Input-specific localization of NMDA receptor GluN2 subunits in thalamocortical neurons.

Molecular and functional diversity among synapses is generated, in part, by differential expression of neurotransmitter receptors and their associated protein complexes. N-methyl-D-aspartate receptors (NMDARs) are tetrameric ionotropic glutamate receptors that most often comprise two GluN1 and two GluN2 subunits. NMDARs generate functionally diverse synapses across neuron populations through cell-type-specific expression patterns of GluN2 subunits (GluN2A - 2D), which have vastly different functional properties and distinct downstream signaling. Diverse NMDAR function has also been observed at anatomically distinct inputs to a single neuron population. However, the mechanisms that generate input-specific NMDAR function remain unknown as few studies have investigated subcellular GluN2 subunit localization in native brain tissue. We investigated NMDAR synaptic localization in thalamocortical (TC) neurons expressing all four GluN2 subunits. Utilizing super resolution imaging and knockout-validated antibodies, we revealed subtype- and input-specific GluN2 localization at corticothalamic (CT) versus sensory inputs to TC neurons in 4-week-old male and female C57Bl/6J mice. GluN2B was the most abundant postsynaptic subunit across all glutamatergic synapses followed by GluN2A and GluN2C, and GluN2D was localized to the fewest synapses. GluN2B was preferentially localized to CT synapses over sensory synapses, while GluN2A and GluN2C were more abundant at sensory inputs compared to CT inputs. Furthermore, postsynaptic scaffolding proteins PSD95 and SAP102 were preferentially localized with specific GluN2 subunits, and SAP102 was more abundant at sensory synapses than PSD95. This work indicates that TC neurons exhibit subtype- and input-specific localization of diverse NMDARs and associated scaffolding proteins that likely contribute to functional differences between CT and sensory synapses.

Dendritically localized RNAs are packaged as diversely composed ribonucleoprotein particles with heterogeneous copy number states.

Localization of mRNAs to dendrites is a fundamental mechanism by which neurons achieve spatiotemporal control of gene expression. Translationally repressed neuronal mRNA transport granules, also referred to as ribonucleoprotein particles (RNPs), have been shown to be trafficked as single or low copy number RNPs and as larger complexes with multiple copies and/or species of mRNAs. However, there is little evidence of either population in intact neuronal circuits. Using single molecule fluorescence in situ hybridization studies in the dendrites of adult rat and mouse hippocampus, we provide evidence that supports the existence of multi-transcript RNPs with the constituents varying in amounts for each RNA species. By competing-off fluorescently labeled probe with serial increases of unlabeled probe, we detected stepwise decreases in Arc RNP number and fluorescence intensity, suggesting Arc RNAs localize to dendrites in both low- and multiple-copy number RNPs. When probing for multiple mRNAs, we find that localized RNPs are heterogeneous in size and colocalization patterns that vary per RNA. Further, localized RNAs that are targeted by the same trans-acting element (FMRP) display greater levels of colocalization compared to an RNA not targeted by FMRP. Simultaneous visualization of a dozen FMRP-targeted mRNA species using highly multiplexed imaging demonstrates that dendritic RNAs are mostly trafficked as heteromeric cargoes of multiple types of RNAs (at least one or more RNAs). Moreover, the composition of these RNA cargoes, as assessed by colocalization, correlates with the abundance of the transcripts even after accounting for the expected differences in colocalization based on expression. Collectively, these results suggest that dendritic RNPs are packaged as heterogeneous co-assemblies of different mRNAs and that RNP contents may be driven, at least partially, by highly abundant dendritic RNAs; a model that favors efficiency over fine-tuned control for sustaining long-distance trafficking of thousands of messenger molecules.

Acute Adenoviral Infection Elicits an Arrhythmogenic Substrate Prior to Myocarditis.

