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The studies presented in this thesis were undertaken to investigate the cellular and molecular mechanisms responsible for sympathetic hyperactivity that is observed in the Spontaneous Hypertensive Rat (SHR) and whether these abnormalities arise even before the onset of hypertension. Moreover, selected molecular candidates related to oxidative state in cardiac autonomic signalling have been explored for their potential therapeutic effects. <b>Chapter One</b> is an overview of (i) the relevance of autonomic dysfunction in cardiovascular disease in both human and animal models, (ii) the physiological basis of cardiac sympathetic neurotransmission, (iii) the neuromodulators of peripheral cardiac sympathetic-vagal balance discussed along with how they may be involved in cardiac adrenergic control of neurotransmission and NO-cGMP signalling. This develops the formulation of the specific aims of the thesis. <b>Chapter Two</b> outlines a detailed rationale for the experimental approach taken to (i) characterise protein expression in the pre-hypertensive animal model with immunohistochemistry and Western blotting, (ii) manipulate selected gene expression to amplify NO-cGMP signalling in vivo and in vitro via viral gene transfer, (iii) investigate calcium handling in cardiac sympathetic stellate neurons with calcium imaging , (iv) measure cardiac noradrenergic neurotransmission from double atria using radioactive-labelled [³H]-noradrenaline. <b>Chapter Three</b> demonstrated abnormal NO-cGMP signalling in pre-hypertensive SHRs. Endogenous nNOS protein residing in both cardiac parasympathetic and sympathetic neurons was significantly lower in the pre-hypertensive SHR compared to aged-matched WKYs. This was associated with lower cGMP levels. An enhanced depolarization evoked [Ca²⁺]i transient was observed in cardiac stellate neurons from pre-hypertensive SHR when compared with the WKY, an effect that was reversed by nNOS or sGC inhibition. <b>Chapter Four</b> investigated the role of nNOS and brain natriuretic peptide (BNP) in cGMP signalling pathways. Gene transfer of nNOS via adenoviral vector in SHR cardiac sympathetic neurons increased cGMP concentration and normalised neuronal calcium handling during depolarization. BNP significantly reduces [3H]- noradrenaline release. Overexpression of PDE2 which facilitates the breakdown of cGMP caused an increase in [³H]- noradrenaline release in response to field stimulation and also prevented the inhibitory action of BNP. <b>Chapter Five</b> examined the role of the nNOS adaptor protein, CAPON in NO-cGMP signalling. Endogenous CAPON protein is present in cardiac sympathetic neurons in the WKY, and is significantly reduced in pre-hypertensive SHR cardiac neurons. Artificial up-regulation of cardiac sympathetic CAPON via targeted gene transfer directly attenuated neuronal Ca²⁺ transients, resulting in decreased noradrenaline release in the SHR. <b>Chapter Six</b> is a concluding discussion summarising the main findings from this thesis, placing them in a physiological context and discussing avenues for further research.

O cérebro não atinge sua completa maturidade até a idade adulta, tornando os adolescentes especialmente vulneráveis aos efeitos do estresse. Nesta etapa da vida, o bullying é um fator de risco que pode levar ao desenvolvimento da depressão, no entanto, não está claro porque alguns indivíduos são mais suscetíveis que outros. O óxido nítrico (NO), uma importante molécula sinalizadora no organismo, é sintetizada principalmente pela enzima óxido nítrico sintase neuronal (nNOS) no sistema nervoso central. Estudos sugerem que o NO e a nNOS poderiam desempenhar um importante papel na fisiopatologia da depressão, no entanto, ainda não foram realizadas pesquisas sobre a participação da nNOS no fenômeno da resiliência e suscetibilidade à depressão no período da adolescência. Por tanto, o objetivo deste trabalho foi estudar os efeitos da subjugação social prolongada em camundongos machos adolescentes C57BL/6 resilientes e suscetíveis a este tipo de estresse sobre comportamentos