L’émergence de la notion selon laquelle la réponse inflammatoire exercerait des effets bénéfiques dans la pathologie ischémique cérébrale, en particulier au cours de la phase de récupération fonctionnelle nous a conduit à étudier l’implication des cellules microgliales dans le déclenchement des mécanismes de neuroplasticité post-ischémique. Notre étude a été réalisée chez le Rat soumis à une ischémie focale permanente induite par photothrombose. L’activation microgliale a été modulée par un traitement au 3-aminobenzamide (3-AB), un inhibiteur spécifique de la poly(ADP-ribose)polymérase-1, jouant un rôle prépondérant dans l’activation de ces cellules. Nos données montrent que le 3-AB entraîne une diminution importante de l’activation microgliale aux temps courts associée à plus long terme à une réduction de l’expression de la synaptophysine et de GAP-43, respectivement marqueurs des processus de synaptogenèse et croissance axonale. L’ensemble de ces données indique donc que les cellules microgliales constituent effectivement des acteurs cellulaires essentiels de la neuroplasticité post-ischémique. Le Brain-derived neurotrophic factor (BDNF) se révélant un candidat potentiellement capable de promouvoir de tels changements, nous avons pu mettre en évidence que ces cellules représentaient de façon précoce une source importante de BDNF. Ces résultats ont été confirmés par la nette diminution des taux de BDNF mesurés dans les zones corticales lésionnelles et péri-lésionnelles des animaux traités par le 3-AB. Dans un deuxième temps, le métabolisme complexe de cette neurotrophine à travers l’existence de deux formes, pro- et mature, aux effets biologiques opposés, nous a conduit à réaliser une étude spatio-temporelle des expressions post-ischémiques du BDNF total (ELISA), pro- et mature (Western blotting). Aux temps courts (4-24 h), les expressions du BDNF total, pro- et mature sont augmentées dans les territoires corticaux lésés, péri-lésionnels et homotopiques tandis qu’aux temps longs (8-30 j), le BDNF total reste accru dans les régions distantes de la zone infarcie (hippocampes et cortex contralatéral). Concernant les expressions des formes pro- et mature, nos résultats indiquent une augmentation entre 8 et 30 j uniquement dans les territoires hippocampiques. D’un point de vue cellulaire, le BDNF est exprimé du côté ipsilatéral dans les neurones et les cellules non neuronales tandis que du côté contralatéral, l’expression est limitée aux neurones. Nos résultats tout en faisant apparaître des divergences importantes dans les variations d’expressions du BDNF total (ELISA) et des différentes formes (Western blotting) indiquent que la mesure du BDNF total doit être couplée à une étude permettant de discriminer les deux formes. De plus, tout en confirmant l’implication de cette neurotrophine dans les mécanismes adaptatifs induits en réponse à une ischémie cérébrale, ces données suggèrent que les territoires distants de la zone lésée jouent un rôle majeur dans ces processus. / Evidences showing that under certain circumstances, inflammatory response could be neuroprotective and could also promote adult neurogenesis are growing. In this context, the objective of this work was to investigate the impact of microglial cells in the neuroplastic events. Rats were subjected to photothrombotic ischemia and microglial cells activation was blocked by the mean of poly(ADP-ribose)polymérase-1 (PARP-1) inhibition using 3- aminobenzamide (3-AB) since this protein has been shown to play a major role in this activation. Our results show that PARP-1 activity reduction was associated with a strong repression of the acute microglial activation. Beside, 3-AB treated animals exhibited a decrease in synaptophysin (synaptogenesis) and GAP-43 (axonal growth) expressions. Taken together, our data argue for a supportive role of microglial in adaptive brain plasticity events. According to the preponderant contribution of BDNF in these events, assessment of its cellular localization was performed, and confirmed that these cells represent a significant source. Beside, BDNF immunoreactivity (IR) in microglial cells and BDNF levels in the lesioned and surrounding lesioned areas were found decreased in 3-AB treated animals. However, since this neurotrophin can exert ambivalent biological actions through pro- versus mature forms, we investigate the proper effect of cerebral ischemia on total (Elisa), pro- and mature (Western blotting) expressions. Our results show that total, pro- and mature BDNF expressions are augmented in the early times (4-24h) of ischemia within the lesioned, the surrounding non lesioned and the contralateral cortical areas. At longer time points, total BDNF was still increased at 8d in regions distant from the lesion (hippocampi and contralateral cortex) while pro- and mature forms rise between 8d to 30d in hippocampic territories only. In term of cellular distribution, BDNF-IR was found in neurons but also in non neuronal cells ipsilaterally whereas in the opposite side BDNF staining was restricted to neurons. Our data while raising the question of the pertinence of total BDNF expression in a context of studying its supportive potential action indicate that such assessment has to be coupled with the discrimination of both forms. In addition, our data confirm the important role of BDNF in post-stroke adaptive mechanisms and argue in favour of an important contribution of the hippocampal territory and of the contralateral hemisphere in BDNF related post-stroke neuronal circuit remodelling suggesting that strategies targeting this hemisphere are likely to mediate functional compensation.

Le facteur neurotrophique dérivé du cerveau (BDNF, Brain-Derived Neurotrophic Factor) est une protéine qui joue un rôle essentiel dans la survie et la différenciation des neurones, ainsi que dans l'induction et l'expression de la plasticité synaptique (Deinhardt and Chao, 2014; Lu et al., 2005). Le BDNF est très exprimé à l'âge adulte et la réduction de son expression est impliquée dans de nombreuses maladies neurodégénératives et troubles psychologiques (Anastasia and Hempstead, 2014). Son action sur la plasticité synaptique est majeure pour la mise en place de fonctions cognitives et pour la mise en place de la mémoire (Bekinschtein et al., 2008; Egan et al., 2003).Le BDNF existant sous deux formes ayant des fonctions opposées, on peut parler d'effet Yin et Yang du BDNF (Lu et al., 2005). En effet, le BDNF est synthétisé en tant que molécule précurseur, le proBDNF, qui a des effets négatifs " Yin ". Par sa liaison au récepteur p75NTR, le proBDNF favorise l'apoptose, la rétraction dendritique et la dépression à long-terme. Au contraire, la forme clivée, le BDNF mature (mBDNF), se lie préférentiellement au récepteur TrkB qui active les voies de signalisation de survie cellulaire, de différentiation et de potentialisation à long terme. On parle alors d'effets positifs ou " Yang ". Le clivage de BDNF joue donc un rôle capital dans la régulation de sa balance fonctionnelle vers l'une ou l'autre voie. Il est généralement admis que le proBDNF est clivé en mBDNF par la furine dans le Golgi ou par la pro-protéine convertase 1/3 dans les vésicules de sécrétion (Mowla et al., 2001; Seidah et al., 1996). Le proBDNF restant n'ayant pas été clivé dans la cellule peut être sécrété et être rapidement clivé par la plasmine ou les métalloprotéases de matrice (MMP7) dans l'espace extracellulaire (Lee et al., 2001). Cependant, si les enzymes de conversion du BDNF sont bien connues, les mécanismes de régulation du clivage ne sont pas encore compris.Nous montrons ici un nouveau mécanisme de régulation de la maturation de BDNF via une phosphorylation qui impacte directement la balance fonctionnelle. En effet, nous montrons que la phosphorylation du résidu S130, localisé à l'interface entre le pro-domaine et le domaine mature, diminue l'efficacité du clivage du BDNF par la furine, régulant ainsi l'équilibre entre les formes immature et mature. Cette phosphorylation au site S130 est catalysée par les ectokinases FJX1 et FAM69B qui sont localisées dans l'appareil de Golgi avec BDNF. De plus, grâce à l'utilisation de souris transgéniques knock-in phospho-mutantes, nous montrons que la phosphorylation de BDNF, en favorisant la forme proBDNF, inhibe la potentialisation à long-terme et diminue la dynamique de plasticité des épines dendritiques après stimulation neuronale. Ces résultats suggèrent ainsi une nouvelle voie de régulation de la balance fonctionnelle de BDNF et suggèrent un rôle critique de la phosphorylation S130 dans les processus d'apprentissage et de la mémoire.En parallèle, la recherche de kinases potentielles de BDNF nous a amenés à identifier une exoPKA, localisée dans le Golgi et qui interagit directement avec BDNF. Cette exoPKA atypique phosphoryle BDNF au niveau de la sérine S130 et régule sa maturation et donc l'équilibre entre les formes pro et matures de BDNF. Cette exoPKA est différente de la PKA cytosolique ce qui suggère l'existence d'un nouveau mécanisme de régulation de la plasticité par PKA via BDNF. Enfin, nous sommes en train de tester le rôle de l'exoPKA sur la régulation négative de la plasticité synaptique via la régulation du clivage de BDNF. Ces résultats permettront de déterminer l'existence d'une dichotomie d'action de PKA sur la plasticité en fonction de la forme activée, cytosolique ou golgienne/sécrétée. / Brain-Derived Neurotrophic Factor (BDNF) is a protein that plays an essential role in the survival and differentiation of neurons, as well as in the induction and expression of synaptic plasticity (Deinhardt and Chao, 2014; Lu et al., 2005). BDNF is highly expressed during adulthood and the reduction of its expression is implicated in many neurodegenerative diseases and psychological disorders (Anastasia and Hempstead, 2014). Its action on synaptic plasticity is critical for the establishment of cognitive functions and for the establishment of memory (Bekinschtein et al., 2008; Egan et al., 2003).BDNF exists as two forms with opposite functions, or Yin and Yang effects (Lu et al., 2005). Indeed, BDNF is synthesized as a precursor molecule, proBDNF, which has negative effects "Yin". By binding to the p75NTR receptor, proBDNF promotes apoptosis, dendritic retraction and long-term depression. In contrast, the cleaved form, mature BDNF (mBDNF), binds preferentially to the TrkB receptor that activates the signaling pathways promoting cell survival, differentiation, and long-term potentiation. Thus, mBDNF has positive effects or "Yang". The cleavage of BDNF therefore plays a key role in regulating its functional balance towards one or the other pathway. It is believed that proBDNF is cleaved into mBDNF by