BACKGROUND: Viral cardiac infection represents a significant clinical challenge encompassing several etiological agents, disease stages, complex presentation, and a resulting lack of mechanistic understanding. Myocarditis is a major cause of sudden cardiac death in young adults, where current knowledge in the field is dominated by later disease phases and pathological immune responses. However, little is known regarding how infection can acutely induce an arrhythmogenic substrate before significant immune responses. Adenovirus is a leading cause of myocarditis, but due to species specificity, models of infection are lacking, and it is not understood how adenoviral infection may underlie sudden cardiac arrest. Mouse adenovirus type-3 was previously reported as cardiotropic, yet it has not been utilized to understand the mechanisms of cardiac infection and pathology. METHODS: We have developed mouse adenovirus type-3 infection as a model to investigate acute cardiac infection and molecular alterations to the infected heart before an appreciable immune response or gross cardiomyopathy. RESULTS: Optical mapping of infected hearts exposes decreases in conduction velocity concomitant with increased Cx43Ser368 phosphorylation, a residue known to regulate gap junction function. Hearts from animals harboring a phospho-null mutation at Cx43Ser368 are protected against mouse adenovirus type-3-induced conduction velocity slowing. Additional to gap junction alterations, patch clamping of mouse adenovirus type-3-infected adult mouse ventricular cardiomyocytes reveals prolonged action potential duration as a result of decreased IK1 and IKs current density. Turning to human systems, we find human adenovirus type-5 increases phosphorylation of Cx43Ser368 and disrupts synchrony in human induced pluripotent stem cell-derived cardiomyocytes, indicating common mechanisms with our mouse whole heart and adult cardiomyocyte data. CONCLUSIONS: Together, these findings demonstrate that adenoviral infection creates an arrhythmogenic substrate through direct targeting of gap junction and ion channel function in the heart. Such alterations are known to precipitate arrhythmias and likely contribute to sudden cardiac death in acutely infected patients.

Ventral posterolateral and ventral posteromedial thalamocortical neurons have distinct physiological properties.

Somatosensory information is propagated from the periphery to the cerebral cortex by two parallel pathways through the ventral posterolateral (VPL) and ventral posteromedial (VPM) thalamus. VPL and VPM neurons receive somatosensory signals from the body and head, respectively. VPL and VPM neurons may also receive cell type-specific GABAergic input from the reticular nucleus of the thalamus. Although VPL and VPM neurons have distinct connectivity and physiological roles, differences in their functional properties remain unclear as they are often studied as one ventrobasal thalamus neuron population. Here, we directly compared synaptic and intrinsic properties of VPL and VPM neurons in C57Bl/6J mice of both sexes aged P25-P32. VPL neurons showed greater depolarization-induced spike firing and spike frequency adaptation than VPM neurons. VPL and VPM neurons fired similar numbers of spikes during hyperpolarization rebound bursts, but VPM neurons exhibited shorter burst latency compared with VPL neurons, which correlated with larger sag potential. VPM neurons had larger membrane capacitance and more complex dendritic arbors. Recordings of spontaneous and evoked synaptic transmission suggested that VPL neurons receive stronger excitatory synaptic input, whereas inhibitory synapse strength was stronger in VPM neurons. This work indicates that VPL and VPM thalamocortical neurons have distinct intrinsic and synaptic properties. The observed functional differences could have important implications for their specific physiological and pathophysiological roles within the somatosensory thalamocortical network.NEW & NOTEWORTHY This study revealed that somatosensory thalamocortical neurons in the VPL and VPM have substantial differences in excitatory synaptic input and intrinsic firing properties. The distinct properties suggest that VPL and VPM neurons could process somatosensory information differently and have selective vulnerability to disease. This work improves our understanding of nucleus-specific neuron function in the thalamus and demonstrates the critical importance of studying these parallel somatosensory pathways separately.

Development of a Dihydroquinoline-Pyrazoline GluN2C/2D-Selective Negative Allosteric Modulator of the N-Methyl-d-aspartate Receptor.

