Estudo de associação entre disfunção neurocognitiva, estresse oxidativa e polimorfismos em pacientes jovens com Transtornos Bipolar tipo I

O Transtorno Bipolar (TB) tipo I é uma doença caracterizada por episódios de mania e depressão recorrentes com importante prejuízo do funcionamento global e comprometimento das funções cognitivas. Além disso, sabe-se que o número de episódios de humor patológico ao longo da vida pode também influenciar o funcionamento cognitivo destes sujeitos. Neste cenário, ocorreu a necessidade de se investigar marcadores genéticos para disfunção cognitiva no TB com o objetivo de estudar este fenômeno. Dentre os potenciais genes responsáveis por influenciar a cognição destacam-se os polimorfismos funcionais do fator neurotrófico derivado do cérebro (BDNF), da catecol-O-metiltransferase (COMT), da apolipoproteína-E (APOE) e do canal de cálcio de baixa voltagem subunidade 1-C (CACNA1C). Sabe-se, também, que no TB os marcadores de estresse oxidativo estão aumentados durante todas as fases da doença, entretanto, não é claro qual impacto destes na disfunção cognitiva de indivíduos com TB. O objetivo dessa tese foi avaliar o desempenho cognitivo de pacientes jovens com bipolaridade tipo I e sua associação com o genótipo de BDNF, COMT, APOE e CACNA1C e também com os níveis plasmáticos de oxidação da guanosina (8-OHdG) e citosina (5-Mec) durante os episódios de humor, eutimia e em controles. Para investigar essa associação foram incluídos 116 pacientes (79 em episódio de humor patológico e 37 eutímicos) com diagnóstico de TB tipo I (DSMIV-TR); 97 controles saudáveis foram submetidos à avaliação neuropsicológica e coleta de sangue para extração de DNA visando genotipagem para BDNF (rs6265), COMT (rs4680; rs165599), APOE (rs429358 e rs7412), CACNA1C (rs1006737), 8-OhdG e 5-Mec. A análise dos dados obtidos revelou que pacientes portadores do genótipo Met/Met rs4680/rs165599 do COMT apresentam comprometimento cognitivo mais grave (função executiva, fluência verbal, memória e inteligência) comparado ao genótipo Val/Met ou Val/Val durante episódios maníacos ou mistos. Na mesma direção destes resultados, verificou-se que pacientes portadores do alelo Met rs4680 do COMT apresentam comprometimento do reconhecimento de emoções faciais em episódios de mania e depressão. Nenhum efeito do COMT foi observado em controles. O alelo de risco Met do CACNA1C se associou a um pior comprometimento executivo independente dos sintomas maníacos ou depressivos no TB, porém nenhum efeito se observou nos controles. O alelo Met do BDNF rs6265 ou a presença do alelo 4 da APOE não representa um fator que identifique um grupo com desempenho cognitivo diferenciado durante as fases do TB ou em controles. Sujeitos com TB apresentaram níveis mais elevados de 8-OHdG e tais níveis eram diretamente proporcionais ao número de episódios maníacos ao longo da vida, sugerindo um papel dos episódios hiperdopaminérgicos na oxidação das bases de DNA. Concluiu-se que a genotipagem para COMT e CACNA1C em pacientes com TB pode identificar um grupo de pacientes associados a pior disfunção cognitiva durante as fases maníacas e mistas do TB. Tal dado pode ser um indicador do envolvimento do sistema dopaminérgico e dos canais de cálcio de baixa voltagem na fisiopatologia da disfunção cognitiva no TB e deve ser explorado em outros estudos

Modifications post-traductionnelles des canaux calciques cardiaques de type L : identification des résidus asparagine qui participent à la glycosylation de la sous-unité auxiliaire CaVα2δ1

