Membrane Traffic at Synapses
Team Leader : David Perrais
The team (May 2022)
Front row, from left to right: Alessandra, Maria, Silvia, Sarka, Kiran
Second row: Vivek, Paul, David, Julie, Vincent, Lou, Etienne
Vesicular trafficking is one of the most salient features of synaptic physiology. In the tiny (less than 1 µm wide) chemical synapses, presynaptic vesicles concentrate and release neurotransmitter molecules which bind to post-synaptic receptors. The exocytosis and recycling of synaptic vesicles is a very prominent and essential feature of neuronal physiology that is highly controlled in time and space. Moreover, post-synaptic membrane trafficking, although not as prominent quantitatively, is pivotal for the maintenance of signal transduction complexes supporting synaptic transmission and plasticity. Most of our knowledge about synapse physiology comes from studying glutamatergic synapses which represent the majority of synapses in the brain. Nevertheless, other types of synapses, such as neuromodulatory dopaminergic synapses, could have a very different molecular composition and operate in a different way. However, because they represent a small minority of synapses formed from a very small number of neurons, their analysis has been difficult through classical cellular and molecular methods.
Our goal in the team is to use the most advanced fluorescence imaging techniques together with refined purification of synaptic elements (synaptosomes) to address the mechanisms regulating synapse function through membrane trafficking events in normal brain physiology or in the course of disease. To achieve this goal, we use, on top of the standard techniques of the modern neuroscience lab (molecular biology, biochemistry, imaging, electrophysiology), two unique expertise developed by the two PIs: first, with David Perrais, we develop methods to detect individual exocytosis and endocytosis events with pH sensitive fluorophores and perform quantitative imaging. Second, with Etienne Herzog, we purify synaptosomes from adult animals with fluorescence activated synaptosome sorting (FASS), which enables powerful proteomics, transcriptomics and functional approaches.
Altogether, we aim at identifying new pathways in specific synapses and test their relevance for synaptic nanostructure and function in the normal and diseased brain.
Post-synaptic exocytosis and synaptic plasticity - Cell Reports Sept 2021
Synapses, the basic building blocks of neural networks, are both very stable and capable of rapid and long-lasting modifications, a phenomenon known as synaptic plasticity. The modification of a synapse often involves the addition of synaptic receptors (long-term potentiation or LTP) or the removal of part of the synaptic receptors (long-term depression or LTD). This rapid plasticity is possible because synaptic receptors are not immobile in the synapse but travel to intracellular compartments called recycling endosomes (RE). The regulation of RE trafficking has thus become an important topic of study for understanding the mechanisms of synaptic plasticity.
Picture of all contributors of this study. David Perrais and May Bakr stand in front of "The paint pipette", taken by May, which was awarded the best IINS picture prize in 2020.
In this study, we searched for the molecules involved in these phenomena, in particular the proteins responsible for exocytosis called SNAREs. The VAMP2 protein, target of the tetanus toxin (released by the bacterium responsible for tetanus and one of the most deadly in humans), was known to block LTP. However, to our surprise, it only marginally affected RE exocytosis. We therefore searched for other SNARE proteins and found VAMP4 to be responsible for the majority of RE exocytosis, whereas VAMP2 is involved in only a small fraction of exocytosis, but plays a major role in the exocytosis of REs containing AMPA-type postsynaptic receptors (see Figure). Furthermore, VAMP4 deletion also alters the trafficking of AMPA receptors that are in greater quantity at the surface of neurons, increasing synaptic transmission and limiting its plasticity by occlusion.
This work shows the great diversity of membrane trafficking mechanisms in the dendrites of neurons that allows receptors to be delivered when and where they are needed to regulate individual synapses. It was the result of a long-term work, over more than eight years, of many students, engineers and researchers of the IINS.
Dopamine hub synapses in the striatum
Dopamine Hub Synapses in the striatum: a new hot spot for dopamine transmission?
How is the conversation between neurons organized in the brain? Through 2 recent articles* we describe part of this organization between the dopamine and surrounding neurons at synapses. Synapses are points of contact between neurons, essential for the proper functioning of the brain. In the brain, neurons are of 2 main types. The effector neurons ensure a rapid and local transmission of information, either excitatory or inhibitory, while the modulatory neurons, few in number, affect large regions of the brain over longer periods of time. Modulatory neurons using dopamine are very important for the tuning of motor control, motivation and reward perception.
