Myelinated axons are crucial for rapid information transmission within the central nervous system (CNS). Myelin injury is a common feature of white matter (WM) pathology in a number of disease states, including ischemic stroke. Myelin disruption can lead to a complete failure in saltatory action potential conduction, resulting in devastating neurological deficits. However, the fundamental mechanism of ischemic myelin injury is controversial. Glutamate-mediated excitotoxicity is now recognised as a crucial event in the development of ischemic WM pathology. This thesis investigates the potential mechanisms of glutamate release in central WM and examines the hypothesis that NMDA receptor over-activation mediates ischemic myelin damage. Using glutamate biosensor microelectrodes and FM-dye imaging, I show that axonal depolarisation in the adult corpus callosum evokes rapid vesicular docking in axons, capable of elevating extracellular glutamate concentration. My findings show that vesicular fusion occurs under the myelin sheath in myelinated axons, which supports the existence of a novel synapse between the axon and overlaying myelin. Simulation of ischemia triggered an early and robust rise in optic nerve extracellular glutamate levels. Unexpectedly, a significant component of ischemic glutamate release also originated from axonal vesicular fusion. Together, these findings show that the axon-myelin synapse represents a significant site of excitotoxic injury during ischemia. Resolving prior conflicting results, I show that NMDA receptor antagonists prevent myelin degradation and improve functional recovery when applied for sufficient time to penetrate the sheath. Finally, I identify a fluorescent myelin stain (QNZ-46) which is a negative allosteric modulator of NR2C/D-containing NMDA receptors. QNZ-46 selectively accumulates in myelinated WM regions of the CNS following systemic administration, and is retained following wash-out. As a result, QNZ-46 provides persistent protection during ischemia by preserving myelin structure and improving functional recovery.

Les mécanismes physiopathologiques liés aux maladies neurodégénératives restent encore largement méconnus. Deux processus semblent être particulièrement en cause dans les phénomènes de neurodégénérescence : la neurotoxicité par afflux massif de calcium due à une activation excessive des récepteurs NMDA (GluN) et la neurotoxicité par déstabilisation du cytosquelette du neurone par le biais de la phosphorylation anormale de la protéine tau. L'imagerie moléculaire par le biais de la tomographie par émission de positons (TEP) et de radiotraceurs, en étudiant les mécanismes moléculaires in vivo, permet de détecter et quantifier ces phénomènes. Ce travail a eu pour objet d'étudier un dérivé de la mémantine, un antagoniste des GluN se fixant sur un site intra-canal accessible uniquement lorsque ces récepteurs sont activés ce qui en fait donc un vecteur d'imagerie intéressant pour étudier leur activation. Nous avons mis au point la synthèse d'un nouveau radiotraceur dérivé de la mémantine : la [18F]-FNM (Fluoroéthylnormémantine). Il s'agit d'une synthèse par substitution nucléophile d'un groupement tosylate par du [18F], suivie d'une hydrolyse acide. Cette synthèse est reproductible avec un rendement de 10%, son activité spécifique est > 355 GBq/µmol. Chez le rat, le traceur passe la barrière hémato-encéphalique et sa distribution cérébrale est bien corrélée avec la localisation des GluN (r=0.622, p<0.0001). Sa cinétique de fixation (40 minutes) est compatible avec son utilisation en TEP. En ce qui concerne les tauopathies, la protéine tau stabilise l'organisation microtubulaire. Lors d'une phosphorylation anormale, l'interaction avec les microtubules diminue et les protéines tau vont s'accumuler en formant des Paires de Filaments en Hélice (PHF). Nous avons optimisé la radiosynthèse de l' [18F]-AV1451 ciblant les PHF. Notre rendement de synthèse est de 30% et l'activité spécifique du traceur > 10 GBq/µmol. Nous avons réalisé des autoradiographies sur des coupes de cerveaux atteints de tauopathie et nous avons constaté la capacité du traceur à différencier les coupes saines des coupes malades. La production de cet outil dans notre centre va nous permettre d'étudier la présence de PHF chez le marmouset, un primate particulièrement intéressant dans l'étude du vieillissement. Nous avons donc réalisé la synthèse de deux radiotraceurs innovants : la [18F]-FNM et le [18F]-AV1451, les synthèses sont reproductibles et les rendements compatibles avec des productions de doses en recherche pré-clinique et clinique. / The pathophysiological