furin in the Golgi network or by pro-protein convertase 1/3 in secretory vesicles (Mowla et al., 2001; Seidah et al., 1996). The remaining proBDNF that has not been cleaved in the cell can be secreted and rapidly cleaved by plasmin or matrix metalloproteases (MMP7) into the extracellular space (Lee et al., 2001).Having diametrically opposite roles, together, mature BDNF and proBDNF allow fine regulation of neuronal survival and differentiation and of their activity-dependent synaptic plasticity (Lu et al., 2014; Yang et al., 2014). Since BDNF has critical roles in neuronal functions, it is not surprising that its expression and action are extremely regulated in time and space. However, even if the enzymes processing BDNF are well known, the mechanisms regulating BDNF cleavage are not yet understood.Here we show a new mechanism for the regulation of BDNF maturation via phosphorylation which directly impacts the functional balance. Indeed, we show that phosphorylation of the S130 residue, located at the interface between the pro- and the mature domain, decreases the efficiency of BDNF cleavage by furin, thus regulating the balance between immature and mature forms. This phosphorylation at the S130 site is catalyzed by ectokinases FJX1 and FAM69B which are localized in the Golgi apparatus with BDNF. Furthermore, by using phospho-mutant knock-in transgenic mice, we show that the phosphorylation of BDNF, by promoting the proBDNF form, inhibits the long-term potentiation and decreases the plasticity dynamics of the dendritic spines after neuronal stimulation. These results suggest a novel regulatory pathway for BDNF functional balance and suggest a critical role for S130 phosphorylation in learning and memory processes.In parallel, the search for potential kinases of BDNF led us to identify an exoPKA, located in the Golgi and interacting directly with BDNF. This atypical exoPKA phosphorylates BDNF at serine S130 and regulates its maturation and therefore the balance between the pro- and mature forms of BDNF. This exoPKA is different from the cytosolic PKA which suggests the existence of a new mechanism of regulation of plasticity by PKA via BDNF. Finally, we are testing the role of exoPKA on the downregulation of synaptic plasticity via the regulation of BDNF cleavage. These results will allow to determine the existence of a dichotomous action of PKA on plasticity whether cytosolic or Golgi/secreted PKA is activated.

Einleitung: Die Ausbildung eines Netzhautödems ist eine Hauptursache für die Verschlechterung des Sehvermögens bei ischämisch-hypoxischen und inflammatorischen Netzhauterkrankungen. Neben der erhöhten Permeabilität der Blut-Retina-Schranke trägt eine Wasserakkumulation in Netzhautzellen zur Ausbildung eines Netzhautödems bei. Müllerzellen regulieren die retinale Ionen- und Osmohomöostase, indem sie einen transzellulären Ionen- und Wassertransport vermitteln. Zudem kontrollieren Müllerzellen die Größe des Extrazellularraumes, indem sie bei neuronaler Aktivität eine Zellkörperschwellung – ausgelöst durch eine Verkleinerung der extrazellulären Osmolarität – verhindern. Unter pathologischen Bedingungen ist die Volumenregulation gestört, sodass Müllerzellen bei Hypoosmolarität anschwellen. Diese Müllerzellschwellung und eine Glutamat-induzierte Schwellung retinaler Neurone tragen zur Ausbildung eines zytotoxischen Netzhautödems bei. Neuroprotektive Faktoren wie BDNF (brain-derived neurotrophic factor) und bFGF (basic fibroblast growth factor) stimulieren das Überleben retinaler Neurone und verzögern so die retinale Degeneration. Zielstellung: Es war zu zu ermitteln, ob BDNF die zytotoxische Schwellung von Müller- und Bipolarzellen der Rattennetzhaut verhindert. Material und Methoden: Es wurden Netzhautschnitte und isolierte Müller- und Bipolarzellen von 55 adulten Long-Evans-Ratten (durchschnittlich 8-15 Zellen pro Versuchsreihe) verwendet. Eine osmotische Schwellung von Müller- und Bipolarzellen wurde durch eine Superfusion der Schnitte oder der Zellen mit einer 60%igen hypoosmolaren Lösung in Ab- oder Anwesenheit von Bariumchlorid induziert. Die maximale Querschnittsfläche von Müller- und Bipolarzellsomata wurde vor und nach einer vierminütigen Superfusion mit einem konfokalen Laserscanningmikroskop aufgezeichnet. Die nach der Superfusion ermittelte Querschnittsfläche wurde zu den anfänglich gemittelten Kontrollwerten in Beziehung gesetzt und prozentual als Mittelwert mit Standardfehler bestimmt. Mit Hilfe des Prism-Statistikprogramms (Graphpad) wurden die Ergebnisse mittels einem one-way ANOVA Test und einem nachfolgenden Bonferroni\'s multiple comparison Test sowie durch einen Mann-Whitney U Test statistisch analysiert. Ergebnisse: Bei Anwesenheit von BDNF wurde die osmotische Schwellung von Müllerzellen konzentrationsabhängig sowohl in Netzhautschnitten als auch in isolierten Zellen inhibiert. Ebenso inhibierte BDNF konzentrationsabhängig die Schwellung von Bipolarzellen in Netzhautschnitten, jedoch nicht in isolierten Zellen. In Schnitten von postischämischen Netzhäuten bewirkte BDNF eine Schwellungsinhibition von Müllerzellen, nicht aber von Bipolarzellen. Mit pharmakologischen Blockern wurde die durch BDNF induzierte Signalkaskade untersucht. Die BDNF-Schwellungsinhibition von Müllerzellen wurde durch eine Aktivierung von TrkB bewirkt. Die TrkB-Aktivierung führte in Müllerzellen zu einer Transaktivierung von FGF-Rezeptoren sowie zu einer Aktivierung einer glutamatergen-purinergen Signalkaskade, von der bekannt ist, dass sie die osmotische Müllerzellschwellung unterdrückt. Da bFGF die osmotische Müllerzellschwellung inhibiert, wird die Transaktivierung der FGF-Rezeptoren wahrscheinlich durch eine BDNF-induzierte Freisetzung von bFGF aus Müllerzellen vermittelt. Die Ergebnisse lassen vermuten, dass BDNF indirekt auf Bipolarzellen wirkt, indem es eine Freisetzung von Faktoren wie bFGF aus Müllerzellen induziert. Schlussfolgerungen: Die Schwellungsinhibition von Müller- und Bipolarzellen könnte ein neuroprotektiver Mechanismus von BDNF in der Netzhaut darstellen. Während BDNF direkt TrkB auf Müllerzellen aktiviert, ist die Inhibition der Bipolarzellschwellung indirekt und durch die Ausschüttung von glialen Faktoren wie bFGF vermittelt. Der Verlust des Effektes von BDNF auf die Bipolarzellschwellung in ischämischen Netzhäuten könnte darauf zurückzuführen sein, dass gliotische Müllerzellen keine glialen Faktoren mehr in Reaktion auf BDNF freisetzen. Der Verlust des glialen Einflusses auf die Bipolarzellvolumenhomöostase könnte zur Neurodegeneration in der ischämischen Netzhaut beitragen. / Introduction: Tissue edema is a major blinding complication of ischemic-hypoxic and inflammatory retinal diseases. In addition to the hyperpemeability of the blood-retinal barrier, water accumulation in retinal cells resulting in cellular swelling may contribute to the development of retinal edema. Müller glial cells regulate the retinal ion and water homeostasis by allowing transcellular ion and water fluxes. During neuronal activity Müller cells control the extracellular space volume by autocrine inhibition of cellular swelling caused by the reduction of extracellular osmolarity. However, under pathological conditions, Müller cells are not capable to regulate their volume so that they swell rapidly under hypoosmolarity. The osmotic swelling of Müller glial cells and the glutamate induced swelling of retinal neurons contribute to the development of cytotoxic retinal edema. Various neuroprotective factors including brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) stimulate the survival of retinal neurons and thus delay the retinal degeneration. Objective: The objective of the study is to determine whether BDNF inhibits the osmotic swelling of Müller and bipolar cells of the rat retina. Material and Methods: Retinal slices and freshly isolated Müller and bipolar cells of 55 adult Long-Evans rats (in average 8-15 cells per trial) were used. Osmotic swelling of Müller and bipolar cells was induced by superfusion of retinal slices or isolated cells with a 60% hypoosmotic extracellular solution in the absence or presence of barium chloride. The maximal cross-sectional area of Müller and bipolar cell somata was recorded before and after a four minute-long superfusion by using a laser scanning microscope. To determine the extent of cell soma swelling, the cross-sectional area of the cell body extent after superfusion was related to the former averaged cross-sectional area. Results were given as means with standard error as percent values. Statistical analysis was made with Prism (Graphpad) and the significance was determined by the One-way ANOVA test followed by Bonferroni\'s multiple comparison test and the Mann-Whitney U test, respectively. Results: We found that BDNF inhibits dose-depending the osmotic swelling of Müller cells in retinal slices and of isolated cells. BDNF also inhibited dose-depending the osmotic swelling of bipolar cells in retinal slices; however, it did not inhibit the osmotic swelling in isolated bipolar cells. In slices of postischemic retinas, BDNF inhibited the swelling of Müller cells but not the swelling of bipolar cells. The BDNF induced signal transduction cascade was examined by simultaneous administration of blocking agents with the receptor agonists in the hypoosmotic solution. The BDNF-induced inhibition of the osmotic Müller cell swelling was mediated by activation of TrkB. Activation of TrkB in Müller cells results in transactivation of FGF receptors and in an activation of a glutamatergic-purinergic signal transduction cascade which is known to inhibit the osmotic swelling of the cells. Since bFGF also inhibits the osmotic swelling of Müller cells, it can be assumed that the transactivation of FGF receptors is mediated by a BDNF-induced release of bFGF from Müller cells. The results suggest that the effect of BDNF on bipolar cells is indirect by inducing a subsequent release of glial factor from Müller cells such as bFGF. Conclusion: The results show that BDNF inhibits the osmotic swelling of Müller and bipolar cells. The inhibition of cytotoxic cell swelling may contribute to the neuroprotective action of BDNF in the retina. While BDNF acts directly in Müller cells, the BDNF-induced inhibition of the bipolar cell swelling is indirect and mediated by the release of glial factors such as bFGF from Müller cells. The abrogation of the BDNF-induced inhibition of the osmotic bipolar cell swelling in the postischemic retina could be explained with the impairment of the release of glial factors by Müller cells. The abrogation of the Müller cell-mediated regulation of the bipolar cell volume could contribute to the neuronal degeneration in the ischemic retina.

