible de réguler la dynamique des microtubules. Nos données suggèrent qu'Eml1 peut jouer un rôle dans la régulation de la longueur du fuseau puisque celle-ci est perturbée dans les cellules de la glie radiaire chez la souris HeCo. Ceci pourrait représenter la cause primaire de leur ectopie. Nous avons analysé le nombre et la taille des cellules en métaphase dans la partie apicale de la zone ventriculaire où ont lieu les mitoses. Nous proposons ici de nouveaux mécanismes qui régissent l'organisation des progéniteurs dans la zone ventriculaire au cours du développement cortical normal et pathologique
The cerebral cortex develops through genetically regulated processes of cellular proliferation, neurogenesis, migration and differentiation. Cortical malformations represent a spectrum of heterogeneous disorders due to abnormalities in these steps, and associated with epilepsy and intellectual disability. We studied the HeCo (heterotopic cortex) mutant mouse, which exhibits bilateral subcortical band heterotopia (SBH), characterized by many aberrantly positioned neurons in the white matter. We found that Eml1 (Echinoderm Microtubule-associated protein-Like 1) is mutated in these mice. Screening of EML1 in heterotopia patients identified mutations giving rise to a severe and rare form of atypical heterotopia. In HeCo embryonic brains, progenitors were identified outside the normal proliferative ventricular zone (VZ), representing a novel cause of this disorder. We studied Eml1 function in radial glial progenitors (RGCs), which are important during corticogenesis generating other subtypes of progenitors and post-mitotic neurons, and serving as guides for migrating neurons. We showed that Eml1 localizes to the mitotic spindle where it might regulate microtubule dynamics. My data suggest a role in the establishment of the steady state metaphase spindle length. Indeed, HeCo RGCs in the VZ showed a perturbed spindle length during corticogenesis, and this may represent one of the primary mechanisms leading to abnormal progenitor behavior. I also analyzed cell number and metaphase cell size at the apical side of the VZ, where mitosis occurs. I thus propose new mechanisms governing normal and pathological VZ progenitor organization and function during cortical development

The Hedgehog (HH) signalling pathway and its primary transducer, GLI2, regulate cardiomyogenesis in vivo and in differentiating P19 embryonal carcinoma (EC) cells. To further assess the role of HH signalling during mouse embryonic stem (mES) cell differentiation, we studied the effects of GLI2 overexpression during mES cell differentiation. GLI2 overexpression resulted in temporal enhancement of cardiac progenitor genes, Mef2c and Nkx2-5, along with enhancement of Tbx5, Myhc6, and Myhc7 in day 6 differentiating mES cells. Mass spectrometric analysis of proteins that immunoprecipitate with GLI2 determined that GLI2 forms a complex with BRG1 during mES cell differentiation. Furthermore, modulation of HH signalling during P19 EC cell differentiation followed by chromatin immunoprecipitation with an anti-BRG1 antibody determined that HH signalling regulates BRG1 enrichment on Mef2c. Therefore, HH signalling accelerates cardiac progenitor gene expression during mES cell differentiation potentially by recruiting a chromatin remodelling factor to at least one cardiac progenitor gene.

