NMDA receptor autoantibodies primarily impair the extrasynaptic compartment.

Autoantibodies directed against the N-methyl-D-aspartate receptor (NMDAR-Ab) are pathogenic immunoglobulins detected in patients suffering from NMDAR encephalitis. NMDAR-Ab alter the receptor membrane trafficking, synaptic transmission and neuronal network properties, leading to patients' neurological and psychiatric symptoms. Patients often have very little neuronal damage but rapid and massive (treatment-responsive) brain dysfunctions related to unknown early mechanism of NMDAR-Ab. Our understanding of this early molecular cascade remains surprisingly fragmented. Here, we used a combination of single molecule-based imaging of membrane proteins to unveil the spatio-temporal action of NMDAR-Ab onto live hippocampal neurons. We first demonstrate that different clones of NMDAR-Ab primarily affect extrasynaptic -and not synaptic- NMDAR. In the first minutes, NMDAR-Ab increase extrasynaptic NMDAR membrane dynamics, de-clustering its surface interactome. NMDAR-Ab also rapidly reshuffle all membrane proteins located at the extrasynaptic compartment. Consistent with this alteration of multiple proteins, NMDAR-Ab effects were not mediated through the sole interaction between NMDAR and EphB2 receptor. At the long-term, NMDAR-Ab reduce NMDAR synaptic pool by slowing down receptor membrane dynamics in a cross-linking independent manner. Remarkably, exposing only extrasynaptic NMDAR to NMDAR-Ab was sufficient to produce their full-blown effect on synaptic receptors. Collectively, we demonstrate that NMDAR-Ab first impair extrasynaptic proteins, and then the synaptic ones. These data shed thus new, and unsuspected, lights on the mode of action of NMDAR-Ab and likely to our understanding of (extra)synaptopathies.

Positive Allosteric Modulation of NMDARs Prevents the Altered Surface Dynamics Caused by Patients' Antibodies.

OBJECTIVES: A positive allosteric modulator of the NMDAR, SGE-301, has been shown to reverse the alterations caused by the antibodies of patients with anti-NMDAR encephalitis (NMDARe). However, the mechanisms involved beyond receptor modulation are unclear. In this study, we aimed to investigate how this modulator affects NMDAR membrane dynamics. METHODS: Cultured hippocampal neurons were treated with SGE-301 or vehicle, alongside with immunoglobulins G (IgG) from patients with NMDARe or healthy controls. NMDAR surface dynamics were assessed with single-molecule imaging by photoactivated localization microscopy. RESULTS: NMDAR trajectories from neurons treated with SGE-301 were less confinement, with increased diffusion coefficients. This effect mainly occurred at synapses because extrasynaptic diffusion and confinement were minimally affected by SGE-301. Treatment with patients' IgG reduced NMDAR surface dynamics and increased their confinement. Remarkably, SGE-301 incubation antagonized patients' IgG effects in both synaptic and extrasynaptic membrane compartments, restoring diffusion and confinement values similar to those from neurons exposed to control IgG. DISCUSSION: We demonstrate that SGE-301 upregulates NMDAR surface diffusion and antagonizes the pathogenic effects of patients' IgG on NMDAR membrane organization. These findings suggest a potential therapeutic strategy for NMDARe.

Converging synaptic and network dysfunctions in distinct autoimmune encephalitis.

Psychiatric and neurological symptoms, as well as cognitive deficits, represent a prominent phenotype associated with variable forms of autoimmune encephalitis, regardless of the neurotransmitter receptor targeted by autoantibodies. The mechanistic underpinnings of these shared major neuropsychiatric symptoms remain however unclear. Here, we investigate the impacts of patient-derived monoclonal autoantibodies against the glutamatergic NMDAR (NMDAR mAb) and inhibitory GABAaR (GABAaR mAb) signalling in the hippocampal network. Unexpectedly, both excitatory and inhibitory synaptic receptor membrane dynamics, content and transmissions are altered by NMDAR or GABAaR mAb, irrespective of the affinity or antagonistic effect of the autoantibodies. The effect of NMDAR mAb on inhibitory synapses and GABAaR mAb on excitatory synapses requires neuronal activity and involves protein kinase signalling. At the cell level, both autoantibodies increase the excitation/inhibition balance of principal cell inputs. Furthermore, NMDAR or GABAaR mAb leads to hyperactivation of hippocampal networks through distinct alterations of principal cell and interneuron properties. Thus, autoantibodies targeting excitatory NMDAR or inhibitory GABAaR trigger convergent network dysfunctions through a combination of shared and distinct mechanisms.