Einleitung: Die Ausbildung eines Netzhautödems ist eine Hauptursache für die Verschlechterung des Sehvermögens bei ischämisch-hypoxischen und inflammatorischen Netzhauterkrankungen. Neben der erhöhten Permeabilität der Blut-Retina-Schranke trägt eine Wasserakkumulation in Netzhautzellen zur Ausbildung eines Netzhautödems bei. Müllerzellen regulieren die retinale Ionen- und Osmohomöostase, indem sie einen transzellulären Ionen- und Wassertransport vermitteln. Zudem kontrollieren Müllerzellen die Größe des Extrazellularraumes, indem sie bei neuronaler Aktivität eine Zellkörperschwellung – ausgelöst durch eine Verkleinerung der extrazellulären Osmolarität – verhindern. Unter pathologischen Bedingungen ist die Volumenregulation gestört, sodass Müllerzellen bei Hypoosmolarität anschwellen. Diese Müllerzellschwellung und eine Glutamat-induzierte Schwellung retinaler Neurone tragen zur Ausbildung eines zytotoxischen Netzhautödems bei. Neuroprotektive Faktoren wie BDNF (brain-derived neurotrophic factor) und bFGF (basic fibroblast growth factor) stimulieren das Überleben retinaler Neurone und verzögern so die retinale Degeneration. Zielstellung: Es war zu zu ermitteln, ob BDNF die zytotoxische Schwellung von Müller- und Bipolarzellen der Rattennetzhaut verhindert. Material und Methoden: Es wurden Netzhautschnitte und isolierte Müller- und Bipolarzellen von 55 adulten Long-Evans-Ratten (durchschnittlich 8-15 Zellen pro Versuchsreihe) verwendet. Eine osmotische Schwellung von Müller- und Bipolarzellen wurde durch eine Superfusion der Schnitte oder der Zellen mit einer 60%igen hypoosmolaren Lösung in Ab- oder Anwesenheit von Bariumchlorid induziert. Die maximale Querschnittsfläche von Müller- und Bipolarzellsomata wurde vor und nach einer vierminütigen Superfusion mit einem konfokalen Laserscanningmikroskop aufgezeichnet. Die nach der Superfusion ermittelte Querschnittsfläche wurde zu den anfänglich gemittelten Kontrollwerten in Beziehung gesetzt und prozentual als Mittelwert mit Standardfehler bestimmt. Mit Hilfe des Prism-Statistikprogramms (Graphpad) wurden die Ergebnisse mittels einem one-way ANOVA Test und einem nachfolgenden Bonferroni\''s multiple comparison Test sowie durch einen Mann-Whitney U Test statistisch analysiert. Ergebnisse: Bei Anwesenheit von BDNF wurde die osmotische Schwellung von Müllerzellen konzentrationsabhängig sowohl in Netzhautschnitten als auch in isolierten Zellen inhibiert. Ebenso inhibierte BDNF konzentrationsabhängig die Schwellung von Bipolarzellen in Netzhautschnitten, jedoch nicht in isolierten Zellen. In Schnitten von postischämischen Netzhäuten bewirkte BDNF eine Schwellungsinhibition von Müllerzellen, nicht aber von Bipolarzellen. Mit pharmakologischen Blockern wurde die durch BDNF induzierte Signalkaskade untersucht. Die BDNF-Schwellungsinhibition von Müllerzellen wurde durch eine Aktivierung von TrkB bewirkt. Die TrkB-Aktivierung führte in Müllerzellen zu einer Transaktivierung von FGF-Rezeptoren sowie zu einer Aktivierung einer glutamatergen-purinergen Signalkaskade, von der bekannt ist, dass sie die osmotische Müllerzellschwellung unterdrückt. Da bFGF die osmotische Müllerzellschwellung inhibiert, wird die Transaktivierung der FGF-Rezeptoren wahrscheinlich durch eine BDNF-induzierte Freisetzung von bFGF aus Müllerzellen vermittelt. Die Ergebnisse lassen vermuten, dass BDNF indirekt auf Bipolarzellen wirkt, indem es eine Freisetzung von Faktoren wie bFGF aus Müllerzellen induziert. Schlussfolgerungen: Die Schwellungsinhibition von Müller- und Bipolarzellen könnte ein neuroprotektiver Mechanismus von BDNF in der Netzhaut darstellen. Während BDNF direkt TrkB auf Müllerzellen aktiviert, ist die Inhibition der Bipolarzellschwellung indirekt und durch die Ausschüttung von glialen Faktoren wie bFGF vermittelt. Der Verlust des Effektes von BDNF auf die Bipolarzellschwellung in ischämischen Netzhäuten könnte darauf zurückzuführen sein, dass gliotische Müllerzellen keine glialen Faktoren mehr in Reaktion auf BDNF freisetzen. Der Verlust des glialen Einflusses auf die Bipolarzellvolumenhomöostase könnte zur Neurodegeneration in der ischämischen Netzhaut beitragen.:Inhaltsverzeichnis ABKÜRZUNGSVERZEICHNIS IX ABBILDUNGSVERZEICHNIS XI TABELLENVERZEICHNIS XIV 1 EINLEITUNG 1 2 LITERATURÜBERSICHT 3 2.1 Sehorgan - Das Auge 3 2.2 Retina beim Mensch und Tier – Aufbau und Funktionalitäten 4 2.2.1 Bildprozessor der Tierwelt – Die Retina 10 2.2.2 Die Sehbahn – Visuelle Verarbeitung 10 2.2.3 Die Müllerzelle – Vorkommen und Funktion 13 2.2.4 Tierartlicher Vergleich der Müllerzelle 14 2.2.5 Netzhaut als Modellgewebe 16 2.3 Ödembildung: Netzhautödem – Hirnödem 17 2.3.1 Allgemein: Die Pathogenese des Ödems 17 2.3.2 Das Gehirnödem – Ursachen und Folge 17 2.3.3 Das Netzhautödem – Ursachen und Folge 18 2.3.4 Volumen- und Osmohomöostase der Retina 19 2.3.5 Osmotische Schwellung und Osmoregulation der Müllerzelle 19 2.4 Neuroprotektion durch Neurotrophine und Wachstumsfaktoren 21 2.4.1 Brain-derived neurotrophic factor (BDNF) 21 2.4.2 Basic Fibroblastic Growth Factor (bFGF) 22 2.4.3 Neuroprotektive Effekte von BDNF und bFGF in der Retina 23 2.5 Zielstellung 24 2.6 Veterinärmedizinische Relevanz 25 2.6.1 Augenerkrankungen in der Klein- und Großtiermedizin 25 2.6.2 Schlussfolgerung für die Tiermedizin 31 3 TIERE, MATERIAL UND METHODEN 32 3.1 Versuchstiere 32 3.2 Material 32 3.2.1 Chemikalien und Reagenzien 32 3.2.2 Lösungen 33 3.2.3 Testsubstanzen 33 3.2.4 Pharmakologische Blocker 33 3.2.5 Antikörper 35 3.3 Verwendete Materialien und Geräte 36 3.4 Versuchsaufbau und Durchführung von Schwellungsversuchen 37 3.4.1 Gewebspräparation: Retina 37 3.4.2 Zellschwellungsversuche 39 3.4.3 Aufnahmetechnik 40 3.4.4 Superfusion von Retinaschnitten 40 3.4.5 Superfusion von isolierten retinalen Zellen 41 3.4.6 Das Physiologie-Modell: Isoosmolarität – Hypoosmolarität 42 3.4.7 Das Pathologie-Modell mit Barium 42 3.4.8 Tiermodell der retinalen Ischämie – Reperfusion 43 3.5 Auswertungsverfahren 43 3.5.1 Morphometrie der Somata 44 3.5.2 Statistische Analyse 44 3.6 Immunozytochemische Färbungen 45 4 ERGEBNISSE 46 4.1 Zellidentifikation in Retinaschnitten 46 4.1.1 Färbemethodik – MitoTracker Orange 47 4.1.2 Morphologie der Müllerzelle 47 4.1.3 Morphologie der Bipolarzelle 48 4.1.4 Identifikation von Müller- und Bipolarzellen 48 4.1.5 Morphologie isolierter Müller – und Bipolarzellen 49 4.2 Kontrollversuche 50 4.3 Untersuchung des Schwellungsverhaltens mit Testsubstanzen 51 4.4 Untersuchung des Schwellungsverhalten unter BDNF 52 4.4.1 Morphologie der Müllerzellen in Retinaschnitte unter BDNF 52 4.4.2 Wirkung von BDNF auf Müllerzellen in Retinaschnitten 53 4.4.3 Wirkung von BDNF auf die Schwellung von isolierten Müllerzellen 57 4.4.4 Wirkung von BDNF auf die osmotische Schwellung von Bipolarzellen in Retinaschnitten 58 4.4.5 Konzentrationsabhängigkeit der Wirkung von BDNF auf die osmotische Schwellung von Bipolarzellen in Retinaschnitten 59 4.4.6 Wirkung von BDNF auf die Schwellung von isolierten Bipolarzellen 61 4.4.7 Wirkung von BDNF auf die osmotische Müller- und Bipolarzellschwellung in der postischämischen Retina 62 4.5 Untersuchung der osmotischen Schwellung von retinalen Zellen unter bFGF 63 4.5.1 Wirkung von bFGF auf Müllerzellen in Retinaschnitten 63 4.5.2 bFGF-induzierte Transaktivierung metabotroper Glutamatrezeptoren in Müllerzellen 65 4.5.3 Wirkung von bFGF auf die Schwellung von Bipolarzellen in Retinaschnitten 65 4.6 Immunozytochemischer Nachweis von BDNF und TrkB in Müller- und Bipolarzellen 66 4.6.1 Kontrollaufnahmen 66 4.6.2 Immunzytochemischer Nachweis von BDNF und TrkB in isolierten Müllerzellen 67 4.6.3 Immunzytochemischer Nachweis von BDNF und TrkB