emocionais, bem como sobre a expressão gênica, proteica e a atividade enzimática da nNOS em algumas regiões cerebrais. Nossos resultados mostraram que alguns animais expostos ao estresse social prolongado desenvolveram esquiva social (56,7%) no teste de interação social e anedonia (50%) no teste de preferência por sacarose. Estes animais foram denominados suscetíveis. No entanto, outra parcela de animais não mostraram estas alterações comportamentais e foram denominados resilientes. Nossas análises moleculares mostraram que somente os camundongos resilientes apresentaram uma diminuição na expressão proteica e gênica da nNOS no hipocampo (HC) e no córtex pré-frontal (CPF), áreas comumente relacionadas com a depressão. Os camundongos suscetíveis apresentaram valores semelhantes aos controle nessas áreas cerebrais. Curiosamente, o estriado dorsal (ED), área utilizada como controle negativo, apresentou alterações na expressão gênica, proteica e na atividade enzimática nos animais resilientes e suscetíveis. Adicionalmente, foram analisadas as concentrações séricas dos nitratos e nitritos sistêmicos (NOx). Os camundongos resilientes apresentaram maiores concentrações destes metabolitos quando comparado com os animais suscetíveis. Em conclusão, nossos dados mostram que o estresse social prolongado é capaz de induzir comportamentos anedônicos e de esquiva social em camundongos machos adolescentes. As análises moleculares indicam que a resiliência a este tipo de estresse está associada à diminuição gênica e proteica da nNOS no HC e no CPF e ao aumento dos níveis séricos dos NOx. Além disso, as alterações moleculares no ED estariam sugerindo um papel para essa área na resposta do organismo frente ao estresse social. / The brain does not reach full maturity until adulthood, making adolescents especially vulnerable to the effects of stress. At this period of life, bullying is a risk factor for developing depression; however, it is unclear why some individuals are more susceptible than others. Nitric oxide (NO), an important neurotransmitter in the organism, is mainly synthetized by the neuronal NO synthase (nNOS) in the central nervous system. Several studies suggest that NO and nNOS can play an important role in the pathophysiology of depression, nevertheless, no research has been done yet on the participation of nNOS in the phenomenon of resilience and susceptibility to depression during adolescence. Therefore, the objective of this work was to study the effects of prolonged social defeat in adolescent male mice C57BL/6 resilient and susceptible to this type of stress on emotional behaviors, as well as on the gene expression, protein and enzymatic activity of nNOS in some brain regions. Our results showed that some animals exposed to prolonged social stress developed social approach-avoidance (56.7%) in social interaction test and anhedonia (50%) in sucrose preference test. These animals were called susceptible. However, remaining animals did not show these behavioral alterations and they were classified as resilient. Our molecular tests showed that resilient animals had a reduction nNOS gene and protein expression in hippocampus (HC) and prefrontal cortex (PFC), areas commonly related with depression. Susceptible mice displayed similar data to controls in these brain areas. Interestingly, dorsal striatum (DS), brain area used as negative control, showed alterations in gene and protein expression and enzymatic activity in resilient and susceptible mice. Additionally, serum nitrate and nitrite (NOx) concentration were evaluated. Resilient mice displayed increase of systemic NOx levels when compared with susceptible animals. Concluding, our data show that prolonged social defeat stress is capable of inducing anhedonic and social approach-avoidance behaviors in adolescent male mice. Molecular tests suggest that resilience to this type of stress is associated with reduction of nNOS gene and protein expression in HC and PFC and with increase of serum NOx levels. Furthermore, molecular alterations in DS would be suggesting a role for this area in organism response to social stress.

