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

Axonal transport regulation : Implication for the development of neuronal network

Pendant le développement, les projections axonales à longue distance se ramifient pour se connecter à leurs cibles. L’établissement et le remodelage de ces connexions est notamment régulé par l’activité neuronale. L’adaptation de la morphologie de l’axone nécessite alors des quantités importantes de matériel sécrétoire et de facteur trophiques comme le BDNF (brain derived neurotrophic factor). Ce matériel est transporté dans des vésicules le long de l’axone depuis le corps cellulaire où il est synthétisé, vers les sites actifs à l’extrémité de l’axone. Si le relargage de vésicules sécrétoires à la synapse est bien étudié, les mécanismes régulant le transport axonal par l’activité sont encore méconnus.Dans ce travail de thèse, nous avons dans un premier temps développé des outils permettant d’étudier les dynamiques intracellulaires dans des réseaux neuronaux. Nous avons ainsi développé une chambre microfluidique permettant de reconstruire in vitro des réseaux neuronaux physiologiques et compatibles avec de la vidéomicroscopie à haute résolution. Nous avons caractérisé l’établissement et la maturation du réseau et validé l’intérêt de ce dispositif microfluidique dans le contexte de la maladie de Huntington. Nous avons ensuite étudié l’évolution des dynamiques intracellulaires avec la maturation du réseau. Nous avons notamment observé une augmentation du transport axonal de vésicules sécrétoires en fonction de l'état de maturation du réseau neuronal. Ces premières observations ont renforcé l’hypothèse d’une régulation directe du transport axonal de vésicules sécrétoires par l’activité neuronale au cours du développement du réseau.Nous avons ainsi fait évoluer la plateforme microfluidique par l’ajout d’un réseau d’électrodes (MEA) qui permet d'étudier les dynamiques intracellulaires tout en contrôlant l’activité neuronale. A l’aide de ce système, nous avons identifié un groupe de vésicules sécrétoires ancré le long de l’axone et recruté en réponse à une haute activité neuronale en direction des sites présynaptiques actifs. Nous avons alors identifié les acteurs impliqués dans ce mécanisme dépendant de l’activité. Nous avons montré que la myosine Va permettait l’attachement des vésicules le long de l’axone dans des structures d’actine dynamique. L’activité neuronale induit une augmentation de calcium le long de l’axone, via l’activation des canaux calciques dépendant du voltage, qui régule la myosine Va et entraine le recrutement des vésicules stockées dans l’axone sur les microtubules. Une fois les acteurs identifiés, nous avons pu mettre en évidence le rôle de ce mécanisme dépendant de l’activité dans la formation de branches axonales pendant le développement. Enfin, nous avons confirmé l’existence de ce groupe de vésicules dépendant de l’activité et résidant dans l’axone in vivo grâce à la mise au point d'un système d’étude du transport axonal sur tranches aigües de cerveau en microscopie biphotonique.L’ensemble de ce travail propose de nouveaux outils in vitro et in vivo pour comprendre les régulations des dynamiques intracellulaires dans des réseaux neuronaux physiologiques. Grâce à ces outils, nous avons identifié un mécanisme de régulation local qui permet l'adressage rapide de facteurs trophiques vers les branches en développement en réponse à l’activité neuronale.During postnatal development, long-distance axonal projections form branches to connect with their targets. Establishment and remodeling of these projections are tightly regulated by neuronal activity and require a large amount of secretory material and trophic factors, such as brain derived neurotrophic factor (BDNF). Axonal transport is responsible for addressing trophic factors packed into vesicles to high demand sites where mechanisms of secretion are well-known. However, mechanisms controlling the preferential targeting of axonal vesicles to active sites in response to neuronal activity are unknown.In this work, we first developed tools to study intracellular dynamics in neuronal networks. We thus developed a microfluidic chamber to reconstruct physiologically-relevant networks in vitro which is compatible with high resolution videomicroscopy. We characterized the formation and maturation of reconstructed networks and we validated the relevance of the microfluidic platform in the context of Huntington’s disease. We then studied the evolution of intracellular dynamics with the maturation of reconstructed neuronal networks in microfluidic chambers. We observed an increase of anterograde axonal transport of secretory vesicles during maturation. These first results lead us to think that neuronal activity could regulate axonal transport of secretory vesicles over maturation of the network.Therefore, we improved the in vitro microfluidic system with a designed microelectrode array (MEA) substrate allowing us to record intracellular dynamics while controlling neuronal activity. Using this system, we identified an axon-resident reserve pool of secretory vesicles recruited upon neuronal activity to rapidly distribute secretory materials to presynaptic sites. We identified the activity-dependent mechanism of recruitment of this axonal pool of vesicles along the axon shaft. We showed that Myosin Va ensures the tethering of vesicles in the axon shaft in axonal actin structures. Specifically, neuronal activity induces a calcium increase after activation of Voltage Gated Calcium Channels along the axon, which regulates Myosin Va and triggers the recruitment of tethered vesicles on microtubules. We then showed the involvement of this activity-dependent pool for axon branches formation during axon development. By developing 2-photon live microscopy of axonal transport in acute slices, we finally confirmed that a pool of axon-resident static vesicles is recruited by neuronal activity in vivo with a similar kinetic.Altogether, this work provides new in vitro and in vivo tools to study intracellular dynamics in physiological networks. Using these tools, we identified the existence of a local mechanism of axonal transport regulation along the axon shaft, allowing rapid supply of trophic factors to developing branches