Le cortex cérébral se développe à partir des zones de prolifération des cellules progénitrices dont le comportement anormal peut donner lieu à des malformations corticales. Des mutations dans Eml1/EML1 ont été identifiées chez la souris HeCo, ainsi que dans trois familles présentant une hétérotopie sous-corticale (SH). La SH se caractérise par une position aberrante des neurones dans la substance blanche. Chez la souris HeCo, des anomalies de position des progéniteurs de la glie radiale apicale (aRG) ont été observées aux stades précoces de la corticogenèse. Je me suis concentré sur la caractérisation de l’aRG dans la zone ventriculaire (VZ) afin d’identifier pourquoi certaines cellules quittent cette région et ainsi mieux comprendre les mécanismes qui sous-tendent l’hétérotopie. En combinant la microscopie confocale et électronique (EM), j'ai découvert des anomalies des centrosomes et des cils primaires dans les aRG mutants pour Eml1 : les cils primaires sont plus courts et souvent mal orientés dans des vésicules. La recherche de partenaires interagissant avec Eml1 à l'aide de la spectrométrie de masse (MS), combinée au séquençage d’exome des ADN de patients SH, nous a permis d'identifier : 1) un partenaire ciliaire interagissant avec Eml1, RPGRIP1L ; 2) des mutations du gène RPGRIP1L chez un patient SH. L’analyse ontologique des gènes sur les données de MS a mis en évidence l’appareil de Golgi et le transport des protéines comme catégories enrichies. En effet, j'ai identifié des altérations de l'appareil de Golgi dans les aRG HeCo. L’ensemble de ces données montre que l'axe appareil de Golgi-cil primaire est perturbé quand Eml1/EML1 est muté et conduit à l’identification de nouvelles voies dans un trouble grave du neurodéveloppement
Cerebral cortical development is a finely regulated process, depending on diverse progenitor cells. Abnormal behavior of the latter can give rise to cortical malformations. Mutations in Eml1/EML1 were identified in the HeCo mouse, as well as in three families presenting severe subcortical heterotopia (SH). SH is characterized by the presence of mislocalized neurons in the white matter. At early stages of corticogenesis, abnormally positioned apical radial glia progenitors (aRG) were found cycling outside the proliferative ventricular zone (VZ) in the HeCo cortical wall. I focused my research on characterizing aRG in the VZ to assess why some cells leave this region and thus to further understand SH mechanisms. Combining confocal and electron microscopy (EM), I uncovered abnormalities of centrosomes and primary cilia in Eml1-mutant aRGs: primary cilia are shorter, and often remain basally oriented within vesicles. Searching for Eml1-interacting partners using mass spectrometry (MS), combined with exome sequencing of SH patient DNAs, allowed us to identify a ciliary Eml1-interacting partner, RPGRIP1L, showing mutations in a SH patient. Gene ontology analyses of MS data pointed to Golgi apparatus and protein transport as enriched categories. Indeed, Golgi abnormalities were identified in HeCo aRGs. Altogether, these data indicate that the Golgi-to-primary cilium axis is perturbed in Eml1mutant conditions, pointing to new intracellular pathways involved in severe neurodevelopmental disorders

Taking into consideration the current knowledge about the subjects treated, several goals were proposed for this PhD thesis in order to further understand PHF8 function in both astrocytes and neural stem cells biology. To do that, we determined the following objectives: 1. To investigate the role of PHF8 histone demethylase during astrocytes differentiation. • Analyse the PHF8-mediated transcriptional profile in astrocytes. • Determine the chromatin bound regions of PHF8 in astrocytes. • Elucidate astrocytes phenotype upon PHF8 depletion. • Examine astrocytic PHF8 function during synaptogenesis. • Determine the molecular mechanism responsible for the PHF8- associated phenotype in astrocytes. 2. To elucidate the function of PHF8 in neural stem cells. • Examine neural stem cells phenotype upon PHF8 depletion. • Determine the PHF8-mediated transcriptional profile in neural stem cells. • Analyse the metabolic impact of PHF8 depletion.

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2014.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 66-74).
Despite the fact that mammalian hair cells and neurons do not naturally regenerate in vivo, progenitor cells exist within the postnatal inner ear that can be manipulated to generate hair cells and neurons. This work reveals the differentiation capabilities of distinct inner ear progenitor populations and pinpoints cell types that can become cochlear hair cells, vestibular hair cells, neurons, and CNS glia. We expanded and differentiated cochlear and vestibular progenitors from mice (postnatal days 1-3) and analyzed the cells for expression of mature properties by RT-PCR, immunostaining, and patch clamping. Whereas previous reports suggested that inner ear stem cells may be pluripotent and/or revert to a more neural stem cell fate, we find that cells from each organ type differentiated into cells with characteristics of the respective organ. Only cochlear-derived cells expressed the outer-hair-cell protein, prestin, while only vestibular derived cells expressed the vestibular extracellular matrix marker, otopetrin. Since Atohi expression is consistently found in new hair cells, we used an Atohl-nGFP mouse line to identify hair cell candidates. We find that cells expressing Atohl also expressed key transduction, hair bundle, and synaptic genes needed for proper function. Whole-cell patch clamp recordings showed that Atoh1-nGFP+ cells derived from both cochlear and vestibular tissue had voltage gated ion channels that were typical of postnatal hair cells. Only vestibular-derived AtohinGFP+ cells, however, had Ih, a hyperpolarization-activated current typical of native vestibular hair cells but not native cochlear hair cells. Lineage tracing studies with known supporting cell and glial cell markers showed that progenitor capacity of cochlear supporting cells positive for Lgr5 (Lgr5+ cells) was limited to differentiation into hair cell-like cells but not neuron-like cells. In contrast, glial cells positive for PLP (PLP1+ cells) from the auditory nerve differentiated into multiple cell types, with properties of neurons, astrocytes, or mature oligodendrocytes but not hair cells. Thus, PLP+ progenitor cells within the auditory nerve are limited to neuronal or glial fates but have greater potency than Lgr5+ progenitors, which only formed hair cell-like cells. In summary, this work identifies distinct populations of post-natal inner ear progenitors and delineates their capacity for differentiation and maturation.