Subunit-selective inhibition of N-methyl-d-aspartate receptors (NMDARs) is a promising therapeutic strategy for several neurological disorders, including epilepsy, Alzheimer's and Parkinson's disease, depression, and acute brain injury. We previously described the dihydroquinoline-pyrazoline (DQP) analogue 2a (DQP-26) as a potent NMDAR negative allosteric modulator with selectivity for GluN2C/D over GluN2A/B. However, moderate (<100-fold) subunit selectivity, inadequate cell-membrane permeability, and poor brain penetration complicated the use of 2a as an in vivo probe. In an effort to improve selectivity and the pharmacokinetic profile of the series, we performed additional structure-activity relationship studies of the succinate side chain and investigated the use of prodrugs to mask the pendant carboxylic acid. These efforts led to discovery of the analogue (S)-(-)-2i, also referred to as (S)-(-)-DQP-997-74, which exhibits >100- and >300-fold selectivity for GluN2C- and GluN2D-containing NMDARs (IC50 0.069 and 0.035 µM, respectively) compared to GluN2A- and GluN2B-containing receptors (IC50 5.2 and 16 µM, respectively) and has no effects on AMPA, kainate, or GluN1/GluN3 receptors. Compound (S)-(-)-2i is 5-fold more potent than (S)-2a. In addition, compound 2i shows a time-dependent enhancement of inhibitory actions at GluN2C- and GluN2D-containing NMDARs in the presence of the agonist glutamate, which could attenuate hypersynchronous activity driven by high-frequency excitatory synaptic transmission. Consistent with this finding, compound 2i significantly reduced the number of epileptic events in a murine model of tuberous sclerosis complex (TSC)-induced epilepsy that is associated with upregulation of the GluN2C subunit. Thus, 2i represents a robust tool for the GluN2C/D target validation. Esterification of the succinate carboxylate improved brain penetration, suggesting a strategy for therapeutic development of this series for NMDAR-associated neurological conditions.

NaV1.1 haploinsufficiency impairs glutamatergic and GABAergic neuron function in the thalamus.

Thalamocortical network dysfunction contributes to seizures and sleep deficits in Dravet syndrome (DS), an infantile epileptic encephalopathy, but the underlying molecular and cellular mechanisms remain elusive. DS is primarily caused by mutations in the SCN1A gene encoding the voltage-gated sodium channel NaV1.1, which is highly expressed in GABAergic reticular thalamus (nRT) neurons as well as glutamatergic thalamocortical neurons. We hypothesized that NaV1.1 haploinsufficiency alters somatosensory corticothalamic circuit function through both intrinsic and synaptic mechanisms in nRT and thalamocortical neurons. Using Scn1a heterozygous mice of both sexes aged P25-P30, we discovered reduced excitability of nRT neurons and thalamocortical neurons in the ventral posterolateral (VPL) thalamus, while thalamocortical ventral posteromedial (VPM) neurons exhibited enhanced excitability. NaV1.1 haploinsufficiency enhanced GABAergic synaptic input and reduced glutamatergic input to VPL neurons, but not VPM neurons. In addition, glutamatergic input to nRT neurons was reduced in Scn1a heterozygous mice. These findings introduce alterations in glutamatergic synapse function and aberrant glutamatergic neuron excitability in the thalamus as disease mechanisms in DS, which has been widely considered a disease of GABAergic neurons. This work reveals additional complexity that expands current models of thalamic dysfunction in DS and identifies new components of corticothalamic circuitry as potential therapeutic targets.

Structure, Function, and Pharmacology of Glutamate Receptor Ion Channels.

Many physiologic effects of l-glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, are mediated via signaling by ionotropic glutamate receptors (iGluRs). These ligand-gated ion channels are critical to brain function and are centrally implicated in numerous psychiatric and neurologic disorders. There are different classes of iGluRs with a variety of receptor subtypes in each class that play distinct roles in neuronal functions. The diversity in iGluR subtypes, with their unique functional properties and physiologic roles, has motivated a large number of studies. Our understanding of receptor subtypes has advanced considerably since the first iGluR subunit gene was cloned in 1989, and the research focus has expanded to encompass facets of biology that have been recently discovered and to exploit experimental paradigms made possible by technological advances. Here, we review insights from more than 3 decades of iGluR studies with an emphasis on the progress that has occurred in the past decade. We cover structure, function, pharmacology, roles in neurophysiology, and therapeutic implications for all classes of receptors assembled from the subunits encoded by the 18 ionotropic glutamate receptor genes. SIGNIFICANCE STATEMENT: Glutamate receptors play important roles in virtually all aspects of brain function and are either involved in mediating some clinical features of neurological disease or represent a therapeutic target for treatment. Therefore, understanding the structure, function, and pharmacology of this class of receptors will advance our understanding of many aspects of brain function at molecular, cellular, and system levels and provide new opportunities to treat patients.

Biased modulators of NMDA receptors control channel opening and ion selectivity.

