Abstract
N-methyl-d-aspartate receptors (NMDARs) are ion channels whose synaptic versus extrasynaptic localization critically influences their functions. This distribution of NMDARs is highly dependent on their lateral diffusion at the cell membrane. Each obligatory subunit of NMDARs (GluN1 and GluN2) contains two extracellular clamshell-like domains with an agonist-binding domain and a distal N-terminal domain (NTD). To date, the roles and dynamics of the NTD of the GluN1 subunit in NMDAR allosteric signaling remain poorly understood. Using single nanoparticle tracking in mouse neurons, we demonstrate that the extracellular neuronal protease tissue-type plasminogen activator (tPA), well known to have a role in the synaptic plasticity and neuronal survival, leads to a selective increase of the surface dynamics and subsequent diffusion of extrasynaptic NMDARs. This process explains the previously reported ability of tPA to promote NMDAR-mediated calcium influx. In parallel, we developed a monoclonal antibody capable of specifically blocking the interaction of tPA with the NTD of the GluN1 subunit of NMDAR. Using this original approach, we demonstrate that the tPA binds the NTD of the GluN1 subunit at a lysine in position 178. Accordingly, when applied to mouse neurons, our selected antibody (named Glunomab) leads to a selective reduction of the tPA-mediated surface dynamics of extrasynaptic NMDARs, subsequent signaling and neurotoxicity, both in vitro and in vivo. Altogether, we demonstrate that the tPA is a ligand of the NTD of the obligatory GluN1 subunit of NMDAR acting as a modulator of their dynamic distribution at the neuronal surface and subsequent signaling.
Highlights
Using single nanoparticle tracking in mouse neurons, we demonstrate that the extracellular neuronal protease tissue-type plasminogen activator, well known to have a role in the synaptic plasticity and neuronal survival, leads to a selective increase of the surface dynamics and subsequent diffusion of extrasynaptic N-methyl-D-aspartate receptors (NMDARs)
NMDAR surface diffusion was recorded in cultured neurons that were incubated with single nanoparticle complexes containing a quantum dot (QD) associated with polyclonal antibodies raised against C-terminal end of GluN1 subunit (GluN1) N-terminal domain (NTD) (Figure 1a)
We measured the phosphorylated and total ERK(1⁄2) levels in primary neuronal cultures subjected to NMDA (50 μM), type plasminogen activator (tPA) (300 nM) and Glunomab (10 μg/ml) exposures either alone or in Discussion In this report, we demonstrate that extrasynaptic GluN1NMDARs surface dynamics and subsequent signaling are increased by the neuronal extracellular serine protease tPA, leading to an enhanced NMDAR signaling and neurotoxicity (Supplementary Figure 5)
Summary
Tissue-type plasminogen activator (tPA), a serine protease of 69 kDa, is expressed in most organs, including the brain and the spinal cord.[11,12,13,14] It consists of five different functional domains through which it interacts with various substrates, binding proteins and receptors.[15,16] In the central nervous system (CNS), tPA can be synthesized and released by virtually all cell types This neuromodulator displays an array of important functions, which are involved in synaptic plasticity,[17] learning and memory processes,[18,19] anxiety 20 and neuronal survival or death.[14,21,22,23] previous studies demonstrated that tPA was a modulator of NMDARs signaling through a possible interaction with the GluN1 NTD,[22,24] the exact molecular mechanism of this function remains under debate.[10]. Received 24.3.16; revised 18.7.16; accepted 02.8.16; Edited by E Baehrecke tPA promotes the dynamics of extrasynaptic NMDAR F Lesept et al
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