Cyclotraxin-B, the First Highly Potent and Selective TrkB Inhibitor, Has Anxiolytic Properties in Mice

In the last decades, few mechanistically novel therapeutic agents have been developed to treat mental and neurodegenerative disorders. Numerous studies suggest that targeting BDNF and its TrkB receptor could be a promising therapeutic strategy for the treatment of brain disorders. However, the development of potent small ligands for the TrkB receptor has proven to be difficult. By using a peptidomimetic approach, we developed a highly potent and selective TrkB inhibitor, cyclotraxin-B, capable of altering TrkB-dependent molecular and physiological processes such as synaptic plasticity, neuronal differentiation and BDNF-induced neurotoxicity. Cyclotraxin-B allosterically alters the conformation of TrkB, which leads to the inhibition of both BDNF-dependent and -independent (basal) activities. Finally, systemic administration of cyclotraxin-B to mice results in TrkB inhibition in the brain with specific anxiolytic-like behavioral effects and no antidepressant-like activity. This study demonstrates that cyclotraxin-B might not only be a powerful tool to investigate the role of BDNF and TrkB in physiology and pathology, but also represents a lead compound for the development of new therapeutic strategies to treat brain disorders

Alix is required for activity-dependent bulk endocytosis at brain synapses

In chemical synapses undergoing high frequency stimulation, vesicle components can be retrieved from the plasma membrane via a clathrin-independent process called activitydependent bulk endocytosis (ADBE). Alix (ALG-2-interacting protein X/PDCD6IP) is an adaptor protein binding to ESCRT and endophilin-A proteins which is required for clathrinindependent endocytosis in fibroblasts. Alix is expressed in neurons and concentrates at synapses during epileptic seizures. Here, we used cultured neurons to show that Alix is recruited to presynapses where it interacts with and concentrates endophilin-A during conditions triggering ADBE. Using Alix knockout (ko) neurons, we showed that this recruitment, which requires interaction with the calcium-binding protein ALG-2, is necessary for ADBE. We also found that presynaptic compartments of Alix ko hippocampi display subtle morphological defects compatible with flawed synaptic activity and plasticity detected electrophysiologically. Furthermore, mice lacking Alix in the forebrain undergo less seizures during kainate-induced status epilepticus and reduced propagation of the epileptiform activity. These results thus show that impairment of ADBE due to the lack of neuronal Alix leads to abnormal synaptic recovery during physiological or pathological repeated stimulations

Développement de modulateurs de l'activité du TRKB, le récepteur du BDNF (applications en neuropsychiatrie)

Le BDNF et son récepteur, le TrkB, jouent un rôle clé dans les fonctions cognitives supérieures et dans la physiopathologie de nombreuses maladies neurodégénératives et psychiatriques. L'absence d'outils pharmacologiques spécifiques nous a conduits à développer différents types de modulateurs fonctionnels. Nous avons tout d'abord produit un petit peptide cyclique, capable d'inhiber spécifiquement l'activité du TrkB et les évènements physiologiques et pathophysiologiques qui lui sont associés. L'injection intraveineuse du peptide à des souris entraine des altérations comportementales spécifiques. A partir d'une approche virtuelle innovante, nous avons également découvert des agonistes et antagonistes du TrkB possédant un fort potentiel d'utilisation in vivo.Enfin, le criblage fonctionnel d'anticorps anti-TrkB du commerce a révélé des propriétés biologiques distinctes, constituant une nouvelle gamme de modulateurs spécifiques. Ces nouveaux outils permettront ainsi l'étude du système BDNF/TrkB dans les fonctions physiologiques et pathologiques et serviront de base pour le développement de stratégies thérapeutiques innovantesPARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Pharmacological characterization of six trkB antibodies reveals a novel class of functional agents for the study of the BDNF receptor

International audienceBACKGROUND AND PURPOSE:By interacting with trkB receptors, brain-derived neurotrophic factor (BDNF) triggers various signalling pathways responsible for neurone survival, differentiation and modulation of synaptic transmission. Numerous reports have implicated BDNF and trkB in the pathogenesis of various central nervous system affections and in cancer, thus representing trkB as a promising therapeutic target. In this study, we used an antibody-based approach to search for trkB-selective functional reagents.EXPERIMENTAL APPROACH:Six commercially available polyclonal and monoclonal antibodies were tested on recombinant and native, human and rodent trkB receptors. Functional and pharmacological characterization was performed using a modified version of the KIRA-elisa method and radioligand binding studies. Western blot analyses and neurite outgrowth assays were carried out to determine the specificity and selectivity of antibody effects. The survival properties of one antibody were further assessed on cultured neurones in a serum-deprived paradigm.KEY RESULTS:The functional trkB-selective antibodies showed distinct pharmacological profiles, ranging from partial agonists to antagonists, acting on trkB receptors through allosteric modulations. The same diversity of effects was observed on the mitogen-activated protein kinase signalling pathway downstream of trkB and on the subsequent neurite outgrowth. One antibody with partial agonist activity demonstrated cell survival properties by activating the Akt pathway. Finally, these antibodies were functionally validated as true trkB-selective ligands because they failed activating trkA or trkC, and contrary to BDNF, none of them bind to p75(NTR).CONCLUSIONS AND IMPLICATIONS:These trkB-selective antibodies represent a novel class of pharmacological tools to explore the pathophysiological roles of trkB and its potential therapeutic relevance for the treatment of various disorders

Balancing the basal ganglia circuitry: a possible new role for dopamine D2 receptors in health and disease