in isolierten Bipolarzellen 69 5 DISKUSSION 71 5.1 Osmotische Volumenregulation bei Müller- und Bipolarzellen 71 5.2 Immunozytochemische Lokalisation von BDNF und TrkB 73 5.3 Inhibition der osmotischen Schwellung von Müller- und Bipolarzellen durch BDNF 75 5.4 TrkB-Aktivierung in Müllerzellen durch BDNF 77 5.5 Abhängigkeit des BDNF-Effekts von einer Transaktivierung von FGF-Rezeptoren 78 5.6 Indirekte Wirkung von BDNF auf die Bipolarzellschwellung durch gliale Faktoren 79 5.7 Abhängigkeit der BDNF-Wirkung von der Transaktivierung weiterer Rezeptoren 80 5.8 BDNF-induzierte Aktivierung von Ionenkanälen in Müllerzellen 81 5.9 BDNF-induzierte Signalkaskade der Inhibition der retinalen Zellschwellung 82 5.10 Neuroprotektive Wirkung von BDNF 84 6 ZUSAMMENFASSUNG 86 7 SUMMARY 88 8 BILDQUELLENVERZEICHNIS 90 9 LITERATURVERZEICHNIS 91 DANKSAGUNG 109 / Introduction: Tissue edema is a major blinding complication of ischemic-hypoxic and inflammatory retinal diseases. In addition to the hyperpemeability of the blood-retinal barrier, water accumulation in retinal cells resulting in cellular swelling may contribute to the development of retinal edema. Müller glial cells regulate the retinal ion and water homeostasis by allowing transcellular ion and water fluxes. During neuronal activity Müller cells control the extracellular space volume by autocrine inhibition of cellular swelling caused by the reduction of extracellular osmolarity. However, under pathological conditions, Müller cells are not capable to regulate their volume so that they swell rapidly under hypoosmolarity. The osmotic swelling of Müller glial cells and the glutamate induced swelling of retinal neurons contribute to the development of cytotoxic retinal edema. Various neuroprotective factors including brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) stimulate the survival of retinal neurons and thus delay the retinal degeneration. Objective: The objective of the study is to determine whether BDNF inhibits the osmotic swelling of Müller and bipolar cells of the rat retina. Material and Methods: Retinal slices and freshly isolated Müller and bipolar cells of 55 adult Long-Evans rats (in average 8-15 cells per trial) were used. Osmotic swelling of Müller and bipolar cells was induced by superfusion of retinal slices or isolated cells with a 60% hypoosmotic extracellular solution in the absence or presence of barium chloride. The maximal cross-sectional area of Müller and bipolar cell somata was recorded before and after a four minute-long superfusion by using a laser scanning microscope. To determine the extent of cell soma swelling, the cross-sectional area of the cell body extent after superfusion was related to the former averaged cross-sectional area. Results were given as means with standard error as percent values. Statistical analysis was made with Prism (Graphpad) and the significance was determined by the One-way ANOVA test followed by Bonferroni\''s multiple comparison test and the Mann-Whitney U test, respectively. Results: We found that BDNF inhibits dose-depending the osmotic swelling of Müller cells in retinal slices and of isolated cells. BDNF also inhibited dose-depending the osmotic swelling of bipolar cells in retinal slices; however, it did not inhibit the osmotic swelling in isolated bipolar cells. In slices of postischemic retinas, BDNF inhibited the swelling of Müller cells but not the swelling of bipolar cells. The BDNF induced signal transduction cascade was examined by simultaneous administration of blocking agents with the receptor agonists in the hypoosmotic solution. The BDNF-induced inhibition of the osmotic Müller cell swelling was mediated by activation of TrkB. Activation of TrkB in Müller cells results in transactivation of FGF receptors and in an activation of a glutamatergic-purinergic signal transduction cascade which is known to inhibit the osmotic swelling of the cells. Since bFGF also inhibits the osmotic swelling of Müller cells, it can be assumed that the transactivation of FGF receptors is mediated by a BDNF-induced release of bFGF from Müller cells. The results suggest that the effect of BDNF on bipolar cells is indirect by inducing a subsequent release of glial factor from Müller cells such as bFGF. Conclusion: The results show that BDNF inhibits the osmotic swelling of Müller and bipolar cells. The inhibition of cytotoxic cell swelling may contribute to the neuroprotective action of BDNF in the retina. While BDNF acts directly in Müller cells, the BDNF-induced inhibition of the bipolar cell swelling is indirect and mediated by the release of glial factors such as bFGF from Müller cells. The abrogation of the BDNF-induced inhibition of the osmotic bipolar cell swelling in the postischemic retina could be explained with the impairment of the release of glial factors by Müller cells. The abrogation of the Müller cell-mediated regulation of the bipolar cell volume could contribute to the neuronal degeneration in the ischemic retina.:Inhaltsverzeichnis ABKÜRZUNGSVERZEICHNIS IX ABBILDUNGSVERZEICHNIS XI TABELLENVERZEICHNIS XIV 1 EINLEITUNG 1 2 LITERATURÜBERSICHT 3 2.1 Sehorgan - Das Auge 3 2.2 Retina beim Mensch und Tier – Aufbau und Funktionalitäten 4 2.2.1 Bildprozessor der Tierwelt – Die Retina 10 2.2.2 Die Sehbahn – Visuelle Verarbeitung 10 2.2.3 Die Müllerzelle – Vorkommen und Funktion 13 2.2.4 Tierartlicher Vergleich der Müllerzelle 14 2.2.5 Netzhaut als Modellgewebe 16 2.3 Ödembildung: Netzhautödem – Hirnödem 17 2.3.1 Allgemein: Die Pathogenese des Ödems 17 2.3.2 Das Gehirnödem – Ursachen und Folge 17 2.3.3 Das Netzhautödem – Ursachen und Folge 18 2.3.4 Volumen- und Osmohomöostase der Retina 19 2.3.5 Osmotische Schwellung und Osmoregulation der Müllerzelle 19 2.4 Neuroprotektion durch Neurotrophine und Wachstumsfaktoren 21 2.4.1 Brain-derived neurotrophic factor (BDNF) 21 2.4.2 Basic Fibroblastic Growth Factor (bFGF) 22 2.4.3 Neuroprotektive Effekte von BDNF und bFGF in der Retina 23 2.5 Zielstellung 24 2.6 Veterinärmedizinische Relevanz 25 2.6.1 Augenerkrankungen in der Klein- und Großtiermedizin 25 2.6.2 Schlussfolgerung für die Tiermedizin 31 3 TIERE, MATERIAL UND METHODEN 32 3.1 Versuchstiere 32 3.2 Material 32 3.2.1 Chemikalien und Reagenzien 32 3.2.2 Lösungen 33 3.2.3 Testsubstanzen 33 3.2.4 Pharmakologische Blocker 33 3.2.5 Antikörper 35 3.3 Verwendete Materialien und Geräte 36 3.4 Versuchsaufbau und Durchführung von Schwellungsversuchen 37 3.4.1 Gewebspräparation: Retina 37 3.4.2 Zellschwellungsversuche 39 3.4.3 Aufnahmetechnik 40 3.4.4 Superfusion von Retinaschnitten 40 3.4.5 Superfusion von isolierten retinalen Zellen 41 3.4.6 Das Physiologie-Modell: Isoosmolarität – Hypoosmolarität 42 3.4.7 Das Pathologie-Modell mit Barium 42 3.4.8 Tiermodell der retinalen Ischämie – Reperfusion 43 3.5 Auswertungsverfahren 43 3.5.1 Morphometrie der Somata 44 3.5.2 Statistische Analyse 44 3.6 Immunozytochemische Färbungen 45 4 ERGEBNISSE 46 4.1 Zellidentifikation in Retinaschnitten 46 4.1.1 Färbemethodik – MitoTracker Orange 47 4.1.2 Morphologie der Müllerzelle 47 4.1.3 Morphologie der Bipolarzelle 48 4.1.4 Identifikation von Müller- und Bipolarzellen 48 4.1.5 Morphologie isolierter Müller – und Bipolarzellen 49 4.2 Kontrollversuche 50 4.3 Untersuchung des Schwellungsverhaltens mit Testsubstanzen 51 4.4 Untersuchung des Schwellungsverhalten unter BDNF 52 4.4.1 Morphologie der Müllerzellen in Retinaschnitte unter BDNF 52 4.4.2 Wirkung von BDNF auf Müllerzellen in Retinaschnitten 53 4.4.3 Wirkung von BDNF auf die Schwellung von isolierten Müllerzellen 57 4.4.4 Wirkung von BDNF auf die osmotische Schwellung von Bipolarzellen in Retinaschnitten 58 4.4.5 