Astrocytes modulate brain phosphate homeostasis via polarized distribution of phosphate uptake transporter PiT2 and exporter XPR1.

Aberrant inorganic phosphate (Pi) homeostasis causes brain calcification and aggravates neurodegeneration, but the underlying mechanism remains unclear. Here, we found that primary familial brain calcification (PFBC)-associated Pi transporter genes Pit2 and Xpr1 were highly expressed in astrocytes, with importer PiT2 distributed over the entire astrocyte processes and exporter XPR1 localized to astrocyte end-feet on blood vessels. This polarized PiT2 and XPR1 distribution endowed astrocyte with Pi transport capacity competent for brain Pi homeostasis, which was disrupted in mice with astrocyte-specific knockout (KO) of either Pit2 or Xpr1. Moreover, we found that Pi uptake by PiT2, and its facilitation by PFBC-associated galactosidase MYORG, were required for the high Pi transport capacity of astrocytes. Finally, brain calcification was suppressed by astrocyte-specific PiT2 re-expression in Pit2-KO mice. Thus, astrocyte-mediated Pi transport is pivotal for brain Pi homeostasis, and elevating astrocytic Pi transporter function represents a potential therapeutic strategy for reducing brain calcification.

Sevoflurane acts as an antidepressant by suppression of GluN2D-containing NMDA receptors on interneurons.

BACKGROUND AND PURPOSE: Sevoflurane, a commonly used inhaled anaesthetic known for its favourable safety profile and rapid onset and offset, has not been thoroughly investigated as a potential treatment for depression. In this study, we reveal the mechanism through which sevoflurane delivers enduring antidepressant effects. EXPERIMENTAL APPROACH: To assess the antidepressant effects of sevoflurane, behavioural tests were conducted, along with in vitro and ex vivo whole-cell patch-clamp recordings, to examine the effects on GluN1-GluN2 incorporated N-methyl-d-aspartate (NMDA) receptors (NMDARs) and neuronal circuitry in the medial prefrontal cortex (mPFC). Multiple-channel electrophysiology in freely moving mice was performed to evaluate sevoflurane's effects on neuronal activity, and GluN2D knockout (grin2d-/-) mice were used to confirm the requirement of GluN2D for the antidepressant effects. KEY RESULTS: Repeated exposure to subanaesthetic doses of sevoflurane produced sustained antidepressant effects lasting up to 2 weeks. Sevoflurane preferentially inhibited GluN2C- and GluN2D-containing NMDARs, causing a reduction in interneuron activity. In contrast, sevoflurane increased action potentials (AP) firing and decreased spontaneous inhibitory postsynaptic current (sIPSC) in mPFC pyramidal neurons, demonstrating a disinhibitory effect. These effects were absent in grin2d-/- mice, and both pharmacological blockade and genetic knockout of GluN2D abolished sevoflurane's antidepressant actions, suggesting that GluN2D is essential for its antidepressant effect. CONCLUSION AND IMPLICATIONS: Sevoflurane directly targets GluN2D, leading to a specific decrease in interneuron activity and subsequent disinhibition of pyramidal neurons, which may underpin its antidepressant effects. Targeting the GluN2D subunit could hold promise as a potential therapeutic strategy for treating depression.

Expression and Purification of Mammalian NMDA Receptor Protein for Functional Characterization.

N-methyl-D-aspartate (NMDA) receptors are critical for brain function and serve as drug targets for the treatment of neurological and psychiatric disorders. They typically form the tetrameric assembly of GluN1-GluN2 (2A to 2D) subtypes, with their diverse three-dimensional conformations linked with the physiologically relevant function in vivo. Purified proteins of tetrameric assembled NMDA receptors have broad applications in the structural elucidation, hybridoma technology for antibody production, and high-throughput drug screening. However, obtaining sufficient quantity and monodisperse NMDA receptor protein is still technically challenging. Here, we summarize a paradigm for the expression and purification of diverse NMDA receptor subtypes, with detailed descriptions on screening constructs by fluorescence size-exclusion chromatography (FSEC), generation of recombinant baculovirus, expression in the eukaryotic expression system, protein purification by affinity chromatography and size-exclusion chromatography (SEC), biochemical and functional validation assays.

Whole-brain in vivo base editing reverses behavioral changes in Mef2c-mutant mice.

Whole-brain genome editing to correct single-base mutations and reduce or reverse behavioral changes in animal models of autism spectrum disorder (ASD) has not yet been achieved. We developed an apolipoprotein B messenger RNA-editing enzyme, catalytic polypeptide-embedded cytosine base editor (AeCBE) system for converting C·G to T·A base pairs. We demonstrate its effectiveness by targeting AeCBE to an ASD-associated mutation of the MEF2C gene (c.104T>C, p.L35P) in vivo in mice. We first constructed Mef2cL35P heterozygous mice. Male heterozygous mice exhibited hyperactivity, repetitive behavior and social abnormalities. We then programmed AeCBE to edit the mutated C·G base pairs of Mef2c in the mouse brain through the intravenous injection of blood-brain barrier-crossing adeno-associated virus. This treatment successfully restored Mef2c protein levels in several brain regions and reversed the behavioral abnormalities in Mef2c-mutant mice. Our work presents an in vivo base-editing paradigm that could potentially correct single-base genetic mutations in the brain.

Structural insights into gating mechanism and allosteric regulation of NMDA receptors.