mechanisms associated with neurodegenerative diseases remain largely unknown. Two processes appear to be particularly involved in the phenomena of neurodegeneration: neurotoxicity induced by massive influx of calcium caused by excessive activation of NMDA receptors (GluN) and neurotoxicity by destabilization of neuron cytoskeleton through abnormal protein tau phosphorylation. Molecular imaging through positron emission tomography (PET) and radiotracers, by studying the molecular mechanisms in vivo, allows to detect and quantify these phenomena. This work was intended to study a memantine derivative, a GluN antagonist. We chose to develop a ligand that selectively binds to the ion channel in the open and active state which therefore makes it an interesting vector to study their overactivation. We have developed the synthesis of a new memantine analogue radiotracer: the [18F]-FNM (Fluoroéthylnormémantine). This is a synthesis by nucleophilic substitution of a tosylate with [18F], followed by acid hydrolysis. This synthesis is reproducible with a yield of 10%, its specific activity was> 355 GBq / µmol. In rats, the tracer cross the blood-brain barrier and brain distribution correlates well with the location of GluN (r = 0.622, p <0.0001). The binding kinetics (40 minutes) is compatible with its use in PET. Regarding tauopathies, the tau protein stabilizes microtubule organization. During abnormal phosphorylation, interaction with microtubules and tau proteins decreases and tau will accumulate to form Paired helical Filament (PHF). We optimized the radiosynthesis of [18F] AV1451 targeting 3 tau PHF. Our yield of synthesis is 30% and the specific activity of the tracer> 10 GBq / µmol. We made autoradiography on brains sections and have shown tracer ability to differentiate healthy and pathological slices. This tool will allow us to study the presence of PHF in marmosets, a particularly interesting primate in the study of aging. So we performed the synthesis of two innovative radiotracers: the [18F]-FNM and [18F]-AV1451, syntheses are reproducible and yields compatible with doses manufacturing in pre-clinical and clinical research.

Les maladies neurodégénératives se caractérisent par une déconnexion synaptique et une dégénérescence des axones (DA) précoces, menant à la mort spécifique d’une population neuronale. Les niveaux intracellulaires de NAD+, co-facteur essentiel dans le maintien de l’intégrité axonale, sont fortement diminués lors de ces pathologies. L’augmentation des taux de NAD+ est ainsi une stratégie thérapeutique dans la prévention de ces maladies. La capacité du nicotinamide riboside (NR) à retarder la DA dans le système nerveux périphérique (SNP) ainsi que la récente mise en évidence d'une conversion extracellulaire du NAD+ en NR dans des lignées cellulaires et dans le SNP soulignent l'intérêt de ce précurseur du NAD+. Mon projet de thèse repose sur la caractérisation des effets du NAD+ et du NR lors de la DA dans des neurones du système nerveux central (SNC). A partir d'un modèle d'excitotoxicité mis au point en dispositifs microfluidiques, nous montrons pour la première fois que le NR protège de la DA dans des neurones corticaux de manière plus efficace que le NAD+. Cet effet différentiel a également été validé dans un modèle ischémique in vivo. De manière surprenante, lors d'une neurodégénérescence induite par une déplétion aigüe en NAD+, un effet protecteur total à la fois du NAD+ et du NR a été mis en évidence. L'analyse de la voie de conversion extracellulaire a ainsi révélée une adaptation du métabolisme du NAD+ et de sa conversion en NR en fonction du paradigme neurotoxique. En conclusion, ce travail démontre un fort effet protecteur du NR dans le SNC et ouvre de nouvelles voies thérapeutiques dans la prévention des processus neurodégénératifs. / Synaptic and axonal degeneration (AxD) are major events in neurodegenerative diseases. Levels of NAD+, an important coenzyme for axonal integrity, are strongly reduced in different degeneration models so enhancing cellular NAD+ is one of the numerous therapeutic strategies against neuronal pathologies. Nicotinamide riboside (NR) is a good NAD+ precursor as it has already been shown to delay AxD in peripheral nervous system (PNS) and extracellular NAD+ conversion to NR was previously described in cell lines and in PNS. During my thesis project, we analyzed the role of NR metabolism to prevent degeneration processes in cortical neurons. Using an excitotoxicity model developed in microfluidic devices, we showed for the first time that both NAD+ and NR delay AxD in cortical neurons, with a more potent effect for NR. We