Non-canonical interplay between glutamatergic NMDA and dopamine receptors shapes synaptogenesis.

Direct interactions between receptors at the neuronal surface have long been proposed to tune signaling cascades and neuronal communication in health and disease. Yet, the lack of direct investigation methods to measure, in live neurons, the interaction between different membrane receptors at the single molecule level has raised unanswered questions on the biophysical properties and biological roles of such receptor interactome. Using a multidimensional spectral single molecule-localization microscopy (MS-SMLM) approach, we monitored the interaction between two membrane receptors, i.e. glutamatergic NMDA (NMDAR) and G protein-coupled dopamine D1 (D1R) receptors. The transient interaction was randomly observed along the dendritic tree of hippocampal neurons. It was higher early in development, promoting the formation of NMDAR-D1R complexes in an mGluR5- and CK1-dependent manner, favoring NMDAR clusters and synaptogenesis in a dopamine receptor signaling-independent manner. Preventing the interaction in the neonate, and not adult, brain alters in vivo spontaneous neuronal network activity pattern in male mice. Thus, a weak and transient interaction between NMDAR and D1R plays a structural and functional role in the developing brain.

Human NMDAR autoantibodies disrupt excitatory-inhibitory balance, leading to hippocampal network hypersynchrony.

Anti-NMDA receptor autoantibodies (NMDAR-Abs) in patients with NMDAR encephalitis cause severe disease symptoms resembling psychosis and cause cognitive dysfunction. After passive transfer of patients' cerebrospinal fluid or human monoclonal anti-GluN1-autoantibodies in mice, we find a disrupted excitatory-inhibitory balance resulting from CA1 neuronal hypoexcitability, reduced AMPA receptor (AMPAR) signaling, and faster synaptic inhibition in acute hippocampal slices. Functional alterations are also reflected in widespread remodeling of the hippocampal proteome, including changes in glutamatergic and GABAergic neurotransmission. NMDAR-Abs amplify network γ oscillations and disrupt θ-γ coupling. A data-informed network model reveals that lower AMPAR strength and faster GABAA receptor current kinetics chiefly account for these abnormal oscillations. As predicted in silico and evidenced ex vivo, positive allosteric modulation of AMPARs alleviates aberrant γ activity, reinforcing the causative effects of the excitatory-inhibitory imbalance. Collectively, NMDAR-Ab-induced aberrant synaptic, cellular, and network dynamics provide conceptual insights into NMDAR-Ab-mediated pathomechanisms and reveal promising therapeutic targets that merit future in vivo validation.

Nanoscale and functional heterogeneity of the hippocampal extracellular space.

The extracellular space (ECS) and its constituents play a crucial role in brain development, plasticity, circadian rhythm, and behavior, as well as brain diseases. Yet, since this compartment has an intricate geometry and nanoscale dimensions, its detailed exploration in live tissue has remained an unmet challenge. Here, we used a combination of single-nanoparticle tracking and super-resolution microscopy approaches to map the nanoscale dimensions of the ECS across the rodent hippocampus. We report that these dimensions are heterogeneous between hippocampal areas. Notably, stratum radiatum CA1 and CA3 ECS differ in several characteristics, a difference that gets abolished after digestion of the extracellular matrix. The dynamics of extracellular immunoglobulins vary within these areas, consistent with their distinct ECS characteristics. Altogether, we demonstrate that ECS nanoscale anatomy and diffusion properties are widely heterogeneous across hippocampal areas, impacting the dynamics and distribution of extracellular molecules.

GluN3A subunit tunes NMDA receptor synaptic trafficking and content during postnatal brain development.