Allosteric modulators of ion channels typically alter the transitions rates between conformational states without changing the properties of the open pore. Here we describe a new class of positive allosteric modulators of N-methyl D-aspartate receptors (NMDARs) that mediate a calcium-permeable component of glutamatergic synaptic transmission and play essential roles in learning, memory and cognition, as well as neurological disease. EU1622-14 increases agonist potency and channel-open probability, slows receptor deactivation and decreases both single-channel conductance and calcium permeability. The unique functional selectivity of this chemical probe reveals a mechanism for enhancing NMDAR function while limiting excess calcium influx, and shows that allosteric modulators can act as biased modulators of ion-channel permeation.

De novo GRIN variants in NMDA receptor M2 channel pore-forming loop are associated with neurological diseases.

N-methyl-D-aspartate receptors (NMDARs) mediate slow excitatory postsynaptic transmission in the central nervous system, thereby exerting a critical role in neuronal development and brain function. Rare genetic variants in the GRIN genes encoding NMDAR subunits segregated with neurological disorders. Here, we summarize the clinical presentations for 18 patients harboring 12 de novo missense variants in GRIN1, GRIN2A, and GRIN2B that alter residues in the M2 re-entrant loop, a region that lines the pore and is intolerant to missense variation. These de novo variants were identified in children with a set of neurological and neuropsychiatric conditions. Evaluation of the receptor cell surface expression, pharmacological properties, and biophysical characteristics show that these variants can have modest changes in agonist potency, proton inhibition, and surface expression. However, voltage-dependent magnesium inhibition is significantly reduced in all variants. The NMDARs hosting a single copy of a mutant subunit showed a dominant reduction in magnesium inhibition for some variants. These variant NMDARs also show reduced calcium permeability and single-channel conductance, as well as altered open probability. The data suggest that M2 missense variants increase NMDAR charge transfer in addition to varied and complex influences on NMDAR functional properties, which may underlie the patients' phenotypes.

Tonic Activation of GluN2C/GluN2D-Containing NMDA Receptors by Ambient Glutamate Facilitates Cortical Interneuron Maturation.

Developing cortical GABAergic interneurons rely on genetic programs, neuronal activity, and environmental cues to construct inhibitory circuits during early postnatal development. Disruption of these events can cause long-term changes in cortical inhibition and may be involved in neurological disorders associated with inhibitory circuit dysfunction. We hypothesized that tonic glutamate signaling in the neonatal cortex contributes to, and is necessary for, the maturation of cortical interneurons. To test this hypothesis, we used mice of both sexes to quantify extracellular glutamate concentrations in the cortex during development, measure ambient glutamate-mediated activation of developing cortical interneurons, and manipulate tonic glutamate signaling using subtype-specific NMDA receptor antagonists in vitro and in vivo We report that ambient glutamate levels are high (≈100 nm) in the neonatal cortex and decrease (to ≈50 nm) during the first weeks of life, coincident with increases in astrocytic glutamate uptake. Consistent with elevated ambient glutamate, putative parvalbumin-positive interneurons in the cortex (identified using G42:GAD1-eGFP reporter mice) exhibit a transient, tonic NMDA current at the end of the first postnatal week. GluN2C/GluN2D-containing NMDA receptors mediate the majority of this current and contribute to the resting membrane potential and intrinsic properties of developing putative parvalbumin interneurons. Pharmacological blockade of GluN2C/GluN2D-containing NMDA receptors in vivo during the period of tonic interneuron activation, but not later, leads to lasting decreases in interneuron morphological complexity and causes deficits in cortical inhibition later in life. These results demonstrate that dynamic ambient glutamate signaling contributes to cortical interneuron maturation via tonic activation of GluN2C/GluN2D-containing NMDA receptors.SIGNIFICANCE STATEMENT Inhibitory GABAergic interneurons make up 20% of cortical neurons and are critical to controlling cortical network activity. Dysfunction of cortical inhibition is associated with multiple neurological disorders, including epilepsy. Establishing inhibitory cortical networks requires in utero proliferation, differentiation, and migration of immature GABAergic interneurons, and subsequent postnatal morphological maturation and circuit integration. Here, we demonstrate that ambient glutamate provides tonic activation of immature, putative parvalbumin-positive GABAergic interneurons in the neonatal cortex via high-affinity NMDA receptors. When this activation is blocked, GABAergic interneuron maturation is disrupted, and cortical networks exhibit lasting abnormal hyperexcitability. We conclude that temporally precise activation of developing cortical interneurons by ambient glutamate is critically important for establishing normal cortical inhibition.