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

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

Reconstituting Corticostriatal Network on-a-Chip Reveals the Contribution of the Presynaptic Compartment to Huntington’s Disease

Summary: Huntington’s disease (HD), a devastating neurodegenerative disorder, strongly affects the corticostriatal network, but the contribution of pre- and postsynaptic neurons in the first phases of disease is unclear due to difficulties performing early subcellular investigations in vivo. Here, we have developed an on-a-chip approach to reconstitute an HD corticostriatal network in vitro, using microfluidic devices compatible with subcellular resolution. We observed major defects in the different compartments of the corticostriatal circuit, from presynaptic dynamics to synaptic structure and transmission and to postsynaptic traffic and signaling, that correlate with altered global synchrony of the network. Importantly, the genetic status of the presynaptic compartment was necessary and sufficient to alter or restore the circuit. This highlights an important weight for the presynaptic compartment in HD that has to be considered for future therapies. This disease-on-a-chip microfluidic platform is thus a physiologically relevant in vitro system for investigating pathogenic mechanisms and for identifying drugs. : Using microfluidics to reconstruct a Huntington’s disease corticostriatal network, Virlogeux et al. identify recurrent pre- and postsynaptic alterations leading to global circuit dysfunctions and hypersynchrony. They further demonstrate that the genetic status of the presynaptic compartment determines integrity of the network. Keywords: microchambers, microfluidics, huntingtin, axonal and dendritic transport, BDNF, mitochondria, glutamate, TrkB, synapse, GCaMP6

ALG-2 interacting protein-X (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 activity dependent bulk endocytosis (ADBE). Alix (ALG-2 interacting protein X)/ PDCD6IP) is an adaptor protein binding to ESCRT and endophilin-A proteins and thereby driving deformation and fission of endosomal and cell surface membranes. In fibroblasts, Alix is required for clathrin-independent endocytosis. Here, using electron microscopy, we show that synapses from mice lacking Alix have subtle defects in presynaptic compartments, translating into flawed short-term synaptic plasticity. Using cultured neurons, we demonstrate that Alix is specifically involved in ADBE. We further demonstrate that in order to perform ADBE, Alix must be recruited to synapses by the calcium binding protein ALG-2 and interact with endophilin-A. Finally, we show that Alix ko mice are resistant to kainaite-induced epileptic seizures. These results thus highlight Alix ko mice as an invaluable model to study the exact role of ADBE at synapses undergoing physiological or pathological stimulations

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 activity-dependent 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 clathrin-independent 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