Orientadores: Iscia Lopes Cendes, Fernando Cendes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T01:52:35Z (GMT). No. of bitstreams: 1 Maurer-Morelli_ClaudiaVianna_D.pdf: 558728 bytes, checksum: dee5aed1e69518c268ddacaa962b5126 (MD5) Previous issue date: 2006 / Resumo: A associação entre epilepsia de lobo temporal e esclerose mesial temporal (EMT) é bem estabelecida, assim como o uso da atrofia hipocampal (AH) e outros sinais indicativos de esclerose hipocampal, visíveis por imagens de ressonância magnética, como marcadores da EMT in vivo. Um dos fatores de risco associados à EMT são as crises epilépticas febris prolongadas na infância. Em 2001, Kobayashi et al. descreveram um tipo distinto de epilepsia de lobo temporal mesial com evidente recorrência familiar associada à AH, mas com baixa freqüência de crises febris, nomeada de epilepsia de lobo temporal mesial familiar (ELTMF). Uma análise prévia dos heredogramas destas famílias sugere que as anormalidades hipocampais podem ser geneticamente determinadas na ELTMF. Para determinar se a ELTMF pode ser explicada por fatores genéticos foi empregada a técnica de análise de segregação complexa baseada no modelo misto de Morton, com o emprego do software POINTER©. Na investigação de genes candidatos, com funções biológicas significantes na fisiologia da epilepsia de lobo temporal ou em modelos animais descritos previamente, foram genotipados marcadores microssatélites que flanqueiam estes genes relevantes e realizada a análise de ligação genética. Finalmente, objetivando identificar a região do genoma responsável por conter o gene principal associado com a AH na ELTMF foi realizada a análise de ligação genética genômica em duas famílias suficientemente informativas, com 57 indivíduos incluindo 27 pacientes. Os resultados obtidos demonstraram que: i) a análise de segregação complexa confirmou a observação prévia de uma predisposição genética para ELTMF, indicando a presença de um gene principal com transmissão Mendeliana e que pode ter um envolvimento na gênese da AH encontrada nestes pacientes; ii) embora a lesão hipocampal encontrada em camundongo transgênico com mutação no gene Scn2a seja similar àquela encontrada na ELTM foi descartada a possibilidade do gene homólogo SCN2A ser um gene candidato na ELTMF; iii) a análise de ligação em genes candidatos codificadores de canais de potássio voltagem-dependente não evidenciou qualquer tipo envolvimento destes genes na determinação das anormalidades hipocampais na ELTMF; iv) foi identificada ligação no cromossomo 18p11.3-11.2 com um LOD score máximo de 3,63 para ?= 0,0 para o marcador D18S976 em uma única família com 11 indivíduos afetados com AH. A análise de multipontos e o haplótipo localizam a região candidata dentro um intervalo de 6 cM flanqueado pelos marcadores D18S976 e D18S452. Além disso, os genes ZFP161 e TGIF que se localizam na região candidata mapeada, não apresentaram mutações em suas regiões codificantes, as quais poderiam estar relacionadas à ELTMF. Estes resultados mostram pela primeira vez, evidências de que a AH pode ser determinada por fatores genéticos, os quais podem ter maiores implicações no estudo dos mecanismos fisiopatológicos que permeiam a EMT e sua relação com a epilepsia de lobo temporal. No entanto, estudos adicionais são necessários para identificar o gene maior responsável pela ELTMF / Abstract: The association between temporal lobe epilepsy and mesial temporal sclerosis (MTS) has been well established; as well as the use of hippocampal atrophy (HA) on magnetic resonance imaging as an in vivo surrogate marker of MTS. One of the risk factors associated to MTS is childhood prolonged febrile seizures. In 2001, Kobayashi et al., described a type of mesial temporal lobe epilepsy with evident familial recurrence associate with HA but low frequency of febrile seizures, named familial mesial temporal lobe epilepsy (FMTLE). Previous pedigree analysis provided evidence that hippocampal abnormalities may be genetically determined in FMTLE. To determine whether FMTLE can be explained by the involvement of genetic factor we employed complex segregation analysis with the POINTER© software. To investigate candidate genes with significant biological functions related to temporal lobe epilepsy, we genotyped microsatellite markers flanking these relevant genes and performed linkage analysis. In addition, we performed a genome wide search in two large families with 57 individuals, including 27 patients. Our results show the following: i) complex analysis segregation strengthened previous evidence for a genetic predisposition in FMTLE, indicating the presence of a major gene with Mendelian transmission, which could be involved in HA development in these patients; ii) we conclusively ruled out the SCN2A gene as candidate in FMTLE, although the hippocampal lesion in the mutant Scn2a transgenic mouse is similar to that found in our FMTLE patients; iii) linkage analysis showed no evidence that voltage-gated potassium channels are involved in the determination of hippocampal abnormalities in FMTLE; iv) we identified linkage to chromosome 18p11.3-11.2, with a maximum LOD score of 3.63 at ?