Triheteromeric GluN1/GluN2A/GluN2C NMDARs with Unique Single-Channel Properties Are the Dominant Receptor Population in Cerebellar Granule Cells.

NMDA-type glutamate receptors (NMDARs) are ligand-gated ion channels that mediate excitatory neurotransmission in the CNS. Here we describe functional and single-channel properties of triheteromeric GluN1/GluN2A/GluN2C receptors, which contain two GluN1, one GluN2A, and one GluN2C subunits. This NMDAR has three conductance levels and opens in bursts similar to GluN1/GluN2A receptors but with a single-channel open time and open probability reminiscent of GluN1/GluN2C receptors. The deactivation time course of GluN1/GluN2A/GluN2C receptors is intermediate to GluN1/GluN2A and GluN1/GluN2C receptors and is not dominated by GluN2A or GluN2C. We show that triheteromeric GluN1/GluN2A/GluN2C receptors are the predominant NMDARs in cerebellar granule cells and propose that co-expression of GluN2A and GluN2C in cerebellar granule cells occludes cell surface expression of diheteromeric GluN1/GluN2C receptors. This new insight into neuronal GluN1/GluN2A/GluN2C receptors highlights the complexity of NMDAR signaling in the CNS.

Synaptic Receptor Diversity Revealed Across Space and Time.

In their 1994 paper, Monyer et al. described unique functional properties and cell type-specific expression of NMDA receptor (NMDAR) GluN2 subunits, suggesting that the roles of NMDAR vary across brain regions and throughout development. This influential work not only provided insight into how molecular diversity impacts synapse function, but also continues to inform new approaches for modulating brain circuits.

De novo mutations in GRIN1 cause extensive bilateral polymicrogyria.

Polymicrogyria is a malformation of cortical development. The aetiology of polymicrogyria remains poorly understood. Using whole-exome sequencing we found de novo heterozygous missense GRIN1 mutations in 2 of 57 parent-offspring trios with polymicrogyria. We found nine further de novo missense GRIN1 mutations in additional cortical malformation patients. Shared features in the patients were extensive bilateral polymicrogyria associated with severe developmental delay, postnatal microcephaly, cortical visual impairment and intractable epilepsy. GRIN1 encodes GluN1, the essential subunit of the N-methyl-d-aspartate receptor. The polymicrogyria-associated GRIN1 mutations tended to cluster in the S2 region (part of the ligand-binding domain of GluN1) or the adjacent M3 helix. These regions are rarely mutated in the normal population or in GRIN1 patients without polymicrogyria. Using two-electrode and whole-cell voltage-clamp analysis, we showed that the polymicrogyria-associated GRIN1 mutations significantly alter the in vitro activity of the receptor. Three of the mutations increased agonist potency while one reduced proton inhibition of the receptor. These results are striking because previous GRIN1 mutations have generally caused loss of function, and because N-methyl-d-aspartate receptor agonists have been used for many years to generate animal models of polymicrogyria. Overall, our results expand the phenotypic spectrum associated with GRIN1 mutations and highlight the important role of N-methyl-d-aspartate receptor signalling in the pathogenesis of polymicrogyria.

A Novel Negative Allosteric Modulator Selective for GluN2C/2D-Containing NMDA Receptors Inhibits Synaptic Transmission in Hippocampal Interneurons.

N-Methyl-d-aspartate receptors (NMDARs) are ionotropic glutamate receptors that mediate excitatory synaptic transmission and have been implicated in numerous neurological disorders. NMDARs typically comprise two GluN1 and two GluN2 subunits. The four GluN2 subtypes (GluN2A-GluN2D) have distinct functional properties and gene expression patterns, which contribute to diverse functional roles for NMDARs in the brain. Here, we present a series of GluN2C/2D-selective negative allosteric modulators built around a N-aryl benzamide (NAB) core. The prototypical compound, NAB-14, is >800-fold selective for recombinant GluN2C/GluN2D over GluN2A/GluN2B in Xenopus oocytes and has an IC50 value of 580 nM at recombinant GluN2D-containing receptors expressed in mammalian cells. NAB-14 inhibits triheteromeric (GluN1/GluN2A/GluN2C) NMDARs with modestly reduced potency and efficacy compared to diheteromeric (GluN1/GluN2C/GluN2C) receptors. Site-directed mutagenesis suggests that structural determinants for NAB-14 inhibition reside in the GluN2D M1 transmembrane helix. NAB-14 inhibits GluN2D-mediated synaptic currents in rat subthalamic neurons and mouse hippocampal interneurons, but has no effect on synaptic transmission in hippocampal pyramidal neurons, which do not express GluN2C or GluN2D. This series possesses some druglike physical properties and modest brain permeability in rat and mouse. Altogether, this work identifies a new series of negative allosteric modulators that are valuable tools for studying GluN2C- and GluN2D-containing NMDAR function in brain circuits, and suggests that the series has the potential to be developed into therapies for selectively modulating brain circuits involving the GluN2C and GluN2D subunits.

