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AMPA-type glutamate receptors (AMPARs) play a critical role in mediating the majority of fast excitatory synaptic transmission in the brain, where alterations in receptor expression, distribution and trafficking have been shown to underlie synaptic plasticity and higher brain function. However, the molecular mechanisms regulating AMPAR surface expression and turnover are still not fully understood. We report that mammalian AMPARs are subject to post-translational modification by ubiquitin, and identify Nedd4 as the E3 ligase responsible for mediating this process. AMPAR ubiquitination enhanced receptor degradation and reduced AMPAR cell-surface expression; conversely, inhibition of proteasomal activity caused AMPAR accumulation. Using site-directed mutagenesis we replaced each of four lysine residues available as putative ubiquitination sites on the AMPAR subunit GluA1 C-terminal with an arginine and identified critical residues for ubiquitination and receptor degradation. Consistent with the role of protein ubiquitination, lysine mutation reduced the efficiency of AMPAR endocytosis. We further investigated the molecular mechanisms involved in the internalization of ubiquitinated AMPARs. We find that the endocytic adaptor protein Eps15 plays a critical role in this process. siRNA-mediated suppression or overexpression of Eps15 results in changes in AMPAR surface expression. Eps15 interaction with AMPARs requires Nedd4-mediated GluA1 ubiquitination along with the ubiquitin interacting motif (UIM) of Eps15. Consistent with ubiquitination-mediated receptor internalization, knockdown of Eps15 suppresses GluA1 internalization of wild-type GluA1, but not a mutant GluA1 lacking ubiquitination sites, indicating a crucial role for Eps15 in the trafficking of ubiquitinated AMPARs. These findings reveal novel regulatory mechanisms in the control of glutamate receptor amount and distribution dynamics, which are key factors implicated in higher brain functions and neurological disorders.

Yes / The interplay between long-term potentiation and long-term depression (LTD) is thought to be involved in learning and memory formation. One form of LTD expressed in the hippocampus is initiated by the activation of the group 1 metabotropic glutamate receptors (mGluRs). Importantly, mGluRs have been shown to be critical for acquisition of new memories and for reversal learning, processes that are thought to be crucial for cognitive flexibility. Here we provide evidence that MAPK-activated protein kinases 2 and 3 (​MK2/3) regulate neuronal spine morphology, synaptic transmission and plasticity. Furthermore, mGluR-LTD is impaired in the hippocampus of ​MK2/3 double knockout (DKO) mice, an observation that is mirrored by deficits in endocytosis of ​GluA1 subunits. Consistent with compromised mGluR-LTD, ​MK2/3 DKO mice have distinctive deficits in hippocampal-dependent spatial reversal learning. These novel findings demonstrate that the ​MK2/3 cascade plays a strategic role in controlling synaptic plasticity and cognition. / BBSRC

Ubiquilin (UBQLN) is a member of type2 ubiquitin-like (UBL) protein family characterized by an UBL domain at the N-terminus and an ubiquitin associated (UBA) domain at the C-terminus. This protein has been shown to play an important role in the regulation of the levels, aggregation and degradation of various neurodegenerative disease-associated proteins. However, the specific functions and mechanisms of UBQLN regulation still remain to be elucidated. In this study, we investigate the effect of UBQLN expression on α-Amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor (AMPAR) degradation and the underlying molecular mechanisms. We show that UBQLN overexpression decreases AMPAR levels in neurons and also reduces GluA1 expression in HEK 293T cells. Moreover, our results indicate that UBQLN can form a complex with GluA1, and this interaction is related to the ubiquitination of AMPARs. In addition, we find a higher expression of UBQLN2 in Alzheimer’s disease (AD) patient brains, which might be a potential pathological mechanism of GluA1 reduction in AD. Given the crucial effect of UBQLN in AMPAR regulation, UBQLN may play an important role in synaptic transmission, brain functions as well as neurodegenerative diseases. / 2018-07-21T00:00:00Z