= 0.0 for the D18S976 marker in a single family (F-10) with 11 affected individuals with HA. Multipoint and haplotype analyses localized the locus within a 6 cM interval flanked by markers D18S976 and D18S452. Furthermore, we failed to find putative pathological mutations related to FMTLE in two candidate genes ZFP161 and TGIF, both mapping within the locus on chromosome 18p11.3-11.2. With our results we have demonstrated the first conclusive evidence that HA may be caused by genetic factors which can have major implications in the study of the pathophysiological mechanisms underlying MTS and its relationship with temporal lobe epilepsy / Doutorado / Neurociencias / Doutor em Fisiopatologia Medica

Hyperkalemic periodic paralysis is characterized by myotonic discharges followed by paralysis. Caused by a mutation in the gene encoding for NaV1.4 channel, patients do not experience symptoms during infancy, but the onset starts between 1-10 years of age. The symptoms severity then increases with age until adolescence. A large increase in gene expression marked by an increase in oxidative capacity of muscles has also been reported in HyperKPP. It is possible that the onset of symptoms is related solely to NaV1.4 channel content/activity reaching a critical level. It is also possible that the onset of some symptoms are due to defective NaV1.4, while other symptoms and the increase in severity with age are related to changes in membrane components as a result of changes in gene expression. To test these possibilities, the progression of paralysis and changes in fiber types were followed with age in HyperKPP mice in relation to changes in NaV1.4 content and activity. Changes in fiber types (index of changes in gene expression), started after the onset of paralysis was observed, which coincided with NaV1.4 channels reaching maximum expression. Therefore, the onset of symptoms was related to defective NaV1.4 channels.

L’objectif de ma thèse était d’identifier, comme agents antinociceptifs potentiels, des toxines de venins originales par leur séquence et/ou leur provenance. Dans cette optique, un criblage à haut débit de deux banques de venins a été réalisé par des méthodes électrophysiologiques de "patch-clamp" automatique, sur des lignées cellulaires exprimant le sous-type neuronal hNaV1.7 de canaux sodium (versus celles exprimant le sous-type cardiaque hNaV1.5), une cible antidouleur validée génétiquement et fortement exprimée au niveau des neurones sensoriels primaires des ganglions de la racine dorsale, premier support de la transmission du message nociceptif.Le criblage de la première banque de venins (appartenant à Smartox Biotechnology) a permis l’identification et la caractérisation, par des approches structurales et fonctionnelles multi-échelles (de la cellule in vitro à l’organisme in vivo), de 2 peptides de venins d’araignées ayant des propriétés potentiellement antinociceptives : (1) la cyriotoxine-1a du venin de Cyriopagopus schioedtei, dont les propriétés fonctionnelles sont proches de celles des peptides appartenant à la famille 1 des toxines d’araignées inhibant les canaux sodium, et (2) la poecitoxine-1a du venin de Poecilotheria subfusca, qui présente une meilleure affinité pour le sous-type hCaV1.2 de canaux calcium que pour le sous-type hNaV1.7. Nous avons également mené une étude de "structure-activité" afin d’améliorer le profil de sélectivité de la phlotoxine-1 d’une araignée Phlogiellus, connue pour son activité antinocieptive. Finalement, nous avons mis en évidence une interaction directe entre l’huwentoxine-IV, déjà connue comme agent antinociceptif potentiel, et le sous-type neuronal NaV1.6, responsable d’effets neuromusculaires indésirables. Le criblage de la deuxième banque de venins (appartenant à SANOFI) a permis d’identifier des hits intéressants provenant de venins d’araignées et de scorpions non étudiés jusqu’à présent et ayant une séquence originale présentant peu d’homologie avec les séquences déjà connues. / The aim of my thesis was to identify original venom toxins, by their sequence and/or origin, as potential antinociceptive agents. In this context, a high-throughput screening of two venom libraries was performed, by automated patch-clamp electrophysiology, on cell lines expressing the hNaV1.7 neuronal subtype of sodium channels (versus those expressing the hNaV1.5 cardiac subtype), a genetically-validated and strongly expressed pain target in the primary sensory neurons of dorsal root ganglia, the first support of nociceptive message transmission.The screening of the first venom library (belonging to Smartox Biotechnology) allowed the identification and characterization, by structural and multiscale functional approaches (from the