Les canaux calciques de type L CaV1.2 sont principalement responsables de l’entrée des ions calcium pendant la phase plateau du potentiel d’action des cardiomyocytes ventriculaires. Cet influx calcique est requis pour initier la contraction du muscle cardiaque. Le canal CaV1.2 est un complexe oligomérique qui est composé de la sous-unité principale CaVα1 et des sous-unités auxiliaires CaVβ et CaVα2δ1. CaVβ joue un rôle déterminant dans l’adressage membranaire de la sous-unité CaVα1. CaVα2δ1 stabilise l’état ouvert du canal mais le mécanisme moléculaire responsable de cette modulation n’a pas été encore identifié. Nous avons récemment montré que cette modulation requiert une expression membranaire significative de CaVα2δ1 (Bourdin et al. 2015). CaVα2δ1 est une glycoprotéine qui possède 16 sites potentiels de glycosylation de type N. Nous avons donc évalué le rôle de la glycosylation de type-N dans l’adressage membranaire et la stabilité de CaVα2δ1. Nous avons d’abord confirmé que la protéine CaVα2δ1 recombinante, telle la protéine endogène, est significativement glycosylée puisque le traitement à la PNGase F se traduit par une diminution de 50 kDa de sa masse moléculaire, ce qui est compatible avec la présence de 16 sites Asn. Il s’est avéré par ailleurs que la mutation simultanée de 6/16 sites (6xNQ) est suffisante pour 1) réduire significativement la densité de surface de! CaVα2δ1 telle que mesurée par cytométrie en flux et par imagerie confocale 2) accélérer les cinétiques de dégradation telle qu’estimée après arrêt de la synthèse protéique et 3) diminuer la modulation fonctionnelle des courants générés par CaV1.2 telle qu’évaluée par la méthode du « patch-clamp ». Les effets les plus importants ont toutefois été obtenus avec les mutants N663Q, et les doubles mutants N348Q/N468Q, N348Q/N812Q, N468Q/N812Q. Ensemble, ces résultats montrent que Asn663 et à un moindre degré Asn348, Asn468 et Asn812 contribuent à la biogenèse et la stabilité de CaVα2δ1 et confirment que la glycosylation de type N de CaVα2δ1 est nécessaire à la fonction du canal calcique cardiaque de type L.

Synthesis and biological evaluation of nonpeptide mimetics of co-conotoxin GVIA

A benzothiazole-derived compound (4a) designed to mimic the C-alpha-C-beta bond vectors and terminal functionalities of Lys2, TyrI3 and Arg17 in omega-conotoxin GVIA was synthesised, together with analogues (4b-d), which had each side-chain mimic systematically truncated or eliminated. The affinity of these compounds for rat brain N-type and P/Q-type voltage gated calcium channels (VGCCs) was determined. In terms of N-type channel affinity and selectivity, two of these compounds (4a and 4d) were found to be highly promising, first generation mimetics of omega-conotoxin. The fully functionalised mimetic (4a) showed low PM binding affinity to N-type VGCCs (IC50 = 1.9 muM) and greater than 20-fold selectivity for this channel sub-type over P/Q-type VGCCs, whereas the mimetic in which the guanidine-type side chain was truncated back to an amine (4d, IC50 = 4.1 muM) showed a greater than 25-fold selectivity for the N-type channel. (C) 2004 Elsevier Ltd. All rights reserved.

Alterations in dihydropyridine receptors in dystrophin-deficient cardiac muscle

The deficiency of dystrophin, a critical membrane stabilizing protein, in the mdx mouse causes an elevation in intracellular calcium in myocytes. One mechanism that could elicit increases in intracellular calcium is enhanced influx via the L-type calcium channels. This study investigated the effects of the dihydropyridines BAY K 8644 and nifedipine and alterations in dihydropyridine receptors in dystrophin-deficient mdx hearts. A lower force of contraction and a reduced potency of extracellular calcium (P < 0.05) were evident in mdx left atria. The dihydropyridine agonist BAY K 8644 and antagonist nifedipine had 2.7- and 1.9-fold lower potencies in contracting left atria (P < 0.05). This corresponded with a 2.0-fold reduction in dihydropyridine receptor affinity evident from radioligand binding studies of mdx ventricular homogenates (P < 0.05). Increased ventricular dihydropyridine receptor protein was evident from both radioligand binding studies and Western blot analysis and was accompanied by increased mRNA levels (P < 0.05). Patch-clamp studies in isolated ventricular myocytes showed no change in L-type calcium current density but revealed delayed channel inactivation (P < 0.05). This study indicates that a deficiency of dystrophin leads to changes in dihydropyridine receptors and L-type calcium channel properties that may contribute to enhanced calcium influx. Increased influx is a potential mechanism for the calcium overload observed in dystrophin-deficient cardiac muscle.

