AMPA receptor inhibition by synaptically released zinc

The vast amount of fast excitatory neurotransmission in the mammalian central nervous system is mediated by AMPA-subtype glutamate receptors (AMPARs). As a result, AMPAR-mediated synaptic transmission is implicated in nearly all aspects of brain development, function, and plasticity. Despite the central role of AMPARs in neurobiology, the fine-tuning of synaptic AMPA responses by endogenous modulators remains poorly understood. Here we provide evidence that endogenous zinc, released by single presynaptic action potentials, inhibits synaptic AMPA currents in the dorsal cochlear nucleus (DCN) and hippocampus. Exposure to loud sound reduces presynaptic zinc levels in the DCN and abolishes zinc inhibition, implicating zinc in experience-dependent AMPAR synaptic plasticity. Our results establish zinc as an activity-dependent, endogenous modulator of AMPARs that tunes fast excitatory neurotransmission and plasticity in glutamatergic synapses.The development, function, and experience-dependent plasticity of the mammalian brain depend on the refined neuronal interactions that occur in synapses. In the majority of excitatory synapses, the release of the neurotransmitter glutamate from presynaptic neurons opens transmembrane ion channels in postsynaptic neurons, the ionotropic glutamate receptors, thereby generating the flow of excitatory signaling in the brain. As a result, these receptors play a fundamental role in normal function and development of the brain, and they are also involved in many brain disorders (1).The ionotropic glutamate receptor family consists of AMPA, kainate, and NMDA receptors (NMDARs). Although kainate receptor-mediated excitatory postsynaptic responses occur in a few central synapses (2), AMPA receptors (AMPARs) and NMDARs are localized in the postsynaptic density of the vast majority of glutamatergic synapses in the brain, mediating most of excitatory neurotransmission (1). NMDAR function is regulated by a wide spectrum of endogenous allosteric neuromodulators that fine-tune synaptic responses (3–5); however, much less is known about endogenous AMPAR neuromodulators [(1, 5), but see refs. 6 and 7]. Recent structural studies revealed that the amino terminal domain (ATD) and ligand-binding domain (LBD) are tightly packed in NMDARs but not AMPARs (8–10). These structural differences explain some of the functional differences in allosteric modulation between AMPARs and NMDARs, such as why the ATD of NMDARs, unlike that of AMPARs, modulates function and contains numerous binding sites for allosteric regulators. Nonetheless, given the importance of fine-tuning both synaptic AMPAR and NMDAR responses for brain function, it is puzzling that there is not much evidence for endogenous, extracellular AMPAR modulation. The discovery and establishment of endogenous AMPAR modulators is crucial both for understanding ionotropic glutamate receptor signaling and for developing therapeutic agents for the treatment of AMPAR-related disorders, such as depression, cognitive dysfunctions associated with Alzheimer’s disease, and schizophrenia (1, 11).Free, or readily chelatable, zinc is an endogenous modulator of synaptic and extrasynaptic NMDARs (12–15). Free zinc is stored in glutamatergic vesicles in many excitatory synapses in the cerebral cortex, limbic, and brainstem nuclei (16). In some brain areas, such as in the hippocampus, 50% of boutons synapsing onto CA1 neurons and all mossy fibers synapsing onto CA3 neurons contain synaptic free zinc (17). Whereas earlier studies demonstrated that exogenous zinc inhibits AMPARs (18–21), more recent work suggests that endogenously released synaptic zinc does not modulate AMPARs in central synapses (14, 22). This conclusion was derived from the inability to efficiently chelate and quantify synaptic zinc with the zinc-selective chelators and probes used (15), in apparent support of the hypothesized low levels of released zinc during synaptic stimulation (14).Recent work in our laboratories used new chemical tools that allowed us to intercept and visualize mobile zinc efficiently (15). These studies revealed modulation of extrasynaptic NMDARs by zinc and led us to reinvestigate whether synaptically released zinc might be an endogenous modulator of AMPARs as well. In the present study, we applied these same tools in electrophysiological, laser-based glutamate uncaging and in imaging experiments using wild type and genetically modified mice that lack synaptic zinc.     

Modulation of extrasynaptic NMDA receptors by synaptic and tonic zinc

Many excitatory synapses contain high levels of mobile zinc within glutamatergic vesicles. Although synaptic zinc and glutamate are coreleased, it is controversial whether zinc diffuses away from the release site or whether it remains bound to presynaptic membranes or proteins after its release. To study zinc transmission and quantify zinc levels, we required a high-affinity rapid zinc chelator as well as an extracellular ratiometric fluorescent zinc sensor. We demonstrate that tricine, considered a preferred chelator for studying the role of synaptic zinc, is unable to efficiently prevent zinc from binding low-nanomolar zinc-binding sites, such as the high-affinity zinc-binding site found in NMDA receptors (NMDARs). Here, we used ZX1, which has a 1 nM zinc dissociation constant and second-order rate constant for binding zinc that is 200-fold higher than those for tricine and CaEDTA. We find that synaptic zinc is phasically released during action potentials. In response to short trains of presynaptic stimulation, synaptic zinc diffuses beyond the synaptic cleft where it inhibits extrasynaptic NMDARs. During higher rates of presynaptic stimulation, released glutamate activates additional extrasynaptic NMDARs that are not reached by synaptically released zinc, but which are inhibited by ambient, tonic levels of nonsynaptic zinc. By performing a ratiometric evaluation of extracellular zinc levels in the dorsal cochlear nucleus, we determined the tonic zinc levels to be low nanomolar. These results demonstrate a physiological role for endogenous synaptic as well as tonic zinc in inhibiting extrasynaptic NMDARs and thereby fine tuning neuronal excitability and signaling.In many excitatory neurons, the zinc transporter ZnT3 loads high levels of free (readily chelatable) zinc into glutamatergic vesicles. Synaptic zinc is coreleased with glutamate into the extracellular space in an activity-dependent manner, where it modulates the function of many targets, including ion channels and receptors (1, 2). It is nonetheless controversial whether or not synaptic zinc acts via a classic phasic release mode defined by short-lived, high concentrations of chelatable zinc that diffuse away from the release site (3, 4). The fact that zinc binds with high affinity to many proteins, and the inconsistent measurements of chelatable zinc after synaptic release, ranging from no detectable to 100 μM levels (1), have led to an alternative hypothesis for the mode of synaptic zinc transmission and action. According to this model, rather than being free to diffuse, synaptic zinc is postulated to remain bound to presynaptic membrane or protein structures forming a “veneer” of zinc in the synaptic cleft (5, 6). This so-called zinc veneer model predicts that synaptic zinc modulation is mediated by slow buildup of an extracellular layer of zinc ions during synaptic activity, referred to as tonic zinc signaling.A recent study suggests a mixed mode of zinc release and action, whereby zinc is phasically released but acts in a tonic-like manner because trains of action potentials are required to inhibit synaptic NMDA receptors (NMDARs) at mossy fiber synapses (7). However, this study directly contradicts previous findings at mossy fiber synapses showing that only a single action potential invading a mossy fiber axon is capable of releasing a sufficient amount of zinc to inhibit NMDARs (8). Such conflicting conclusions prevail in many imaging and electrophysiological studies addressing the mode of mobile zinc transmission and action (5, 7–13), obfuscating the physiological role of zinc in neurons.The inability to resolve fundamental questions about synaptic zinc transmission is due in part to the lack of zinc-sensing fluorescent probes and zinc-chelating agents optimized for quantifying and interrupting localized, rapidly released synaptic zinc ions. To address the quantitation issue, we synthesized an extracellular ratiometric fluorescent zinc sensor (LZ9) for accurate measurement of chelatable zinc levels. To identify the most appropriate zinc chelator we compared the affinity and kinetics of zinc chelation of three widely-used extracellular zinc chelators, ZX1, CaEDTA, and tricine (7, 8). We subsequently chose ZX1 and applied it together with LZ9 in electrophysiological, laser-based glutamate uncaging and imaging investigations of mobile zinc in WT and genetically modified mice that lack ZnT3, the transporter that loads presynaptic vesicles with zinc. We studied zinc-dependent neuronal activity in cartwheel cells, a class of inhibitory interneurons in the molecular layer of the dorsal cochlear nucleus (DCN) that receive glutamatergic input from synaptic zinc-rich parallel fibers (13–15). The DCN has a cerebellum-like organization, and cartwheel cells share common morphological, ontological, and physiological features with cerebellar neurons (15, 16). One of the major physiological properties of cerebellar molecular layer interneurons is the expression of extrasynaptic NMDARs that are activated by glutamate spillover in response to short trains of parallel fiber action potentials (17, 18). Because zinc inhibits NMDARs with high affinity (19, 20), and because synaptic zinc must act in a phasic manner and diffuse away from the release site to inhibit extrasynaptic NMDARs, we investigated whether synaptic zinc modulates extrasynaptic NMDARs in cartwheel cells.     