confirm this differential effect in an in vivo ischemic model. Moreover, NR effect is mainly restricted to the axonal compartment and intracellular NAD+ depletion is reverted after NR application, suggesting that axonal integrity is totally dependent on NAD+ local metabolism. Furthermore, in a complete NAD+ depletion paradigm, NAD+ and NR have surprisingly the same strong effect, protecting equally neuronal death and AxD. Examination of the extracellular pathway suggest that NAD+ conversion to NR is limited in excitotoxicity but effective in the NAD+ depletion model. These results reveal that NR and NAD+ metabolism depend on the neurotoxic paradigm. Our results demonstrate that NR has a strong and local neuroprotective effect on AxD in several neurotoxic processes. These findings open new therapeutic strategies to prevent neurodegenerative diseases.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / O cérebro infantil humano submetido à hipóxia-isquemia (HI) apresenta perda de oligodendrócitos, hipomielinização, astrogliose, alterações no desenvolvimento cortical e no comportamento motor, incluindo a paralisia cerebral. O cerebelo desempenha um importante papel no controle motor e diversos danos vêm sendo observados em humanos e animais que sofreram HI. A excitotoxicidade glutamatérgica é frequentemente associada à HI e junções celulares podem ser responsáveis pela transferência de moléculas capazes de modular os danos decorrentes. Dados prévios de nosso grupo utilizando um modelo de HI pré-natal em ratos demonstraram danos permanentes na estrutura cerebelar, indicando que os efeitos deletérios da HI pré-natal podem ser mantidos até a vida adulta. O objetivo deste trabalho foi caracterizar os níveis de conexinas, receptores e transportadores de glutamato ao longo do desenvolvimento do cerebelo HI, e avaliar a configuração das junções celulares em culturas de astrócitos derivadas do cerebelo de ratos submetidos a esse modelo. Ratas no 18 dia de gestação, após anestesia, tiveram as quatro artérias uterinas obstruídas por 45 minutos (Grupo HI). Animais controle tiveram os úteros expostos sem sofrer a obstrução (Grupo SH). A gestação prosseguiu e apenas filhotes nascidos a termo foram utilizados. Os animais foram decapitados aos 2 (P2), 9 (P9), 16 (P16),23 (P23), 30 (P30), 45 (P45) e 90 (P90) dias pós-natal. Os cerebelos foram submetidos à técnica de Western blotting utilizando os anticorpos anti-NR2B, anti-GluR3, anti-EAAT1, anti-GFAP e anti-Cx43. Para a cultura de astrócitos foram utilizados cerebelos de animais P2. Após terem atingido confluência, as células foram fixadas e imunomarcadas com os anticorpos anti-Cx43, anti-GFAP, anti-nestina e anti-A2B5. Nossos resultados demonstram diferenças nos níveis de GluR3 durante o desenvolvimento do cerebelo SH e HI, havendo uma redução significativa da expressão desta subunidade no grupo HI em P9. Por outro lado, não foram verificadas alterações nos níveis de NR2B e de GFAP entre os grupos nas diferentes idades. Observou-se redução significativa de Cx43 em animais HI em P2 bem como nos astrócitos HI em cultura, os quais também apresentaram alterações morfológicas e diferenças na expressão do marcador A2B5. A alteração referente a GluR3 no grupo HI pode ser causada pela redução da arborização das células de Purkinje e pela redução no número de precursores de oligodendrócitos no cerebelo de animais HI em P9, já observadas em nosso laboratório. A diminuição de Cx43 indica que a passagem de substâncias por canais astrocitários pode estar reduzida e contribuir para a expansão dos danos persistentes descritos em HI. Alterações morfológicas e na expressão de marcadores da diferenciação de astrócitos podem refletir os potenciais efeitos de HI sobre a maturação destas células a longo-prazo. Nossos resultados apontam que a HI sistêmica pré-natal pode ser responsável por alterações que caracterizam a excitotoxicidade glutamatérgica. Ressaltamos também a importância da comunicação entre astrócitos como estratégia neuroprotetora nesta lesão. / Infant human brains submitted to hypoxia-ischemia show oligodendrocyte loss, hypomelination, astrogliosis, cortical development and motor behavior impairments, including cerebral palsy. Cerebellum plays a critical role in motor control and many damages have been demonstrated in humans and animals who suffered HI. Glutamatergic excitotoxicity is usually associated to HI and cellular junctions may be responsible for molecular traffic, being able to modulate HI harm effects. Previous data from our group using a modified model of prenatal HI in rats have shown long-lasting damages in cerebellar structure, indicating