Pain is a major public health issue throughout the world. Increased understanding of the different forms of pain and identification of susceptibility genes could contribute to improved treatments. The main aims of this thesis were to identify the underlying genetic causes of pain by studying two large families affected with migraine and pain insensitivity, respectively. Migraine is one of the most common neurovascular disorders, affecting over 12% of the western population. The genetic contribution to migraine is about 50% according to family and twin studies. To identify novel susceptibility loci for migraine, we performed a genome-wide screen in a large family with migraine from northern Sweden. Linkage analysis revealed significant evidence of linkage (LOD=5.41) on chromosome 6p12.2-p21.1. A predisposing haplotype spanning 10 Mb was inherited with migraine in all affected members of the pedigree. Further fine-mapping of multiple SNP markers restricted the disease critical region to 8.5 Mb. Nine candidate genes were sequenced, revealing no disease-associated polymorphisms in SLC29A1, CLIC5, PLA2G7, IL17, SLC25A27 and TNFRSF21, but rare novel polymorphisms segregating with the disease haplotype in EFHC1, RHAG and MEP1A. EFHC1 has recently been shown to be involved in epilepsy, which is interesting considering the link between migraine and epilepsy. However, association analysis of EFHC1 revealed no difference between patients and controls, suggesting that this gene is not a risk factor for migraine. The combination of the two polymorphisms in RHAG and MEP1A could, however, not be found in any control individuals, indicating that they might be involved in genetic predisposition to migraine in this family. Disorders with reduced pain sensitivity are very rare, since pain perception is essential for survival. A number of disorders have still been identified with pain insensitivity and peripheral nerve degeneration as major clinical signs, including the hereditary sensory and autonomic neuropathies (HSAN). In order to identify novel susceptibility genes for HSAN V, we performed a genome-wide screen in a large consanguineous pedigree from a small village in northern Sweden. A homozygous region identical-by-descent was identified on chromosome 1p11.2-p13.2 in the three most severely affected patients. Subsequent analysis of candidate genes revealed a missense mutation in a conserved region of the nerve growth factor beta (NGFB) gene, causing a drastic amino acid change (R211W) in the NGF protein. NGF is important for the development and maintenance of the sympathetic and sensory nervous system and is therefore likely to be involved in disease. Functional analysis revealed that mutant NGF failed to induce neurite outgrowth and cell differentiation in PC12 cells. Furthermore, almost no mutant NGF was secreted by COS-7 cells, indicating that the processing and/or secretion of the protein might be disrupted. In conclusion, these findings present a novel migraine locus on chromosome 6 and identification of two rare polymorphisms that might be risk factors for migraine. Furthermore, a mutation in NGFB was found to cause complete loss of deep pain perception, which represents a very interesting model system to study pain mechanisms.

Teneurin C-terminal associated peptide-1 (TCAP-1) is a recently characterized peptide that may act as one potential neuroprotective agent as it has been shown to regulate several stress-associated behaviours in rodents and possesses a number of protective actions on cells, however the mechanism remains unknown. Brain-derived neurotrophic factor (BDNF) is a neurotrophin recognized for mediating survival, differentiation, and proliferation. TCAP-1 may act, in part, via BDNF to provide neuroprotection via modulation of BDNF expression. The aim of this research was to further investigate the mechanism of TCAP’s neuroprotective actions. I show that TCAP-1 is neuroprotective and a potent enhancer of cell numbers under varying levels of oxygen. I also establish that TCAP-1 is able to influence neuronal behaviour by differentially regulating neurite growth. In addition, I indicate that TCAP-1 is able to regulate BDNF expression in immortalized mouse hypothalamic N38 cells, which suggests that TCAP-1’s neuroprotective mechanism may involve BDNF.

Teneurin C-terminal associated peptide-1 (TCAP-1) is a recently characterized peptide that may act as one potential neuroprotective agent as it has been shown to regulate several stress-associated behaviours in rodents and possesses a number of protective actions on cells, however the mechanism remains unknown. Brain-derived neurotrophic factor (BDNF) is a neurotrophin recognized for mediating survival, differentiation, and proliferation. TCAP-1 may act, in part, via BDNF to provide neuroprotection via modulation of BDNF expression. The aim of this research was to further investigate the mechanism of TCAP’s neuroprotective actions. I show that TCAP-1 is neuroprotective and a potent enhancer of cell numbers under varying levels of oxygen. I also establish that TCAP-1 is able to influence neuronal behaviour by differentially regulating neurite growth. In addition, I indicate that TCAP-1 is able to regulate BDNF expression in immortalized mouse hypothalamic N38 cells, which suggests that TCAP-1’s neuroprotective mechanism may involve BDNF.