Conformational signaling required for synaptic plasticity by the NMDA receptor complex

The NMDA receptor (NMDAR) is known to transmit important information by conducting calcium ions. However, some recent studies suggest that activation of NMDARs can trigger synaptic plasticity in the absence of ion flow. Does ligand binding transmit information to signaling molecules that mediate synaptic plasticity? Using Förster resonance energy transfer (FRET) imaging of fluorescently tagged proteins expressed in neurons, conformational signaling is identified within the NMDAR complex that is essential for downstream actions. Ligand binding transiently reduces FRET between the NMDAR cytoplasmic domain (cd) and the associated protein phosphatase 1 (PP1), requiring NMDARcd movement, and persistently reduces FRET between the NMDARcd and calcium/calmodulin-dependent protein kinase II (CaMKII), a process requiring PP1 activity. These studies directly monitor agonist-driven conformational signaling at the NMDAR complex required for synaptic plasticity.Agonist binding to the NMDAR is required for two major forms of synaptic plasticity: long-term potentiation (LTP) and long-term depression (LTD) (1). Surprisingly, activation of NMDARs can produce plasticity in opposite directions, with LTP enhancing transmission and LTD reducing transmission. A model was developed (2, 3) to explain how activation of NMDAR could produce these opposing phenomena: strong stimuli during LTP induction drive a large flux of Ca²⁺ through NMDARs, leading to a large increase in intracellular calcium ion concentration ([Ca²⁺]_i) that activates one series of biochemical steps leading to synaptic potentiation; a weaker stimulus during LTD induction drives a more reduced flux of Ca²⁺ through NMDARs, producing a modest increase in [Ca²⁺]_i that activates a different series of biochemical steps, leading to synaptic depression. However, this model is not consistent with recent studies suggesting that no change in [Ca²⁺]_i is required for LTD, and instead invokes metabotropic signaling by the NMDAR (4). Studies supporting an ion-flow-independent role for NMDARs in LTD (4–7) and other processes (7–13) stand in contrast to studies proposing that flow of Ca²⁺ through NMDAR is required for LTD (14) (see ref. 15 for additional references). An important test of an ion-flow-independent model would be to measure directly signaling actions by NMDARs in the absence of ion flow.     