cell in vitro to the organism in vivo), of 2 peptides from spider venoms having potential antinociceptive properties : (1) cyriotoxin-1a from Cyriopagopus schioedtei venom, whose functional properties are close to those of peptides belonging to family 1 of spider toxins inhibiting sodium channels, and (2) poecitoxin-1a from Poecilotheria subfusca venom, which has a better affinity for the hCaV1.2 subtype of calcium channels than for the hNaV1.7 subtype. We also conducted a "structure-activity" study to improve the selectivity profile of phlotoxin-1 from a Phlogiellus spider, known for its antinociceptive activity. Finally, we evidenced a direct interaction between huwentoxin-IV, already known as a potential antinociceptive agent, and the NaV1.6 neuronal subtype which is the main cause of undesirable neuromuscular effects. The screening of the second venom library (belonging to SANOFI) allowed to identify interesting hits from spider and scorpion venoms, not studied until now, having an original sequence with little homology with already known sequences.

This project examined the role of the cytoskeleton in regulatory mechanisms of the amiloride-sensitive Na⁺ channels in isolated frog skin epithelium. The epithelium from ventral frog skin is a model tissue which has proved significant in our understanding of the basic principles involved in water and Na⁺ homeostasis. In particular, this project examines ways in which local (non-hormonal) and hormonal regulatory mechanisms adjust the Na⁺ permeability of apical membranes of frog skin epithelium. Both mechanisms contain factors that are known to increase the apical membrane Na⁺ permeability mainly by increases in the number of open channels. The origin of these new open channels is unknown but, it is postulated that they could arise either by activation of quiescent channels already present in the apical membrane, or by recruitment of channels from cytoplasmic stores. Regarding the latter hypothesis, we also examined the idea that the cytoskeleton might somehow be involved in the insertion of Na⁺ channels within vesicles, into the apical membrane. This is based on the fact that the cytoskeleton is involved in a similar mechanism whereby, in the toad urinary bladder, anti-diuretic hormone (ADH) causes the insertion of aggregates with water channels. Much current interest focuses on the role of the cytoskeleton in the regulation of epithelial Na⁺ channels. To test this hypothesis, we used noise analysis to examine the effects of disrupting the cytoskeleton, on two different mechanisms which bring about changes in open channel densities. The mechanisms are: (1) lowering mucosal Na⁺ concentration (non-hormonal), and (2) addition of arginine-vasopressin (A VP) (hormonal). Non-hormonal, autoregulatory changes in apical membrane Na⁺ conductance were examined by investigating the effects of reducing the mucosal Na⁺ concentration. Our results showed that lowering the mucosal Na⁺ concentration induced large increases in the open channel density in order to stabilise the transport rate. In addition, we observed an average 55-60% increase in the open channel probability, which implies that in epithelium from Rana fuscigula, changes of channel open probability are also an important mechanism in the autoregulation of channel densities in response to a reduction in mucosal Na⁺. The hormonal control of Na⁺ channels by A VP has been intensively studied by noise analysis and the patch clamp. Our results confirmed previous reports that A VP increases the Na⁺ transport rate by increasing the number of open Na⁺ channels, primarily through large changes in the total number of channels, without a significant change in open probability. Regarding the role of the cytoskeleton in regulation of Na⁺ channels and/or its possible role in control of inserting putative vesicles with Na⁺ channels, we studied the effects of disrupting the cytoskeleton on the two regulatory mechanisms. Disrupting microtubules with colchicine had no, or very little effect on either of the regulatory mechanisms. On the other hand, the integrity of the microfilaments was very important for the autoregulatory changes in the number of open channels. After cytochalasin B treatment, lowering the mucosal Na⁺ concentration did not result in the usual compensatory changes in channel densities. There was no prior evidence that cytochalasin B had any actual effect on the F-actin network in the frog skin epithelium. Accordingly, modified cytochemical techniques were designed to demonstrate and localise F-actin in the epithelial granular cells. The direct immunofluorescent method proved useful, but did not allow sufficient resolution to examine the changes to different populations of actin in the cells. We then modified an immunogold method to suit our conditions, and the results demonstrated the localisation of different pools of F-actin and showed the effects of the cytochalasin B and vasopressin.