GRIN1 mutation associated with intellectual disability alters NMDA receptor trafficking and function.

N-methyl-d-aspartate receptors (NMDARs) play important roles in brain development and neurological disease. We report two individuals with similar dominant de novo GRIN1 mutations (c.1858 G>A and c.1858 G>C; both p.G620R). Both individuals presented at birth with developmental delay and hypotonia associated with behavioral abnormalities and stereotypical movements. Recombinant NMDARs containing the mutant GluN1-G620R together with either GluN2A or GluN2B were evaluated for changes in their trafficking to the plasma membrane and their electrophysiological properties. GluN1-G620R/GluN2A complexes showed a mild reduction in trafficking, a ~2-fold decrease in glutamate and glycine potency, a strong decrease in sensitivity to Mg2+ block, and a significant reduction of current responses to a maximal effective concentration of agonists. GluN1-G620R/GluN2B complexes showed significantly reduced delivery of protein to the cell surface associated with similarly altered electrophysiology. These results indicate these individuals may have suffered neurodevelopmental deficits as a result of the decreased presence of GluN1-G620R/GluN2B complexes on the neuronal surface during embryonic brain development and reduced current responses of GluN1-G620R-containing NMDARs after birth. These cases emphasize the importance of comprehensive functional characterization of de novo mutations and illustrates how a combination of several distinct features of NMDAR expression, trafficking and function can be present and influence phenotype.

Molecular Mechanism of Disease-Associated Mutations in the Pre-M1 Helix of NMDA Receptors and Potential Rescue Pharmacology.

N-methyl-D-aspartate receptors (NMDARs), ligand-gated ionotropic glutamate receptors, play key roles in normal brain development and various neurological disorders. Here we use standing variation data from the human population to assess which protein domains within NMDAR GluN1, GluN2A and GluN2B subunits show the strongest signal for being depleted of missense variants. We find that this includes the GluN2 pre-M1 helix and linker between the agonist-binding domain (ABD) and first transmembrane domain (M1). We then evaluate the functional changes of multiple missense mutations in the NMDAR pre-M1 helix found in children with epilepsy and developmental delay. We find mutant GluN1/GluN2A receptors exhibit prolonged glutamate response time course for channels containing 1 or 2 GluN2A-P552R subunits, and a slow rise time only for receptors with 2 mutant subunits, suggesting rearrangement of one GluN2A pre-M1 helix is sufficient for rapid activation. GluN2A-P552R and analogous mutations in other GluN subunits increased the agonist potency and slowed response time course, suggesting a functionally conserved role for this residue. Although there is no detectable change in surface expression or open probability for GluN2A-P552R, the prolonged response time course for receptors that contained GluN2A-P552R increased charge transfer for synaptic-like activation, which should promote excitotoxic damage. Transfection of cultured neurons with GluN2A-P552R prolonged EPSPs, and triggered pronounced dendritic swelling in addition to excitotoxicity, which were both attenuated by memantine. These data implicate the pre-M1 region in gating, provide insight into how different subunits contribute to gating, and suggest that mutations in the pre-M1 helix can compromise neuronal health. Evaluation of FDA-approved NMDAR inhibitors on the mutant NMDAR-mediated current response and neuronal damage provides a potential clinical path to treat individuals harboring similar mutations in NMDARs.

Mechanistic Insight into NMDA Receptor Dysregulation by Rare Variants in the GluN2A and GluN2B Agonist Binding Domains.