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

The postsynaptic density (PSD)-95 family of membrane-associated guanylate kinases (MAGUKs) are major scaffolding proteins at the PSD in glutamatergic excitatory synapses, where they maintain and modulate synaptic strength. How MAGUKs underlie synaptic strength at the molecular level is still not well understood. Here, we explore the structural and functional roles of MAGUKs at hippocampal excitatory synapses by simultaneous knocking down PSD-95, PSD-93, and synapse-associated protein (SAP)102 and combining electrophysiology and transmission electron microscopic (TEM) tomography imaging to analyze the resulting changes. Acute MAGUK knockdown greatly reduces synaptic transmission mediated by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-d-aspartate receptors (NMDARs). This knockdown leads to a significant rise in the number of silent synapses, diminishes the size of PSDs without changes in pre- or postsynaptic membrane, and depletes the number of membrane-associated PSD-95–like vertical filaments and transmembrane structures, identified as AMPARs and NMDARs by EM tomography. The differential distribution of these receptor-like structures and dependence of their abundance on PSD size matches that of AMPARs and NMDARs in the hippocampal synapses. The loss of these structures following MAGUK knockdown tracks the reduction in postsynaptic AMPAR and NMDAR transmission, confirming the structural identities of these two types of receptors. These results demonstrate that MAGUKs are required for anchoring both types of glutamate receptors at the PSD and are consistent with a structural model where MAGUKs, corresponding to membrane-associated vertical filaments, are the essential structural proteins that anchor and organize both types of glutamate receptors and govern the overall molecular organization of the PSD.The postsynaptic density (PSD) at excitatory glutamatergic synapses, appearing in electron micrographs as a prominent electron-dense thickening lining the postsynaptic membrane (1) is a complex macromolecular machine positioned across from synaptic vesicle release sites at the presynaptic active zone. The PSD clusters and organizes neurotransmitter receptors and signaling molecules at the postsynaptic membrane, transmits and processes synaptic signals, and can undergo structural changes to encode and store information (2–5). Two types of ionotropic glutamate receptors, AMPA receptors (AMPARs) and NMDA receptors (NMDARs), present at PSDs of excitatory synapses (6–10) mediate almost all synaptic transmission in the brain (11). Biochemistry and mass spectrometry of the detergent-extracted cellular fraction of PSDs have additionally identified many proteins associated with AMPAR and NMDAR complexes (12, 13).The membrane-associated guanylate kinases (MAGUKs), a class of abundant scaffold proteins consisting of PSD-95, PSD-93, synapse-associated protein (SAP)102, and SAP97, interact directly with NMDARs (14–18). These MAGUK proteins share conserved modular structures consisting of three PDZ domains (19, 20) and one SH3-GK supermodule (21). PDZ domains of MAGUKs bind to a conserved motif at the extreme C-terminal region of GluN2 subunits of NMDARs (16, 22). PSD-95 controls the number of AMPARs at the PSD through interactions with auxiliary proteins, such as Stargazin/TARPs in complex with AMPARs (23–25). Single-particle tracking of AMPARs provides evidence that AMPAR/Stargazin complexes are stabilized by PSD-95 at the membrane (26), where PSD-95 is thought to provide hot spots for accumulating AMPARs at synapses (27, 28). Germ-line knockout of PSD-95 reduces AMPAR transmission with little effects on NMDARs (29), whereas acute loss of single members of the MAGUK family decreases primarily AMPAR-mediated synaptic transmission (30–32), and removal of multiple MAGUKs results in greater losses of transmission mediated by both AMPARs and NMDARs (30).PSD-95 and PSD-93 include N-terminal palmitoylation sites that enable PSD-95 and PSD-93 to associate with membrane lipids. N-terminal palmitoylation of PSD-95 is necessary for its synaptic localization, clustering of receptors (33–35), and stability at the PSD (36). PSD-95 palmitoylation regulates synaptic strength by controlling the accumulation of AMPARs at the PSD (35). Consistent with these results, a recent immunogold electron microscopy (immuno-EM) mapping of the positions of the two ends of the PSD-95 molecule at the PSD shows that its N terminus is located at the membrane, whereas its C terminus is farther away from the membrane in a relatively extended configuration, where it is vertically oriented with respect to the membrane (3, 4, 37). In contrast, neither SAP102 nor SAP97 has palmitoylation sites. SAP97 contains a L27 domain at the N terminus (31, 38), which might be involved in self-association, and has a role in sorting and trafficking of AMPARs and NMDARs (39) but is not required for basal synaptic transmission (40).The MAGUK family proteins interact with a host of other proteins in the PSD, such as GKAP (41, 42), which binds to the GK domain of the MAGUKs, whereas GKAPs in turn bind Shank and Homer (43–45). Both Shank and Homer can interact with actin-associated proteins, thus indirectly linking the core PSD structure to the actin system prevalent in the cytoplasm of dendritic spines (45). MAGUKs interact with signaling complexes such as AKAPs (46, 47), K channels (48), and postsynaptic adhesion molecules such as neuroligin (49, 50). With an average density of 300–400 molecules per PSD (51, 52), the MAGUKs outnumber glutamate receptors by a significant margin. With so many potential binding partners, the MAGUKs are positioned to play an important role in organizing glutamate receptors as well as other scaffolding and signaling molecules at the PSD (53).We have examined the consequences of knocking down three key MAGUKs on excitatory synaptic transmission and found an ∼80% reduction in both AMPAR and NMDAR synaptic responses (54). Interestingly, despite the rather ubiquitous distribution of MAGUKs at excitatory synapses, the reduction in synaptic AMPAR-mediated transmission appeared to be attributable primarily to an all-or-none loss of functional synapses. We present evidence that after the knockdown, there is an initial uniform decrease in AMPARs across all synapses, but over a 4-d period, a consolidation process in which a “winner-take-all” phenomenon occurs (54).Here, we have used EM tomography (3, 4) to study the structural effects of knocking down the three key MAGUKs at the PSD to develop a molecular model of the organization of the core PSD structure in intact hippocampal spine synapses. PSDs in intact synapses show numerous regularly spaced and membrane-associated vertical filaments containing PSD-95 in extended conformation connecting with NMDAR and AMPAR-type complexes. These vertical structures in turn contact horizontal elements, resulting in a molecular scaffold supporting a core PSD structure (3, 4, 37). Thus, vertical filaments appear to be of crucial importance in sustaining the core PSD structure. Here, we show that knocking down three key synaptic MAGUKs results in a profound loss of vertical filaments and the electron-dense materials manifested by the PSD. The loss of MAGUKs is accompanied by a dramatic loss of both NMDAR- and AMPAR-type structures at the PSD.     

Disruption of hippocampal–prefrontal cortex activity by dopamine D2R-dependent LTD of NMDAR transmission

Functional connectivity between the hippocampus and prefrontal cortex (PFC) is essential for associative recognition memory and working memory. Disruption of hippocampal–PFC synchrony occurs in schizophrenia, which is characterized by hypofunction of NMDA receptor (NMDAR)-mediated transmission. We demonstrate that activity of dopamine D2-like receptors (D2Rs) leads selectively to long-term depression (LTD) of hippocampal–PFC NMDAR-mediated synaptic transmission. We show that dopamine-dependent LTD of NMDAR-mediated transmission profoundly disrupts normal synaptic transmission between hippocampus and PFC. These results show how dopaminergic activation induces long-term hypofunction of NMDARs, which can contribute to disordered functional connectivity, a characteristic that is a hallmark of psychiatric disorders such as schizophrenia.The hippocampus to medial prefrontal cortex (PFC) projection is important for executive function and working and long-term memory (1, 2). Glutamatergic neurons of the ventral hippocampal cornu ammonis 1 (CA1) region project directly to layers 2–6 of ipsilateral PFC, and this connection synchronizes PFC and hippocampal activity during particular behavioral conditions (3–5). Disruption of hippocampal–PFC synchrony is associated with cognitive deficits that occur in disorders such as schizophrenia (6). Hippocampal–PFC uncoupling can be achieved by NMDA receptor (NMDAR) antagonism (7), and NMDAR hypofunction is a recognized feature of schizophrenia (8). However, it is unclear, first, how changes in NMDAR function at this synapse may arise, and second, how NMDAR hypofunction affects hippocampal–PFC synaptic transmission.Canonically, NMDARs are considered to contribute little to single synaptic events, but the slow kinetics of NMDARs contribute to maintaining depolarization, leading to the generation of bursts of action potentials (9–13). Furthermore, NMDARs coordinate spike timing relative to the phase of field potential oscillations (14, 15). NMDAR transmission itself undergoes synaptic plasticity (16, 17), and this can have a profound effect on sustained depolarization, burst firing, synaptic integration, and metaplasticity (9, 11, 18, 19). In PFC, NMDARs are oppositely regulated by dopamine receptors; D1-like receptors (D1Rs) potentiate and D2-like receptors (D2Rs) depress NMDAR currents (20). Interestingly, NMDAR hypofunction (8, 21) and dopamine D2 receptor activity (22) are potentially converging mechanisms contributing to schizophrenia (23).We now examine the contribution of NMDARs to transmission at the hippocampal–PFC synapse. We show that NMDAR activity provides sustained depolarization that can trigger action potentials during bursts of hippocampal input to PFC. We next demonstrate that dopamine D2 receptor-dependent long-term depression (LTD) of NMDAR transmission profoundly attenuates summation of synaptic transmission and neuronal firing at the hippocampal–PFC input. These findings allow for a mechanistic understanding of how alterations in dopamine and NMDAR function can lead to the disruption of hippocampal–PFC functional connectivity, which characterizes certain psychiatric disorders.     