Introdução: O óxido nítrico (NO) tem sido relacionado como um importante neuromodulador envolvido com a neurobiologia da adaptação ao estresse e da depressão. De fato, a administração sistêmica ou intra-hipocampal de inibidores da NO sintase neuronial (nNOS) induz efeitos do tipo antidepressivo em modelos animais. Evidências recentes indicam que os efeitos sistêmicos dos inibidores da nNOS são dependentes dos níveis de serotonina no encéfalo. O sistema serotoninérgico do hipocampo dorsal, por ativação dos receptores serotoninérgicos do tipo 1A (5HT1A), facilita a adaptação ao estresse e contribui para os efeitos comportamentais de drogas antidepressivas. Portanto, o objetivo do presente estudo foi testar a hipótese de que o efeito do tipo antidepressivo induzido pela administração hipocampal de inibidores da nNOS seria mediado pela facilitação da neurotransmissão serotoninérgica local e subseqüente ativação de 5HT1A. Métodos: Após cirurgia estereotáxica, ratos Wistar com cânulas-guia direcionadas ao hipocampo dorsal foram submetidos a sessão de pré-teste (PT 15 minutos de nado) e receberam administrações locais das drogas: N-propil-L-arginina (NPA, inibidor seletivo da nNOS: 0,00001 - 1 nmol/0,5 µL), fluoxetina (SSRI: 1, 3 e 10 nmol/0,5 µL), WAY100635 (antagonista seletivo para 5HT1A: 1, 3 e 10 nmol/0,5 µL) ou veículo (0,5 µL). 24h depois, o tempo de imobilidade foi registrado em uma sessão de 5 minutos de nado. Todos os protocolos foram aprovados por um comitê de ética local (Proc. No 08.1.1133.53.4) Resultados: A administração intra hipocampal de NPA ou fluoxetina reduziu significativamente o tempo de imobilidade em animais submetidos ao teste do nado forçado, um efeito tipo antidepressivo. A administração de WAY100635 não induziu efeito por si, mas foi capaz de bloquear os efeitos induzidos por fluoxetina ou NPA. Conclusões: A inibição da nNOS, pelo NPA, ou a inibição da recaptação de serotonina, pela fluoxetina, no DH induziu efeito do tipo antidepressivo de similar magnitude. O fato de que o pré-tratamento com WAY100635 foi capaz de bloquear os efeitos induzidos por NPA e fluoxetina indica que ambos os efeitos são mediados por facilitação da neurotransmissão serotoninérgica local e subseqüente ativação de 5HT1A. Assim, esses resultados sugerem que níveis aumentados de NO no DH poderiam levar a um déficit na neurotransmissão serotoninérgica local e, portanto, predispor ao desenvolvimento das conseqüências comportamentais do estresse. / Introduction: Nitric oxide (NO) has been suggested to play an important role in the neurobiology of stress adaptation and depression. In fact, systemic or hippocampal administration of neuronal NO synthase (nNOS) inhibitors induces antidepressant-like effects in animal models. Recent evidence indicates that the systemic effects of nNOS inhibitors are dependent on serotonin levels in the brain. The serotonergic system of the dorsal hippocampus (DH), through the activation of serotonin 1A (5-HT1A) receptors, is proposed to mediate stress adaptation and the behavioral effects of antidepressant drugs. Therefore, the aim of the present study was to test the hypothesis that the antidepressant-like effects induced by nNOS inhibition into the hippocampus would be mediated by a facilitation of the local serotonergic neurotransmission and subsequent 5-HT1A receptor activation. Methods: Male Wistar rats with guide-cannulae aimed at the DH were submitted to the pretest session (PT - 15 minutes of swimming) and received local administrations of the drugs: n-propyl-L-arginine (NPA, selective nNOS inhibitor: 0.00001 - 1 nmol/0.5 µL), fluoxetine (SSRI: 1, 3 and 10 nmol/0.5 µL), WAY100635 (selective 5-HT1A antagonist: 1, 3 and 10 nmol/0.5 µL) or vehicle (0.5 µL). One day later, the immobility time was registered at a 5 minutes swimming test session. All protocols were approved by a local ethical committee (Proc. N. 08.1.1133.53.4.) Results: The intrahippocampal administration of NPA or fluoxetine reduced the immobility time in animals submitted to the forced swimming test, an antidepressant-like effect in this model. WAY100635 did not induce any effect per se, but it was able to block the effects induced by fluoxetine or NPA. Conclusions: Inhibition of nNOS, by NPA, or inhibition of serotonin reuptake, by fluoxetine, in the DH induced antidepressant-like effects of similar magnitude. The fact that pretreatment with WAY100635 was able to block NPA- and fluoxetine-induced effects indicates that both effects are mediated by a facilitation of the local serotonergic neurotransmission and subsequent activation of 5-HT1A receptors. Therefore, these results suggest that increased NO levels in the DH could impair local serotonergic neurotransmission and, thus, predisposes to the development of stress-induced behavioral consequences, such as depressive-like behaviors.