The imidazoline I1 receptor (I1-R) is a novel receptor found primarily in the brain and nervous tissue where it modulates neurotransmission. It is named for its high affinity for compounds with an imidazoline structure such as the anti-hypertensive drugs, clonidine and moxonidine. The imidazoline receptor antisera-selected protein (IRAS) is the putative clone of the I1-R. IRAS has a unique structure, which does not resemble any other receptor protein. IRAS is present throughout the body with highest levels in the brain. There is a growing body of research examining the physiological roles of IRAS as an I1-R, in cell survival, migration and protein trafficking. However, there is little research into its neuronal functions. IRAS interacts with other membrane receptors: the mouse homologue of IRAS reorganises the actin cytoskeleton through interaction with the α5β1 fibronectin receptor. IRAS also binds insulin receptor substrate 4 and enhances insulin-induced extracellular signal-regulated kinase1/2 (ERK1/2) activation. Actin reorganisation and ERK1/2 activation are important for the development of neurites during neuronal differentiation. Therefore, the work described in this thesis aimed to investigate the effects of IRAS on neuronal differentiation. Studies reported in this thesis also aimed to investigate whether IRAS affected ERK1/2 signalling of other receptors involved in neuronal differentiation such as the NGF receptor, TrkA, and lysophospholipid receptors. The above aims were carried out in neuronal model PC12 cells transfected with either IRAS or a vector plasmid. Fluorescence microscopy and Western blotting techniques were used to examine the effect of IRAS on cell morphology and ERK1/2 signalling. The work described in this thesis found that IRAS reorganises the actin cytoskeleton and enhances growth cone development in PC12 cells. This study also shows that IRAS differentially enhances or inhibits NGF-induced PC12 cell differentiation depending on the presence or absence of serum in the media. In full-serum conditions, IRAS enhanced neurite outgrowth and this was accompanied by an increase in ERK1/2 activation. In serum-starved cells, IRAS inhibited neurite outgrowth with similar levels of ERK1/2 activation observed in vector- and IRAS-transfected cells. Finally, studies presented in this thesis found that IRAS enhances lysophosphatidic acid-induced ERK1/2 activation and that IRAS interacting with lysophospholipid receptor agonists present in serum is a potential mechanism by which it enhances NGF-induced ERK1/2 activation in full-serum conditions. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1345359 / Thesis (Ph.D.) - University of Adelaide, School of Medical Sciences, 2008

La croissance neuritique est un mécanisme complexe qui fait toujours l’objet d’intenses investigations. Les donnés actuelles ont permis de mettre en évidence l’implication de trois mécanismes principaux dans la croissance neuritique : i) la dynamique du cytosquelette, ii) le trafic intracellulaire et l’apport membranaire au niveau du cône de croissance et iii) la signalisation cellulaire, principalement via la voie MAPK-ERK1/2, qui abouti à la régulation de la transcription.La PLD1 et son produit l’acide phosphatidique semblent être au centre de voies majeures impliquées dans le développement neuronal. Mes travaux ont permis d’approfondir nos connaissances sur le rôle cellulaire de la PLD1 au cours de la croissance neuritique. J’ai montré que la PLD1 en collaboration avec la kinase RSK2 régule la fusion des vésicules positives pour Ti-VAMP/VAMP7 au cours de la croissance neuritique. D’autre part, j’ai établi que la PLD1 joue un rôle important dans le maintien de la signalisation endosomale de la voie MAPK-ERK1/2-RSK2-CREB induite par les neurotrophines. J’ai également montré que la PLD1 régule l’activation de mTOR/p70S6K en réponse au BDNF. La dérégulation des voies MAPK-ERK1/2 et mTOR/p70-S6K pourraient être à la base de la réduction de l’arborisation dendritique et de la maturation des épines dendritique observée dans les neurones corticaux Pld1-/- en culture. En plus de l’implication de RSK2 dans la régulation de la PLD1, j’ai également montré que la PLD1 régule l’activation de RSK2 en réponse aux neurotrophines, probablement via une boucle de rétrocontrôle. Ainsi les donnés obtenus suggèrent un lien fort entre les deux protéines au cours du développement neuronal. A la lumière de ces donnés, un dysfonctionnement de ce mécanisme pourrait expliquer le retard mental observé chez les patients atteints du syndrome de Coffin-Lowry causé par la perte de l’activité kinase de RSK2. D’autre part, les résultats obtenus suggerent un rôle de la PLD1 dans l’exocytose des vésicules. / Neurite outgrowth is a complex mechanism that is still the subject of intense investigation. Current given helped to highlight the involvement of three main mechanisms in neurite growth : i) the dynamics of