Taxonomy of Australian Funnel-web spiders using rp-HPLC/ESI-MS profiling techniques

The complex nature of venom from spider species offers a unique natural source of potential pharmacological tools and therapeutic leads. The increased interest in spider venom molecules requires reproducible and precise identification methods. The current taxonomy of the Australian Funnel-web spiders is incomplete, and therefore, accurate identification of these spiders is difficult. Here, we present a study of venom from numerous morphologically similar specimens of the Hadronyche infensa species group collected from a variety of geographic locations in southeast Queensland. Analysis of the crude venoms using online reversed-phase high performance liquid chromatography/electrospray ionisation mass spectrometry (rp-HPLC/ESI-MS) revealed that the venom profiles provide a useful means of specimen identification, from the species level to species variants. Tables defining the descriptor molecules for each group of specimens were constructed and provided a quick reference of the relationship between one specimen and another. The study revealed that the morphologically similar specimens from the southeast Queensland region are a number of different species/species variants. Furthermore, the study supports aspects of the current taxonomy with respect to the H. infensa species group. Analysis of Australian Funnel-web spider venom by rp-HPLC/ESI-MS provides a rapid and accurate method of species/species variant identification. (c) 2006 Elsevier Ltd. All rights reserved.

Investigating the effects of antiepileptic drugs on the electrophysiology of vision

The principal aim of this work was to examine the effects of antiepileptic drugs (AEDs) on vision. Vigabatrin acts by increasing GABA at brain inhibitory synapses by irreversibly binding to GABA-transaminase. Remacemide is a novel non-competitive NMDA receptor antagonist and fast sodium channel inhibitor that results in the inhibition of the NMDA receptors located in the neuronal membrane calcium channels increasing glutamate in the brain. Vigabatrin has been shown to cause a specific pattern of visual field loss, as one in three adults taking vigabatrin have shown a bilateral concentric constriction. Remacemide has unknown effects on vision. The majority of studies of the effects of AEDs on vision have not included the paediatric population due to difficulties assessing visual field function using standard perimetry testing. Evidently an alternative test is required to establish and monitor visual field problems associated with AEDs both in children and in adults who cannot comply with perimetry. In order to test paediatric patients exposed to vigabatrin, a field-specific visual evoked potential was developed. Other tests performed on patients taking either vigabatrin or remacemide were electroretinograms, electro-oculograms, multifocal VEPs and perimetry. Comparing these tests to perimetry results from vigabatrin patients the field specific VEP was found to have a high sensitivity and specificity, as did the 30Hz flicker amplitude. The modified VEP was also found to provide useful results in vigabatrin patients. Remacemide did not produce a similar visual field loss to vigabatrin although macular vision was affected. The field specific VEP is a useful method for detecting vigabatrin associated visual field loss that is well tolerated by young children. This technique combined with the ERG under light adapted (30Hz flicker) condition is presently the superior method for detecting vigabatrin-attributed peripheral field defects present in children below the developmental age of 9. The effects of AEDs on vision should be monitored carefully and the use of multifocal stimulation allows for specific areas of the retina and visual pathway to be monitored.

The impact of the CACNA1C gene polymorphism on frontolimbic function in bipolar disorder

Genome-wide association studies in bipolar disorder (BD)1 have implicated a single-nucleotide polymorphism (rs1006737, G right arrow A) in the CACNA1C gene, which encodes for the alpha 1c (CAV1.2) subunit of the voltage-gated, L-type calcium channel. Neuroimaging studies of healthy individuals report that this risk allele modulates brain function within limbic (amygdala, anterior cingulate gyrus) and hippocampal regions during tasks of reward processing2, 3 and episodic memory. Moreover, animal studies suggest that the CaV1.2 L-type calcium channels influence emotional behaviour through enhanced neurotransmission via the lateral amygdala pathway. On the basis of this evidence, we tested the hypotheses that the CACNA1C rs1006737 risk allele will modulate neural responses within predefined prefrontal and subcortical regions of interest during emotional face processing and that this effect would be amplified in BD patients.

Differential control of two forms of glutamate release by group III metabotropic glutamate receptors at rat entorhinal synapses

Neurotransmitter release at CNS synapses occurs via both action potential-dependent and independent mechanisms, and it has generally been accepted that these two forms of release are regulated in parallel. We examined the effects of activation of group III metabotropic glutamate receptors (mGluRs) on stimulus-evoked and spontaneous glutamate release onto entorhinal cortical neurones in rats, and found a differential regulation of action potential-dependent and independent forms of release. Activation of presynaptic mGluRs depressed the amplitude of stimulus-evoked excitatory postsynaptic currents, but concurrently enhanced the frequency of spontaneous excitatory currents. Moreover, these differential effects on glutamate release were mediated by pharmacologically separable mechanisms. Application of the specific activator of adenylyl cyclase, forskolin, mimicked the effect of mGluR activation on spontaneous, but not evoked release, and inhibition of adenylyl cyclase with 9-tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) blocked mGluR-mediated enhancement of spontaneous release, but not depression of evoked release. Occlusion studies with calcium channel blockers suggested that the group III mGluRs might depress evoked release through inhibition of both N and P/Q, but not R-type calcium channels. We suggest that the concurrent depression of action potential-evoked, and enhancement of action potential-independent glutamate release operate through discrete second messenger/effector systems at excitatory entorhinal terminals in rat brain. © 2007 IBRO.