Introdução: O óxido nítrico (NO) tem sido relacionado como um importante neuromodulador envolvido com a neurobiologia da adaptação ao estresse e da depressão. De fato, a administração sistêmica ou intra-hipocampal de inibidores da NO sintase neuronial (nNOS) induz efeitos do tipo antidepressivo em modelos animais. Evidências recentes indicam que os efeitos sistêmicos dos inibidores da nNOS são dependentes dos níveis de serotonina no encéfalo. O sistema serotoninérgico do hipocampo dorsal, por ativação dos receptores serotoninérgicos do tipo 1A (5HT1A), facilita a adaptação ao estresse e contribui para os efeitos comportamentais de drogas antidepressivas. Portanto, o objetivo do presente estudo foi testar a hipótese de que o efeito do tipo antidepressivo induzido pela administração hipocampal de inibidores da nNOS seria mediado pela facilitação da neurotransmissão serotoninérgica local e subseqüente ativação de 5HT1A. Métodos: Após cirurgia estereotáxica, ratos Wistar com cânulas-guia direcionadas ao hipocampo dorsal foram submetidos a sessão de pré-teste (PT 15 minutos de nado) e receberam administrações locais das drogas: N-propil-L-arginina (NPA, inibidor seletivo da nNOS: 0,00001 - 1 nmol/0,5 µL), fluoxetina (SSRI: 1, 3 e 10 nmol/0,5 µL), WAY100635 (antagonista seletivo para 5HT1A: 1, 3 e 10 nmol/0,5 µL) ou veículo (0,5 µL). 24h depois, o tempo de imobilidade foi registrado em uma sessão de 5 minutos de nado. Todos os protocolos foram aprovados por um comitê de ética local (Proc. No 08.1.1133.53.4) Resultados: A administração intra hipocampal de NPA ou fluoxetina reduziu significativamente o tempo de imobilidade em animais submetidos ao teste do nado forçado, um efeito tipo antidepressivo. A administração de WAY100635 não induziu efeito por si, mas foi capaz de bloquear os efeitos induzidos por fluoxetina ou NPA. Conclusões: A inibição da nNOS, pelo NPA, ou a inibição da recaptação de serotonina, pela fluoxetina, no DH induziu efeito do tipo antidepressivo de similar magnitude. O fato de que o pré-tratamento com WAY100635 foi capaz de bloquear os efeitos induzidos por NPA e fluoxetina indica que ambos os efeitos são mediados por facilitação da neurotransmissão serotoninérgica local e subseqüente ativação de 5HT1A. Assim, esses resultados sugerem que níveis aumentados de NO no DH poderiam levar a um déficit na neurotransmissão serotoninérgica local e, portanto, predispor ao desenvolvimento das conseqüências comportamentais do estresse. / Introduction: Nitric oxide (NO) has been suggested to play an important role in the neurobiology of stress adaptation and depression. In fact, systemic or hippocampal administration of neuronal NO synthase (nNOS) inhibitors induces antidepressant-like effects in animal models. Recent evidence indicates that the systemic effects of nNOS inhibitors are dependent on serotonin levels in the brain. The serotonergic system of the dorsal hippocampus (DH), through the activation of serotonin 1A (5-HT1A) receptors, is proposed to mediate stress adaptation and the behavioral effects of antidepressant drugs. Therefore, the aim of the present study was to test the hypothesis that the antidepressant-like effects induced by nNOS inhibition into the hippocampus would be mediated by a facilitation of the local serotonergic neurotransmission and subsequent 5-HT1A receptor activation. Methods: Male Wistar rats with guide-cannulae aimed at the DH were submitted to the pretest session (PT - 15 minutes of swimming) and received local administrations of the drugs: n-propyl-L-arginine (NPA, selective nNOS inhibitor: 0.00001 - 1 nmol/0.5 µL), fluoxetine (SSRI: 1, 3 and 10 nmol/0.5 µL), WAY100635 (selective 5-HT1A antagonist: 1, 3 and 10 nmol/0.5 µL) or vehicle (0.5 µL). One day later, the immobility time was registered at a 5 minutes swimming test session. All protocols were approved by a local ethical committee (Proc. N. 08.1.1133.53.4.) Results: The intrahippocampal administration of NPA or fluoxetine reduced the immobility time in animals submitted to the forced swimming test, an antidepressant-like effect in this model. WAY100635 did not induce any effect per se, but it was able to block the effects induced by fluoxetine or NPA. Conclusions: Inhibition of nNOS, by NPA, or inhibition of serotonin reuptake, by fluoxetine, in the DH induced antidepressant-like effects of similar magnitude. The fact that pretreatment with WAY100635 was able to block NPA- and fluoxetine-induced effects indicates that both effects are mediated by a facilitation of the local serotonergic neurotransmission and subsequent activation of 5-HT1A receptors. Therefore, these results suggest that increased NO levels in the DH could impair local serotonergic neurotransmission and, thus, predisposes to the development of stress-induced behavioral consequences, such as depressive-like behaviors.