that deleterious effects of prenatal HI may be sustained until adult life. The objective of this study was to characterize connexin (Cx) and glutamate receptors and transporters levels during the development of HI cerebellum and to evaluate cellular junctions in astrocyte cultures derived from the cerebella of rats submitted to this same model. Rats on the 18th gestation day were anesthetized, had their uterine horns exposed and the four uterine arteries were clamped for 45 minutes (HI group). Control animals had the uterine horns exposed but no arteries were clamped (SH group). Gestation proceeded after surgery and only pups born at term were used. The animals were decapitated at 2 (P2), 9 (P9), 16 (P16), 23 (P23), 30 (P30), 45 (P45) e 90 (P90) postnatal days. Cerebella were submitted to Western blotting using anti-NR2B, anti-GluR3, anti-EAAT1, anti-GFAP and anti-Cx43 antibodies. P2 cerebella were used in astrocyte primary cultures. After they had achieved confluence, the cells were fixed and immunostained with anti-Cx43, anti-GFAP, anti-nestin and anti-A2B5 antibodies. Our results demonstrate differences in GluR3 levels along cerebellum development of SH and HI animals, with a significant decrease of this subunit expression in HI group at P9. On the other hand, we did not observe any variation in NR2B and GFAP levels between groups at different ages. We also observed a significant decreased Cx43 expression in HI group at P2 as well as in cultured astrocytes, which had morphological modifications and different A2B5 marker expression. The modification related to GluR3 receptor in HI group may be caused by impaired dendritic arborization or by a reduced number of oligodendrocyte progenitors in the cerebellum of HI animals at P9, already described in our laboratory. Cx43 reduction indicates that substances traffic through astrocytic channels may be impaired and contribute to lesion expansion of permanent damages observed in HI. Morphological and markers expression changes related to astrocyte differentiation may reflect potential effects of HI on cell maturation at long-term. Our results confirm that prenatal systemic HI may be responsible for changes that characterize glutamatergic excitotoxicity. We also reassure the importance of astrocyte communication as a neuroprotective strategy in this kind of lesion.

Orientador: Prof. Dr. Alexandre Hiroaki Kihara / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Neurociência e Cognição, 2016. / Anóxia é uma das maiores causas de morbidade e mortalidade neonatal, especialmente em neonatos pré-maturos, constituindo um importante problema de saúde pública devido às sequelas neurológicas permanentes em pacientes. A privação de oxigênio dispara uma série de cascatas, culminando em morte celular em regiões cerebrais mais vulneráveis, como o hipocampo. Neste processo de morte celular causada pela privação de oxigênio, o cálcio citosólico possui um papel crucial. Receptores intracelulares de inositol 1,4,5-trifosfato (IP3Rs) são importantes reguladores de níveis de deste cálcio, no entanto, não se sabe sobre sua função na anóxia. O objetivo deste estudo é analisar se os IP3Rs do tipo 1 (IP3R1) participam no processo de morte no hipocampo de ratos após a anóxia neonatal. A análise quantitativa de real-time PCR revelou uma diminuição da expressão gênica de IP3R1 24 horas após a anóxia neonatal. Na análise da distribuição de células IP3R1-positivas foi observada uma densidade de IP3R1 na região de CA1 em ambos os grupos, porém, não se observou diferença entre os grupos controle e anóxia. Interessantemente os animais anóxia apresentaram uma alta colocalização de IP3R1 e marcador de núcleo (DAPI), sugerindo que a anóxia causa uma translocação de IP3R1 para o núcleo nas células hipocampais. Além disso, o padrão de marcação mostrou diferentes tamanhos de clusters dos receptores, indicando uma organização diferente entre os grupos. Foi injetado 2-APB, um bloqueador de IP3R1, ou veículo, no hipocampo de forma bilateral após a anóxia. Foi utilizado metodologias de marcação de células degeneradas e foi visto que no grupo 2APB houve uma diminuição do número de células FJC-positivas e TUNEL-positivas em comparação ao grupo veículo anóxia. Porém, não foi observado nenhuma diferença de marcação entre os grupos na imunofluorescência de caspase-3 ativada. Não foi detectada nenhuma diferença entre os grupos no teste de labirinto de Barnes. No teste de campo aberto, observou-se que o grupo 2APB apresentam maiores níveis de ansiedade. Desta forma, este estudo pode contribuir com novas perspectivas na investigação de mecanismos de neurodegeneração ativadas