Epilepsy and intellectual disability are associated with rare variants in the GluN2A and GluN2B (encoded by GRIN2A and GRIN2B) subunits of the N-methyl-D-aspartate receptor (NMDAR), a ligand-gated ion channel with essential roles in brain development and function. By assessing genetic variation across GluN2 domains, we determined that the agonist binding domain, transmembrane domain, and the linker regions between these domains were particularly intolerant to functional variation. Notably, the agonist binding domain of GluN2B exhibited significantly more variation intolerance than that of GluN2A. To understand the ramifications of missense variation in the agonist binding domain, we investigated the mechanisms by which 25 rare variants in the GluN2A and GluN2B agonist binding domains dysregulated NMDAR activity. When introduced into recombinant human NMDARs, these rare variants identified in individuals with neurologic disease had complex, and sometimes opposing, consequences on agonist binding, channel gating, receptor biogenesis, and forward trafficking. Our approach combined quantitative assessments of these effects to estimate the overall impact on synaptic and non-synaptic NMDAR function. Interestingly, similar neurologic diseases were associated with both gain- and loss-of-function variants in the same gene. Most rare variants in GluN2A were associated with epilepsy, whereas GluN2B variants were associated with intellectual disability with or without seizures. Finally, discerning the mechanisms underlying NMDAR dysregulation by these rare variants allowed investigations of pharmacologic strategies to correct NMDAR function.

Altered motility of plaque-associated microglia in a model of Alzheimer's disease.

Alzheimer's disease (AD), the most common form of dementia in the elderly, is characterized by the presence of extracellular plaques composed of amyloid ß (Aß) peptides and intracellular tau aggregates. The plaques are surrounded by microglia, the brain's resident immune cells, which likely participate in the clearance of Aß by phagocytosis. The microglia that are associated with plaques display an abnormal ameboid morphology and do not respond to tissue damage, in contrast to microglia in healthy brains. Here, we used time lapse confocal microscopy to perform a detailed real-time examination of microglial motility in acute hippocampal brain slices from the 5xFAD mouse model of AD, which was crossed to Cx3cr1(GFP/GFP) mice to achieve microglia-specific GFP expression for visualization. During baseline conditions, microglia around plaques appeared hypermotile, moving the processes that were pointing away from plaques at higher speed than microglia not associated with plaques. Yet, neither plaque-associated, nor plaque-free microglia were able to extend processes toward sites of modest mechanical damage. Application of the selective adenosine A2A receptor antagonist preladenant, which restores microglial response to cellular damage in a mouse model of Parkinson's disease, reduced the hypermotility of plaque-associated microglia, but did not restore motility toward damaged cells in slices from 5xFAD mice. Our results suggest that process hypermotility and resistance to A2A antagonism during response to tissue damage may represent unique functional phenotypes of plaque-associated microglia that impair their ability to function properly in the AD brain.

NMDA Receptors Containing the GluN2D Subunit Control Neuronal Function in the Subthalamic Nucleus.

The GluN2D subunit of the NMDA receptor is prominently expressed in the basal ganglia and associated brainstem nuclei, including the subthalamic nucleus (STN), globus pallidus, striatum, and substantia nigra. However, little is known about how GluN2D-containing NMDA receptors contribute to synaptic activity in these regions. Using Western blotting of STN tissue punches, we demonstrated that GluN2D is expressed in the rat STN throughout development [age postnatal day 7 (P7)-P60] and in the adult (age P120). Immunoelectron microscopy of the adult rat brain showed that GluN2D is predominantly expressed in dendrites, unmyelinated axons, and axon terminals within the STN. Using subunit-selective allosteric modulators of NMDA receptors (TCN-201, ifenprodil, CIQ, and DQP-1105), we provide evidence that receptors containing the GluN2B and GluN2D subunits mediate responses to exogenously applied NMDA and glycine, as well as synaptic NMDA receptor activation in the STN of rat brain slices. EPSCs in the STN were mediated primarily by AMPA and NMDA receptors and GluN2D-containing NMDA receptors controlled the slow deactivation time course of EPSCs in the STN. In vivo recordings from the STN of anesthetized adult rats demonstrated that the spike firing rate was increased by the GluN2C/D potentiator CIQ and decreased by the GluN2C/D antagonist DQP-1105, suggesting that NMDA receptor activity can influence STN output. These data indicate that the GluN2B and GluN2D NMDA receptor subunits contribute to synaptic activity in the STN and may represent potential therapeutic targets for modulating subthalamic neuron activity in neurological disorders such as Parkinson's disease.