Rôle de deux groupes de vésicules dans la transmission synaptique

Les synapses formées par les fibres moussues (FM) sur les cellules principales de la région CA3 (FM-CA3) jouent un rôle crucial pour la formation de la mémoire spatiale dans l’hippocampe. Une caractéristique des FM est la grande quantité de zinc localisée avec le glutamate dans les vésicules synaptiques recyclées par la voie d’endocytose dépendante de l’AP3. En combinant l’imagerie calcique et l’électrophysiologie, nous avons étudié le rôle des vésicules contenant le zinc dans la neurotransmission aux synapses FM-CA3. Contrairement aux études précédentes, nous n’avons pas observé de rôle pour le zinc dans l’induction des vagues calciques. Nos expériences ont révélé que les vagues calciques sont dépendantes de l’activation des récepteurs métabotropiques et ionotropiques du glutamate. D’autre part, nos données indiquent que les vésicules dérivées de la voie dépendante de l’AP3 forment un groupe de vésicules possédant des propriétés spécifiques. Elles contribuent principalement au relâchement asynchrone du glutamate. Ainsi, les cellules principales du CA3 de souris n’exprimant pas la protéine AP3 avaient une probabilité inférieure de décharge et une réduction de la synchronie des potentiels d’action lors de la stimulation à fréquences physiologiques. Cette diminution de la synchronie n’était pas associée avec un changement des paramètres quantiques ou de la taille des groupes de vésicules. Ces résultats supportent l’hypothèse que deux groupes de vésicules sont présents dans le même bouton synaptique. Le premier groupe est composé de vésicules recyclées par la voie d’endocytose utilisant la clathrine et participe au relâchement synchrone du glutamate. Le second groupe est constitué de vésicules ayant été recyclées par la voie d’endocytose dépendante de l’AP3 et contribue au relâchement asynchrone du glutamate. Ces deux groupes de vésicules sont nécessaires pour l’encodage de l’information et pourraient être importants pour la formation de la mémoire. Ainsi, les décharges de courte durée à haute fréquence observées lorsque les animaux pénètrent dans les places fields pourraient causer le relâchement asynchrone de glutamate. Finalement, les résultats de mon projet de doctorat valident l’existence et l’importance de deux groupes de vésicules dans les MF qui sont recyclées par des voies d’endocytoses distinctes et relâchées durant différents types d’activités.Mossy fiber-CA3 pyramidal cell synapses play a crucial role in the hippocampal formation of spatial memories. These synaptic connections possess a number of unique features substantial for its role in the information processing and coding. One of these features is presence of zinc co-localized with glutamate within a subpopulation of synaptic vesicles recycling through AP3-dependent bulk endocytosis. Using Ca2+ imaging and electrophysiological recordings we investigated role of these zinc containing vesicles in the neurotransmission. In contrast to previous reports, we did not observe any significant role of vesicular zinc in the induction of large postsynaptic Ca2+ waves triggered by burst stimulation. Moreover, our experiments revealed that Ca2+ waves mediated by Ca2+ release from internal stores are dependent not only on the activation of metabotropic, but also ionotropic glutamate receptors. Nevertheless, subsequent experiments unveiled that the vesicles derived via AP3-dependent endocytosis primary contribute to the asynchronous, but not synchronous mode of glutamate release. Futhermore, knockout mice lacking adaptor protein AP3 had a reduced synchronization of postsynaptic action potentials and impaired information transfer; this was not associated with any changes in the synchronous release quantal parameters and vesicle pool size. These findings strongly support the idea that within a single presynaptic bouton two heterogeneous pools of releasable vesicles are present. One pool of readily releasable vesicles forms via clathrin mediated endocytosis and mainly participates in the synchronous release; a second pool forms through bulk endocytosis and primarily supplies asynchronous release. The existence of two specialized pools is essential for the information coding and transfer within hippocampus. It also might be important for hippocampal memory formation. In contrast to low firing rates at rest, dentate gyrus granule cells tend to fire high frequency bursts once an animal enters a place field. These burst activities, embedded in the lower gamma frequency, should be especially efficient in the triggering of substantial asynchronous glutamate release. Therefore, the results of my PhD project for the first time provide strong evidence for the presence and physiological importance of two vesicle pools with heterogeneous release and recycling properties via separate endocytic pathways within the same mossy fiber bouton

Toxines et Transferts ioniques

Collection Rencontres en Toxinologie, ISSN 1760-6004 ; http://sfet.asso.fr/international/images/stories/SFET/pdf/Ebook-RT19-2011-signets.pdfInternational audienc

Functional analysis of ryanodine receptor 2 mutations in induced pluripotent stem cell-derived cardiomyocytes from CPVT patients