by Will McLean.
Ph. D.

Stem/progenitor cells are present in the adult brain; they undergo constantproliferation and differentiate into mature neurons in certain brain areas, a phenomenoncalled neurogenesis. This study investigated the effects of GDNF, a potent trophic factorof dopaminergic neurons, on neurogenesis in the brain. Nestin and 5-Bromo-2'-deoxyuridine (BrdU) were used as stem/progenitor cells markers.First, we observed extensive bilateral increases of stem/progenitor cells in thedentate gyrus and substantia nigra after continuous infusion of GDNF into the normal ratbrain. However, none of the BrdU+ cells showed neuronal features in the substantia nigraas characterized by immunocytochemical procedures. Next, we identified themorphology of BrdU+ cells after infusing the marker into the brain. While the proceduresincreased the BrdU labeling, neurogenesis was not observed in the basal ganglia. Underelectron microscope, the BrdU+ cells either were undifferentiated or showedcharacteristics of astrocytes. This observation is consistent with suggestions thatastrocytes serve as multipotent progenitors. Later, we repeated GDNF intrastriatalinfusion one month after a severe 6-hydroxydopamine (6-OHDA) lesion. The number ofBrdU+ cells was significantly higher in the GDNF recipients in the ipsilateral substantianigra and both sides of the dentate gyrus. However, no neurogenesis was observed. Inaddition, motor functions were not improved by GDNF treatment. Thus, we measured theeffects of GDNF administration directly into the substantia nigra six hours before apartial 6-OHDA lesion. HPLC measurements of dopamine and its metabolites showed asignificant increase of tissue level in the substantia nigra and striatum, respectively.Despite this, no newly generated dopaminergic neurons was detected in the basal ganglia.Taken together, our studies investigated the effects of GDNF on adultstem/progenitor cells in normal and lesioned rat brain. For the first time, we demonstratedthat GDNF promoted their proliferation in the dentate gyrus, suggesting it has a role inneurogenesis and the function of learning and memory. In each scenario, GDNFpromoted stem/progenitor cell proliferation, but failed to induce neurogenesis in thesubstantia nigra. We believed that the local microenvironment in the substantia nigra mayprevent the stem/progenitor cells to mature into functional neurons.

Proper central nervous system development is critical for survival and depends on complex intracellular and extracellular signaling to regulate neural progenitor cell growth and differentiation; however, the mechanisms that mediate molecular crosstalk between pathways during neurogenesis are not fully understood. Here, we explored the integration of the Hedgehog (Hh) signaling pathway with the two critical developmental pathways, Receptor Tyrosine Kinase (RTK) and Notch signaling, in the growth and maintenance of neural progenitors in the developing neuroretina. We found combined and sustained RTK and Hh signaling was sufficient to establish long-term retinal progenitor cell (RPC) cultures and these cells maintained neurogenic and gliogenic, but not retinogenic, competence in vitro and in vivo. In addition, we identified crosstalk between Notch and Hh signaling, where Notch is required for Hh-mediated proliferation and Gli protein accumulation, and gain-of-function of Notch is sufficient to extend the window of Hh responsiveness in a subset of Müller glia. Both Hh-RPC monolayer establishment and Notch mediated Hh-responsiveness required Gli2. Taken together, we identified molecular cross-communication between the Hh pathway and two major pathways, Notch and RTK, during retinogenesis, advancing our understanding of mechanisms that influence Hh to control neural progenitor growth.