Yes / The activity-regulated cytoskeleton-associated (Arc) protein control synaptic strength by facilitating AMPA receptor (AMPAR) endocytosis. Here we demonstrate that Arc targets AMPAR to be internalized through a direct interaction with the clathrin-adaptor protein 2 (AP-2). We show that Arc overexpression overexpression in dissociated hippocampal neurons obtained from C57BL/6 mouse reduces the density of AMPAR GluA1 subunits at the cell surface and reduces the amplitude and rectification of AMPAR-mediated miniature-excitatory postsynaptic currents (mEPSC). Mutations of Arc, that prevent the AP-2 interaction reduce Arc-mediated endocytosis of GluA1 and abolish the reduction in AMPAR-mediated mEPSC amplitude and rectification. Depletion of the AP-2 subunit µ2 blocks the Arc-mediated reduction in mEPSC amplitude, effect that is restored by re-introducing µ2. The Arc/AP-2 interaction plays an important role in homeostatic synaptic scaling as the Arc-dependent decrease in mEPSC amplitude, induced by a chronic increase in neuronal activity, is inhibited by AP-2 depletion. This data provides a mechanism to explain how activity-dependent expression of Arc decisively controls the fate of AMPAR at the cell surface and modulates synaptic strength, via the direct interaction with the endocytic clathrin adaptor AP-2. / This work was supported by the BBSRC_FAPPA BB/J02127X/1 and BBSRC-BB/H018344/1 to SALC and by the FAPESP_RCUK_FAPPA 2012/50147-5 and FAPESP_Young Investigator’s grant 2009/50650-6 to LLdS. SCW was a PhD Student supported be the BBSRC/GSK PhD-CASE Studentship, LPdA is a postdoc fellow supported by FAPESP, YCJ was supported by a FAPESP scientific initiation scholarship.

Les récepteurs ionotropiques AMPA (AMPAR) activés par le glutamate sont les principaux acteurs de la transmission synaptique excitatrice rapide du cerveau. Ils jouent également un rôle crucial dans les processus de plasticité synaptique, reconnus pour être à la base des fonctions cognitives. Les récepteurs canaux P2X sont activés par l'adénosine-5'-triphosphate (ATP) extracellulaire libéré par les neurones ou les cellules gliales. Ils sont exprimés dans le cerveau en périphérie des synapses glutamatergiques, où ils participent à l’excitabilité neuronale et modulent la transmission synaptique ainsi que la plasticité synaptique. Bien que la signalisation purinergique ait de multiples effets sur la transmission et la plasticité synaptique, la fonction des récepteurs P2X au niveau des synapses du cerveau reste à établir. Ici, nous montrons dans les neurones d'hippocampe en culture que l'activation des récepteurs P2X postsynaptiques par l'ATP exogène ou via la libération d'ATP endogène par les cellules gliales diminue l'amplitude des courants miniatures et évoqués des AMPAR postsynaptiques. En utilisant des approches d’électrophysiologie, de biochimie et d'imagerie en temps réel, nous démontrons que l'afflux de calcium passant par les canaux P2X déclenche l’internalisation des AMPAR par un mécanisme d’endocytose clathrine et dynamine dépendante. Cette diminution de surface altère par conséquent la transmission synaptique médiée par les AMPAR. Nous avons aussi démontré par des approches moléculaires et pharmacologiques la cascade de signalisation engagée dans l’altération du trafic des AMPAR de surface après activation des récepteurs P2X. Cette inhibition par les récepteurs P2X, serait dépendante de l’activation de kinases et des phosphatases qui régulent le niveau de phosphorylation des AMPAR. Nos travaux de recherche suggèrent ainsi, que les récepteurs postsynaptiques P2X jouent un rôle essentiel dans la régulation de l'expression de surface des AMPAR et régulent ainsi la force et la plasticité synaptique. / Ionotropic AMPA receptors (AMPAR) activated by glutamate are the main actors of the fast excitatory synaptic transmission in the brain. They also play a crucial role in the process of synaptic plasticity that are widely recognized to be the basis cognitive functions. P2X receptors are ATP-gated cation channels widely expressed in the brain where they mediate action of extracellular adenosine-5’-triphosphate (ATP) released by neurons or glia. P2X receptors are located et the periphery of glutamatergic synapses and although purinergic signaling has multiple effects on synaptic transmission and plasticity, the function of P2X receptors at brain synapses remains to be established.Here, we show in cultured hippocampal neurons that activation of postsynaptic P2X receptors by exogenous ATP or glial release of endogenous ATP decreases the amplitude of miniature excitatory postsynaptic currents and AMPA-evoked currents. Using a combination of electrophysiology, surface or internalization assays and real time imaging, we demonstrate that the calcium influx through the ATP-gated channels triggers AMPA receptor internalization through clathrin-mediated dynamin-dependent endocytosis leading to reduced surface AMPA receptors and therefore, altered AMPA-mediated current. We also identified by molecular and pharmacological approaches the signaling cascade involved in the P2X-mediated alteration of surface AMPAR trafficking. P2X-mediated AMPAR internalization is dependent on the activation of kinases CamKII and phosphatases which regulate the phosphorylation level of AMPARs. Our finding indicates that postsynaptic P2X receptors play a critical role in regulating the surface expression of AMPAR and thereby regulate the synaptic strength.