Konzentrationsabhängigkeit der Wirkung von BDNF auf die osmotische Schwellung von Bipolarzellen in Retinaschnitten 59 4.4.6 Wirkung von BDNF auf die Schwellung von isolierten Bipolarzellen 61 4.4.7 Wirkung von BDNF auf die osmotische Müller- und Bipolarzellschwellung in der postischämischen Retina 62 4.5 Untersuchung der osmotischen Schwellung von retinalen Zellen unter bFGF 63 4.5.1 Wirkung von bFGF auf Müllerzellen in Retinaschnitten 63 4.5.2 bFGF-induzierte Transaktivierung metabotroper Glutamatrezeptoren in Müllerzellen 65 4.5.3 Wirkung von bFGF auf die Schwellung von Bipolarzellen in Retinaschnitten 65 4.6 Immunozytochemischer Nachweis von BDNF und TrkB in Müller- und Bipolarzellen 66 4.6.1 Kontrollaufnahmen 66 4.6.2 Immunzytochemischer Nachweis von BDNF und TrkB in isolierten Müllerzellen 67 4.6.3 Immunzytochemischer Nachweis von BDNF und TrkB in isolierten Bipolarzellen 69 5 DISKUSSION 71 5.1 Osmotische Volumenregulation bei Müller- und Bipolarzellen 71 5.2 Immunozytochemische Lokalisation von BDNF und TrkB 73 5.3 Inhibition der osmotischen Schwellung von Müller- und Bipolarzellen durch BDNF 75 5.4 TrkB-Aktivierung in Müllerzellen durch BDNF 77 5.5 Abhängigkeit des BDNF-Effekts von einer Transaktivierung von FGF-Rezeptoren 78 5.6 Indirekte Wirkung von BDNF auf die Bipolarzellschwellung durch gliale Faktoren 79 5.7 Abhängigkeit der BDNF-Wirkung von der Transaktivierung weiterer Rezeptoren 80 5.8 BDNF-induzierte Aktivierung von Ionenkanälen in Müllerzellen 81 5.9 BDNF-induzierte Signalkaskade der Inhibition der retinalen Zellschwellung 82 5.10 Neuroprotektive Wirkung von BDNF 84 6 ZUSAMMENFASSUNG 86 7 SUMMARY 88 8 BILDQUELLENVERZEICHNIS 90 9 LITERATURVERZEICHNIS 91 DANKSAGUNG 109

La méthylation de l’ADN est une modification majeure du génome des eucaryotes permettant de moduler l’expression génique et contrôler le développement des mammifères. La protéine Mecp2 (Methyl CpG binding protein 2), dont le gène est situé sur le chromosome X, appartient à la famille des protéines de liaison à l’ADN méthylé. Sur la base de sa structure et de ses interactions Mecp2 a été décrit comme un répresseur de l’expression des gènes. A l’heure actuelle, son implication en tant qu’activateur de la transcription et organisateur de la structure chromatinienne lui confère un rôle plus global dans la régulation de l’épigénome. Des mutations de MECP2 conduisent à des troubles neurologiques dont le principal est le syndrome de Rett (RTT). Cette pathologie dominante liée à l’X affecte principalement les jeunes filles (incidence: 1/15000 naissances). Même si les causes précises du phénotype RTT ne sont pas connues, le profil d’expression de Mecp2 est en lien avec la synaptogenèse, la maturation et la maintenance des réseaux neuronaux. A mon arrivée en thèse l’équipe qui m’a accueilli venait d’identifier des déficits neuronaux, affectant notamment les groupes catécholaminergiques bulbaires et périphériques, à l’origine de troubles respiratoires chez un modèle murin de cette pathologie. Mon travail de thèse a permis de caractériser l’évolution postnatale des déficits moteurs et physiologiques affectant la souris Mecp2-déficiente. L’étude de structures catécholaminergiques d’intérêt telles que la Substantia Nigra et le Locus Coeruleus a révélé que les neurones dopaminergiques et noradrénergiques centraux ont un métabolisme affecté. Le nombre de neurones immunomarqués apparait significativement réduit dans ces groupes ce qui résulterait d’une perte progressive du phénotype « catécholaminergique », en l’absence de mort cellulaire. Nos données suggèrent que ces atteintes constituent un corrélat neuropathologique aux troubles comportementaux observés chez les souris Mecp2-déficientes. Ainsi certains troubles moteurs ont pu être améliorés, à l’aide d’un agent pharmacologique pro-dopaminergique, la L-Dopa. En relation avec les déficits en Bdnf (Brain-derived neurotrophic factor) décrits chez les patientes et les souris Mecp2-déficientes, nous avons identifié qu’une modification du dosage de Mecp2 induit une dérégulation de gènes (Htt, Hap1) codant des protéines impliquées dans le transport intracellulaire des vésicules de Bdnf. Nos travaux nous permettent de postuler que chez la souris Mecp2-déficiente, une altération de la dynamique de transport des vésicules chargées en Bdnf pourrait exacerber le déficit d’expression de cette neurotrophine. Notre traitement des souris Mecp2-déficientes par la cystéamine, une molécule capable d’agir sur les contenus, la libération et la sécrétion du Bdnf permet d’augmenter la survie des animaux et de réduire leurs troubles moteurs. Nos résultats montrent que les déficiences en Mecp2 entrainent des déficits de transport axonal du Bdnf qui s’ajoutent aux déficits de production du Bdnf. Par ailleurs, avec l’utilisation d’agents pharmacologiques agissant sur ce transport, nous offrons de nouvelles perspectives thérapeutiques. / DNA methylation is the major modification of eukaryotic genomes and plays an essential role in mammalian development. The protein Mecp2 (Methyl CpG binding protein 2), encoded by a gene located on the X chromosome, belongs to the ‘Methyl Binding domain’ protein family. Based on its structure and its interactions Mecp2 has historically been described as a repressor of expression for many genes. Currently, its involvement as an activator of transcription and its role in chromatin architecture suggests that it could be a global regulator of the epigenome. Mutations in MECP2 lead to neurological disorders, among which Rett syndrome (RTT). This dominant X-linked pathology mainly affects girls (incidence: 1/15000 live births). Although the precise causes of the RTT phenotype are unknown, the pattern of Mecp2 expression is related to synaptogenesis, maturation and neuromaintenance. Before my integration in the ‘Human Neurogenetics’ team, this group identified neural deficits, affecting brainstem and peripheral catecholaminergic cell groups, causing respiratory disturbances in a mouse model of this disease. My thesis work enabled the characterization of the postnatal physiological and motor deficits affecting the Mecp2-deficient mice. The study of catecholaminergic structures of interest such as the substantia nigra pars compacta and the locus coeruleus has revealed that the central noradrenergic and dopaminergic neurons are affected in their metabolism. The number of immunolabelled neurons of these groups appears significantly reduced and would result in a gradual loss of the mature ‘catecholaminergic’ phenotype, in the absence of cell death. Our data suggest that these defects are a neuropathological correlate for behavioral disorders observed in Mecp2-deficient mice. Some motor deficits have been improved, with L-Dopa, a pro-dopaminergic drug. In relation with Bdnf (Brain-derived neurotrophic factor) reduction described in patients and Mecp2-deficient mice, we identified that a change in the dosage of Mecp2 deregulates genes (Htt, Hap1) encoding proteins involved in the intracellular transport of Bdnf. Our work allows to postulate that in the Mecp2-deficient neurons, an altered dynamics of Bdnf vesicles transport could exacerbate the deficit of expression of this neurotrophin. Our treatment of Mecp2-deficient mice with cysteamine, a molecule able to increase Bdnf contents and enhancing its release and secretion, increased the survival of the animals and reduced their motor defects. Our results show that the Mecp2-deficiencies lead to alteration in the axonal transport of Bdnf in addition to deficits in Bdnf production. In addition, by the use of pharmacological agents that affect this transport, we offer new therapeutic perspectives.