Identity of the NMDA receptor coagonist is synapse specific and developmentally regulated in the hippocampus

NMDA receptors (NMDARs) require the coagonists d-serine or glycine for their activation, but whether the identity of the coagonist could be synapse specific and developmentally regulated remains elusive. We therefore investigated the contribution of d-serine and glycine by recording NMDAR-mediated responses at hippocampal Schaffer collaterals (SC)–CA1 and medial perforant path–dentate gyrus (mPP–DG) synapses in juvenile and adult rats. Selective depletion of endogenous coagonists with enzymatic scavengers as well as pharmacological inhibition of endogenous d-amino acid oxidase activity revealed that d-serine is the preferred coagonist at SC–CA1 mature synapses, whereas, unexpectedly, glycine is mainly involved at mPP–DG synapses. Nevertheless, both coagonist functions are driven by the levels of synaptic activity as inferred by recording long-term potentiation generated at both connections. This regional compartmentalization in the coagonist identity is associated to different GluN1/GluN2A to GluN1/GluN2B subunit composition of synaptic NMDARs. During postnatal development, the replacement of GluN2B- by GluN2A-containing NMDARs at SC–CA1 synapses parallels a change in the identity of the coagonist from glycine to d-serine. In contrast, NMDARs subunit composition at mPP–DG synapses is not altered and glycine remains the main coagonist throughout postnatal development. Altogether, our observations disclose an unprecedented relationship in the identity of the coagonist not only with the GluN2 subunit composition at synaptic NMDARs but also with astrocyte activity in the developing and mature hippocampus that reconciles the complementary functions of d-serine and glycine in modulating NMDARs during the maturation of tripartite glutamatergic synapses.The glutamate-gated N-methyl-d-aspartate receptors (NMDARs) play a critical role in structural and functional plasticity at synapses during postnatal brain development and in adulthood (1) and are therefore central to many cognitive functions such as learning and memory (2). Disturbances of their functions have been associated to a broad range of neurological and psychiatric disorders (3). NMDARs are heterotetramers typically composed of GluN1 and GluN2 subunits (3, 4), and the precise subunit composition determines NMDAR functional and trafficking properties (3, 4).NMDARs are unique among neurotransmitter receptors because their activation requires the binding of both glutamate and a coagonist initially thought to be glycine (5, 6). Nevertheless, subsequent studies have shown that d-serine synthesized by serine racemase (SR) (7) would be the preferred endogenous coagonist for synaptic NMDARs in many areas of the mature brain (8), raising controversies about “where, when, and how” glycine and d-serine might regulate NMDARs at synapses in the brain. This controversy is highlighted by the recent findings showing that d-serine and glycine both released by neurons come into play to regulate synaptic NMDAR-dependent functions at the hippocampal Schaffer collateral (SC)–CA1 synapses of adult brain (9), whereas others found no evidence for a function of glycine at this connection (10). Possible explanations for the relative contribution of d-serine and glycine in gating mature NMDARs were recently given by two recent studies. First, it was shown at hippocampal SC–CA1 synapses that d-serine would target GluN1/GluN2A-containing NMDARs, which are preferentially present within the synapse, whereas glycine would rather target GluN1/GluN2B-containing NMDARs located extrasynaptically (10). Second, it was proposed that the identity of the effective coagonist at synapses could depend on synaptic activity levels with tonic activation of NMDARs under low-activity conditions supported by ambient d-serine, whereas glycine will contribute in response to enhanced afferent activity (11).So far, most studies have explored the functions of d-serine vs. glycine at excitatory synapses in the adult brain or during aging (8) where GluN2A-expressing NMDARs prevail (1). Intriguingly, the respective role of the coagonists during postnatal development awaits to be addressed. Considerable evidence indicates that the NMDAR composition at excitatory synapses undergo an experience-dependent developmental switch from primarily GluN2B to GluN2A subunits during the first 2 wk of maturation and refinement of cortical circuits in the postnatal brain (1, 12–14). However, how this developmental switch is controlled is still elusive, and we do not know how it could be regulated or associated to the action of a preferred coagonist.The present study aimed at investigating the relative synapse specificity and the time window at which d-serine and glycine enter in function to drive NMDAR activity of developing and mature excitatory synapses in the hippocampus. In particular, we sought to elucidate whether the preference for one of the two coagonists could be related to any of the GluN2 subtypes of NMDARs.     

Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

Dopamine is a powerful modulator of glutamatergic neurotransmission and NMDA receptor-dependent synaptic plasticity. Although several intracellular cascades participating in this functional dialogue have been identified over the last few decades, the molecular crosstalk between surface dopamine and glutamate NMDA receptor (NMDAR) signaling still remains poorly understood. Using a combination of single-molecule detection imaging and electrophysiology in live hippocampal neurons, we demonstrate here that dopamine D1 receptors (D1Rs) and NMDARs form dynamic surface clusters in the vicinity of glutamate synapses. Strikingly, D1R activation or D1R/NMDAR direct interaction disruption decreases the size of these clusters, increases NMDAR synaptic content through a fast lateral redistribution of the receptors, and favors long-term synaptic potentiation. Together, these data demonstrate the presence of dynamic D1R/NMDAR perisynaptic reservoirs favoring a rapid and bidirectional surface crosstalk between receptors and set the plasma membrane as the primary stage of the dopamine–glutamate interplay.Hippocampal dopaminergic neuromodulation participates in several cognitive functions including novelty detection and long-term memory storage (1, 2). As a consequence, impairments in hippocampal neuromodulatory transmission affect synaptic plasticity at glutamatergic synapses, prevent learning and memory formation, and have been proposed to be a cellular substrate for neurodevelopmental psychiatric disorders such as schizophrenia (3). In the hippocampus and cortex, pyramidal neurons express mostly dopamine D1 and D5 receptors along their dendritic tree (4–6). Their recruitment affects the trafficking and surface expression of glutamate NMDA receptors (NMDARs), two processes that are essential for excitatory neurotransmission and synaptic plasticity. Indeed, activating dopamine D1 receptors (D1Rs) promotes the surface expression and function of NMDAR and thereby favors the long-term potentiation of excitatory glutamate synapses (7–10). Reciprocally, the activation of NMDAR modulates D1R surface expression and signaling (11). The bidirectional dialogue between dopamine and glutamate NMDAR-associated signaling thus involves changes in membrane receptor content and trafficking.Although this functional interaction is usually considered as relying on intracellular protein kinase signaling cascades (7, 10, 12), physical interactions between D1R and NMDAR at the plasma membrane were recently reported to stabilize laterally diffusing surface D1R in spines, modulate D1R- and NMDAR-mediated signaling, and influence working memory (13–16). Thus, direct interactions between these receptors could contribute to the regulation of their surface distributions and play a major role in the dopamine–glutamate interplay (15, 17). In particular, because the regulation of NMDAR synaptic content involves surface diffusion processes in and out of synaptic and extrasynaptic compartments (18), the possibility emerges that dopamine might modulate NMDAR-dependent synaptic transmission by tuning NMDAR lateral dynamics through D1R–NMDAR physical interactions. To address this question and investigate the role of the D1R/NMDAR surface crosstalk in synaptic physiology, we here assessed the surface distribution and trafficking of D1 and NMDA receptors in rat hippocampal neurons using a combination of high-resolution single-nanoparticle tracking, bulk imaging, and electrophysiology.     