Human immunodeficiency virus (HIV) infected patients with a history of injection opiate abuse have higher incidences of acquired immunodeficiency syndrome (AIDS) and neurological dysfunction. The use of combined anti-retroviral therapy has significantly reduced the prevalence of mortality and progression to AIDS. Due to extended life expectancy, these patients are still at a great risk for HIV-associated neurological disorders and impairment in their later life. Neural progenitor cells (NPCs) play critical roles in brain growth and repair after injury and insult. Pediatric HIV patients whose glial populations are still developing are especially at risk for central nervous system (CNS) damage. Our previous reports suggest that HIV-1 transactivator of transcription (Tat) can directly cause pathology in neural progenitors and oligodendroglia (OLs) (Hauser et al. 2009). Thus, we have hypothesized that NPCs and/or glial progenitors may be targets of HIV proteins ± opiates drugs of abuse. To determine whether progenitors are targets of HIV-1, a multi-plex assay was performed to assess chemokine/cytokine expression after treatment with viral proteins Tat or glycoprotein 120 (gp120) with/without morphine. Murine striatal progenitors released increased amount of the beta-chemokines CCL5/regulated upon activation, normal T cell expressed and secreted (RANTES), CCL3/macrophage inflammatory protein-1α (MIP-1α), and CCL4/macrophage inflammatory protein-1β (MIP-1β) after 12 h exposure to HIV-1 Tat, but no to gp120. Secreted factors from Tat-treated progenitors were chemoattractive towards microglia, an effect blocked by 2D7 anti- C-C chemokine receptor type 5 (CCR5) antibody pre-treatment. Tat and opiates have interactive effects on astroglial chemokine secretion, but this interaction did not occur in progenitors. We also examined effects of Tat and morphine on proliferation and lineage progression of NPCs in vitro and in vivo. In vitro, Tat and morphine independently reduced the proliferation and population of Sox2+ and Olig2+ cells in the absence of cell death. The interactive effects of morphine and either Tat or supernatant from HIV-1SF162 infected monocytes varied depending on outcome measure and time of exposure, but interactive effects occurred primarily on proliferation. In rare instances, viable human progenitors were associated with p24 immunolabeling suggesting that progenitors may be infected, a concept that is still controversial. To investigate effects of Tat and morphine on NPCs in vivo, we used a mouse model in which HIV-1 Tat1-86 is conditionally expressed in astroglia. In vivo results in neonatal striata were similar to those in cell cultures. We extended the experiments into adult mice with inducing Tat expression for 3 month and the effect of sexes was examined in these animals. Intriguingly, males showed more Tat-induced impairment in behavioral tests (rotarod, grip strength, light-dark box) than females. Tat+ males also showed a greater reduction in the proportion of NeuN+ cells and NeuN immunoreactivity in the striatum, accompanied by greater microglial activation (3-nitrotyrosine+/Iba-1+). Unbiased stereological estimation in Nissl staining revealed that the depletion of NeuN immunoreactivity in these mice was not due to neuron cell death or loss, because the total neuron number in striatum and total striatal volume were not affected by long-term Tat induction. Tat exposure appears to selectively reduce levels of NeuN in living neurons, although the reason is not known. Therefore, both the enhanced microglial reactivity and depletion of NeuN levels in males may help to explain sex-specific behavioral outcomes. Sox2+ and Olig2+ cells showed equivalent reduction in their population in both sexes. Overall, our findings show that CNS progenitors, including both undifferentiated NPCs and glial progenitors, are vulnerable to individual or combined effects of HIV-1 or Tat and opiates. Changes in progenitor dynamics may alter the balance of cell populations in both the developing and adult CNS. We speculate that such changes may contribute to the behavioral abnormalities that we observed in Tat+ mice and which appear to model aspects of motor, cognitive and anxiety deficits in HIV-infected patients.