Made available in DSpace on 2016-03-22T17:26:26Z (GMT). No. of bitstreams: 1 Dissertacao_Marta.pdf: 316598 bytes, checksum: 4827ddafff08a3c37d59d9f11b39691c (MD5) Previous issue date: 2010-12-11 / BACKGROUND: Postpartum depression (PPD) is the most common psychiatric complication observed in women after birth. Some women have a particular sensitivity to hormonal changes, starting in early menarche, which increases their vulnerability to psychological stressing agents that are triggered by environment and physiological factors during life. Decreased serum brain-derived neurotrophic factor (BDNF) levels have been associated to different neuropsychiatric conditions and BDN has been considered as a candidate marker for such disfunctions. The goal of this study was to compare the levels of BDNF between mothers with PPD and control mothers as well as to seek for associations between BDNF levels and the severity of PPD. METHODS: This is a case-control study including 36 mothers with PPD and 36 control mothers. PPD was defined according to the Beck Depression Inventory (BDI). Serum BDNF was assayed with the sandwich ELISA method. RESULTS: Serum levels of BDNF were significantly lower in women with PPD than in control mothers (p = 0.026). No significant correlation between BDI score and serum BDNF levels was observed (r= 0.16, p = 0.09). CONCLUSIONS: Our study suggests that low BDNF levels are associated with PPD, although not with PPD severity. This result point out to the potential usage of BDNF in the screening of PPD, which could promote early treatment and, therefore, reduce the burden to the PPD women and to the health system / O BDNF (brain-derived neurotrophic factor) é um fator neurotrófico que parece estar envolvido na fisiopatologia de muitos transtornos psiquiátricos, incluindo depressão 1. O pós-parto é um período de risco psiquiátrico aumentado no ciclo de vida da mulher. A depressão pós-parto pode se manifestar com intensidade variável, tornando-se um fator que dificulta o estabelecimento de um vínculo afetivo seguro entre mãe e filho, podendo interferir nas futuras relações interpessoais estabelecidas pela criança. Fatores neurotróficos, ou neurotrofinas são famílias de proteínas, nas quais são capazes de promover o desenvolvimento, modulação e sobrevivência dos neurônios no sistema nervoso central (SNC) e periférico. Estes fatores são secretados pelos tecidos e vão atuar de diversas maneiras tais como na modulação sináptica, na apoptose, na diferenciação celular, dentre outras. O BDNF é crítico no desenvolvimento e modulação do SNC 2. As diversas funções das neurotrofinas são mediadas via duas classes de receptores: o Trk (cinase do receptor tropomiosina), da família dos RTKs (receptor da tirosina cinase), e o p75NTR (receptor da neurotrofina p75). Os subtipos dos receptores Trk ligam-se a neurotrofinas maduras com diferentes especificidades. O TrkB preferencialmente liga-se ao BDNF e ao NT4 (neurotrofina-4). O p75NTR liga-se a todas neurotrofinas maduras com baixa afinidade. O p75NTR também pode interagir com outros receptores, incluindo os receptores Trks 3. Para a mulher, o ciclo gravídico-puerperal é considerado período de risco para o psiquismo devido à intensidade da experiência vivida 4. A prevalência deste transtorno pode variar de acordo com os critérios utilizados para o diagnóstico, como o período de início dos sintomas e o método utilizado para a avaliação. Uma meta-análise estimou uma prevalência de 10 13% de depressão pós-parto (DPP) em mulheres 5. Recentemente, Moraes et al. (2006) e Pinheiro et al. (2006) encontraram prevalência de DPP de 19,1% em mulheres na cidade de Pelotas/RS 6,7. A partir da primeira semana após o parto 80% das mulheres apresentam um estado de tristeza materna (baby blues), podendo vir a ser confundido com a depressão. O quadro é benigno e regride por si só por volta do primeiro mês. Os sintomas mais freqüentes são: irritabilidade, mudanças bruscas de humor, indisposição, tristeza, insegurança, baixa auto-estima, sensação de incapacidade de cuidar do bebê, entre outros 8. Recentemente, o envolvimento do BDNF, tem sido foco de interesse na pesquisa relacionada com a regulação dos transtornos de humor e depressão. Interessantemente, diversos estudos caso-controle têm encontrado níveis séricos de BDNF diminuídos em pacientes deprimidos. Nos casos em que a depressão é induzida por alterações nos níveis de BDNF, os sintomas desaparecem após o final do tratamento ou com o uso de antidepressivos 1,9,10. A descoberta de novos marcadores pode não só auxiliar no diagnóstico, monitoramento, como também na escolha do tratamento a ser seguido, bem como no desenvolvimento de novos fármacos para o tratamento da depressão. Existem fortes evidências do envolvimento do BDNF na fisiopatologia da depressão, como esses achados envolveram exclusivamente amostras clínicas oriundas de sistema terciário, o fato de utilizarmos uma amostra comunitária aumenta a representatividade dos achados, e é relevante ao entendimento da neurobiologia e conseqüentemente abre caminho à pesquisa de novas abordagens no tratamento da DPP. Sendo assim o presente estudo pretende utilizar medidas mais sofisticadas para o diagnóstico e intervenção no tratamento da DPP, relacionando essa patologia com a concentração sérica de BDNF

Les astrocytes joueraient un rôle central dans la physiopathologie des troubles anxio-dépressifs et dans l’activité thérapeutique des antidépresseurs. En effet, différentes études in vitro suggèrent que les inhibiteurs sélectifs de recapture de la sérotonine (ISRS) stimulent la synthèse de substances neuroactives par ces cellules gliales nécessaires à la prolifération, la maturation et la survie neuronale mais également au maintien de la plasticité synaptique. Le Brain-derived neurotrophic factor (BDNF) fait partie de ces substances mais son origine, notamment astrocytaire, doit encore être démontrée dans les systèmes intégrés tel que l’animal vivant. A partir de ce constat, dans une première partie de ce travail de thèse, nous avons donc voulu préciser si les astrocytes constituent une source cellulaire participant à la synthèse et/ou à la libération de BDNF en réponse à l’administration prolongée d’un ISRS, la fluoxétine d’une part chez des souris naïves et d’autre part chez des souris exposées à la corticostérone pendant plusieurs semaines (modèle « CORT »). Pour cela, nous avons utilisé une stratégie de transfection virale induisant la surexpression de BDNF spécifiquement dans les astrocytes de l'hippocampe. Nos résultats mettent en lumière que cette surexpression provoque des effets de types anxiolytiques-antidépresseurs dépendant de la neurogenèse hippocampique chez des souris naïves soumises au test d’hypophagie induite par la nouveauté mais pas dans le modèle CORT. Nous avons également mis en évidence que le BDNF pouvait agir en retour sur les cellules qui l’ont libéré pour renforcer le réseau astrocytaire (mécanisme autocrine) mais également sur les neurones sérotoninergiques pré-synaptiques (mécanisme paracrine) pour exercer un frein sur la libération de sérotonine dans l’hippocampe. Différents arguments de la littérature suggèrent d’ailleurs que ce dernier mécanisme pourrait être favorable à l’activité anxiolytique de la fluoxétine tel que nous l’avons observé dans ce travail. Sachant que les astrocytes expriment à leur surface, une grande variété de récepteurs sérotoninergiques, nous nous sommes ensuite intéressé à la possibilité que le sous-type de récepteur 5-HT2A pourrait être un élément clé dans la synthèse et/ou la libération de BDNF et de ce fait moduler la réponse au stress et celle à la fluoxétine. Lors de cette seconde étude nous avons appliqué une approche génétique utilisant des souris mutées, privées de manière constitutive du récepteur 5-HT2A (5-HT2A-/-). A l’opposé de la surexpression de BDNF dans les astrocytes, nous avons montré que les souris 5-HT2A-/- sont plus sensibles au stress, modélisé par l’exposition chronique à la corticostérone, et semblent résistantes à la fluoxétine comparées aux souris 5-HT2A+/+. Afin de préciser le mécanisme pouvant rendre compte de ces observations, nous avons mis en évidence que l’inactivation du récepteur 5-HT2A s’accompagne d’une hypersensibilité du frein inhibiteur exercé par les autorécepteurs 5-HT1A somatodendritiques sur le tonus sérotoninergique. / Growing evidence demonstrates that astrocytes could play a crucial role in the pathophysiologies of anxiety and depression as well as in the therapeutic activity of antidepressant drugs. Indeed, in vitro studies suggest that the selective serotonin reuptake inhibitors (SSRIs) stimulate the synthesis of neuroactive substances by these glial cells which are necessary for the proliferation and maturation of neuronal progenitors but also for the maintenance of the neuronal survival. Brain-derived neurotrophic factor (BDNF) is one of these substances, but its cellular origin has yet to be demonstrated in embedded systems such as living