Metabotropic glutamate receptor signaling is required for NMDA receptor-dependent ocular dominance plasticity and LTD in visual cortex

A feature of early postnatal neocortical development is a transient peak in signaling via metabotropic glutamate receptor 5 (mGluR5). In visual cortex, this change coincides with increased sensitivity of excitatory synapses to monocular deprivation (MD). However, loss of visual responsiveness after MD occurs via mechanisms revealed by the study of long-term depression (LTD) of synaptic transmission, which in layer 4 is induced by acute activation of NMDA receptors (NMDARs) rather than mGluR5. Here we report that chronic postnatal down-regulation of mGluR5 signaling produces coordinated impairments in both NMDAR-dependent LTD in vitro and ocular dominance plasticity in vivo. The data suggest that ongoing mGluR5 signaling during a critical period of postnatal development establishes the biochemical conditions that are permissive for activity-dependent sculpting of excitatory synapses via the mechanism of NMDAR-dependent LTD.Temporary monocular deprivation (MD) sets in motion synaptic changes in visual cortex that result in impaired vision through the deprived eye. The primary cause of visual impairment is depression of excitatory thalamocortical synaptic transmission in layer 4 of visual cortex (1–3). The study of long-term depression (LTD) of synapses, elicited in vitro by electrical or chemical stimulation, has revealed many of the mechanisms involved in deprived-eye depression (4). In slices of visual cortex, LTD in layer 4 is induced by NMDA receptor (NMDAR) activation and expressed by posttranslational modification and internalization of AMPA receptors (AMPARs) (5, 6). MD induces identical NMDAR-dependent changes in AMPARs, and synaptic depression induced by deprivation in vivo occludes LTD in visual cortex ex vivo (6–8). Manipulations of NMDARs and AMPAR trafficking that interfere with LTD also prevent the effects of MD (7, 9–11).Although NMDAR-dependent LTD is widely expressed in the brain (12, 13), it is now understood that different circuits use different mechanisms for long-term homosynaptic depression (14). For example, in the CA1 region of hippocampus, synaptic activation of either NMDARs or metabotropic glutamate receptor 5 (mGluR5) induces LTD. In both cases, depression is expressed postsynaptically as a reduction in AMPARs, but these forms of LTD are not mutually occluding and have distinct signaling requirements (15). A defining feature of mGluR5-dependent postsynaptic LTD in CA1 is a requirement for the immediate translation of synaptic mRNAs (16). In visual cortex, there is evidence that induction of LTD in layers 2–4 requires NMDAR activation, whereas induction of LTD in layer 6 requires activation of mGluR5 (17, 18).The hypothesis that mGluRs, in addition to NMDARs, play a key role in visual cortical plasticity can be traced back more than 25 y to observations that glutamate-stimulated phosphoinositide turnover, mediated in visual cortex by mGluR5 coupled to phospholipase C, is elevated during the postnatal period of heightened sensitivity to MD (19). Early attempts to test this hypothesis were inconclusive owing to the use of weak and nonselective orthosteric compounds (20–22); however, subsequent experiments did confirm that NMDAR-dependent LTD occurs normally in layers 2/3 of visual cortex in Grm5 knockout mice (23).The idea that mGluR5 is critically involved in visual cortical plasticity in vivo was rekindled with the finding that deprived-eye depression fails to occur in layer 4 of Grm5^+/− mutant mice (24). This finding was unexpected because, as reviewed above, a considerable body of evidence has implicated the mechanism of NMDAR-dependent LTD in deprived-eye depression. In the present study, we reexamined the role of mGluR5 in LTD and ocular dominance plasticity in layer 4, using the Grm5^+/− mouse and a highly specific negative allosteric modulator, 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP), that has proven suitable for chronic inhibition of mGluR5 (25, 26). Our data show that NMDAR-dependent LTD and deprived-eye depression in layer 4 require mGluR5 signaling during postnatal development.     

GluN3A expression restricts spine maturation via inhibition of GIT1/Rac1 signaling

NMDA-type glutamate receptors (NMDARs) guide the activity-dependent remodeling of excitatory synapses and associated dendritic spines during critical periods of postnatal brain development. Whereas mature NMDARs composed of GluN1 and GluN2 subunits mediate synapse plasticity and promote spine growth and stabilization, juvenile NMDARs containing GluN3A subunits are thought to inhibit these processes via yet unknown mechanisms. Here, we report that GluN3A binds G protein-coupled receptor kinase-interacting protein (GIT1), a postsynaptic scaffold that assembles actin regulatory complexes, including the Rac1 guanine nucleotide exchange factor βPIX, to promote Rac1 activation in spines. Binding to GluN3A limits the synaptic localization of GIT1 and its ability to complex βPIX, leading to decreased Rac1 activation and reduced spine density and size in primary cultured neurons. Conversely, knocking out GluN3A favors the formation of GIT1/βPIX complexes and increases the activation of Rac1 and its main effector p21-activated kinase. We further show that binding of GluN3A to GIT1 is regulated by synaptic activity, a response that might restrict the negative regulatory effects of GluN3A on actin signaling to inactive synapses. Our results identify inhibition of Rac1/p21-activated kinase actin signaling pathways as an activity-dependent mechanism mediating the inhibitory effects of GluN3A on spine morphogenesis.During the development of neural circuits, a phase of intense synaptogenesis is followed by a period of activity-dependent remodeling (or “synaptic refinement”) in which more than half of the initially formed synapses are eliminated, whereas other connections will mature and be kept (1, 2). The subunit composition of NMDA-type glutamate receptors (NMDARs) expressed by individual synapses during this critical period is a key factor influencing functional and structural synaptic plasticity and, in turn, synapse fate (3). Mature NMDARs composed of GluN1 and GluN2 subunits drive the maturation of active synapses by detecting coincident pre- and postsynaptic activity and coupling this activity to signaling pathways that trigger the enlargement and stabilization of synapses and associated dendritic spines (4–6). This structural plasticity is critical for coupling the wiring of neural circuits to experience and supporting the long-term maintenance of spines and memories. During the refinement stage, NMDARs additionally contain GluN3A subunits that serve as a brake on synapse maturation and stabilization, which might provide a counterbalance to limit synapse numbers. Supporting this idea, loss of GluN3A increases spine density and size (7) and accelerates the expression of markers of synaptic maturation (8), whereas overexpression reduces synapse and spine density and yields a higher proportion of smaller, immature spines (9). However, the downstream mechanisms by which GluN3A inhibits synapse and spine maturation remain unknown.Spines are actin-rich, and their structural remodeling relies on rearrangements of the actin cytoskeleton (10–13). Cytoskeletal rearrangements are regulated by the Rho family of small GTPases, and two members of this family, Rac1 and RhoA, are major regulators of spine remodeling (14, 15). Rho-GTPases act as molecular switches that cycle between an inactive GDP-bound conformation and an active GTP-bound conformation (16). Their activation state is controlled by guanine exchange factors (GEFs), which promote the exchange of GDP for GTP, and GTPase-activating proteins (GAPs), which catalyze GTP hydrolysis. Several Rac1-specific GEFs, including Kalirin7, Tiam1, and βPIX, are targeted to synapses via interactions with scaffolding proteins, which allows local regulation of actin remodeling in spines and its coupling to synaptic activity (12, 17–22). Although many studies have shown that NMDAR activation induces cytoskeletal and spine remodeling by activating Rac1-GEFs (23–25), much less is known about pathways that restrict excitatory synapse maturation and/or promote elimination.Here, we identify a physical association between the intracellular C-terminal domain of GluN3A subunits and G protein-coupled receptor kinase-interacting protein (GIT1), a postsynaptic scaffold that assembles a multiprotein signaling complex with Rac1 and the Rac1-GEF βPIX to regulate actin dynamics in spines (19, 26). GIT1 selectively bound juvenile NMDARs containing GluN3A but not mature NMDAR subtypes, and binding was regulated by activity because it could be enhanced or reduced, respectively, by brief episodes of synaptic inactivity or synaptic stimulation. A functional analysis demonstrates that binding to GluN3A interferes with the synaptic localization of GIT1 and its ability to recruit βPIX, leading to decreased Rac1 activation. We finally show that GluN3A-induced reductions in spine density and size critically require GIT1 binding. We propose that the coupling of GIT1/GluN3A binding to synapse use might provide an effective mechanism with which to restrict the maturation and growth of inactive synapses in a selective manner.     