Signaling via N-methyl-d-aspartate receptors (NMDARs) is critical for the maturation of glutamatergic synapses, partly through a developmental switch from immature synapses expressing primarily GluN2B- and GluN3A-containing subtypes to GluN2A-rich mature ones. This subunit switch is thought to underlie the synaptic stabilization of NMDARs necessary for neural network consolidation. However, the cellular mechanisms controlling the NMDAR exchange remain unclear. Using a combination of single-molecule and confocal imaging and biochemical and electrophysiological approaches, we show that surface GluN3A-NMDARs form a highly diffusive receptor pool that is loosely anchored to synapses. Remarkably, changes in GluN3A subunit expression selectively alter the surface diffusion and synaptic anchoring of GluN2A- but not GluN2B-NMDARs, possibly through altered interactions with cell surface receptors. The effects of GluN3A on NMDAR surface diffusion are restricted to an early time window of postnatal development in rodents, allowing GluN3A subunits to control the timing of NMDAR signaling maturation and neuronal network refinements.

NMDA receptor functions in health and disease: Old actor, new dimensions.

N-Methyl-D-aspartate ionotropic glutamate receptors (NMDARs) play key roles in synaptogenesis, synaptic maturation, long-term plasticity, neuronal network activity, and cognition. Mirroring this wide range of instrumental functions, abnormalities in NMDAR-mediated signaling have been associated with numerous neurological and psychiatric disorders. Thus, identifying the molecular mechanisms underpinning the physiological and pathological contributions of NMDAR has been a major area of investigation. Over the past decades, a large body of literature has flourished, revealing that the physiology of ionotropic glutamate receptors cannot be restricted to fluxing ions, and involves additional facets controlling synaptic transmissions in health and disease. Here, we review newly discovered dimensions of postsynaptic NMDAR signaling supporting neural plasticity and cognition, such as the nanoscale organization of NMDAR complexes, their activity-dependent redistributions, and non-ionotropic signaling capacities. We also discuss how dysregulations of these processes may directly contribute to NMDAR-dysfunction-related brain diseases.

Multi-Dimensional Spectral Single Molecule Localization Microscopy.

Single molecule localization (SML) and tracking (SPT) techniques, such as (spt)PALM, (u/DNA)PAINT and quantum dot tracking, have given unprecedented insight into the nanoscale molecular organization and dynamics in living cells. They allow monitoring individual proteins with millisecond temporal resolution and high spatial resolution (<30 nm) by precisely localizing the point spread function (PSF) of individual emitters and tracking their position over time. While SPT methods have been extended to study the temporal dynamics and co-organization of multiple proteins, conventional experimental setups are restricted in the number of proteins they can probe simultaneously and usually have to tradeoff between the number of colors, the spatio-temporal resolution, and the field of view. Yet, localizing and tracking several proteins simultaneously at high spatial and temporal resolution within large field of views can provide important biological insights. By employing a dual-objective spectral imaging configuration compatible with live cell imaging combined with dedicated computation tools, we demonstrate simultaneous 3D single particle localization and tracking of multiple distinct species over large field of views to be feasible without compromising spatio-temporal resolution. The dispersive element introduced into the second optical path induces a spectrally dependent displacement, which we used to analytically separate up to five different fluorescent species of single emitters based on their emission spectra. We used commercially available microscope bodies aligned one on top of the other, offering biologists with a very ergonomic and flexible instrument covering a broad range of SMLM applications. Finally, we developed a powerful freely available software, called PALMTracer, which allows to quantitatively assess 3D + t + λ SMLM data. We illustrate the capacity of our approach by performing multi-color 3D DNA-PAINT of fixed samples, and demonstrate simultaneous tracking of multiple receptors in live fibroblast and neuron cultures.

Near-Infrared Carbon Nanotube Tracking Reveals the Nanoscale Extracellular Space around Synapses.

We provide evidence of a local synaptic nanoenvironment in the brain extracellular space (ECS) lying within 500 nm of postsynaptic densities. To reveal this brain compartment, we developed a correlative imaging approach dedicated to thick brain tissue based on single-particle tracking of individual fluorescent single wall carbon nanotubes (SWCNTs) in living samples and on speckle-based HiLo microscopy of synaptic labels. We show that the extracellular space around synapses bears specific properties in terms of morphology at the nanoscale and inner diffusivity. We finally show that the ECS juxta-synaptic region changes its diffusion parameters in response to neuronal activity, indicating that this nanoenvironment might play a role in the regulation of brain activity.