pela privação de oxigênio. / Anoxia is one of the most prevalent causes of neonatal morbidity and mortality, especially in preterm neonates, constituting an important public health problem due to permanent neurological sequelae observed in patients. Oxygen deprivation triggers a series of simultaneous cascades, culminating in cell death mainly located in more vulnerable metabolic brain regions, such as the hippocampus. In the process of cell death by oxygen deprivation, cytosolic calcium plays crucial roles. Intracellular inositol 1,4,5-trisphosphate receptors (IP3Rs) are important regulators of cytosolic calcium levels, although the role of these receptors in neonatal anoxia is completely unknown. This study focused on the functional role of inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) in rat hippocampus after neonatal anoxia. Quantitative real-time PCR analysis revealed a decrease of IP3R1 gene expression 24 hours after neonatal anoxia. Distribution analysis of IP3R-positive cells was performed and we observed higher IP3R1 pixels quantity in CA1 of both groups; however, we were not able to observe alterations between control and anoxia animals. Interestingly, we observed that anoxia animals present a higher colocalization of IP3R1 and nucleus marker (DAPI), suggesting that neonatal anoxia may cause IP3R1 translocation to the nucleus in hippocampal cells. Furthermore, puncta-labelling pattern showed different cluster sizes, larger in control group, indicating different organization between groups. We injected 2-APB, an IP3R1 blocker, or vehicle in hippocampus bilaterally after anoxia. Labelling techniques of degenerate cells was performed and we observed that 2APB group decrease the number of FJC-positive cells compared to vehicle anoxia group. In contrast, TUNEL labelling and active caspase-3 immunofluorescence showed no difference between groups. Barnes maze test showed no differences between 2APB group and anoxia vehicle group. On the other hand, the open field test showed that 2APB group presents higher anxiety levels than vehicle group. In this way, this study may contribute to new perspectives in the investigation of neurodegenerative mechanisms triggered by oxygen deprivation.

Orientadores: Fabio Rogério, Carlos Amilcar Parada / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T17:39:33Z (GMT). No. of bitstreams: 1 Ignarro_RaffaelaSilvestre_M.pdf: 3644274 bytes, checksum: 6e13f812b2d525e18878656d3ec27815 (MD5) Previous issue date: 2011 / Resumo: A injeção de ácido quinolínico (AQ), um agonista glutamatérgico do receptor N-metil-D-aspartato, no estriado de roedores induz morte seletiva de neurônios espinhosos médios, gliose reativa e neurogênese na zona subventricular, acompanhada da migração dos neurônios recém-gerados para o estriado lesado. Tais achados são também descritos na doença de Huntington (DH). Há indícios de que a via de sinalização JAK/STAT esteja envolvida no mecanismo de ação do AQ, bem como na patogênese da DH. A interação das citocinas da família da IL-6 com seus receptores desencadeia a ativação de enzimas da família das Janus-Quinases (JAKs), que por sua vez permitem o recrutamento e a ativação de fatores de transcrição da família das proteínas transdutoras de sinais e ativadoras da transcrição (STATs). Embora as principais características da DH sejam a presença da coréia e déficits na execução de movimentos voluntários, poucos testes são realizados abordando o comportamento locomotor dos animais no modelo de lesão por AQ. Neste trabalho, estudamos o efeito do AG490, um inibidor da JAK2, na gliose, perda neuronal e neurogênese no estriado de camundongos adultos C57BL/6J após a administração estereotáxica unilateral de AQ (30nmol). Imediatamente após a lesão, os animais receberam uma injeção subcutânea de AG490 (10mg/kg) ou veículo (PBS+DMSO), e injeções diárias por 6 dias adicionais. Além disso, investigamos o possível efeito da lesão por AQ na atividade física voluntária diária (AFVD) em rodas de atividade. A distância percorrida pelos camundongos foi monitorada por 28 dias após a injeção unilateral de QA (30nmol) ou PBS no estriado. Cortes coronais do cérebro (40?m) obtidos em criostato foram utilizados para quantificação de neurônios por estereologia e para a análise de expressão protéica, através de imunoistoquímica e Western Blotting para GFAP e doublecortina, marcadores de gliose e neuroblastos, respectivamente. A área total de células doublecortina-positivas (ACDP) e o número de neurônios (NN) no lado lesado (L) e contralateral à lesão (CL) foram avaliados. O Índice de Neurogênese (IN=ACDP(L)/ACDP(CL)) e o Índice de Sobrevivência