Optical control of an ion channel gate

The powerful optogenetic pharmacology method allows the optical control of neuronal activity by photoswitchable ligands tethered to channels and receptors. However, this approach is technically demanding, as it requires the design of pharmacologically active ligands. The development of versatile technologies therefore represents a challenging issue. Here, we present optogating, a method in which the gating machinery of an ATP-activated P2X channel was reprogrammed to respond to light. We found that channels covalently modified by azobenzene-containing reagents at the transmembrane segments could be reversibly turned on and off by light, without the need of ATP, thus revealing an agonist-independent, light-induced gating mechanism. We demonstrate photocontrol of neuronal activity by a light-gated, ATP-insensitive P2X receptor, providing an original tool devoid of endogenous sensitivity to delineate P2X signaling in normal and pathological states. These findings open new avenues to specifically activate other ion channels independently of their natural stimulus.Optogenetic approaches in neuroscience rely on the heterologous expression of engineered light-gated ion channels or pumps to trigger or inhibit electrical activity of selected neurons (1, 2). This powerful and revolutionizing technique provides an exquisite remote control of neuronal circuits that drive behavior in animals. Of special interest is the optogenetic pharmacology (also known as optochemical genetic) (3, 4), which allows the control of an ion channel or receptor function by a photoswitchable ligand that is irreversibly tethered to the genetically modified protein through cysteine substitution (3, 4). Ligands are pharmacologically active substances targeting either competitive (5–7), or noncompetitive binding sites (8–10), and light sensitivity is mostly conferred by substituting a photoisomerizable azobenzene derivative, which interconverts reversibly between a long trans-isomer and a short cis-isomer (11). However, this approach is technically demanding, as it requires the design of site-specific ligands for each target. The development of versatile methods with generic photoswitchable molecules would thus improve the optochemical strategy.Here, we present optogating, a unique method for the optical control of ATP-activated P2X channel gating. P2X receptors are a family of trimeric, cation-selective channels (12, 13) comprising seven mammalian subtypes that mediate a variety of physiological responses, including fast synaptic transmission, contraction of smooth muscle, modulation of neurotransmitter release, and pain sensation (12, 14, 15). P2X receptors are considered as emerging therapeutic targets because of their link to cancer (16), inflammatory (17), and neuronal diseases, including neuropathic pain (18). Inspired by previous works showing that chemical modification of the transmembrane (TM) pore region of the P2X2 receptor affects channel gating through labeling of single cysteine mutants by positively charged methanethiosulphonate reagents (19–21), we sought to optically control the gating machinery of the receptor by covalently introducing into the same region positively charged, azobenzene-containing photoswitches. We found that engineered channels could be reversibly turned on and off by light, without the need of ATP, revealing a unique ATP-independent, light-induced gating mechanism. We applied our system in cultured hippocampal neurons, and showed that a light-gated P2X2 receptor that was made genetically insensitive to endogenous ATP rapidly and reversibly photomodulated synaptic activity and action potential firing. These findings establish original tools to delineate P2X receptor signaling in vivo, and provide new opportunities to specifically activate by light other ion channels in which the endogenous sensitivity of the natural signal can be genetically removed.     

Targeting Wnt-driven cancer through the inhibition of Porcupine by LGK974

Wnt signaling is one of the key oncogenic pathways in multiple cancers, and targeting this pathway is an attractive therapeutic approach. However, therapeutic success has been limited because of the lack of therapeutic agents for targets in the Wnt pathway and the lack of a defined patient population that would be sensitive to a Wnt inhibitor. We developed a screen for small molecules that block Wnt secretion. This effort led to the discovery of LGK974, a potent and specific small-molecule Porcupine (PORCN) inhibitor. PORCN is a membrane-bound O-acyltransferase that is required for and dedicated to palmitoylation of Wnt ligands, a necessary step in the processing of Wnt ligand secretion. We show that LGK974 potently inhibits Wnt signaling in vitro and in vivo, including reduction of the Wnt-dependent LRP6 phosphorylation and the expression of Wnt target genes, such as AXIN2. LGK974 is potent and efficacious in multiple tumor models at well-tolerated doses in vivo, including murine and rat mechanistic breast cancer models driven by MMTV–Wnt1 and a human head and neck squamous cell carcinoma model (HN30). We also show that head and neck cancer cell lines with loss-of-function mutations in the Notch signaling pathway have a high response rate to LGK974. Together, these findings provide both a strategy and tools for targeting Wnt-driven cancers through the inhibition of PORCN.Wnt signaling is a key oncogenic pathway in multiple cancers (1, 2). On binding to its receptors LRP5/6 and Frizzled (FZD) at the plasma membrane, Wnt ligand triggers the disruption of the β-catenin degradation machinery (consisting of AXIN2, GSK3β, APC, and others), leading to the accumulation of β-catenin in the cytoplasm (3). Elevated levels of β-catenin ultimately lead to its translocation into the nucleus to form a complex with LEF/TCF and drive downstream gene expression (3). Dysregulation of Wnt signaling (1) can occur through mutations of downstream components, such as APC and β-catenin, which are well-documented in colon cancer (1). In addition, overexpression of Wnt ligands or costimulants, such as R-Spondin 2/3 (RSPO2/3), or silencing of Wnt inhibitor genes has been reported in various cancers (1, 4). Mutations of pan-Wnt pathway components, such as AXIN1/2 or the RSPO coreceptors RNF43/ZNFR3, may potentially play key roles in pancreatic, colon, and hepatocellular carcinoma (4–6).The Wnt gene was originally identified as an oncogene in murine mammary tumors 30 y ago (2) and confirmed to be a key oncogenic pathway in many studies, including an unbiased insertional mutagenesis screen, with Wnt1, Wnt3, and Wnt3A comprising 38% of all unbiased hits (7). In human breast cancer, overexpression of Wnt or silencing of Wnt inhibitor genes has been observed in up to one-half of breast cancer samples (8). Cytoplasmic and nuclear β-catenin have also been correlated with triple-negative and basal-like breast cancer subtypes (9, 10), and Wnt signaling has also been implicated in cancer-initiating cells in multiple cancer types (11–14). Wnt pathway signaling activity is dependent on Wnt ligand. During the biosynthesis of Wnt ligands, Wnt undergoes posttranslational acylation (palmitoylation) that is mediated by Porcupine (PORCN), a membrane bound O-acyltransferase (3, 15). PORCN is specific and dedicated to Wnt posttranslational acylation, which is required for subsequent Wnt secretion (16). Loss of PORCN leads to inhibition of Wnt ligand-driven signaling activities in KO mouse models (17, 18). In humans, loss-of-function (LoF) mutations in the PORCN gene cause focal dermal hypoplasia, an X-linked dominant disorder associated with a variety of congenital abnormalities in both heterozygotes and those individuals with mosaicism for the PORCN gene. This phenotype is consistent with the role of the Wnt signaling pathway during embryogenesis and development (15).Given the key role of Wnt signaling in cancer, targeting this pathway has been an attractive therapeutic approach. However, success has been limited because of the lack of effective therapeutic agents for targets in the Wnt pathway and the lack of a defined patient population that would be sensitive to a Wnt inhibitor. Herein, we describe a cellular high-throughput screen for small molecules that block Wnt secretion. This effort led to the discovery of LGK974, a potent and specific PORCN inhibitor. We show that LGK974 potently inhibits Wnt signaling in vitro and in vivo and has strong efficacy in tumor models in vivo. We also show that head and neck squamous cell carcinoma cell lines with LoF mutations in the Notch signaling pathway have a high response rate to LGK974. These findings provide a path forward to target Wnt-driven cancer through the inhibition of PORCN.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.     