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an ion channel disorder in the heart, which is characterized by abnormal calcium handling, ventricular arrhythmias, and sudden cardiac death. This inherited disease is predominantly caused by mutations in the ryanodine receptor type 2 (RYR2). Most of the identified mutations are clustered into four distinct domains of the RYR2 channel. Although heterologous expression systems and animal models have brought important insights in the CPVT pathogenesis, the underlying electrophysiological mechanisms have not been completely understood. The aim of the study was to take cells from CPVT patients with specific RYR2 mutations, to create patient-specific induced pluripotent stem cells (iPSCs), to differentiate these cells into cardiomyocytes (CMs) and then to model the disease in vitro for a better understanding of the disease mechanism and for the investigation of novel therapeutic applications for CPVT patients. Somatic cells from skin biopsies of CPVT patients carrying the RYR2 mutation in domain a (R420W), domain b (A2254V), domain c (E4076K) or domain d (H4742Y) were isolated and reprogrammed into patient-specific iPSCs. The CPVT as well as healthy control (Ctrl) iPSC lines were differentiated into CMs. The CPVT- and Ctrl-iPSC-derived CMs were investigated for their biological, electrophysiological, and pharmacological differences between the RYR2 mutations in different domains and healthy controls. The differences in 3',5'-cyclic adenosine monophosphate (cAMP) dynamics were investigated as well. Electrophysiological analyses showed that the CPVT-CMs recapitulated the phenotype of CPVT both by patch-clamp assessment and by multielectrode array assessment. The single CPVTdCMs showed a unique early afterdepolarization (EAD) phenotype in basal condition and isoproterenol- (ISO-) challenged condition. However, CPVTa-, CPVTb- and CPVTc-CMs exhibited delayed afterdepolarization (DAD) and DAD-induced triggered activities (TAs), which were significantly enhanced after the ISO treatment. In the monolayer cultures, all CPVT-CMs revealed a significantly increased number of premature ventricular complex- (PVC-) like events and prolonged duration of ventricular tachycardia- (VT-) like events after the ISO treatment. In contrast, no increased appearance of arrhythmic events (DADs, EADs, DAD- or EAD-induced TAs, and PVC- and VT-like events) was observed in Ctrl-CMs after the ISO treatment. Four antiarrhythmic drugs (flecainide, dantrolene, rycal1 and rycal2) showed antiarrhythmic effects on CPVTa-, CPVTb- and CPVTc-CMs, but no or minor antiarrhythmic effect on CPVTd-CMs. FRET measurement revealed that the contribution of phosphodiesterase 2 (PDE2) to cAMP degradation in all CPVTa- (15.41%), b- (9.48%), c- (15.07%), d-CMs (7.9%) were significantly lower than in Ctrl-CMs (27.5%) in cytosol. The contribution of PDE2 to cAMP degradation at the RYR2 compartment in all CPVTa- (14.19%), b- (25.21%), c- (17.32%), d-CMs (8.6%) were also significantly lower than in Ctrl-CMs (39.98%). Similar to PDE2, the contribution of PDE3 to cAMP degradation in all CPVT-CMs were significantly lower than in Ctrl-CMs both in cytosol and at the RYR2 compartment. However, for PDE4, there were lower activities in cytosol in CPVTb- and CPVTc-CMs when compared to Ctrl-CMs, and lower activities at the RYR2 compartment in CPVTc- and CPVTd-CMs when compared to Ctrl-CMs. Furthermore, the data indicate that PDE4 is the major regulator of cAMP level in CPVT-CMs both in cytosol and at the RYR2 compartment after ISO stimulation and that PDE2 and PDE3 have a smaller contribution to regulate the cAMP level. Taken together, this study reveals that mutation-specific CPVT-iPSCs can be used to model the disease in vitro, to investigate the disease pathophysiological and molecular mechanisms and to optimize drug therapies