Interplay between NMDA receptor dynamics and the synaptic proteasome.

Proteasome activity at the excitatory synapse plays an important role in neuronal communication. The proteasome translocation to synapses is mediated by neuronal activity, in particular the activation of N-methyl-d-aspartate receptors (NMDARs). These receptors are composed of different subunits with distinct trafficking properties that provide various signalling and plasticity features to the synapse. Yet whether the interplay between the proteasome and NMDAR relies on specific subunit properties remain unclear. Using a combination of single molecule and immunocytochemistry imaging approaches in rat hippocampal neurons, we unveil a specific interplay between GluN2B-containing NMDARs (GluN2B-NMDARs) and the synaptic proteasome. Sustained proteasome activation specifically increases GluN2B-NMDAR (not GluN2A-NMDAR) lateral diffusion. In addition, when GluN2B-NMDAR expression is downregulated, the proteasome localization decreases at glutamatergic synapses. Collectively, our data fuel a model in which the cellular dynamics and location of GluN2B-NMDARs and proteasome are intermingled, shedding new lights on the NMDAR-dependent regulation of synaptic adaptation.

Alteration of NMDA receptor trafficking as a cellular hallmark of psychosis.

A dysfunction of the glutamatergic transmission, especially of the NMDA receptor (NMDAR), constitutes one of the main biological substrate of psychotic disorders, such as schizophrenia. The NMDAR signaling hypofunction, through genetic and/or environmental insults, would cause a neurodevelopmental myriad of molecular, cellular, and network alterations that persist throughout life. Yet, the mechanisms underpinning NMDAR dysfunctions remain elusive. Here, we compared the membrane trafficking of NMDAR in three gold-standard models of schizophrenia, i.e., patient's cerebrospinal fluids, genetic manipulations of susceptibility genes, and prenatal developmental alterations. Using a combination of single nanoparticle tracking, electrophysiological, biochemical, and behavioral approaches in rodents, we identified that the NMDAR trafficking in hippocampal neurons was consistently altered in all these different models. Artificial manipulations of the NMDAR surface dynamics with competing ligands or antibody-induced receptor cross-link in the developing rat brain were sufficient to regulate the adult acoustic startle reflex and compensate for an early pathological challenge. Collectively, we show that the NMDAR trafficking is markedly altered in all clinically relevant models of psychosis, opening new avenues of therapeutical strategies.

Restoring glutamate receptosome dynamics at synapses rescues autism-like deficits in Shank3-deficient mice.

Shank3 monogenic mutations lead to autism spectrum disorders (ASD). Shank3 is part of the glutamate receptosome that physically links ionotropic NMDA receptors to metabotropic mGlu5 receptors through interactions with scaffolding proteins PSD95-GKAP-Shank3-Homer. A main physiological function of the glutamate receptosome is to control NMDA synaptic function that is required for plasticity induction. Intact glutamate receptosome supports glutamate receptors activation and plasticity induction, while glutamate receptosome disruption blocks receptors activity, preventing the induction of subsequent plasticity. Despite possible impact on metaplasticity and cognitive behaviors, scaffold interaction dynamics and their consequences are poorly defined. Here, we used mGlu5-Homer interaction as a biosensor of glutamate receptosome integrity to report changes in synapse availability for plasticity induction. Combining BRET imaging and electrophysiology, we show that a transient neuronal depolarization inducing NMDA-dependent plasticity disrupts glutamate receptosome in a long-lasting manner at synapses and activates signaling pathways required for the expression of the initiated neuronal plasticity, such as ERK and mTOR pathways. Glutamate receptosome disruption also decreases the NMDA/AMPA ratio, freezing the sensitivity of the synapse to subsequent changes of neuronal activity. These data show the importance of a fine-tuning of protein-protein interactions within glutamate receptosome, driven by changes of neuronal activity, to control plasticity. In a mouse model of ASD, a truncated mutant form of Shank3 prevents the integrity of the glutamate receptosome. These mice display altered plasticity, anxiety-like, and stereotyped behaviors. Interestingly, repairing the integrity of glutamate receptosome and its sensitivity to the neuronal activity rescued synaptic transmission, plasticity, and some behavioral traits of Shank3∆C mice. Altogether, our findings characterize mechanisms by which Shank3 mutations cause ASD and highlight scaffold dynamics as new therapeutic target.