La dystrophine est une très grande protéine codée le gène DMD et située sous la membrane plasmique des fibres musculaires. Elle joue un rôle essentiel dans le maintien de l’intégrité de la cellule musculaire lors des cycles de contraction/relaxation. Cette protéine filamenteuse est composée de quatre domaines structuraux dont le domaine central composé de 24 répétitions homologues à la spectrine. Chaque répétition est organisée en faisceau de trois α-hélices appelé « coiled-coil ». Des mutations du gène DMD sont à l’origine des myopathies de Duchenne (DMD) et de Becker (BMD) qui s’accompagnent d’un déficit total ou d’une dystrophine mutée et induisent de ruptures fréquentes de la membrane des cellules musculaires. La connaissance de la structure de la dystrophine est nécessaire au développement de thérapies à ce jour inexistantes pour les myopathies. Au laboratoire, des données structurales du domaine central de la dystrophine ont été acquises par diffusion des rayons X aux petits angles (SAXS, Small Angles X-ray Scattering). Cette thèse présente le développement d’une approche multi-échelle combinant des données expérimentales SAXS et des données in silico pour la reconstruction de modèles haute-résolution des fragments du domaine central de la dystrophine et d’un fragment muté observé dans une mutation BMD fréquente. Nous avons également cartographié l’interaction de ce domaine central avec deux de ses partenaires fonctionnels importants, l’actine filamenteuse et avec la nitroxyde synthase neuronale (nNOS) et proposé les premiers modèles atomiques des complexes macromoléculaires correspondants. L’ensemble de ces résultats permettra à terme l’optimisation de thérapies pour le traitement des dystrophies musculaires. / Dystrophin is a large protein encoded by DMD gene and located under the plasma membrane of muscle fibers. It plays an essential role in maintaining the integrity of muscle cells during contraction/relaxation cycles. This filamentous protein is composed of four structural domains including the central domain consisting of 24 spectrin-like repeats and four hinges. Each repetition is folded in three α-helices in a ‘coiled-coil’ assembly. Mutations in the DMD gene leads to Duchenne muscular dystrophy (DMD) and Becker (MDBs), which are accompanied by frequent plasma membrane ruptures, due to the loss or modification of dystrophin protein. There are very few structural data available concerning the central domain of dystrophin, which is subject to many mutations involved in DMD and BMD diseases. However, the description and the understanding to an atomic level of dystrophin structure and its interaction is essential for optimization of therapies. Given the impossibility to solve its structure by X-ray crystallography or NMR, structural data of the dystrophin central domain were acquired by small angles X-rays scattering (SAXS, Small Angles X-ray Scattering). This thesis presents the development of an innovative multi-scale approach combining experimental SAXS and in silico derived data, allowing the reconstruction of high-resolution models of dystrophin central domain fragments. Structural data were also obtained on a mutated dystrophin frequently observed in BMDs. Furthermore, we also mapped the interactions of the central domain with two of its majors functional partners, Filamentous actin and neuronal nitroxyde synthase (nNOS) and proposed models of the related macromolecular complexes. At long-term, all of these results will allow optimization of therapies for the treatment of muscular dystrophies.

Name of the thesis Effect of nitric oxide on cerebral blood flow during neuronal activity Aim of the thesis The aim of this thesis is to determine whether the application of 7-nitroindazole, relatively specific inhibitor of neuronal nitric oxide synthase, affects the baseline blood pressure. Furthermore, to determine whether the application of the substance affects the baseline cerebral blood flow and whether it influences blood flow in brain during transcallosal stimulation with increasing frequency. Research method The research took place at the premises of the Institute of Physiology, Academy of Sciences of the Czech Republic. Experiments were carried out on laboratory albino Wistar rats. The group contained both experimental and control sample. General anesthesia was performed to rats, stimulating and sensing electrodes were implanted in epidural area of sensorimotor cortex and Laser Doppler flow probe was implanted into the contralateral hemisphere. A plastic catheter was applied in the carotid artery for measuring systemic blood pressure. In the first part of the experiment, we tested the effects of 7-nitroindazole on the systemic blood pressure. In the second part of the experiment, we investigated the effects of 7-nitroindazole on baseline cerebral blood flow. The third part of the...