Characterisation of inter-subunit interactions within cardiac ryanodine receptor

Ryanodine receptors (RyRs) are the largest known ion channels composed of four identical subunits. Interactions between structural/functional domains have been proposed to regulate channel activity and play an important role in the pathogenesis of RyR-associated disorders. RyR2 mediates the release of calcium form sarcoplasmic reticulum of cardiac myocytes and its dysfunction is associated with life-threatening arrhythmias. The principal aim of this study was to characterise the self-association of the RyR2 N-terminus biochemically and evaluate its impact on channel function. Moreover, its role in channel dysfunction observed in arrhythmia-susceptible individuals was tested together with dantrolene’s ability to rescue the disease phenotype. RyR2 N-terminus self-association is mediated by multiple sites with two critical oligomerisation determinants located in the loops connecting strands β8-β9 and β20-β21, predicted to reside at the inter-subunit interface. N-terminus self-association is further stabilised by disulphide bonds most likely involving multiple cysteine residues with cysteine 361 contributing to this process. Normal N-terminal inter-subunit interactions within the full-length RyR2 appear to prevent spontaneous activation of the channel at diastolic calcium. Channel hypersensitivity is a common feature of the arrhythmia-associated phenotype suggesting that abnormal N-terminus self-interaction might be involved in RyR2 pathology. Indeed the presence of arrhythmia-linked mutations (L433P and R176Q) compromises the ability of the RyR2 N-terminus to oligomerise. Defective N-terminus self-association appears to underlie the functional impairment of RyR2L433P. The mutated channel displays compromised [3H]ryanodine binding and reduced stability of tetrameric assembly, both of which can be rescued by dantrolene at clinically relevant concentrations. Notably, dantrolene’s primary mode of action appears to involve stabilisation of the N-terminal inter-subunit interactions. In summary, the work presented here provides important insights into a novel domain-domain interaction and its role in the regulation of RyR2 function

Design and synthesis of polycyclic amine derivatives for sigma receptor activity

>Magister Scientiae - MScNew therapeutic strategies are needed for a diverse array of poorly understood neurological impairments. These include neurodegenerative disorders such as Parkinson’s disease and Alzheimer’s disease, and the psychiatric disorders such as depression, anxiety and drug dependence. Popular neuropharmacotherapies have focused on dopamine (DA), serotonin (5HT), γ-aminobutric acid (GABA) and glutamate systems (Jupp & Lawrence, 2010). However recent research points to the sigma receptor (σR) as a possible neuromodulatory system. Due to its multi-receptor action, the σR can trigger several significant biological pathways. This indicates its ideal potential as a drug target to effectively minimise drug dosage and potential side effects. Currently there are a limited number of σR ligands available and few possess the selectivity to significantly show σR’s role in neurological processes. Polycyclic amines have shown notable sigma activity and provide an advantageous scaffold for drug design that can improve pharmacodynamic and pharmacokinetic properties (Banister et al., 2010; Geldenhuys et al., 2005). Aryl-heterocycle amine groups were also shown to improve σR activity (Piergentili et al., 2009)

Calcium Regulation of Cell-Cell Communication and Extracellular Signaling

As a highly versatile signal, Ca2+ operates over a wide temporal range to regulate many different cellular processes, impacting nearly every aspect of cellular life including excitability, exocytosis, motility, apoptosis, and transcription. While it has been well recognized that Ca2+ acts as both a second messenger to regulate cell-cell communication upon external stimuli and as a first messenger to integrate extracellular with intracellular signaling in various cell types. Molecular bases for such regulation and related human diseases are largely hampered by the challenges related to key membrane proteins. In the present study, we first investigated the regulatory role of intracellular Ca2+ ([Ca2+]i) on Connexin45 (Cx45) gap junction through a ubiquitous Ca2+ sensor protein-Calmodulin (CaM). Using bioluminescence resonance energy transfer assay, this study provides the first evidence of direct association of Cx45 and CaM in a Ca2+-dependent manner in cells. Complementary approaches including bioinformatics analysis and various biophysical methods identified a putative CaM-binding site in the intracellular loop of Cx45 with high Ca2+/CaM-binding affinity and Ca2+-dependent binding mode that is different from alpha family of connexins. To understand the role of extracellular calcium in regulation of gap junction hemichannels, we would like to prove a possible Ca2+-binding site predicted by our computational algorithm MUGSR in Connexin 26 (Cx26) through mutagenesis study, metal binding affinity measurement, conformational changes examination of purified Cx26 protein from Sf9; however, we failed to achieve this goal due to either the limitation of available methods or lethal effect of mutating the predicted Ca2+-binding ligand. Additionally, in this study, we identified a putative Ca2+-binding site in metabotropic glutamate receptor 5 (mGluR5) and demonstrated the importance of this Ca2+-binding site in activation of mGluR5 and modulating the actions of other orthosteric ligands on mGluR5. In addition, we successfully solved the first crystal structure of the extracellular domain of Ca2+-sensing receptor (CaSR) bound with Mg2+ and an unexpected Trp derivative. The extensive study of mechanism of CaSR function specifically through Mg2+-binding site and the unexpected ligand-binding site was done using several cell-based assays in wild type CaSR and mutants. Studies in this dissertation provides more information on how Ca2+ regulates gap junction channels, modulates mGluR5 activities and structural basis for regulation of CaSR by Mg2+ and an unexpected Trp derivative co-agonist