No description available.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / The cardiac voltage-gated sodium channel α subunit Nav1.5 generates the cardiac sodium current, which is essential for the initiation and propagation of the cardiac action potentials. Mutations of SCN5A, the gene that encodes Nav1.5, have been well documented to cause long-QT syndrome (LQTs) by disrupting channel inactivation and increasing late sodium current. Previous studies have revealed the importance of the intracellular loop region between transmembrane domain III and IV of sodium channel α subunit in regulating the fast inactivation. A recent clinical case study reported an infant patient with LQTs carrying a phenylalanine (F) deletion at amino acid 1486 of the Nav1.5 channel. This study reported that the patient showed severe cardiac arrhythmia reflected as LQTs and subsequent ventricular tachycardia, which was refractory to antiarrhythmic drug lidocaine treatment. Therefore, it was hypothesized that the deletion of F1486 on Nav1.5 would substantially alter electrophysiological properties of the channel and reduce the potency of lidocaine on sodium channel. Using HEK293 cells and neonatal rat cardiomyocytes, the F1486del channel was functionally characterized by whole-cell patch clamp techniques. Studies revealed that the deletion of F1486 causes a combination of changes including a loss-of-function alteration reflected as a substantial reduction of peak current density and a number of gain-of-function alterations including reduced channel inactivation, substantial augmentation of late sodium current, and an increase in ramp current. In addition, lidocaine sensitivity was dramatically reduced. By contrast, the voltage for half maximal activation (V1/2) and the time constant for channel deactivation for the F1486del channel were identical to the wild type channels. Using neonatal rat cardiomyocytes, we were able to study the functional consequence of F1484del on action potential duration (APD). Cardiomyocytes expressing F1486del channel have substantial APD prolongation and prominent spontaneous early afterdepolarizations, which likely underlie the subsequent LQTs in the patient. Taken together, despite the reduction in peak current density, the substantial gain-of-function changes are sufficient to cause the APD prolongation, which is a prominent characteristic of LQTs. These findings provide knowledge for understanding the relationships between sodium channel structure, pharmacology and the physiological consequence of sodium channel mutations that underlie LQT3.

Yes / Sublethal carbon monoxide (CO) exposure is frequently associated with myocardial arrhythmias, and our recent studies have demonstrated that these may be attributable to modulation of cardiac Na+ channels, causing an increase in the late current and an inhibition of the peak current. Using a recombinant expression system, we demonstrate that CO inhibits peak human Nav1.5 current amplitude without activation of the late Na+ current observed in native tissue. Inhibition was associated with a hyperpolarizing shift in the steady-state inactivation properties of the channels and was unaffected by modification of channel gating induced by anemone toxin (rATX-II). Systematic pharmacological assessment indicated that no recognized CO-sensitive intracellular signaling pathways appeared to mediate CO inhibition of Nav1.5. Inhibition was, however, markedly suppressed by inhibition of NO formation, but NO donors did not mimic or occlude channel inhibition by CO, indicating that NO alone did not account for the actions of CO. Exposure of cells to DTT immediately before CO exposure also dramatically reduced the magnitude of current inhibition. Similarly, L-cysteine and N-ethylmaleimide significantly attenuated the inhibition caused by CO. In the presence of DTT and the NO inhibitor Nω-nitro-L-arginine methyl ester hydrochloride, the ability of CO to inhibit Nav1.5 was almost fully prevented. Our data indicate that inhibition of peak Na+ current (which can lead to Brugada syndrome-like arrhythmias) occurs via a mechanism distinct from induction of the late current, requires NO formation, and is dependent on channel redox state. / This work was supported by the British Heart Foundation

Voltage-gated sodium channels are composed of pore-forming α-subunits, and modulatory and multifunctional associated β subunits. While much of the field of cardiac electrophysiology and pathology has focused on treating and preventing cardiac arrhythmias by targeting the α subunit, there is also evidence that targeting the β subunits, particularly SCN1B, the gene that encodes β1 and an alternatively spliced variant β1B, has therapeutic potential. The first attempt at targeting the β1 subunit was with the generation of and treatment with an SCN1B Ig domain mimetic peptide βadp1. Here we describe further investigation into the function and mode-of-action of both βadp1 and novel peptides derived from the original βadp1 sequence. We find that in a heterologous expression system