animals. In this context, the first part of this thesis was aimed at clarifying whether astrocytes constitute a source of BDNF in response to the chronic administration of the SSRI, fluoxetine, in both naive and anxio-depressive mice exposed to corticosterone (" CORT" model). In this prospect, we used a novel and efficient gene transfer strategy inducing BDNF overexpression specifically in the astrocytes of the hippocampus. Our results indicated that BDNF overexpression produced anxiolytic-/antidepressant-like activity in the novelty suppressed feeding paradigm in relation with the stimulation of hippocampal neurogenesis in naive mice but not in the CORT model. We also showed that BDNF could act on astrocytes themselves (autocrine mechanism) to improve the hippocampal astrocytic network but also on the pre-synaptic serotonergic nerve terminals (paracrine mechanism) to limit the local serotonin release. Different arguments from the literature suggest that the latter mechanism may be favorable to an anxiolytic-like activity of fluoxetine. Given that astrocytes express at the surface a variety of serotonin receptors, we then raised the possibility that the 5-HT2A receptor subtype may be a key element in the synthesis and/or release of BDNF and thereby modulating the vulnerability to depression and/or the response of fluoxetine. In the second part of this thesis, a genetic approach with mutant mice constitutively lacking the 5-HT2A receptor (5-HT2A-/-) was applied. In contrast to Lenti-BDNF mice, we showed that 5-HT2A-/- mice were more prone to develop anxio-depressive-like symptoms in response to the chronic exposure to corticosterone. Moreover, these mutants were resistant to the chronic administration of fluoxetine compared to 5-HT2A+/+ wild type mice. In order to clarify the mechanism underpinning these observations, we demonstrated that the inactivation of the 5-HT2A receptor was associated with a hypersensitivity of a negative feedback exerted by the somatodendritic 5-HT1A autoreceptors on serotonergic tone.Together these data suggest that astrocytes act in concert with neurons to regulate mood and antidepressant drug response, notably through the synthesis and/or release of BDNF following the activation of the 5-HT2A receptor. More generally, our results illustrate the concept of the tripartite synapse in which the bidirectional communication between astrocytes and monoaminergic neurons would be essential in the regulation of higher brain functions.

Introdução - Os efeitos na saúde de dietas vegetarianas (DV) apontam principalmente para a diminuição do risco de Doenças Crônicas Não Transmissíveis (DCNT), uma vez que modula positivamente parâmetros bioquímicos, particularmente aqueles relacionados ao controle da glicemia e lipemia, além de ser uma medida para o controle de peso. Estudos recentes em adultos demonstram que a dieta possa também modular parâmetros moleculares. Nesse cenário, atenta-se para o papel do Fator Neurotrófico Derivado do Encéfalo (BDNF) o qual parece estar relacionado com a DV em relação à redução de triglicerídeos e colesterol e aumento da sensibilidade à insulina. Objetivo - Avaliar adultos que adotaram uma DV, em relação ao estado nutricional, aos parâmetros bioquímicos e moleculares comparados aos adultos com dieta onívora. Métodos - A população avaliada foi constituída por 96 indivíduos, 56 vegetarianos e 40 onívoros, adultos e de ambos os sexos, em um estudo do tipo transversal. Para o levantamento dos dados sociodemográficos e de estilo de vida foi aplicado questionário e aferidas às medidas de peso corporal (PC) e altura, para posterior cálculo de Índice de Massa Corporal (IMC) e circunferência de cintura (CC). Foi realizada também coleta de sangue para estudos bioquímicos e expressão de BDNF plasmático. Os índices de Castelli 1 e 2 (razões lipídicas) são indicadores de risco cardiovascular (RCV) com maior valor preditivo do que parâmetros isolados, por isso, também foram calculados. A resistência à insulina (IR) foi avaliada pelo índice HOMA_IR. As análises foram conduzidas pelo software SPSS (Statistical Package for Social Sciences) versão 20.0 e para todas foi considerado um nível de significância de 5 por cento .Foi realizada análise de regressão logística para identificar se a DV e outros fatores podem prever a redução da chance de ter RCV, determinado pelos índices de Castelli 1 e 2.Resultados Em relação às variáveis de estilo de vida, os VEG têm uma tendência a praticar mais atividade física (64,3 por cento vs 42,5 por cento , p = 0,056) e ingerir suplementos alimentares (48,1 por cento vs 20,5 por cento , p = 0,012), embora o número de fumantes se apresente semelhante em ambos os grupos. Não houve diferença estatisticamente significante para as variáveis: idade, sexo, prática de fumar, triglicerídeos (TG), Colesterol Total (CT) e lipoproteína de baixa densidade (LDL- c) entre os dois grupos. Já os valores dos índices de Castelli 1 (3,23 ± 0,84 vs 3,90 ± 0,99, p =0,001)e 2 (1,91 ± 0,69 vs 2,42 ± 0,79, p = 0,001) foram menores em vegetarianos (VEG) do que em onívoros (ONV). O grupo VEG tinha significativamente menor PC (63,9 ± 10,4 vs 69,4 ± 14,6 kg, p = 0,032); IMC (22,5 ± 2,6 vs 25,0 ± 3,9 kg/m2, p = 0,001); CC( 81,8 ± 8,2 vs 87,8 ± 10,9 cm, p = 0,003 ) e maior lipoproteína de alta densidade (HDL-c) (54,88 ± 14,44 vs 47,30 ± 12,27 mg / dl , p = 0,008) . Os VEG também apresentaram menor HOMA-IR (1,17 ± 0,70 vs 1,48 ± 0,8, p = 0,02) em comparação com ONV. Quanto a variável BDNF, não houve diferença entre os grupos VEG e ONV (662,8 + 276,5pg/ml vs 698,1 + 314,9 pg/ml, p=0,563). Conclusão - Sugere-se, portanto, que a DV pode ter efeitos protetores na saúde cardiovascular e no metabolismo desses indivíduos. / Introduction - The effects of vegetarian diets (VD) on health points out mainly to a decrease in the risk for noncomunnicable chronic disease (NCDs) once it positively modulates the biochemical parameters, particularly those related to the control of glicemic and lipemia being also a way of controlling weight. Recent studies have shown that diet can also modulate molecular parameters. In this scenario, one must pay attention to the role of the brain-derived neurotrophic factor (BDNF) which seems to be related to the VG in what regards the reduction of triacylglycerol and cholesterol, and the increase of insulin sensitivity. Objective - To assess adults that adopted a VD in what regards their nutritional status, biochemical and molecular parameters, in comparison to adults that adopted an omnivorous diet. Methods- A cross-sectional study assessed a population composed of 96 individuals, 56 vegetarians and 40 omnivores, adults of both genders. A questionnaire was administered in order to gather sociodemographic and life-style related data, body weight (BW), height and waist circumference (WC) were surveyed. In order to carry out the biochemical study and the expression of plasmatic BDNF, blood was collected. The Castelli indexes 1 and 2 (lipid ratios) are indicators of cardiovascular risk (CVR) with a higher predictive value than isolated parameters and therefore were calculated. Insulin resistance (IR) was calculated by the HOMA_IR index. The analyses were carried out through the SPSS (Statistical Package for Social Sciences) software, 20.0 version, taking into account a 5 per cent significance level. An analysis of logistic regression was done in order to identify if the VD and other factors are able to prevent the reduction of CVR, determined by the Castelli indexes 1 and 2. Results - There was no statistically significant difference between both groups regarding the following variables: age, gender, smoking habits, triglyceride (TG), Total Cholesterol (TC) low-density lipoprotein cholesterol (LDL- col). On the other hand, the values of the Castelli indexes 1 (3,23 ± 0,84 vs 3,90 ± 0,99, p =0,001) and 2 (1,91 ± 0,69 vs 2,42 ± 0,79, p = 0,001) were lower in vegetarians (VEG) than in omnivores (OMV). The VEG-groups showed significant lower BW (63,9 ± 10,4 vs 69,4 ± 14,6 kg, p = 0,032); BMI (22,5 ± 2,6 vs 25,0 ± 3,9 kg/m2, p = 0,001); WC ( 81,8 ± 8,2 vs 87,8 ± 10,9 cm, p = 0,003 ) and more high-density lipoprotein cholesterol (HDL col) (54,88 ± 14,44 vs 47,30 ± 12,27 mg / dl , p = 0,008). The VEG-group also presented lower HOMA-IR (1,17 ± 0,70 vs 1,48 ± 0,8, p = 0,02) in comparison to the OMV-group. Regarding life-style parameters, the individuals of the VEG-group displayed a tendency for practicing more physical activity (64,3 per cent vs 42,5 per cent , p = 0,056) and for ingesting food supplement (48,1 per cent vs 20,5 per cent , p = 0,012), although the number of smokers was quite similar between both groups. Regarding the BDNF variable, there was no difference between the VEG-group and the OMVGroup (662,8 + 276,5 pg/ml vs 698,1 + 314,9 pg/ml, p=0,563).Conclusion - In relation to these results it is to be suggested that a VD may exert protective effects on cardiovascular health and on the metabolism of the individuals that adopt it.