TLR-dependent phagosome tubulation in dendritic cells promotes phagosome cross-talk to optimize MHC-II antigen presentation

Dendritic cells (DCs) phagocytose large particles like bacteria at sites of infection and progressively degrade them within maturing phagosomes. Phagosomes in DCs are also signaling platforms for pattern recognition receptors, such as Toll-like receptors (TLRs), and sites for assembly of cargo-derived peptides with major histocompatibility complex class II (MHC-II) molecules. Although TLR signaling from phagosomes stimulates presentation of phagocytosed antigens, the mechanisms underlying this enhancement and the cell surface delivery of MHC-II–peptide complexes from phagosomes are not known. We show that in DCs, maturing phagosomes extend numerous long tubules several hours after phagocytosis. Tubule formation requires an intact microtubule and actin cytoskeleton and MyD88-dependent phagosomal TLR signaling, but not phagolysosome formation or extensive proteolysis. In contrast to the tubules that emerge from endolysosomes after uptake of soluble ligands and TLR stimulation, the late-onset phagosomal tubules are not essential for delivery of phagosome-derived MHC-II–peptide complexes to the plasma membrane. Rather, tubulation promotes MHC-II presentation by enabling maximal cargo transfer among phagosomes that bear a TLR signature. Our data show that phagosomal tubules in DCs are functionally distinct from those that emerge from lysosomes and are unique adaptations of the phagocytic machinery that facilitate cargo exchange and antigen presentation among TLR-signaling phagosomes.Professional phagocytes take up large particles, such as bacteria, by phagocytosis and submit them to an increasingly harsh environment during phagosome maturation (1). Phagocytes concomitantly alert the immune system that an invader is present via signaling programs initiated by pattern recognition receptors, such as Toll-like receptors (TLRs) (2). Conventional dendritic cells (DCs) also alter and optimize phagosome maturation and TLR-signaling programs to preserve bacterial antigens for loading onto MHC class I and class II (MHC-II) molecules and optimize cytokine secretion to stimulate and direct T-cell responses to the invading agent (3, 4). DC presentation of soluble antigen is facilitated by TLR-driven tubulation of lysosomes that harbor MHC-II–peptide complexes and by consequent fusion of tubulovesicular structures with the plasma membrane (5–7); however, little is known about the mechanism by which signaling pathways influence the formation or presentation of phagosome-derived MHC-II–peptide complexes, key processes in the adaptive immunity to bacterial pathogens.TLRs respond to microbial ligands at the plasma membrane and in intracellular stores (8). TLR stimulation at the plasma membrane, endosomes, or phagosomes elicits distinct signaling pathways via two sets of adaptors, TIRAP (or MAL)-MyD88 and TRAM-TRIF (8, 9), which induce proinflammatory cytokine secretion and other downstream responses. TLRs such as TLR2 and TLR4 are recruited to macrophage and DC phagosomes at least partly from an intracellular pool (10–13), and signal autonomously from phagosomes independent of plasma membrane TLRs (11, 14, 15). Autonomous phagosomal signaling from TLRs or Fcγ receptors enhances the degradation of phagocytosed proteins and assembly of MHC-II with their derived peptides (14–16). Phagosomal TLR signaling has been proposed to also promote the reorganization of phagosome-derived MHC-II-enriched compartments (MIICs) to favor the delivery of MHC-II–peptide complexes to the plasma membrane (17), analogous to TLR-stimulated formation of tubules from MIICs/lysosomes (18–20) that fuse with the plasma membrane (7) and extend toward the immunologic synapse with T cells (5). Tubules emerge from phagosomes in macrophages shortly after phagocytosis and likely function in membrane recycling during early phagosome maturation stages (21–23), but tubules at later stages that might facilitate the presentation of phagosome-derived MHC-II–peptide complexes have not been reported previously. Moreover, a role for TLR signaling in formation of phagosome-derived tubules has not been established.Herein we show that in DCs, maturing phagosomes undergo extensive tubulation up to several hours after phagocytosis, and that tubulation requires TLR and MyD88 signaling and an intact actin and microtubule cytoskeleton. Unlike lysosome tubulation, phagosome tubulation is not essential for MHC-II–peptide transport to the cell surface. Rather, it contributes to content exchange among phagosomes that carry a TLR signature, and thereby enhances presentation of phagocytosed antigens from potential pathogens.     

Parameter-free methods distinguish Wnt pathway models and guide design of experiments