O complexo da esclerose tuberosa (TSC) é um transtorno clínico, com expressividade variável, caracterizado por hamartomas que podem ocorrer em diferentes órgãos. Tem herança autossômica dominante e é devido a mutações em um de dois genes supressores de tumor, TSC1 ou TSC2. Estes codificam para as proteínas hamartina e tuberina, respectivamente, que se associam formando um complexo macromolecular que regula funções como proliferação, diferenciação, crescimento e migração celular. As lesões cerebrais podem ser muito graves em pacientes com TSC e caracterizam-se por nódulos subependimários (SEN), astrocitomas subependimários de células gigantes (SEGA), tuberosidades corticais e heterotopias neuronais, podendo relacionar-se clinicamente à epilepsia refratária à terapia medicamentosa, deficiência intelectual, desordens do comportamento e hidrocefalia. O potencial de crescimento de SEGA até os 21 anos de idade dos pacientes exige acompanhamento periódico por exame de imagem e condutas clínicas ou cirúrgicas, conforme indicação médica. As lesões subependimárias têm sido explicadas por déficits de controle da proliferação, crescimento e diferenciação de precursores neuro-gliais na zona subventricular telencefálica. Embora a capacidade da tuberina em inibir a proliferação celular pela repressão do alvo da rapamicina em mamíferos (mTOR) esteja bem documentada, outros aspectos celulares do desenvolvimento de SEGA ainda não foram examinados. Assim, é importante estabelecer um sistema in vitro para o estudo de células da zona subventricular e testá-lo na análise das proteínas hamartina e tuberina. Neste sentido, o cultivo de neuroesferas em suspensão é muito apropriado. Neste estudo, buscamos relacionar a expressão e distribuição subcelular da hamartina e tuberina à capacidade proliferativa e de diferenciação das células de neuroesferas cultivadas in vitro a partir da dissociação da vesícula telencefálica de embriões de ratos normais. Analisamos a expressão e distribuição subcelular da hamartina e tuberina por imunofluorescência indireta em células entre a primeira e a quarta passagens das neuroesferas, sincronizadas nas fases G1 ou S do ciclo celular e após a reentrada no ciclo celular, através da incorporação de 5-bromo-2\'-desoxiuridina (BrdU) e imunofluorescência com anticorpo anti-BrdU. Em geral, células de neuroesferas apresentaram baixa colocalização entre hamartina e tuberina in vitro. A expressão da tuberina foi elevada em basicamente todas as células das esferas e fases do ciclo celular; ao contrário, a hamartina apresentou-se principalmente nas células da periferia das esferas. A colocalização entre hamartina e tuberina foi observada em células mais periféricas das esferas, sobretudo no citoplasma e, em G1, no núcleo celular. A proteína rheb, que conhecidamente interage diretamente com a tuberina, apresentou distribuição subcelular muito semelhante à desta. Ao carenciamento das células visando à parada do ciclo celular na transição G1/S, tuberina distribuiu-se ao núcleo celular em quase todas as células avaliadas e, de forma menos frequente, a hamartina também. À reentrada no ciclo celular pelo reacréscimo dos fatores de crescimento, avaliaram-se células com incorporação de BrdU ao seu núcleo celular, após 72 e 96 horas. Nestas, tuberina mostrou-se novamente no citoplasma de forma preponderante e hamartina manteve-se citoplasmática, em geral subjacente à membrana plasmática, em níveis mais baixos. Os grupos cujas células reciclaram por 72 ou 96 horas diferiram quanto ao aumento significativo da expressão da hamartina em células proliferativas no último. À diferenciação neuronal, aumentaram-se os níveis de expressão de hamartina observáveis à imunofluorescência indireta, tornando-se equivalentes àqueles da tuberina. Concluímos que as células de neuroesferas cultivadas em suspensão apresentam-se como um sistema apropriado ao estudo da distribuição das proteínas hamartina e tuberina e sua relação com o ciclo celular
The tuberous sclerosis complex (TSC) is a clinical disorder with variable expressivity, characterized by hamartomas that can occur in different organs. It has autosomal dominant inheritance and is due to mutations in one of two tumor suppressor genes, TSC1 or TSC2. These encode for the proteins hamartin and tuberin, respectively, which are associated in a macromolecular complex which functions as a regulator of cell proliferation, differentiation, growth and migration. TSC brain lesions may be severe and are characterized by subependymal nodules (SEN), subependymal giant cell astrocytomas (SEGA), neuronal heterotopias and cortical tubers, and may be clinically related to refractory epilepsy, intellectual disability, behavioral disorders and hydrocephaly. The growth potential of SEGA up to 21 years of age in TSC patients requires regular monitoring