Contribution of rare damaging variants in Autism Spectrum Disorder

Autism Spectrum Disorder (ASD) is a heterogeneous and highly heritable neurodevelopmental disorder with a complex genetic architecture, consisting of a combination of common low-risk and more penetrant rare variants. This PhD project aimed to explore the contribution of rare variants in ASD susceptibility through NGS approaches in a cohort of 106 ASD families including 125 ASD individuals. Firstly, I explored the contribution of inherited rare variants towards the ASD phenotype in a girl with a maternally inherited pathogenic NRXN1 deletion. Whole exome sequencing of the trio family identified an increased burden of deleterious variants in the proband that could modulate the CNV penetrance and determine the disease development. In the second part of the project, I investigated the role of rare variants emerging from whole genome sequencing in ASD aetiology. To properly manage and analyse sequencing data, a robust and efficient variant filtering and prioritization pipeline was developed, and by its application a stringent set of rare recessive-acting and ultra-rare variants was obtained. As a first follow-up, I performed a preliminary analysis on de novo variants, identifying the most likely deleterious variants and highlighting candidate genes for further analyses. In the third part of the project, considering the well-established involvement of calcium signalling in the molecular bases of ASD, I investigated the role of rare variants in voltage-gated calcium channels genes, that mainly regulate intracellular calcium concentration, and whose alterations have been correlated with enhanced ASD risk. Specifically, I functionally tested the effect of rare damaging variants identified in CACNA1H, showing that CACNA1H variation may be involved in ASD development by additively combining with other high risk variants. This project highlights the challenges in the analysis and interpretation of variants from NGS analysis in ASD, and underlines the importance of a comprehensive assessment of the genomic landscape of ASD individuals.

Characterization of calcium-activated chloride channels in patches excised from the dendritic knob of mammalian olfactory receptor neurons

We investigated the properties of calcium-activated chloride channels in inside-out membrane patches from the dendritic knobs of acutely dissociated rat olfactory receptor neurons. Patches typically contained large calcium-activated currents, with total conductances in the range 30-75 nS. The dose response curve for calcium exhibited an EC50 of about 26 mu M. In symmetrical NaCl solutions, the current-voltage relationship reversed at 0 mV and was linear between -80 and +70 mV. When the intracellular NaCl concentration was progressively reduced from 150 to 25 mM, the reversal potential changed in a manner consistent with a chloride-selective conductance. Indeed, modeling these data with the Goldman-Hodgkin-Katz equation revealed a P-Na/P-Cl of 0.034. The halide permeability sequence was P-Cl > P-F > P-I > P-Br indicating that permeation through the channel was dominated by ion binding sites with a high field strength. The channels were also permeable to the large organic anions, SCN-, acetate(-), and gluconate(-), with the permeability sequence P-Cl > P-SCN > gluconaie. Significant permeation to gluconate ions suggested that the channel pore had a minimum diameter of at least 5.8 Angstrom.

Upregulation of intermediate calcium-activated potassium channels counterbalance the impaired endothelium-dependent vasodilation in stroke-prone spontaneously hypertensive rats

