8 research outputs found
Consequences of human pathological L1CAM mutations for protein maturation, function and interaction
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
L1 syndrome diagnosis complemented with functional analysis of L1CAM variants located to the two N-terminal Ig-like domains.
L1CAM gene mutations cause neurodevelopmental disorders collectively termed L1 syndrome. Insufficient information about L1CAM variants complicates clinical prognosis, genetic diagnosis and genetic counseling. We combined clinical data, in silico effect predictions and functional analysis of four L1CAM variants, p.I37N, p.D202Y, p.M172I and p.T38M, located to the two N-terminal Ig-like domains present in five families with symptoms of L1 syndrome. Software tools predicted destabilizing effects of p.I37N and p.D202Y but results for p.T38M and p.M172I were inconsistent. Cell surface expression of mutant proteins L1-T38M, L1-M172I and L1-D202Y was normal. Conversely, L1-I37N accumulated in the endoplasmic reticulum and showed temperature-sensitive protein maturation suggesting that p.I37N induces protein misfolding. L1CAM-mediated cell-cell aggregation was severely impaired by L1CAM variants p.I37N, p.M172I and p.D202Y but was preserved by the variant p.T38M. Our experimental data indicate that protein misfolding and accumulation in the endoplasmic reticulum affect function of the L1CAM variant p.I37N whereas the variants p.M172I and p.D202Y impair homophilic interaction at the cell surface
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