Neuronal (ISN=NN(L)/NN(CL)) foram calculados. Após a administração de AQ, o estriado ipsilateral apresentou intensa gliose e células doublecortina positivas com características de células migratórias. O Western Blotting para GFAP mostrou uma redução ipsilateral de 19% nos animais tratados com AG490, em comparação aos animais do grupo tratado apenas com veículo (0.82±0.05; 1.010±0.06, n=9, p<0.05). O ISN foi 25% maior nos camundongos que receberam AG490 em comparação aos animais controles (0.75 ± 0.07; 0.60 ± 0.03; n=8, p<0.05). O IN mostrou uma diminuição de 21% no grupo AG490 em relação ao grupo de animais tratados apenas veículo de diluição (1.08±0.06; 1.37±0.09, n=5, p<0.05). A AFVD média, medida em quilômetros por dia, não se alterou nos animais que receberam injeção intra-estriatal de QA (30nmol) em comparação aos animais do grupo controle (3.97±0.34; 3.90±0.21, n=8, p>0.05). Portanto, nossos resultados suportam um papel para a JAK2 na morte neuronal, gliose, e neurogênese estriatais após lesão com AQ. O tratamento com o inibidor AG490 causou neuroproteção e diminuição da gliose, sugerindo que a reação astrocitária pode prejudicar a sobrevivência neuronal neste modelo experimental / Abstract: Injection of quinolinic acid (QA), a N-methyl-D-aspartate receptor agonist, in murine striatum induces death of medium spiny neurons, gliosis and neurogenesis in the subventricular zone with migration of newly synthesized neurons to damaged striatum. Such findings are also described in Huntington's disease (HD). The Janus-kinase (JAK) pathway would take part in QA mechanism of action and HD pathogenesis as well. The interaction of interleukin-6 family of cytokines with its receptor triggers the activation of enzymes of the family of JAKs, which in turn allow the recruitment and activation of transcription factors, known as signal transducers and activators of transcription (STATs). Although the main features of HD are the presence of chorea and deficits in performing voluntary movements, few tests are realized regarding locomotor behavioral on QA model. We studied the effect of AG490, an inhibitor of JAK isoform 2 (JAK2), on gliosis, neuronal loss and neurogenesis in the striatum of adult C57BL/6J mice after unilateral estereotaxic administration of QA (30 nmol). Immediately after injury, animals received a subcutaneous injection of AG490 (10 mg/kg) or vehicle (PBS + DMSO), and then once daily injections for 6 days. Furthermore, in a parallel experiment, we investigated the possible effect of the lesion by AQ on the voluntary daily physical activity (VDPA) in running wheels. The distance traveled by mice was monitored daily for 28 days after unilateral injection of QA (30 nmol) or PBS into the striatum. Frozen brain sections (40?m) were used for neuronal stereological quantification and immunohistochemical and Western Blotting analyses for GFAP and doublecortin, markers of gliosis and neuroblasts, respectively. The total area of doublecortin-positive cells (ADPC) and the number of neurons (NN) in the lesioned (L) and contralateral (CL) sides were evaluated. Neurogenesis index (NI = ADPC in L/ ADPC in CL) and neuronal survival ratio (NSR = NN in L/ NN in CL) were calculated. After QA administration, ipsilateral striatum showed intense gliosis and doublecortin-positive cells with few processes and ovoid bodies, morphological features corroborating a migratory activity. Western Blotting for GFAP showed an ipsilateral decrease of 19% in AG490- vs vehicle-treated animals (0.82 ± 0.05 vs 1.010 ± 0.06; n=9, p<0.05). NSR was 25% higher in mice given AG490 vs controls given vehicle (0.75 ± 0.07 vs 0.60 ± 0.03; n=8, p<0.05). NI showed a decrease of 21% in AG490- vs vehicle-treated mice (1.08 ± 0.06, 1.37 ± 0.09; n=5, p<0.05). The average VDPA, measured in kilometers per day for 28 days, has not changed in animals that received intrastriatal injection of QA (30nmol) compared to animals that received PBS (3.97 ± 0.34, 3.90 ± 0.21, n = 8, p> 0.05). In conclusion, our results support a role for JAK2 in striatal neuronal death, gliosis and neurogenesis determined by QA. AG490 caused neuroprotection and reduced gliosis suggesting that astrocytic reaction may impair neuronal survival in the present experimental model / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Implications for the endocannabinoid system in global cerebral ischemia has not been clearly defined. Ischemia produces an excitotoxic environment that is severely damaging to neurons, causing degradation of cell membrane and ultimately cell death. Contradicting research suggests both the benefits and adverse effects of