AMPA-Rezeptoren sind entscheidend für ein funktionsfähiges Nervensystem von Säugetieren und spielen eine Schlüsselrolle bei exzitatorischen Neurotransmitter-Reaktionen im zentralen Nervensystem. Insbesondere die post-transkriptionale Modifizierung des GluA2-Untertyps beeinflusst die Permeation von Kalzium. Diese Arbeit untersucht den Permeationsmechanismus verschiedener Kationentypen durch die Kanäle des AMPA-Rezeptors mittels Molekulardynamik-Simulationen (MD). Die Untersuchung konzentriert sich auf die Q/R modifizierten Untereinheiten des AMPAR-Tetramers und zeigt, dass die Permeation von monovalenten Kationen wie Kalium und Caesium ähnliche Eigenschaften in Simulationen wie im Labor aufweist. Jedoch stellen Natrium und Kalzium aufgrund hoher Hydrationsenergien eine Herausforderung dar, die mit einem neu entwickelten multi-site Modell für Kalzium überwunden wird. Die Simulationen zeigen, dass monovalente Kationen während der Permeation etwa die Hälfte ihrer Wasserschale verlieren, während Kalzium seine erste Hydrathülle aufrechterhält. Dies legt einen wassermediierten Mechanismus für die Kationenpermeation nahe. Interessanterweise blockiert die Q/R Modifikation die Kalziumpermeation und während sie die Leitfähigkeit für monovalente Kationen nur verringert. Die Ergebnisse weisen jedoch auf eine eingeschränkte einwärtsgerichtete Permeationsrate im Vergleich zu Labormessungen und auswärtsgerichteten Permeationen hin, was in der Arbeit eingehend diskutiert wird. / The excitatory neurotransmission mediated by AMPA receptors (AMPARs) is essential for numerous higher brain functions, and abnormalities in their function have been implicated in severe neurodevelopmental disorders. This study employs molecular dynamics (MD) simulations to investigate the mechanisms underlying cation permeation through AMPAR channels with various compositions, shedding light on their functional dynamics. By conducting simulations under transmembrane potentials and utilizing advanced force fields, we explore the permeation of potassium, cesium, and calcium ions. Our results show that advanced models accurately capture calcium conductance, providing insights into the behavior of cations and water molecules during permeation, particularly highlighting the crucial role of the selectivity filter in facilitating ion passage. Additionally, we investigate the impact of natural editing of AMPAR subunits, revealing that the replacement of glutamine with arginine diminishes calcium conductance while allowing monovalent cation permeation. Moreover, simulations with edited subunit compositions demonstrate the differential effects on potassium and calcium permeation, providing further understanding of the structural determinants governing ion selectivity. Furthermore, our investigations into permeation dynamics in both inward and outward directions uncover intriguing differences that warrant further exploration. Overall, this thesis contributes to elucidating the intricate mechanisms underlying cation permeation through AMPAR channels, offering valuable insights into their role in neurotransmission and potential implications for therapeutic interventions targeting neurodevelopmental disorders.