the cytoskeleton, ii) the intracellular membrane trafficking and membrane supply at the growth cone and iii) cell signaling , mainly via the MAPK-ERK1 / 2, which resulted in the regulation of transcription. The PLD1 and its product the phosphatidic acid (PA) appear to be at the center of the major pathways involved in neuronal development. My work has deepened our understanding of the cellular role of PLD1 during neurite outgrowth. I showed that PLD1 together with the protein kinase RSK2 regulates the fusion of vesicles positive for Ti-VAMP/VAMP7 during neurite outgrowth. On the other hand, I have determined that PLD1 plays an important role in maintaining the endosomal signaling pathwayMAPK-ERK1/2-RSK2-CREB induced by neurotrophin. I also showed that PLD1 regulates the activation of mTOR/p70S6K in response to BDNF. Deregulation of MAP -ERK1 / 2 and mTOR/p70-S6K pathways could be the basis for the reduction of dendritic arborization and maturation of dendritic spines observed in cortical neurons Pld1-/- culture. In addition to the involvement of RSK2 in the regulation of PLD1, I also showed that PLD1 regulates RSK2 activation in response to neurotrophin, possibly via a feedback loop. Thus given obtained suggest a strong link between the two proteins during neuronal development. In the light of these data, alteration of this mechanism could explain the mental retardation observed in patients with Coffin -Lowry syndrome caused by loss of the kinase activity of RSK2. On the other hand, our results suggest a role for PLD1 in exocytosis of vesicles.

Orientador: Leonilda Maria Barbosa dos Santos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-07T03:41:32Z (GMT). No. of bitstreams: 1 Castro_FabianoRobertode_M.pdf: 943470 bytes, checksum: 271c21deb3b25015f9737d33adf49814 (MD5) Previous issue date: 2006 / Resumo: A Síndrome de Guillain-Barré (SGB) é uma doença desmielinizante do sistema nervoso periférico (SNP). Baseado principalmente nas similaridades clínicas e histopatológicas a Neurite Experimental Auto-imune (EAN) tem sido extensivamente usada como modelo de estudo da SGB. A EAN é uma doença auto-imune, que pode ser experimentalmente induzida em ratos geneticamente suscetíveis através da imunização com os componentes da mielina de nervos periféricos tais como os peptídeos P0 e P2 , ou ainda por transferência adotiva de lifócitos T CD4+ do tipo Th1. Diferentes tentativas de tratamentos para a SGB têm sido estudadas, dentre elas pode-se citar a plasmaferese, o uso de anticorpos monoclonais, administração de corticóides e a imunossupressão global através da administração de intérferon ß. A utilização de venenos totais de serpentes, ou frações deles, já demonstrou bons resultados na tentativa de tratamento de alguns modelos de doenças auto-imunes como a diabetes auto-imune insulino dependente, lúpus e encefalomielite experimental auto-imune (EAE). No presente trabalho foi estudado o efeito de uma fração do veneno da cascavel sul americana Crotalus durissus terrificus (Cdt), a crotapotina, no modelo de EAN. São apresentadas evidências de que tanto a administração intraperitoneal (IP) como a oral de crotapotina reduz significativamente a gravidade da EAN induzida em ratos Lewis, associada a um significativo declínio na resposta proliferativa das células T neuritogênicas, assim como diminuição de infiltrados de células mononucleares no nervo ciático dos os animais / Abstract: Biomedical research in which venom components are being investigated for their potential as novel therapeutic agents has emerged as an interesting option. Crotapotin which is a fraction of the venom of the rattlesnake Crotalus durissus terrificus, has been described as an antinflammatory that acts on the innate arm of the immune response. Here we have demonstrated that intraperitoneal (IP), as well as oral administration of crotapotin significantly reduces the severity of experimental autoimmune neuritis (EAN), an experimental model for Guillain-Barré Syndrome. The reduction of the severity of the disease is associated with a reduction in the mononuclear cells infiltrating in the sciatic nerve and a significant decrease in the lymphocyte proliferative response to neuritogenic peptide / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-05-06T14:33:30Z No. of bitstreams: 1 rubensmurilogibailesoares.pdf: 2619186 bytes, checksum: a84a49cdc9fab5886335802ef1c22da4 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-06-07T16:00:23Z (GMT) No. of bitstreams: 1 rubensmurilogibailesoares.pdf: 2619186 bytes, checksum: a84a49cdc9fab5886335802ef1c22da4 (MD5) / Made available in DSpace on 2016-06-07T16:00:23Z (GMT). No. of bitstreams: 1 rubensmurilogibailesoares.pdf: 2619186 bytes, checksum: a84a49cdc9fab5886335802ef1c22da4 (MD5) Previous issue date: 2013-06-19 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / O modelo de Encefalomielite Autoimune Experimental (EAE) é o mais utilizado no estudo da neurite