The canonical Wnt signaling pathway, mediated by β-catenin, is crucially involved in development, adult stem cell tissue maintenance, and a host of diseases including cancer. We analyze existing mathematical models of Wnt and compare them to a new Wnt signaling model that targets spatial localization; our aim is to distinguish between the models and distill biological insight from them. Using Bayesian methods we infer parameters for each model from mammalian Wnt signaling data and find that all models can fit this time course. We appeal to algebraic methods (concepts from chemical reaction network theory and matroid theory) to analyze the models without recourse to specific parameter values. These approaches provide insight into aspects of Wnt regulation: the new model, via control of shuttling and degradation parameters, permits multiple stable steady states corresponding to stem-like vs. committed cell states in the differentiation hierarchy. Our analysis also identifies groups of variables that should be measured to fully characterize and discriminate between competing models, and thus serves as a guide for performing minimal experiments for model comparison.The Wnt signaling pathway plays a key role in essential cellular processes ranging from proliferation and cell specification during development to adult stem cell maintenance and wound repair (1). Dysfunction of Wnt signaling is implicated in many pathological conditions, including degenerative diseases and cancer (2–4). Despite many molecular advances, the pathway dynamics are still not well understood. Theoretical investigations of the Wnt/β-catenin pathway serve as testbeds for working hypotheses (5–12).We focus on models of canonical Wnt pathway processes with the aim of elucidating mechanisms, predicting function, and identifying key pathway components in adult tissues, such as colonic crypts. We compare four preexisting ordinary differential equation models (5–8) and find, using injectivity theory, that for any given conditions and parameter values, none of the models is capable of multiple cellular responses.In many tissues Wnt plays a crucial role in cell fate specification (3). At the base of colonic crypts, cells exist in a stem-like, proliferative phenotype in the presence of Wnt. As these cells’ progeny move up the crypt axis they enter a Wnt-low environment and change fate (perhaps reversibly), becoming differentiated, specialized gut cells (13). In neuronal and endocrinal tissues, Wnt/β-catenin data suggest cell fate plasticity under different environmental conditions (14, 15). Here, we introduce a new model motivated by experimental findings not described in previous models (16–18) to investigate bistable switching in the Wnt pathway. We find the new model to be capable of multiple cellular responses; furthermore, our parameter-free techniques identify that molecular shuttling (between cytoplasm and nucleus) and degradation together may serve as a possible mechanism for governing bistability in the pathway, corresponding to, for example, a committed cell state and a stem-like cell state.Comparison of models (and mechanisms) requires data; the type of comparison performed depends on the data at hand. If data show bistability (two distinct response states), then we could rule out all models that preclude bistability; however the converse is not true (a graded response may be compatible with all models). Experimental studies in Xenopus extracts have been performed to validate a model of Wnt signaling (5), with further pathway elucidation in refs. 19, 20; however, the parameters identified in these studies may differ markedly from those involved in mammalian Wnt signaling (21, 22). With the aim of discriminating between models, we present the five Wnt models under a unifying framework, with standardized notation to facilitate comparison. We fit parameters to recently published mammalian β-catenin signaling time-course data using Bayesian inference (22) and find that all of the studied models can describe the data well, demonstrating that additional data are required to compare models.To determine which sets of protein species should be measured for carrying out a comparison between models and data, we introduce matroid theory to systems biology. A matroid is a combinatorial structure from mathematics, and in our case, it provides all of the steady-state invariants (23, 24) that have minimal sets of variables. The algebraic matroid associated with the steady-state ideal determines specific sets of species that should be measured to perform model discrimination without knowledge of parameter values. We demonstrate this parameter-free analysis with experimental data for two Wnt models.In the next section, we introduce the previous models and new shuttle model. We perform injectivity–multistability analysis and classify the shuttle model as the only one capable of multistability. Next we infer the parameters of five competing models for time-course β-catenin data, revealing that all of the models fit the data. Finally we introduce algebraic matroids to inform experimental design for discriminating between models and data.     

Agonist binding to the NMDA receptor drives movement of its cytoplasmic domain without ion flow

The NMDA receptor (R) plays important roles in brain physiology and pathology as an ion channel. Here we examine the ion flow-independent coupling of agonist to the NMDAR cytoplasmic domain (cd). We measure FRET between fluorescently tagged cytoplasmic domains of GluN1 subunits of NMDARs expressed in neurons. Different neuronal compartments display varying levels of FRET, consistent with different NMDARcd conformations. Agonist binding drives a rapid and transient ion flow-independent reduction in FRET between GluN1 subunits within individual NMDARs. Intracellular infusion of an antibody targeting the GluN1 cytoplasmic domain blocks agonist-driven FRET changes in the absence of ion flow, supporting agonist-driven movement of the NMDARcd. These studies indicate that extracellular ligand binding to the NMDAR can transmit conformational information into the cell in the absence of ion flow.The NMDA receptor (R) is a ligand-gated ion channel precisely positioned at synapses to act as a coincidence detector (1, 2), which may permit this molecule to mediate the association of memories in the brain (3, 4). Despite the importance of the NMDAR in the development, function, and dysfunction of the brain (5–8), the molecular link between its agonist-driven activation to its signaling function is not well understood. Recent studies suggested that activation of NMDARs can transmit signals in the absence of ion flux through the channel (9–12). An important test of this view is to measure directly agonist-driven changes in the cytoplasmic domain of NMDARs in the absence of ion flow through the NMDAR.Transmembrane signaling by transmembrane receptors in the absence of ion flux is common in cells; less common are classic ion channels with a secondary metabotropic function. G protein-coupled receptors (GPCRs) act by transducing extracellular agonist binding to a conformational change that activates signaling molecules controlling intracellular physiology (13). Such conformational changes induced by agonist binding to GPCRs have been measured directly using spectroscopic methods, including FRET (14, 15). For the NMDAR, a tetrameric receptor composed of two GluN1 and two GluN2 subunits in the hippocampus (7, 16), intrareceptor FRET signals from fluorescent molecules connected to the extracellular domain of subunits have been measured and used to monitor the assembly of the receptors (17). However, detection of transmembrane transduction that couples agonist binding to conformational changes at the cytoplasmic domain of the NMDAR has not been attempted.     

Aβ induces astrocytic glutamate release,extrasynaptic NMDA receptor activation,and synaptic loss

Synaptic loss is the cardinal feature linking neuropathology to cognitive decline in Alzheimer’s disease (AD). However, the mechanism of synaptic damage remains incompletely understood. Here, using FRET-based glutamate sensor imaging, we show that amyloid-β peptide (Aβ) engages α7 nicotinic acetylcholine receptors to induce release of astrocytic glutamate, which in turn activates extrasynaptic NMDA receptors (eNMDARs) on neurons. In hippocampal autapses, this eNMDAR activity is followed by reduction in evoked and miniature excitatory postsynaptic currents (mEPSCs). Decreased mEPSC frequency may reflect early synaptic injury because of concurrent eNMDAR-mediated NO production, tau phosphorylation, and caspase-3 activation, each of which is implicated in spine loss. In hippocampal slices, oligomeric Aβ induces eNMDAR-mediated synaptic depression. In AD-transgenic mice compared with wild type, whole-cell recordings revealed excessive tonic eNMDAR activity accompanied by eNMDAR-sensitive loss of mEPSCs. Importantly, the improved NMDAR antagonist NitroMemantine, which selectively inhibits extrasynaptic over physiological synaptic NMDAR activity, protects synapses from Aβ-induced damage both in vitro and in vivo.Emerging evidence suggests that the injurious effects of amyloid β peptide (Aβ) in Alzheimer’s disease (AD) may be mediated, at least in part, by excessive activation of extrasynaptic or perisynaptic NMDARs (eNMDARs) containing predominantly NR2B subunits (1, 2). In contrast, in several neurodegenerative paradigms, physiological synaptic NMDAR (sNMDAR) activity can be neuroprotective (refs. 3–8, but see ref. 9). Soluble oligomers of Aβ_1–42 are thought to underlie dementia, mimic extracellular glutamate stimulation of eNMDARs, and disrupt synaptic plasticity and long-term potentiation, eventually leading to synaptic loss (1, 6, 10, 11). However, mechanistic insight into the action of Aβ that causes excessive eNMDAR stimulation and the potential link between eNMDARs and synaptic damage remain to be elucidated. Here, we examine the cascade involved in eNMDAR activation by oligomeric Aβ and its consequences on miniature excitatory postsynaptic currents (mEPSCs). We found that eNMDAR activation is triggered by extrasynaptic glutamate released from astrocytes in response to Aβ peptide. In turn, eNMDAR stimulation is followed rapidly by a decrease in mEPSC frequency with accompanying generation of nitric oxide (NO), hyperphosphorylation of tau, and activation of caspase-3. Pharmacological blockade of eNMDARs with relative sparing of sNMDARs abrogated NO production, tau phosphorylation, caspase activation, and subsequent synaptic loss. These results suggest a glutamate-mediated cascade triggered by Aβ in which early eNMDAR activation may contribute to subsequent synaptic damage and consequent cognitive decline in AD.