Antidepressants are generally believed to exert their clinical efficacy by enhancing 5-HT transmission. Interestingly, sustained administration of selective serotonin (5-HT) reuptake inhibitors (SSRIs) strongly suppresses in the first few days the firing activity of 5-HT neurons in the dorsal raphe nucleus (DRN), thereby severely hampering the increase of 5-HT in target regions. Remarkably, the firing activity of 5-HT neurons gradually recovers over the time course of treatment and this recovery is believed to be accounted for by the desensitization of 5-HT1A somatodendritic autoreceptors. Here, we sought to investigate whether additional mechanisms might contribute to the dynamic regulation of excitability of 5-HT neurons during the course of SSRI treatments. Borrowing from the well-described homeostatic strengthening of glutamatergic synapses onto cortical pyramidal neurons following prolonged periods of inactivity, we hypothesized that a similar homeostatic-like regulation of synaptic strength might be operant on 5-HT cells during an SSRI treatment. To test this possibility, we used whole-cell electrophysiological recordings on acute midbrain slices to monitor glutamatergic synapses onto 5-HT neurons. We found that a two-day treatment with the SSRI citalopram induced a robust reduction in both the amplitude and frequency of AMPAR-mediated mEPSCs. We also show that this depression in synaptic strength, induced by an SSRI, is transient since excitatory drive onto 5-HT neurons was enhanced by 7 days of treatments. Altogether, these results document a dynamic regulation of glutamatergic synaptic transmission during the time course of a prolonged treatment with an SSRI. Further elucidation of the cellular and molecular mechanisms driving this synaptic plasticity might identify novel pharmacological target to shorten the delay of antidepressant action.

Ubiquilin (UBQL) is a member of type 2 ubiquitin-like (UBL) protein family. They structurally contain an N-terminal ubiquitin-like domain and a C-terminal ubiquitin-associated (UBA) domain. Ubiquilin 2 (UBQL2) physically associates with poly ubiquitinated proteins and delivers them to the proteasome for degradation. This protein has been shown to play an important role in the regulation of aggregation and degradation of various neurodegenerative disease-associated proteins. In this study, we looked into the role of the ubiquilin-2 proteins in the AMPA receptor ubiquitination and proteasomal degradation pathway. Our results indicate that UBQL2 overexpression decreases AMPAR levels in neurons and also reduces GluA1 expression in HEK 293T cells. Moreover, by co-immunoprecipitation we found that UBQL2 interacts with ubiquitinated AMPARs. We, therefore propose that UBQL2 brings AMPARs to the proteasome for degradation. Consistent with this notion, expression of UBQL2 P497H, a mutant form incapable of interaction with proteasome, causes accumulation of AMPA receptors. These results indicate a role for UBQL2 in associating with and directing ubiquitinated AMPA receptors to the proteasome for degradation. / 2020-08-09T00:00:00Z

Antidepressants are generally believed to exert their clinical efficacy by enhancing 5-HT transmission. Interestingly, sustained administration of selective serotonin (5-HT) reuptake inhibitors (SSRIs) strongly suppresses in the first few days the firing activity of 5-HT neurons in the dorsal raphe nucleus (DRN), thereby severely hampering the increase of 5-HT in target regions. Remarkably, the firing activity of 5-HT neurons gradually recovers over the time course of treatment and this recovery is believed to be accounted for by the desensitization of 5-HT1A somatodendritic autoreceptors. Here, we sought to investigate whether additional mechanisms might contribute to the dynamic regulation of excitability of 5-HT neurons during the course of SSRI treatments. Borrowing from the well-described homeostatic strengthening of glutamatergic synapses onto cortical pyramidal neurons following prolonged periods of inactivity, we hypothesized that a similar homeostatic-like regulation of synaptic strength might be operant on 5-HT cells during an SSRI treatment. To test this possibility, we used whole-cell electrophysiological recordings on acute midbrain slices to monitor glutamatergic synapses onto 5-HT neurons. We found that a two-day treatment with the SSRI citalopram induced a robust reduction in both the amplitude and frequency of AMPAR-mediated mEPSCs. We also show that this depression in synaptic strength, induced by an SSRI, is transient since excitatory drive onto 5-HT neurons was enhanced by 7 days of treatments. Altogether, these results document a dynamic regulation of glutamatergic synaptic transmission during the time course of a prolonged treatment with an SSRI. Further elucidation of the cellular and molecular mechanisms driving this synaptic plasticity might identify novel pharmacological target to shorten the delay of antidepressant action.