A Ouabaína (OUA), esteroide cardiotônico endógeno, ao ligar-se a enzima NKA pode sinalizar modulando a atividade da Src quinase e os níveis intracelulares de Ca2+, desencadeando a ativação de diversas vias de sinalização, dentre elas a via do NFκB, que uma vez ativada, é capaz de ativar outras vias de sinalização, como a via canônica da WNT. O objetivo dessa tese foi verificar os efeitos da administração intrahipocampal de OUA, em ratos, na sinalização das vias NFκB, BDNF/CREB e canônica da WNT, assim como os possíveis efeitos morfológicos e funcionais. Os resultados sugerem que a OUA altera o microambiente celular favorecendo a ativação das vias propostas levando ao aumento da arborização dendrítica de neurônios do CA1 e do GD, com melhora da memória de referência espacial dos animais e piora do processo de extinção de memória de longa duração, também conhecido como flexibilidade comportamental, além de proteger os neurônios da apoptose frente a um estímulo excitotóxico. / It is well established that Ouabain (OUA), an endogenous cardiotonic steroid, can bind to NKA and trigger activation of signaling pathways dependent on Src kinase activation and intracellular Ca2+ levels oscillation, what can lead to NFκB activation, and in turn, can transactivate other signaling pathways, such as the canonical WNT pathway. The aim of this thesis was to investigate the effects of intrahippocampal administration of OUA in adult rats, whether it was able to activate NFκB, BDNF/CREB and the canonical WNT signaling pathways, as well as the possible morphological and functional effects of this injection. Results suggested that OUA changed the cellular microenvironment in favor to the activation of the proposed pathways leading to increased dendritic branching in CA1 and DG neurons, with spatial reference memory improvement and worsening of long-term memory extinction. Furthermore, OUA protected neurons from apoptosis stimulus triggered by excitotoxicity.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Traumatic brain injury (TBI) is a major cause of morbidity and mortality in industrialized countries leading to the motor and cognitive deficits. Evidence demonstrated that exercise is neuroprotective in traumatic brain injury. However, the effects of exercise before of the TBI at the cognitive function are unknown. Role of excitotoxicity and oxidative damage in secondary damage of TBI, however, until this moment, were not demonstrated if exists a relationship between early phase of damage and the late cognitive deficit. In the current study, we proposed that improvement cognitive response induced by exercise prior in rats after a TBI can be associated with the neuroprotection of early phase after injury. To demonstrate this hypotheses, adult rats practice swimming exercise during 6 weeks followed for TBI operation. We assessed the motor alterations of early phase, the glutamate uptake and antioxidant defense in twenty four hours (24 h) and 15 days after TBI. Acquisition of memory was assessed by recognition object task on days 15 post TBI. Moreover, we evaluated the brain-derived neurotrophic factor (BDNF) to assessement the synaptic plastic. In the present study, we showed that TBI induced by fluid percussion injury (FPI) in adult male Wistar rats induced early motor impairment 24 h, followed by learning retention deficit (2 weeks after neuronal injury). Previous swimming training improved the memory in object recognition task per se and protected against FPI-related disabilities. Although the FPI did not alter hippocampal expression of glutamate transporters (EAAT1 / EAAT2) and brain-derived neurotrophic factor (BDNF), the alterations in the redox status, herein characterized by DCFH-DA oxidation and SOD activity inhibition, led to marked impairment of protein functionally (Na+, K+-ATPase activity inhibition) and glutamate uptake inhibition 24 h after neuronal injury in sedentary injured rats. Indeed, the early increase of nuclear factor erythroid 2-related factor (pNRF2/NRF2 ratio) followed by a repair mechanism (protein HSP70 expression), 24 h and 2 weeks after neuronal injury, suggests that FPI-induced signal transduction may exert compensatory effect on pathophysiological processes. In this report we showed that previous physical exercise induced the increase of immune content of glutamate transporters (EAAT1/ EAAT2), pNrf2/Nrf2 ratio, SOD enzyme and HSP70 per se besides preventing against FPI-induced Na+, K+ - ATPase activity, glutamate uptake inhibition DCFH-DA oxidation 24 h after neuronal injury. The enhancement of hippocampal pNrf2/Nrf2 and HSP70 immune content in trained injured when compared with sedentary rats suggest that protein expression modulation associated to antioxidant defense elicited by previous physical exercise prevent against toxicity induced by TBI. The significant increase of BDNF levels in trained injured rats 24 h and 2 weeks strongly reinforce the idea that physical activity alters neuronal functions and thus delays or prevents secondary cascades that leave the neurobehavioral disability after TBI. / O traumatismo cranioencefálico (TCE) é uma das maiores causas de morte e morbidade nos países industrializados podendo levar ao comprometimento motor e déficits cognitivos. Evidências demonstram que o exercício físico é neuroprotetor na recuperação após o TCE. Porém, os efeitos do exercício físico antes do TCE na função cognitiva não são totalmente conhecidos. Sabe-se da participação da excitotoxicidade e do estresse oxidativo na cascata do dano secundário após o TCE, entretanto até o momento não foi demonstrado qual a relação da fase inicial após o TCE com os déficits cognitivos tardios. Portanto, no presente estudo, nós propomos que a melhora cognitiva tardia induzida pelo exercício prévio em ratos após o TCE pode estar associada com a neuroproteção da fase inicial após o dano. Para demonstrar esta hipótese, ratos adultos praticaram treinamento de natação durante 6 semanas e posteriormente foram submetidos a cirurgia para o TCE. Nós avaliamos as alterações motoras iniciais, a captação de glutamato e a defesa antioxidante em 24 horas (24 h) e 15 dias após o TCE. Aquisição da memória foi avaliada pela tarefa de reconhecimento de objetos em 15 dias após o TCE. Além disso, nós avaliamos o fator neurotrófico derivado do encéfalo (BDNF) para avaliar a plasticidade sináptica. No presente estudo, nós mostramos que o TCE induzido pela lesão de percussão de fluido (LPF) em ratos Wistar machos adultos induziu déficit motor inicial 24 h, seguido por déficit de aprendizagem (15 dias após o dano neuronal). O treinamento de natação prévio melhorou a memória na tarefa de reconhecimento de objeto per se e protegeu contra desabilidades relacionadas ao LPF. Embora o LPF não tenha alterado a expressão dos transportadores de glutamato (EAAT1/EAAT2) e de BDNF, causou uma alteração no estado redox, caracterizado pela oxidação de DCFH-DA e inibição da atividade da SOD. O LPF também causou prejuízo acentuado da funcionalidade de proteínas (inibição da atividade da enzima Na+, K+-ATPase) e inibição da captação de glutamato 24 h após o dano neuronal em ratos sedentários lesionados. De fato, o aumento inicial do fator de transcrição Nrf2 (relação pNrf2/Nrf2), 24 h após o TCE, seguido por um mecanismo de reparo (expressão da proteína Hsp70), 24 h e 15 dias após o dano neuronal, sugerem que a transdução de sinal induzida pelo LPF pode exercer um efeito compensatório em processos patofisiológicos. Neste trabalho, nós mostramos que o exercício físico prévio induziu o aumento do imunoconteúdo dos transportadores de glutamato (EAAT1/EAAT2), relação pNrf2/Nrf2, enzima SOD e a proteína Hsp70 per se, além de prevenir contra inibição da atividade da Na+, K+-ATPase, inibição da captação de glutamato e oxidação de DCFH-DA induzida pelo LPF, 24 h após o dano neuronal. O aumento do imunoconteúdo hipocampal de pNrf2/Nrf2 e Hsp70 em ratos treinados e lesionados quando comparado com ratos sedentários, sugerem que a modulação da expressão das proteínas associadas às defesas antioxidantes induzidas pelo exercício físico prévio preveniu contra a excitotoxicidade induzida pelo TCE. O significante aumento nos níveis de BDNF em ratos treinados e lesionados 24 h e 15 dias, reforçam fortemente a ideia que a atividade física altera a função neuronal e assim retarda ou previne as cascatas do dano secundário que levam a desabilidade neuronal após o TCE.