endocannabinoids on neurological injury. Due to the excitotoxic nature of ischemic injury, and the mechanisms at play with endocannabinoid agonists, such as increased transmission of dopamine and glutamate, it is suspected that endocannabinoid antagonists, such as AM251, may a provide cell protection.40 male Wistar rats were separated into 4 groups (n=10/group). The first group of rats were administered AM251 (2 mg/kg, i.p) 30 minutes prior to global cerebral ischemia (four vessel occlusion), while the second group were given AM251, 30 minutes prior to sham surgery. Finally the last two groups were given a vehicle control instead of AM251 and given either ischemia or the sham surgery. Behavioural testing, open field test and elevated plus maze, took place after a five day recovery period following ischemia. Immunohistochemical analyses were performed using to mark tyrosine hydroxylase (TH) and dopamine receptor 1(DRD1) to compare dopamine function amongst groups. Cell survival was also evaluated using thionin staining. Ischemia induced significant reduction in dopamine within the mesolimbic circuit, including: ventral tegmental area, nucleus accumbens, CA3 & CA1 of the hippocampus, and basolateral amygdala. These reductions in dopamine transmission by global ischemia were partially or fully reversed when AM251 was given beforehand. Furthermore, cell survival was increased in the CA1 from treatment of AM251. Behavioural results show similar results that AM251 reversed emotional irregularities associated with ischemia insult. The endocannabinoid antagonist AM251 improves deficits in dopamine function, prevents cell death and regulates emotionality when given prior global cerebral ischemia.

An increasing body of evidence points to a key role of endoplasmic reticulum (ER) stress in chronic and acute neurodegenerative diseases. Indeed, markers of ER stress are common features of neurons destined to die in these conditions. In the present study we demonstrate that PUMA, a BH3-only member of the Bcl-2 family is essential for ER stress-induced cell death. PUMA is known to be a key transcriptional target of p53, however we have found that ER stress triggers PUMA induction and cell death through a p53-independent mechanism involving instead the ER stress inducible transcription factor ATF4. Specifically, we demonstrate that ectopic expression of ATF4 sensitizes neurons to ER stress induced apoptosis, and that ATF4-deficient neurons exhibit markedly reduced levels of PUMA expression and cell death. However, chromatin immunoprecipitation experiments suggest that ATF4 does not directly regulate the PUMA promoter. Rather, we found that ATF4 induces expression of the transcription factor CHOP, and that CHOP in turn directly activates PUMA induction. Specifically, we demonstrate that CHOP binds to the PUMA promoter during ER stress and that CHOP knockdown attenuates PUMA induction and neuronal apoptosis. In summary, we have identified a key signaling pathway in ER stress induced neuronal death involving ATF4-CHOP mediated transactivation of the pro-apoptotic Bcl-2 family member PUMA. Protein aggregates and markers of ER stress response have also been observed in dying neurons in several animal models of cerebral ischemia. Therefore, to decipher the significance of the ER stress apoptotic response, we investigate the role of ATF4-CHOP signaling pathway in ischemic neuronal injury. Ischemic stroke results from a transient or permanent reduction in cerebral blood flow in the brain. In spite of much research in trying to develop therapeutic strategies, most clinical trials have failed. These failures demonstrate that effective treatments require a more complete understanding of molecular signals that lead to neuronal death. However, stroke is a complex scenario since distinct mechanisms may involve in rapid and/or delayed neuronal death. The signaling pathways regulating these mechanisms however are not fully defined. Previous studies had suggested that ER stress playing a pivotal role in post-ischemic neuronal death. Yet, the relevance of ER stress signals was not fully known in ischemic neuronal injury. Accordingly, this thesis research attempts to explore the functional role of ER stress -inducible pathway, ATF4-CHOP axis, in different models of neuronal death (delayed and excitotoxic cell death) evoked by ischemia. The data indicates that ATF4 is essential in delayed type of death in vitro. In focal ischemia model (tMCAO) ATF4 also plays a role as a mediator of death signal in vivo. However, CHOP function looks more complex, and our data did not support the role of CHOP in ischemic neuronal death.