In our studies, we established the first selective purification of dopaminergic synapses in the striatum that allowed us to identify 2650 proteins, 57 of which were specifically enriched. In contrast, few messenger RNAs (encoding proteins) are selectively detected, suggesting that local translation of proteins is not a major mechanism at the axons of dopaminergic neurons. In addition, we have identified a new structure where dopaminergic synapses physically interact with other classical synapses and affect the composition of the latter. These "Dopamine Hub Synapses" may mediate dopamine neuromodulation on striatal neuronal circuits, fueling the debate between volume and synaptic models of modulatory transmission. Within this new conceptual framework, future research will provide a detailed understanding of the cellular mechanisms by which dopamine modulates voluntary movements or reward-prediction based learning. This is crucial as many pathologies such as Parkinson's disease, addiction and schizophrenia are linked to dopamine dysfunction.
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* January 2022: Hobson, BD., et al. Subcellular and regional localization of mRNA translation in midbrain dopamine neurons. Cell Reports, 38-2, (2022)
* June 2022: Paget-Blanc, V., Pfeffer, M.E., Pronot, M. et al. A synaptomic analysis reveals dopamine hub synapses in the mouse striatum. Nat Commun 13, 3102 (2022).
Endocytosis in the axon initial segment maintains neuronal polarity - Nature, August 2022
Neurons are highly polarized cells that face the fundamental challenge of compartmentalizing a vast and diverse repertoire of proteins in order to function properly. The axon initial segment (AIS) is a specialized domain that separates a neuron's morphologically, biochemically and functionally distinct axon and dendrite compartments. How the AIS maintains polarity between these compartments is not fully understood. Here we find that in Caenorhabditis elegans, mouse, rat and human neurons, dendritically and axonally polarized transmembrane proteins are recognized by endocytic machinery in the AIS, robustly endocytosed and targeted to late endosomes for degradation. Forcing receptor interaction with the AIS master organizer, ankyrinG, antagonizes receptor endocytosis in the AIS, causes receptor accumulation in the AIS, and leads to polarity deficits with subsequent morphological and behavioural defects. Therefore, endocytic removal of polarized receptors that diffuse into the AIS serves as a membrane-clearance mechanism that is likely to work in conjunction with the known AIS diffusion-barrier mechanism to maintain neuronal polarity on the plasma membrane. Our results reveal a conserved endocytic clearance mechanism in the AIS to maintain neuronal polarity by reinforcing axonal and dendritic compartment membrane boundaries.
Authors: Kelsie Eichel, Takeshi Uenaka, Vivek Belapurkar, Rui Lu, Shouqiang Cheng, Joseph S Pak, Caitlin A Taylor, Thomas C Südhof, Robert Malenka, Marius Wernig, Engin Özkan, David Perrais, Kang Shen
- Nature. 2022 Aug 17 - doi: 10.1038/s41586-022-05074-5.
- Contact: David Perrais
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May Bakr (PhD student 2017-2021 - Now Assistant Professor at American University of Cairo, Egypt)
Florencia Angelo (Lab Manager, 2015-2020 - Now Lab Manager at NutriNeuro Lab, Bordeaux, France)
Magalie Martineau (post-doc, 2015-2019 - Now Research and Development specialist at EIF Innovation, Paris, France)
Léa Claverie (PhD student, 2015-2019 - Now Project Manager at Euroquality, Bordeaux, France)
Domenico Azarnia Tehran (Guest, 2019)
Anne-Sophie Hafner (Guest, 2017-2018)
Thi Nhu Ngoc Van (post-doc, 2014-2016 - Now Project Manager at Sys2dia, Montpellier, France)
Julia Krapivkina (PhD student, 2012-2016 - Now Project Manager at Assystem, Paris, France)
Xiao Min Zhang (PhD Student, 2012-2016 - Now Assistant Professor at Sun Yat Sen University - China)
Morgane Rosendale (PhD student and post-doc, 2011-2016 - Now post-doctoral fellow at Institute for Molecular Sciences, Bordeaux)
Damien Jullié (PhD student, 2008-2012 - Now Research Specialist at UCSF Weill Institute for Neurosciences, San Francisco, USA)