óptica. Este trabalho tem como objetivo avaliar a cinética da neurite óptica em modelo animal de EAE induzido por duas diferentes concentrações de Glicoproteína dos Oligodendrócitos da Mielina (MOG). Para a indução da EAE foram utilizadas fêmeas de camundongos da linhagem C57BL/6, divididas em dois grupos, um grupo induzido com 100 μg de MOG35-55 e um segundo grupo induzido com 300 μg de MOG35-55. Os animais foram diariamente avaliados por meio da análise do escore clínico entre os dias zero e 58 pós-imunização. Nos dias 7, 10, 14, 21 ou 58 pósimunização, os animais foram submetidos a eutanásia, e os nervos ópticos, dissecados em seu trajeto desde a parte posterior do globo ocular até o quiasma óptico. Posteriormente, foram avaliados os aspectos morfológico e imuno-histoquímico dos nervos ópticos. As alterações histopatológicas observadas em um ou em ambos os nervos ópticos consistiram de infiltrado celular inflamatório, tendo a neurite óptica gravidade diferente nos dois grupos estudados. A quimiocina CCL5 foi avaliada no dia 10 pós-imunização, primeiro dia em que foi detectado o infiltrado inflamatório. Os resultados sugerem que duas diferentes concentrações de MOG35-55 utilizadas na indução do modelo animal de EAE induzem duas diferentes formas de evolução da neurite óptica. / The model of Experimental Autoimmune Encephalomyelitis (EAE) is the most used model in the study of optic neuritis. This study aims to evaluate the kinetics of optic neuritis in the EAE animal model induced by two different concentrations of Oligodendrocytes Myelin Glycoprotein (MOG). For induction of EAE were used female mice of the C57BL/6 lineage, divided into two groups, one group induced with 100 μg of MOG35-55 and a second group induced with 300 μg of MOG35-55. The animals were evaluated daily by analysis of clinical score between zero and 58 days after immunization. On days 7, 10, 14, 21 or 58 post-immunization, the animals were euthanized. The optic nerves were dissected from the back of the eyeball to the optic chiasm; subsequently the morphological and immunohistochemical aspects of the optic nerves were evaluated. The histopathological changes observed in one or in both optic nerves consisted of inflammatory cell infiltrate. Optic neuritis had different levels of severity in the two groups. The chemokine CCL5 was evaluated on day 10 post-immunization, the first day when the inflammatory infiltrate was detected. The results suggest that two different concentrations of MOG35-55 used in the induction of EAE animal model induce two different forms of optic neuritis evolution.

Orientadores: Alessandro dos Santos Farias, Leonilda Maria Barbosa dos Santos / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T01:58:27Z (GMT). No. of bitstreams: 1 Pradella_Fernando_M.pdf: 4468527 bytes, checksum: 63d6760bd0ea06c5fcab94d1421da291 (MD5) Previous issue date: 2013 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic document / Mestrado / Imunologia / Mestre em Genética e Biologia Molecular

Development of functional neural circuits involves a series of complicated steps, including neurogenesis and neuronal morphogenesis. To understand the molecular mechasnims of neurite complexity, especially neurite branching/arborization, the Drosophila brain, especially MBNs (mushroom body neurons) and PNs (projection neurons) in olfactory circuitry, was used in this dissertation work as the model system to study how two molecules, Dscam and Kr-h1 affect neurite complexity in the Drosophilabrain. For the Drosophila Dscam, through alternative splicing it could encode up to 152,064 distinct immunoglobulin/fibronectin type cell adhesion molecules. Each Dscam isoform is derived from one of the 19,008 ectodomain variants connected with one of the two alternative transmembrane segments and one of the four possible endodomain portions. Recent studies revealed that Dscam was widely required for neurite branching/arborizaiton. However, due to the technical difficulty, the functional roles of Dscam transmembrane variants and ectodomain variants remain unclear. In this thesis work, a microRNA based RNA interference was used to knock down distinct subsets of Dscam isoform. First, loss of Dscam[TM1] versus Dscam[TM2], two distinct Dscam transmembrane variants, disrupted the dendritic versus axonal morphogenesis, respectively. Furthermore, structural analysis suggested that the juxtamembrane portion of transmembrane segment was required for the Dscam protein targeting in dendrites/axons and this differential protein targeting might account for the functional distinction between Dscam[TM1] and Dscam[TM2]. Second, to further address the functional significance of having two Dscam transmembrane variants in axons versus dendrites, the possibility that there might be different usage of Dscam repertoire between axons and dendrites that lead to different levels of morphological complexity between axons and dendrites in the