Amyotrophic lateral sclerosis (ALS) is an incurable and fatal neurodegenerative disease characterized by motor neuron loss. Although pathological mutations exist in >15 genes, the mechanism(s) underlying ALS are unknown. FUS is one such gene and encodes the nuclear RNA-binding protein (RBP), fused in sarcoma (FUS), which actively shuttles between the nucleus and cytoplasm. Intriguingly, nearly half of the ALS mutations identified in FUS cause this protein to mislocalize, suggesting that FUS localization is relevant to disease. Here, we found that excitotoxicity, a neuronal stress caused by aberrant glutamate signaling, induces the rapid redistribution of FUS and additional disease-linked RBPs from the nucleus to the cytoplasm. As excitotoxicity is pathologically associated with ALS, it was notable that the nuclear egress of FUS was particularly robust. Further, ALS-FUS variants that predominantly localize to the nucleus also undergo redistribution. Thus, we sought to understand the purpose underlying FUS translocation and the potential relevance of this response to disease. As calcium dysregulation is strongly associated with neurodegenerative disorders, we examined the contribution of calcium to FUS egress. In addition to global changes to nucleocytoplasmic transport following excitotoxic insult, we observed that FUS translocation caused by excitotoxicity is calcium mediated. Moreover, we found that dendritic expression of Gria2, a transcript encoding an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit responsible for regulating calcium permeability, is FUS-dependent under conditions of stress. Together, these observations support the premise that FUS has a normal function during excitotoxic stress and that glutamatergic signaling may be dysregulated in FUS-mediated ALS.

Stroke is serious neurological disease and is a major cause of death as well as disability throughout the globe. Stroke has a complex pathophysiology that involves inflammatory pathways, excitotoxicity mechanisms, oxidative damage, apoptosis, ionic imbalances, angiogenesis and neuroprotection. 85% of strokes are ischemic and occurs when a cerebral vessel, or any vessel supplying the brain, narrows or loses pressure resulting in subsequent brain ischemia and infarction downstream to the site of obstruction depriving tissues of vital oxygen and nutrients. This may be caused by either atherosclerotic thrombi or distant emboli defined as a mass of clotted blood or other material. It is estimated that over a billion people currently smoke cigarettes or use other tobacco products, seeing as smoking is a major risk factor for stroke this is of major concern. Platelets are hematopoietic cells produced by bone marrow megakaryocytes. Platelets play a role in the development of ischemic stroke primarily by means of their participation in the formation of thromboemboli, the presence of abnormal platelet function may predispose patients to a pro-thrombotic, pro-inflammatory state. The reorganization of the cytoskeleton in platelets is an important factor in the complex mechanisms found in thrombosis and haemostasis. The platelet membrane contains a large number of receptors which specifically bind agonists that stimulate the physiological platelet response. Oxidative stress is one of the mechanisms involved in the neuronal damage of stroke. Oxidative stress is a state of imbalance between free radical production, in particular, reactive oxygen species (ROS), and the ability of the organism to neutralize them, leading to progressive oxidative damage. Smoking is known to result in the generation of various free radicals. Flow cytometric analysis of the platelets of thrombo-embolic ischemic stroke patients and smokers revealed that the membranes of the two groups were altered in some form as well as an increased activation in both groups when compared to healthy individuals. Superoxide levels in the platelets were higher in smokers when compared to stroke patients, while hydrogen peroxide levels were elevated in the platelets of both groups. Superoxide was elevated in the whole blood samples of both groups. The production and subsequent reactions of reactive oxygen species appear to be influential in stroke and smoking and may likely be a crucial factor in the development of a pro-thrombotic, pro-inflammatory state which may prove to be a hallmark in the pathophysiology of stroke and smoking. Confocal microscopy and Scanning electron microscopy showed that platelets of stroke patients and smokers appear to be more activated and more prone to form tight clots. Furthermore an increased amount of superoxide is present in the platelets of stroke patients and smokers, specifically in the centre of clots. This may be an indication that once platelets have aggregated and started to fuse together, the mitochondria are expelled from the platelets and “trapped” within the clot. Atomic force microscopy also indicated both the stroke patients and smoker‟s platelets appear to be in a more activated state than the control group. Here it is apparent that some form of cytoskeletal rearrangement takes place to a more severe extent in the stroke group than in the smokers. Necrosis may be present in the platelets of stroke patients while neither apoptosis nor necrosis can be identified in the platelets of smokers however some form of membrane alteration is likely present. All the techniques used showed an increase in platelet activation in stroke patients and smokers, necrotic platelets may be present in the stroke patients while the platelet membrane of smokers seems to be altered. ROS is present and alters the platelet function of smokers and stroke patients in some way. It appears as if thrombo-embolic ischemic stroke patients and smokers‟ platelets have similar trends in activation but the processes involved to achieve this differ as there are structural differences present. These differences may prove a useful tool to further understand the pathophysiology behind thrombo-embolic ischemic stroke as well as to discover new therapeutic pathways. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Physiology / Unrestricted