Characterization of the Functional Cross-Talk between Surface GABAA and Dopamine D5 Receptors.

The γ-aminobutyric acid type A receptor (GABAAR) plays a major role in fast inhibitory synaptic transmission and is highly regulated by the neuromodulator dopamine. In this aspect, most of the attention has been focused on the classical intracellular signaling cascades following dopamine G-protein-coupled receptor activation. Interestingly, the GABAAR and dopamine D5 receptor (D5R) have been shown to physically interact in the hippocampus, but whether a functional cross-talk occurs is still debated. In the present study, we use a combination of imaging and single nanoparticle tracking in live hippocampal neurons to provide evidence that GABAARs and D5Rs form dynamic surface clusters. Disrupting the GABAAR-D5R interaction with a competing peptide leads to an increase in the diffusion coefficient and the explored area of both receptors, and a drop in immobile synaptic GABAARs. By means of patch-clamp recordings, we show that this fast lateral redistribution of surface GABAARs correlates with a robust depression in the evoked GABAergic currents. Strikingly, it also shifts in time the expression of long-term potentiation at glutamatergic synapses. Together, our data both set the plasma membrane as the primary stage of a functional interplay between GABAAR and D5R, and uncover a non-canonical role in regulating synaptic transmission.

The diverse and complex modes of action of anti-NMDA receptor autoantibodies.

NMDA receptors are ligand-gated ion channels that are found throughout the brain and are required for both brain development and many higher order functions. A variety of human patients with diverse clinical phenotypes have been identified that carry autoantibodies directed against NMDA receptor subunits. Here we focus on two general classes of autoantibodies, anti-GluN1 antibodies associated with anti-NMDA receptor encephalitis and anti-GluN2 antibodies associated with systemic lupus erythematosus (SLE). These two general classes of anti-NMDA receptor autoantibodies display a wide range of pathophysiological mechanisms from altering synaptic composition to gating of NMDARs. While we have made progress in understanding how these autoantibodies work at the molecular and cellular level, many unanswered questions remain including their long-term actions on brain function, the significance of clonal variations, and their effects on different NMDA receptor-expressing cell types in local circuits. This information will be needed to define fully the transition from anti-NMDA receptor autoantibodies to a clinical phenotype.

A Bottom-Up Approach to Red-Emitting Molecular-Based Nanoparticles with Natural Stealth Properties and their Use for Single-Particle Tracking Deep in Brain Tissue.

Fluorescent nanoparticles dedicated to bioimaging applications should possess specific properties that have to be maintained in the aqueous, reactive, and crowded biological environment. These include chemical and photostability, small size (on the scale of subcellular structures), biocompatibility, high brightness, and good solubility. The latter is a major challenge for inorganic nanoparticles, which require surface coating to be made water soluble. Molecular-based fluorescent organic nanoparticles (FONs) may prove a promising, spontaneously water-soluble alternative, whose bottom-up design allows for the fine-tuning of individual properties. Here, the critical challenge of controlling the interaction of nanoparticles with cellular membranes is addressed. This is a report on bright, size-tunable, red-emitting, naturally stealthy FONs that do not require the use of antifouling agents to impede interactions with cellular membranes. As a proof of concept, single FONs diffusing up to 150 µm deep in brain tissue are imaged and tracked.

CaMKII activation persistently segregates postsynaptic proteins via liquid phase separation.

Transient information input to the brain leads to persistent changes in synaptic circuits, contributing to the formation of memory engrams. Pre- and postsynaptic structures undergo coordinated functional and structural changes during this process, but how such changes are achieved by their component molecules remains largely unknown. We found that activated CaMKII, a central player of synaptic plasticity, undergoes liquid-liquid phase separation with the NMDA-type glutamate receptor subunit GluN2B. Due to CaMKII autophosphorylation, the condensate stably persists even after Ca2+ is removed. The selective binding of activated CaMKII with GluN2B cosegregates AMPA receptors and the synaptic adhesion molecule neuroligin into a phase-in-phase assembly. In this way, Ca2+-induced liquid-liquid phase separation of CaMKII has the potential to act as an activity-dependent mechanism to crosslink postsynaptic proteins, which may serve as a platform for synaptic reorganization associated with synaptic plasticity.

