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.

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

In 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.

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.

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.