International audienceCurrent therapies for treating movement disorders such as Parkinson's disease are effective but limited by undesirable and intractable side effects. Developing more effective therapies will require better understanding of what causes basal ganglia dys-regulation and why medication-induced side effects develop. Although basal ganglia have been extensively studied in the last decades, its circuit anatomy is very complex, and significant controversy exists as to how the interplay of different basal ganglia nuclei process motor information and output. We have recently identified the importance of an underappreciated collateral projection that bridges the striatal output direct pathway with the indirect pathway. These bridging collaterals are extremely plastic in the adult brain and are involved in the regulation of motor balance. Our findings add a new angle to the classical model of basal ganglia circuitry that could be exploited for the development of new therapies against movement disorders. In this Scientific Perspective, we describe the function of bridging collaterals and other recent discoveries that challenge the simplicity of the classical basal ganglia circuit model. We then discuss the potential implication of bridging collaterals in the pathophysiology of Parkinson's disease and schizophrenia. Because dopamine D2 receptors and striatal neuron excitability have been found to regulate the density of bridging collaterals, we propose that targeting these projections downstream of D2 receptors could be a possible strategy for the treatment of basal ganglia disorders

An integrated microfluidic/microelectrode array for the study of activity-dependent intracellular dynamics in neuronal networks

International audienceIn the central nervous system, neurons are organized in specific neural networks with distinct electrical patterns, input integration capacities, and intracellular dynamics. In order to better understand how neurons process information, it is crucial to keep the complex organization of brain circuits. However, performing subcellular investigations with high spatial and temporal resolution in vivo is technically challenging, especially in fine structures, such as axonal projections. Here, we present an on-a-chip system that combines a microfluidic platform with a dedicated matrix of electrodes to study activity-dependent dynamics in the physiological context of brain circuits. Because this system is compatible with high-resolution video-microscopy, it is possible to simultaneously record intracellular dynamics and electrical activity in presynaptic axonal projections and in their postsynaptic neuronal targets. Similarly, specific patterns of electrical activity can be applied to both compartments in order to investigate how intrinsic and network activities influence intracellular dynamics. The fluidic isolation of each compartment further allows the selective application of drugs at identified sites to study activity-dependent synaptic transmission. This integrated microfluidic/microelectrode array (microMEA) platform is a valuable tool for studying various intracellular and synaptic dynamics in response to neuronal activity in a physiologically relevant context that resembles in vivo brain circuits

Striatal D2 Receptors Regulate Dendritic Morphology of Medium Spiny Neurons via Kir2 Channels

International audienceStructural plasticity in the adult brain is essential for adaptive behaviors and is thought to contribute to a variety of neurological and psychiatric disorders. Medium spiny neurons of the striatum show a high degree of structural plasticity that is modulated by dopamine through unknown signaling mechanisms. Here, we demonstrate that overexpression of dopamine D2 receptors in medium spiny neurons increases their membrane excitability and decreases the complexity and length of their dendritic arbors. These changes can be reversed in the adult animal after restoring D2 receptors to wild-type levels, demonstrating a remarkable degree of structural plasticity in the adult striatum. Increased excitability and decreased dendritic arborization are associated with downregulation of inward rectifier potassium channels (Kir2.1/2.3). Downregulation of Kir2 function is critical for the neurophysiological and morphological changes in vivo because virally mediated expression of a dominant-negative Kir2 channel is sufficient to recapitulate the changes in D2 transgenic mice. These findings may have important implications for the understanding of basal ganglia disorders, and more specifically schizophrenia, in which excessive activation of striatal D2 receptors has long been hypothesized to be of pathophysiologic significance

B41 HD on chip : reconstituting the cortico-striatal network on microfluidics to study intracellular trafficking and synaptic transmission

International audienceMost of the cellular or molecular studies in HD used so far separated cultures of striatal or cortical neurons. However, in the brain these neurons are connected and form a particular network that is defective in HD. The polarised nature of neurons and the size and density of synapses complicates the manipulation and visualisation of specific events taking place in axons or dendrites and of specific synaptic transmission within the cortico-striatal network.To overcome these limitations, we developed several microfluidic systems compatible with high-resolution videomicroscopy and connected to microelectrode arrays (MEA) to reconstitute and identify each component of the corticostriatal network. The microfluidic system directs the formation of identified synapses separately between cortical axons and striatal dendrites and soma. In parallel, a multielectrode substrate monitors and controls presynaptic and postsynaptic activity independently. Using this multicomplex system we are investigating how the trafficking of synaptic vesicles or mitochondria along axons is regulated by presynaptic and postsynaptic patterns in the corticostriatal network in health and HD. In addition, the system allows modifying the genetic status of the cortical or striatal neurons as a way to selectively investigate how disease neurons differentially affect pre or post-synaptic events in HD and overall alter synapse function

Neuronal network maturation differently affects secretory vesicles and mitochondria transport in axons

Abstract Studying intracellular dynamics in neurons is crucial to better understand how brain circuits communicate and adapt to environmental changes. In neurons, axonal secretory vesicles underlie various functions from growth during development to plasticity in the mature brain. Similarly, transport of mitochondria, the power plant of the cell, regulates both axonal development and synaptic homeostasis. However, because of their submicrometric size and rapid velocities, studying the kinetics of these organelles in projecting axons in vivo is technically challenging. In parallel, primary neuronal cultures are adapted to study axonal transport but they lack the physiological organization of neuronal networks, which in turn may bias observations. We previously developed a microfluidic platform to reconstruct a physiologically-relevant and functional corticostriatal network in vitro that is compatible with high-resolution videorecording of axonal trafficking. Here, using this system we report progressive changes in axonal transport kinetics of both dense core vesicles and mitochondria that correlate with network development and maturation. Interestingly, axonal flow of both types of organelles change in opposite directions, with rates increasing for vesicles and decreasing for mitochondria. Overall, our observations highlight the need for a better spatiotemporal control for the study of intracellular dynamics in order to avoid misinterpretations and improve reproducibility