βadp1 initially disrupts β1-mediated trans-homophilic adhesion, but after approximately 30 hours eventually increases adhesion. Novel mimetic dimers increase β1 adhesion up to 48 hours post-treatment. Furthermore, it appears that βadp1 may increase β1 adhesion by upregulating the intramembrane proteolysis of β1, a process which has important downstream implications and effects on translation. Despite these exciting findings, we were unable to translate them into a primary culture of cardiac cells with endogenous expression of β1 because we found that both neonatal rat cardiomyocytes and isolated adult mouse cardiomyocytes do not express β1 at detectable levels, whereas they do appear to express β1B. In summary, we show exciting findings on the function and mode-of-action of SCN1B mimetic peptides and their therapeutic potential in targeting the β1 subunit, but further work is needed to determine the translatability of our findings to in vivo models and eventually to humans. / Doctor of Philosophy / Voltage-gated sodium channels have two main parts: the pore-forming α-subunits and the modulatory β subunits. Most research in heart function and issues has focused on fixing problems with the α subunit. However, there's evidence that working on the β subunits, specifically the SCN1B gene that makes β1 and another version called β1B, could be helpful. Previously, researchers used a peptide that is designed exactly like a part of β1, called βadp1, to target the β1 subunit. In our study, we explore more about how βadp1 works and test new peptides based on βadp1. We found that βadp1 initially disrupts trans-homophilic adhesion, where 2 β1 subunits interact with each other across the space between 2 cells, but after about 30 hours, it actually increases adhesion. New mimetic dimers also boost adhesion up to 48 hours later. It seems like βadp1 might enhance adhesion by triggering a process called intramembrane proteolysis of β1, which has important effects on translation. Despite these exciting findings, we couldn't confirm the presence of this protein in heart cells because we discovered that certain heart cells don't have enough β1, although they do have β1B. In conclusion, our study shows promising results about how SCN1B mimetic peptides work and their potential for treating arrhythmia. However, more research is needed to see if these findings apply to real-life situations and eventually to help people with cardiac conduction abnormalities.

Sulfatide is a galactolipid and a major lipid component of the myelin sheath. Its production is catalyzed by the enzyme cerebroside sulfotransferase (CST). To determine the functions of sulfatide, the gene encoding CST was genetically disrupted resulting in mice incapable of sulfatide synthesis. Using these mice, it has been shown in the central nervous system (CNS) that sulfatide is essential for normal myelin synthesis and stability even though the onset of myelination is not impaired. Additionally, proper initial clustering of paranodal proteins and cluster maintenance of nodal proteins is impaired suggesting that paranodal domains are important for long-term node stability. In contrast to the CNS, a requirement for sulfatide in the initiation of myelination, and in initiation of paranodal and nodal clustering or in the long-term maintenance of these clusters in the peripheral nervous system (PNS) has not been analyzed. Therefore, we have employed a combination of electron microscopic, immunocytochemical, and confocal microscopic analyses of the CST KO mice to determine the role of sulfatide in PNS myelination and onset of protein domain formation and maintenance. For these studies we have quantified myelin thickness, paranodal structural integrity, and the number of paranodal and nodal protein clusters in the CST KO and wild type mice at 4 days, 7 days, and 10 months of age. Our findings indicate that myelination onset is not delayed in the absence of sulfatide and that both the node and paranode are grossly normal; however, closer analysis reveals that paranodal junctions are compromised, Schwann cell microvilli are disoriented and the myelin-axon interface along the internodal region is transiently disrupted. In addition, we report that the paranodal myelin protein neurofascin 155 (Nfasc155) shows a transient decrease in initial clustering in the CST null mice at 4 days of age that is restored to WT levels by 7 days of age that is also maintained in the adult mice. Whereas nodal clustering of neuronal voltage-gated sodium channels is initially normal, cluster number is significantly but also transiently reduced by 7 days of age. By 10 months of age, the number of sodium channel clusters is restored to normal levels. In contrast, clustering of neither the paranodal neuronal protein contactin nor the myelin nodal protein gliomedin is altered at any of the ages studied. Together our findings suggest that sulfatide is not essential for PNS myelination or for protein domain formation in contrast to its more vital role in the development and maintenance of the CNS.