by imaging. Clinical and surgical interventions may be medically indicated. Subependymal lesions have been explained by deficient control of proliferation, growth and differentiation of neuro-glial progenitors from the telencephalic subventricular zone. While tuberin ability to inhibit cell proliferation by repressing the mammalian target of rapamycin (mTOR) has been well documented, other cell aspects of SEGA development have not been thoroughly examined. Therefore, it is important to establish conditions for an in vitro system to study the cells from the subventricular zone and to test its suitability for the study of the TSC proteins. In this regard, the neurosphere suspension culture is very appropriate. We evaluated the expression and subcellular distribution of hamartin and tuberin in relation to the proliferation and differentiation capability of neurosphere cells derived in vitro from the dissociation of the telencephalic vesicle of normal E14 rat embryos. These analyses were performed by indirect immunofluorescence in cells from first through fourth passages of neurospheres, synchronized in G1 or S phases of the cell cycle, and after reentry into the cell cycle by the addition of 5-brome-2\'-desoxyuridine (BrdU) and immunolabeling with anti-BrdU antibody. In general, neurosphere cells presented low colocalization between hamartin and tuberin in vitro. Tuberin expression was relatively high in basically all neurosphere cells and cell cycle phases, whereas hamartin distributed mainly to cells from the periphery of the spheres. In these cells, hamartin and tuberin colocalization was evident mostly in the cytoplasm and, in G1, also in the cell nucleus. Rheb, which is known to interact directly with tuberin, had subcellular distribution very similar to tuberin. Cell starvation indicating cell cycle arrest at G1/S redistributed tuberin to the cell nucleus in virtually all cells examined, what was accompanied by nuclear location of hamartin in a small subset of cells. When cells were allowed to reenter cell cycle by adding growth factors, we evaluated BrdU-labeled nuclei 72 and 96 hours later. In the two groups, tuberin was shown to move back to the cytoplasm as well as hamartin, which apparently maintained its lower expression levels distribution underneath the plasma membrane. Group of cells that recycled for 96 hours had significantly more expression of hamartin than those cells that cycled for only 72 hours. After neuronal differentiation, hamartin expression levels observed by immunofluorescence were similar to those of tuberin. We conclude that neurosphere cells cultured in suspension showed to be an appropriate cell system to study hamartin and tuberin distribution in respect to the cell cycle

Gliomas are the most common primary tumors of the central nervous system believed to arise from glial cells. Invasive growth and inherent propensity for malignant progression make gliomas incurable despite extensive treatment. I have developed a life-like orthotopic glioma model and used this and other in vivo models to study basic mechanisms of glioma development and treatment. Previous studies had indicated that experimental gliomas could arise from glial stem cells and astrocytes. The present thesis describes the making and characterization of a novel mouse model, Ctv-a, where gliomas are induced from oligodendrocyte progenitor cells (OPCs). Our study shows that OPCs have the capacity to give rise to gliomas and suggests in light of previous data that the differentiation state of the cell of origin affects tumor malignancy. CDKN2A encodes p16INK4a and p14ARF (p19Arf in mouse) commonly inactivated in malignant glioma. Their roles in experimental glioma have been extensively studied and both proteins have tumor suppressor functions in glial stem cells and astrocytes. Here, we demonstrate that p19Arf only could suppress gliomagenesis in OPCs while p16Ink4a had no tumor suppressive effect. Functional DNA repair is pivotal for maintaining genome integrity, eliminating unsalvageable cells and inhibiting tumorigenesis. We have studied how RAD51, a central protein of homology-directed repair, affected experimental glioma development and have found that expression of RAD51 may protect against genomic instability and tumor development. Angiogenesis, the formation of new blood vessels from pre-existing ones, is a central feature of malignant progression in glioma. Antiangiogenic treatment by inhibition of vascular endothelial growth factor receptor signaling is used in the clinic for treatment of some cancers. We have investigated the effect of an alternative antiangiogenic protein, histidine-rich glycoprotein (HRG), on glioma development and found that HRG could inhibit the formation of malignant gliomas and completely prevent the formation of glioblastoma.