Objectives. Experimental stroke studies have shown that endothelial (eNOS) and neuronal (nNOS) nitric oxide synthase isoforms exhibit opposite effects on brain injury. nNOS has been identified recently in endothelial cells, however, its functional significance is unclear. Our objective was to identify the nNOS in brain microvascular endothelial cells (BMECs) and characterize its functional role. Methods and Results. Primary BMECs from humans (hBMECs) and rats (rBMECs) were used in our studies. Immunocytochemistry identified von Willebrand factor, eNOS, and nNOS in hBMECs. Western blot analysis using antibodies targeting N-terminal domain of nNOS revealed an approximately 130 kD immunoband of a potential nNOS splice variant in hBMECs as opposed to 160 kD nNOS specific immunoband in cultured rat cortical neurons. In contrast, antibodies targeting C-terminal domain of nNOS failed to show nNOS specific immunoband. PCR experiments using the species specific primers identified the mRNA of nNOS in hBMECs. Electron Spin Resonance (ESR) spectroscopy revealed reduction of superoxide levels in hBMECs by two structurally different nNOS inhibitors, (N-ω-Propyl-L-arginine; NPA and ARL-17477), compared with vehicle (ethanol) treated cells. In contrast, treatment with eNOS inhibitor (L-N5-(1-Iminoethyl) ornithine; NIO) significantly enhanced the superoxide levels in hBMECs compared with vehicle (DMSO) treated cells. Supplementation of tetrahydrobiopterin (BH4) resulted in reduced superoxide levels in the hBMECs whereas BH4 co-treatment had no effect on the superoxide levels in the NPA or ARL-17477 treated hBMECs. NO measurements in hBMECs by ESR spectroscopy showed greatly diminished magnitude of the NO signal by the treatment with NIO, compared to vehicle (DMSO) treatment, and whereas, treatment with NPA enhanced NO signal intensity compared to vehicle (ethanol) treated cells. Conclusions. We identified a constitutively active nNOS splice variant in hBMECs that is distinct from the nNOS expressed in neurons. In addition, we found that uncoupled nNOS, significantly contributes to basal superoxide generation in hBMECs that reduces the NO bioavailability. In contrast, eNOS is the only source of NO produced by the hBMECs. We conclude that hBMECs express a unique nNOS isoform distinct from the nNOS expressed in neurons and also exhibiting effects which are distinctly opposite of eNOS. / acase@tulane.edu

Myoclonus Dystonia (MD) is an autosomal dominant movement disorder characterized by bilateral myoclonic jerks paired with dystonia 1. Mutations have been mapped to the ε-sarcoglycan (SGCE) gene in about 40% of patients 2,92. The purpose of this project was to examine the properties of SGCE in the central nervous system (CNS) and use this knowledge to elucidate the pathology of MD. Although Sgce is a member of the sarcoglycan complex (SGC) in other tissues, little is known about its interactions in the CNS. The vast majority of mutations in SGCE alter the translational reading frame. Proteins arising from these rare mutations are less stable than the wild type (WT) and undergo preferential degradation via the ubiquitin proteasome system 3. As this locus is maternally imprinted, patients with MD are effectively null for sgce expression 73,91. Therefore, Sgce knock out (KO) models should approximate MD conditions both in vivo and in vitro. As there are no current treatments for MD, in sight into the pathology of the disease will aid in eventual treatments and help bring patients some relief by finally understanding their disease. Since a large percentage of MD patients are without the sgce protein, identifying what this protein’s function is and how its absence effects normal processing in the brain should help to identify the underlying cellular pathology which produces the MD phenotype. This research was performed under the hypothesis that, in neuronal cells, sgce interacts with a group of proteins that together play a role in stabilization and localization of ion channels and signaling proteins at the cell membrane. The aims were to: (1) Build a MD mouse model with either a conditional knock-out (cKO) or a conditional gene repair (cGR) mutation; (2) Use neuroblastoma cells to identify the other proteins which interact with sgce in neurons, and; (3) Determine if there is a disruption of the localization of the sgce-complex members due to the loss of sgce. Recombineering was used to complete the constructs for two transgenic mouse models: One model for the KO of exon 4 of sgce and one for the cGR in intron 1. Primary neurosphere lines from two previously generated chimeras were developed, as well as from a WT mouse. These neurosphere cell lines allowed comparisons of RT-PCR results from a heterogeneous neurological cell population to neuroblastoma cell lines. mRNA is present in neuronal cells for many of the DGC associated proteins. It was confirmed that the KD of sgce results in a reduction of nNOS protein and in increased proliferation of NIE cells. By using a nitrite/nitrate assay as well as studies with L-NAME, it was confirmed that this increased proliferation was in fact due to a lack of nNOS function. These proliferation changes did not occur in N2A cells, which do not express high levels of nNOS during proliferation, further confirming nNOS’s role in the proliferation changes. Using qRT-PCR, KD of sgce was shown to result in significant changes in the transcript levels for many DGC associated proteins. This suggests that a DGC-like complex is forming in neuronal cells. Also, as a result of difficulties with the research, it became clear that over-expression of sgce causes cell death. This observation was quantified using cell counts and TUNEL staining, both showing significant results. Additionally, several new constructs were created which will hopefully be of use for future students wanting to study sgce’s functions. New shRNA targeting sgce and sgcb have been made and both constructs result in reducing the expression of sgce. Seven different flag-tagged sgces have been created and some of these have been transferred into a tet-inducible system, which should circumvent the problem of over-expression. Finally GFP-tagged constructs for sgce and sgcb have been made and pooled clones have been developed. These tools will hopefully enable future students to continue to tease apart sgce’s function(s).