NMDA receptor membrane dynamics tunes the firing pattern of midbrain dopaminergic neurons.

KEY POINTS: NMDA receptors (NMDARs) expressed by dopamine neurons of the ventral tegmental area (VTA) play a central role in glutamate synapse plasticity, neuronal firing and adaptative behaviours. The NMDAR surface dynamics shapes synaptic adaptation in hippocampal networks, as well as associative memory. We investigated the basic properties and role of the NMDAR surface dynamics on cultured mesencephalic and VTA dopamine neurons in rodents. Using a combination of single molecule imaging and electrophysiological recordings, we demonstrate that NMDARs are highly diffusive at the surface of mesencephalic dopamine neurons. Unexpectedly, the NMDAR membrane dynamics per se regulates the firing pattern of VTA dopaminergic neurons, probably through a functional interplay between NMDARs receptors and small-conductance calcium-dependent potassium (SK) channels. ABSTRACT: Midbrain dopaminergic (DA) neurons play a central role in major physiological brain functions, and their dysfunctions have been associated with neuropsychiatric diseases. The activity of midbrain DA neurons is controlled by ion channels and neurotransmitter receptors, such as the glutamate NMDA receptor (NMDAR) and small-conductance calcium-dependent potassium (SK) channels. However, the cellular mechanisms through which these channels tune the firing pattern of midbrain DA neurons remain unclear. Here, we investigated whether the surface dynamics and distribution of NMDARs tunes the firing pattern of midbrain DA neurons. Using a combination of single molecule imaging and electrophysiological recordings, we report that NMDARs are highly diffusive at the surface of cultured midbrain DA neurons from rodents and humans. Reducing acutely the NMDAR membrane dynamics, which leaves the ionotropic function of the receptor intact, robustly altered the firing pattern of midbrain DA neurons without altering synaptic glutamatergic transmission. The reduction of NMDAR surface dynamics reduced apamin (SK channel blocker)-induced firing change and the distribution of SK3 channels in DA neurons. Together, these data show that the surface dynamics of NMDAR, and not solely its ionotropic function, tune the firing pattern of midbrain DA neurons partly through a functional interplay with SK channel function.

Relationship Between Serum NMDA Receptor Antibodies and Response to Antipsychotic Treatment in First-Episode Psychosis.

BACKGROUND: When psychosis develops in NMDA receptor (NMDAR) antibody encephalitis, it usually has an acute or subacute onset, and antipsychotic treatment may be ineffective and associated with adverse effects. Serum NMDAR antibodies have been reported in a minority of patients with first-episode psychosis (FEP), but their role in psychosis onset and response to antipsychotic treatment is unclear. METHODS: Sera from 387 patients with FEP (duration of psychosis <2 years, minimally or never treated with antipsychotics) undergoing initial treatment with amisulpride as part of the OPTiMiSE (Optimization of Treatment and Management of Schizophrenia in Europe) trial (ClinicalTrials.gov number NCT01248195) were tested for NMDAR IgG antibodies using a live cell-based assay. Symptom severity was assessed using the Positive and Negative Syndrome Scale and the Clinical Global Impressions Scale at baseline and again after 4 weeks of treatment with amisulpride. RESULTS: At baseline, 15 patients were seropositive for NMDAR antibodies and 372 were seronegative. The seropositive patients had similar symptom profiles and demographic features to seronegative patients but a shorter duration of psychosis (median 1.5 vs. 4.0 months; p = .031). Eleven seropositive and 284 seronegative patients completed 4 weeks of amisulpride treatment: after treatment, there was no between-groups difference in improvement in Positive and Negative Syndrome Scale scores or in the frequency of adverse medication effects. CONCLUSIONS: These data suggest that in FEP, NMDAR antibody seropositivity alone is not an indication for using immunotherapy instead of antipsychotic medications. Further studies are required to establish what proportion of patients with FEP who are NMDAR antibody seropositive have coexisting cerebrospinal fluid inflammatory changes or other paraclinical evidence suggestive of a likely benefit from immunotherapy.