Endothelial dysfunction has been linked to a decrease in nitric oxide (NO) bioavailability and attenuated endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation. The small (SK(Ca)) and intermediate (IK(Ca)) calcium-activated potassium channels play a key role in endothelium-dependent relaxation. Because the repressor element 1-silencing transcription factor (REST) negatively regulates IK(Ca) expression, we hypothesized that augmented REST and decreased IK(Ca) expression contributes to impaired endothelium-dependent vasodilation associated with hypertension. Acetylcholine (ACh) responses were slightly decreased in small mesenteric arteries from male stroke-prone spontaneously hypertensive rats (SHRSPs) versus arteries from Wistar Kyoto (WKY) rats. Incubation with N-nitro-L-arginine methyl ester (L-NAME; 100 mu mol/L) and indomethacin (100 mu mol/L) greatly impaired ACh responses in vessels from SHRSP. lberiotoxin (0.1 mu mol/L), which is a selective inhibitor of large-conductance K(Ca) (BK(Ca)) channels, did not modify EDHF-mediated vasodilation in SHRSP or WKY. UCL-1684 (0.1 mu mol/L.), which is a selective inhibitor of SKCa channels, almost abolished EDHF-mediated vasodilation in WKY and decreased relaxation in SHRSP. 1-((2-chlorophenyl)diphenylmethyl)-1H-pyrazole (TRAM-34; 10 mu mol/L) and charybdotoxin (0.1 mu mol/L), which are both IKCa inhibitors, produced a small decrease of EDHF relaxation in WKY but completely abrogated EDHF vasodilation in SHRSP. EDHF-mediated relaxant responses were completely abolished in both groups by simultaneous treatment with UCL-1684 and TRAM-34 or charybdotoxin. Relaxation to SK(Ca)/IK(Ca) channels agonist NS-309 was decreased in SHRSP arteries. The expression of SK(Ca) was decreased, whereas IK(Ca) was increased in SHRSP mesenteric arteries. REST expression was reduced in arteries from SHRSP. Vessels incubated with TRAM-34 (10 mu mol/L) for 24h displayed reduced REST expression and demonstrated no differences in IK(Ca). In conclusion, IK(Ca) channel upregulation, via decreased REST, seems to compensate deficient activity of SK(Ca) channels in the vasculature of spontaneously hypertensive rats. (Translational Research 2009; 154:183-193)

Large-conductance calcium-activated potassium channels in neonatal rat intracardiac ganglion neurons

The properties of single Ca2+-activated K+ (BK) channels in neonatal rat intracardiac neurons were investigated using the patch-clamp recording technique. In symmetrical 140 mM K+, the single-channel slope conductance was linear in the voltage range -60/+60 mV. and was 207+/-19 pS. Na+ ions were not measurably permeant through the open channel. Channel activity increased with the cytoplasmic free Ca2+ concentration ([Ca2+],) with a Hill plot giving a half-saturating [Ca2+] (K-0.5) of 1.35 muM and slope of congruent to3. The BK channel was inhibited reversibly by external tetraethylammonium (TEA) ions, charybdotoxin, and quinine and was resistant to block by 4-aminopyridine and apamin. Ionomycin (1-10 muM) increased BK channel activity in the cell-attached recording configuration. The resting activity was consistent with a [Ca2+](i)

The role of calcium, calcium-activated K+ channels, and tyrosine/kinase in psoralen-evoked responses in human melanoma cells

8-Methoxy psoralen (8-MOP) exerts a short-term (24 h) mitogenic action, and a long-term (48-72 h) anti-proliferative and melanogenic action on two human melanoma cell lines, SK-Mel 28 and C32TG. An increase of intracellular calcium concentration was observed by spectrofluorometry immediately after the addition of 0.1 mM 8-MOP to both cell lines, previously incubated with calcium probe fluo-3 AM (5 µM). The intracellular Ca2+ chelator BAPTA/AM (1 µM) blocked both early (mitogenic) and late (anti-proliferative and melanogenic) 8-MOP effects on both cell lines, thus revealing the importance of the calcium signal in both short- and long-term 8-MOP-evoked responses. Long-term biological assays with 5 and 10 mM tetraethylammonium chloride (TEA, an inhibitor of Ca2+-dependent K+ channels) did not affect the responses to psoralen; however, in 24-h assays 10 mM TEA blocked the proliferative peak, indicating a modulation of Ca2+-dependent K+ channels by 8-MOP. No alteration of cAMP basal levels or forskolin-stimulated cAMP levels was promoted by 8-MOP in SK-Mel 28 cells, as determined by radioimmunoassay. However, in C32TG cells forskolin-stimulated cAMP levels were further increased in the presence of 8-MOP. In addition, assays with 1 µM protein kinase C and calcium/calmodulin-dependent kinase inhibitors, Ro 31-8220 and KN-93, respectively, excluded the participation of these kinases in the responses evoked by 8-MOP. Western blot with antibodies anti-phosphotyrosine indicated a 92% increase of the phosphorylated state of a 43-kDa band, suggesting that the phosphorylation of this protein is a component of the cascade that leads to the increase of tyrosinase activity.