A lesão medular (LM) é uma patologia incapacitante que resulta em déficits sensoriais e motores. No Brasil, a incidência anual é de 30 novos casos de lesão medular a cada 1 milhão de indivíduos e, infelizmente, a LM permanece sem um tratamento eficaz. Células-tronco derivadas do dente decíduo humano estão entre as potenciais fontes de células-tronco para transplante após a lesão medular, cujo objetivo é de promover a proteção ou a recuperação da lesão na medula espinal. Buscou-se nesta tese avaliar os efeitos do transplante, uma hora após a lesão, das células tronco de dente decíduo humano (SHED) no período agudo, subagudo e crônico sobre a neuroproteção, proteção tecidual e recuperação funcional em ratos Wistar submetidos à lesão medular por contusão. Os principais objetivos foram: a) investigar os efeitos do transplante das SHED sobre a recuperação funcional, volume da lesão e morte neuronal; b) verificar os efeitos do transplante sobre as células progenitoras, formação da cicatriz glial e modificações astrocitárias após o modelo de contusão medular Observou-se a melhora na recuperação funcional, redução do volume da lesão e morte neuronal na medula espinal dos animais que receberam o transplante de SHED após a lesão medular. As SHED aumentam o número de células precursoras na medula espinal, no período subagudo, reduzem a expressão da proteína fibrilar glial ácida (GFAP) e aumentam a expressão do canal retificador de influxo de potássio 4.1, ambas proteínas astrocitárias. Concluímos que o transplante de células-tronco derivadas do dente decíduo humano após a lesão medular promove a recuperação funcional a partir do efeito neuroprotetor iniciado na fase aguda, confirmado pelo maior número de neurônios motores presentes seis semanas após a contusão. As SHED são capazes de aumentar o número de células precursoras e de produzir modificações astrocitárias na medula espinal de ratos lesados na fase subaguda, reduzindo a formação da cicatriz glial.
Spinal cord injury (SCI) is a disabling condition that results in sensory and motor deficits. The estimated annual incidence in Brazil is of 30 new cases of spinal cord injury per 1 million of individuals; unfortunately SCI remains without an effective treatment. Stem cells from human exfoliated deciduous teeth (SHED) are one among potential sources of stem cells for transplantation after spinal cord injury in order to promote protection or tissue and functional recovery after spinal cord injury. The aim of this Thesis was to evaluate the effects of stem cells from human exfoliated deciduous teeth (SHED) transplantation, one hour after lesion, in the acute, subacute and chronic phases on neuroprotection, tissue protection and functional recovery in Wistar rats submitted to spinal cord injury by contusion The main goals were: a) to investigate the effects of SHED transplantation on functional recovery, lesion volume, and neuronal death; b) to verify the effects of the transplantation on the progenitor cells number, glial scar formation and astrocytic modifications after spinal cord contusion. Improvement of functional recovery, reduction of lesion volume and neuronal death were observed in the spinal cord of animals submitted to spinal cord injury and SHED transplantation. SHEDs increased the number of precursor cells in the spinal cord in the subacute period, reduced the expression of glial fibrillary acidic protein (GFAP) and increased the expression of the potassium influx rectifier channel 4.1, both astrocyte proteins. We conclude that transplantation of stem cells from human exfoliated deciduous teeth after spinal cord injury promotes functional recovery from the neuroprotection effect, which starts in the acute phase and is confirmed six weeks after the contusion with a higher number of motor neurons in the ventral horn of spinal cord. SHEDs are able to increase the number of precursor cells and produce astrocyte modifications in the spinal cord of injured rats in the subacute phase, reducing glial scar formation.

Yes
Expression of the transmembrane NG2 chondroitin sulphate proteoglycan (CSPG) defines a distinct population of NG2-glia. NG2-glia serve as a regenerative pool of oligodendrocyte progenitor cells in the adult central nervous system (CNS), which is important for demyelinating diseases such as multiple sclerosis, and are a major component of the glial scar that inhibits axon regeneration after CNS injury. In addition, NG2-glia form unique neuron–glial synapses with unresolved functions. However, to date it has proven difficult to study the importance of NG2-glia in any of these functions using conventional transgenic NG2 ‘knockout’ mice. To overcome this, we aimed to determine whether NG2-glia can be targeted using an immunotoxin approach. We demonstrate that incubation in primary anti-NG2 antibody in combination with secondary saporin-conjugated antibody selectively kills NG2-expressing cells in vitro. In addition, we provide evidence that the same protocol induces the loss of NG2-glia without affecting astrocyte or neuronal numbers in cerebellar brain slices from postnatal mice. This study shows that targeting the NG2 CSPG with immunotoxins is an effective and selective means for killing NG2-glia, which has important implications for studying the functions of these enigmatic cells both in the normal CNS, and in demyelination and degeneration.