same neuron was examined. To this end, end-in targeting approaches were used to exchange Dscam populations between axons and dendrites. Though the genetic data suggested that Dscam populations were exchanged between axons and dendrites, the phenotypic analysis in various neuronal types revealed that depending on the neuronal types, exchange of Dscam populations between axons and dendrites might primarily affect either axonal or dendritic morphology, suggesting that different usage of Dscam population between axons and dendrites might regulate complex patterns of neurite morphology. Finally, the functions of Dscam exon 4 variants had been addressed in different model neurons in the Drosophilabrain. First, 12 Dscam exon 4 variants were divided into three groups based on their phylogenetic distance. Then, three miRNA constructs were engineered to knock down one group at a time. The genetic data suggested that different Dscam exon 4 variants are differentially required in different neurons to support their proper neuronal morphogenesis. In summary, this part of my thesis work identified and characterized previously unrecognized functions of all these distinct Dscam variants and provided novel insights into how diverse Dscam isoforms regulate the different aspects of neuronal morphogenesis. In the honey bee brain, Kr-h1 is upregulated during the behavioral shift from nursing to foraging when there is increased neurite branching in the brain. To directly examine the hypothesis that altered Kr-h1 expression might regulate morphological complexity of neurites, this research work involved the MARCM (mosaic analysis with a repressible cell marker) and TARGET (temporal and regional gene expression targeting) techniques to analyze the roles of Kr-h1 in Drosophila neuronal morphogenesis. Interestingly, increased expression of Kr-h1 blocked the axon branching and further disrupted the lobe formation in the mushroom body whereas the loss-of-Kr-h1 did not show any apparent neuronal morphogenetic defects. In addition, it was observed that Kr-h1 was expressed when MB (mushroom body) did not undergo active morphogenesis, suggesting its potential anti-morphogenetic activity. Indeed, loss of Kr-h1 (Kruppel homolog 1) enhanced the neuronal morphogenesis that was otherwise delayed due to the defective TGF-beta signaling. Furthermore, Kr-h1 expression was closely linked to ecdysone dependent signaling: Kr-h1 was first regulated by usp (ultraspiracle), which dimerized with various ecdysone receptors and then Kr-h1 expression was essential for proper ecdysone patterning in the larval CNS (central nervous system). Together, though Kr-h1could potentially regulate the neurite complexity, it seems primarily involved in the coordinating ecdysone signaling. In conclusion, the powerful genetic toolkit available in the Drosophila has allowed the investigation in the molecular mechanisms of neuronal morphogenesis and understanding of these mechanisms will enhance our understanding of how the complex nervous system is wired to perform the delicate behaviors.

<p> The urgency for deeper knowledge about nervous system function and dysfunction has never been greater. With increasing rates of mental disorders and expanding healthcare costs, deciphering details of axonal development is essential to meeting this imperative. Models of neuronal growth are improving as roles of microtubules and motor proteins surface. However, traditional motor protein studies focus on intracellular cargo transport, leaving deficits in knowledge about how these proteins organize cytoskeletal filaments in the axon and growth cone during neuronal development. Inconsistent findings on microtubule activity in growing axons also leave gaps in quantitative assessments of microtubule translocation and assembly, limiting the ability to construct a comprehensive model of axonal elongation.</p> The goal of this study was to provide a more complete neuronal growth cone model by determining how individual microtubule translocation and assembly, mass microtubule movements, and motor protein activity contribute to axonal elongation. The underlying mechanisms of these processes were investigated by testing the roles of dynein and microtubule dynamics in axonal elongation of <i>Aplysia </i><i>californica </i>neurons using transillumination, fluorescent speckle, and super-resolution microscopy. Pharmacologically inhibiting either dynein activity or microtubule assembly reduced both bulk and individual microtubule anterograde translocation and neurite elongation rates. Suppressing both processes simultaneously had compensatory rather than additive effects. Super-resolution imaging also revealed fewer dynein motors co-localized with microtubules when microtubule assembly was inhibited. These results strongly suggest that disrupting microtubule assembly blocks neurite outgrowth partly because it inhibits dynein-mediated bulk microtubule translocation.