Myoclonus Dystonia (MD) is an autosomal dominant movement disorder characterized by bilateral myoclonic jerks paired with dystonia 1. Mutations have been mapped to the ε-sarcoglycan (SGCE) gene in about 40% of patients 2,92. The purpose of this project was to examine the properties of SGCE in the central nervous system (CNS) and use this knowledge to elucidate the pathology of MD. Although Sgce is a member of the sarcoglycan complex (SGC) in other tissues, little is known about its interactions in the CNS. The vast majority of mutations in SGCE alter the translational reading frame. Proteins arising from these rare mutations are less stable than the wild type (WT) and undergo preferential degradation via the ubiquitin proteasome system 3. As this locus is maternally imprinted, patients with MD are effectively null for sgce expression 73,91. Therefore, Sgce knock out (KO) models should approximate MD conditions both in vivo and in vitro. As there are no current treatments for MD, in sight into the pathology of the disease will aid in eventual treatments and help bring patients some relief by finally understanding their disease. Since a large percentage of MD patients are without the sgce protein, identifying what this protein’s function is and how its absence effects normal processing in the brain should help to identify the underlying cellular pathology which produces the MD phenotype. This research was performed under the hypothesis that, in neuronal cells, sgce interacts with a group of proteins that together play a role in stabilization and localization of ion channels and signaling proteins at the cell membrane. The aims were to: (1) Build a MD mouse model with either a conditional knock-out (cKO) or a conditional gene repair (cGR) mutation; (2) Use neuroblastoma cells to identify the other proteins which interact with sgce in neurons, and; (3) Determine if there is a disruption of the localization of the sgce-complex members due to the loss of sgce. Recombineering was used to complete the constructs for two transgenic mouse models: One model for the KO of exon 4 of sgce and one for the cGR in intron 1. Primary neurosphere lines from two previously generated chimeras were developed, as well as from a WT mouse. These neurosphere cell lines allowed comparisons of RT-PCR results from a heterogeneous neurological cell population to neuroblastoma cell lines. mRNA is present in neuronal cells for many of the DGC associated proteins. It was confirmed that the KD of sgce results in a reduction of nNOS protein and in increased proliferation of NIE cells. By using a nitrite/nitrate assay as well as studies with L-NAME, it was confirmed that this increased proliferation was in fact due to a lack of nNOS function. These proliferation changes did not occur in N2A cells, which do not express high levels of nNOS during proliferation, further confirming nNOS’s role in the proliferation changes. Using qRT-PCR, KD of sgce was shown to result in significant changes in the transcript levels for many DGC associated proteins. This suggests that a DGC-like complex is forming in neuronal cells. Also, as a result of difficulties with the research, it became clear that over-expression of sgce causes cell death. This observation was quantified using cell counts and TUNEL staining, both showing significant results. Additionally, several new constructs were created which will hopefully be of use for future students wanting to study sgce’s functions. New shRNA targeting sgce and sgcb have been made and both constructs result in reducing the expression of sgce. Seven different flag-tagged sgces have been created and some of these have been transferred into a tet-inducible system, which should circumvent the problem of over-expression. Finally GFP-tagged constructs for sgce and sgcb have been made and pooled clones have been developed. These tools will hopefully enable future students to continue to tease apart sgce’s function(s).