The major goals of my research were to characterize the hypothalamic neural progenitors and to understand how Hh/Gli signaling plays a role in regulating cell proliferation in the hypothalamic neurogenic zone. In contrast to mammals, the zebrafish brain has a life-long potential to grow continuously. Thus, for comparative neurogenesis studies, zebrafish become an indispensible model organism to understand adult neurogenesis and regulatory signaling pathways. Identification of the regulatory mechanisms underlying the controlled cell proliferation in adult zebrafish brain will pave the way to manipulate the healing potential of the mammalian brain. Using immunohistochemistry and in situ hybridization techniques to label known markers for neural stem/ and progenitor cells I have identified three different populations of cells with radial glia (RG) like morphology in the adult zebrafish hypothalamic ventricular zone. In adult zebrafish, cells with RG-like morphology in the ventricular regions are thought to be the neurogenic population. The first population of cells I identified was positive for the neural stem cell marker NESTIN and showed additional characteristics of neural stem cells. Using a label retention assay we showed that Nestin(+) cells are slow cycling. The second population of RG-like cells was Hh responsive, and expressed markers of neural progenitor/transit amplifier cells. Double labeling experiments reveal that the Hh responsive cells were distinct from the Nestin(+) cells These cells were proliferative and cycled faster compared to nestin(+) neural stem cells. The third population of cells with RG morphology in the hypothalamic ventricular zone expressed shh ligand, indicating a regulatory role for Hh signaling in the hypothalamic ventricular zone. Down-regulation of Hh signaling at larval and adult stages reduced proliferation in the hypothalamic ventricle, indicating that Hh acts as a positive regulator of proliferation, as in the dorsal brain. According to our working model, nestin(+) cells are slow cycling, and/or quiescent neural stem cell population in the hypothalamic ventricular zone, whereas Hh responsive cells are the fast cycling transit amplifier cells which proliferate and give rise to new neurons and glia in the adult. My comprehensive analysis of the neural stem/progenitors in the adult zebrafish hypothalamic ventricular zone provides a starting point for the continued study of the mammalian hypothalamic ventricular zone. This study also demonstrates Hh signaling functions as a positive regulator of cell proliferation in the post-embryonic zebrafish hypothalamus consistent with its role in the dorsal brain. (Abstract shortened by UMI.)

Müller cells are major glial cells in the retina and have a broad range of functions that are vital for the retinal neurons. During retinal injury gliotic response either leads to Müller cell dedifferentiation and formation of a retinal progenitor or to maintenance of mature Müller cell functions. The overall aim of this thesis was to investigate the intra- and extracellular signaling of Müller cells, to understand how Müller cells communicate during an injury and how their properties can be regulated after injury. Focus has been on the α2-adrenergic receptor (α2-ADR) and endothelin receptor (EDNR)-induced modulation of Müller cell-properties after injury. The results show that α2-ADR stimulation by brimonidine (BMD) triggers Src-kinase mediated ligand-dependent and ligand-independent transactivation of epidermal growth factor receptor (EGFR) in both chicken and human Müller cells. The effects of this transactivation in injured retina attenuate injury-induced activation and dedifferentiation of Müller cells by attenuating injury-induced ERK signaling. The attenuation was concomitant with a synergistic up-regulation of negative ERK- and RTK-feedback regulators during injury. The data suggest that adrenergic stress-signals modulate glial responses during retinal injury and that α2-ADR pharmacology can be used to modulate glial injury-response. We studied the effects of this attenuation of Müller cell dedifferentiation on injured retina from the perspective of neuroprotection. We analyzed retinal ganglion cell (RGC) survival after α2-ADR stimulation of excitotoxically injured chicken retina and our results show that α2-ADR stimulation protects RGCs against the excitotoxic injury. We propose that α2-ADR-induced protection of RGCs in injured retina is due to enhancing the attenuation of the glial injury response and to sustaining mature glial functions. Moreover, we studied endothelin-induced intracellular signaling in Müller cells and our results show that stimulation of EDNRB transactivates EGFR in Müller cells in a similar way as seen after α2-ADR stimulation. These results outline a mechanism of how injury-induced endothelins may modulate the gliotic responses of Müller cells. The results obtained in this thesis are pivotal and provide new insights into glial functions, thereby uncovering possibilities to target Müller cells by designing neuroprotective treatments of retinal degenerative diseases or acute retinal injury.