N-methyl-d-aspartate receptors (NMDARs) belong to the ionotropic glutamate receptors (iGluRs) superfamily and act as coincidence detectors that are crucial to neuronal development and synaptic plasticity. They typically assemble as heterotetramers of two obligatory GluN1 subunits and two alternative GluN2 (from 2A to 2D) and/or GluN3 (3A and 3B) subunits. These alternative subunits mainly determine the diverse biophysical and pharmacological properties of different NMDAR subtypes. Over the past decade, the unprecedented advances in structure elucidation of these tetrameric NMDARs have provided atomic insights into channel gating, allosteric modulation and the action of therapeutic drugs. A wealth of structural and functional information would accelerate the artificial intelligence-based drug design to exploit more NMDAR subtype-specific molecules for the treatment of neurological and psychiatric disorders.

Distinct structure and gating mechanism in diverse NMDA receptors with GluN2C and GluN2D subunits.

N-methyl-D-aspartate (NMDA) receptors are heterotetramers comprising two GluN1 and two alternate GluN2 (N2A-N2D) subunits. Here we report full-length cryo-EM structures of the human N1-N2D di-heterotetramer (di-receptor), rat N1-N2C di-receptor and N1-N2A-N2C tri-heterotetramer (tri-receptor) at a best resolution of 3.0 Å. The bilobate N-terminal domain (NTD) in N2D intrinsically adopts a closed conformation, leading to a compact NTD tetramer in the N1-N2D receptor. Additionally, crosslinking the ligand-binding domain (LBD) of two N1 protomers significantly elevated the channel open probability (Po) in N1-N2D di-receptors. Surprisingly, the N1-N2C di-receptor adopted both symmetric (minor) and asymmetric (major) conformations, the latter further locked by an allosteric potentiator, PYD-106, binding to a pocket between the NTD and LBD in only one N2C protomer. Finally, the N2A and N2C subunits in the N1-N2A-N2C tri-receptor display a conformation close to one protomer in the N1-N2A and N1-N2C di-receptors, respectively. These findings provide a comprehensive structural understanding of diverse function in major NMDA receptor subtypes.

NMDA receptors as therapeutic targets for depression treatment: Evidence from clinical to basic research.

Ketamine, functioning as a channel blocker of the excitatory glutamate-gated N-methyl-d-aspartate (NMDA) receptors, displays compelling fast-acting and sustained antidepressant effects for treatment-resistant depression. Over the past decades, clinical and preclinical studies have implied that the pathology of depression is associated with dysfunction of glutamatergic transmission. In particular, the discovery of antidepressant agents modulating NMDA receptor function has prompted breakthroughs for depression treatment compared with conventional antidepressants targeting the monoaminergic system. In this review, we first summarized the signalling pathway of the ketamine-mediated antidepressant effects, based on the glutamate hypothesis of depression. Second, we reviewed the hypotheses of the synaptic mechanism and network of ketamine antidepressant effects within different brain areas and distinct subcellular localizations, including NMDA receptor antagonism on GABAergic interneurons, extrasynaptic and synaptic NMDA receptor-mediated antagonism, and ketamine blocking bursting activities in the lateral habenula. Third, we reviewed the different roles of NMDA receptor subunits in ketamine-mediated cognitive and psychiatric behaviours in genetically-manipulated rodent models. Finally, we summarized the structural basis of NMDA receptor channel blockers and discussed NMDA receptor modulators that have been reported to exert potential antidepressant effects in animal models or in clinical trials. Integrating the cutting-edge technologies of cryo-EM and artificial intelligence-based drug design (AIDD), we expect that the next generation of first-in-class rapid antidepressants targeting NMDA receptors would be an emerging direction for depression therapeutics. This article is part of the Special Issue on 'Ketamine and its Metabolites'.

Mechanisms of PiT2-loop7 Missense Mutations Induced Pi Dyshomeostasis.

PiT2 is an inorganic phosphate (Pi) transporter whose mutations are linked to primary familial brain calcification (PFBC). PiT2 mainly consists of two ProDom (PD) domains and a large intracellular loop region (loop7). The PD domains are crucial for the Pi transport, but the role of PiT2-loop7 remains unclear. In PFBC patients, mutations in PiT2-loop7 are mainly nonsense or frameshift mutations that probably cause PFBC due to C-PD1131 deletion. To date, six missense mutations have been identified in PiT2-loop7; however, the mechanisms by which these mutations cause PFBC are poorly understood. Here, we found that the p.T390A and p.S434W mutations in PiT2-loop7 decreased the Pi transport activity and cell surface levels of PiT2. Furthermore, we showed that these two mutations attenuated its membrane localization by affecting adenosine monophosphate-activated protein kinase (AMPK)- or protein kinase B (AKT)-mediated PiT2 phosphorylation. In contrast, the p.S121C and p.S601W mutations in the PD domains did not affect PiT2 phosphorylation but rather impaired its substrate-binding abilities. These results suggested that missense mutations in PiT2-loop7 can cause Pi dyshomeostasis by affecting the phosphorylation-regulated cell-surface localization of PiT2. This study helps understand the pathogenesis of PFBC caused by PiT2-loop7 missense mutations and indicates that increasing the phosphorylation levels of PiT2-loop7 could be a promising strategy for developing PFBC therapies.

Identification of a Subtype-Selective Allosteric Inhibitor of GluN1/GluN3 NMDA Receptors.

N-methyl-D-aspartate receptors (NMDARs) are Ca2+-permeable ionotropic glutamate receptors (iGluRs) in the central nervous system and play important roles in neuronal development and synaptic plasticity. Conventional NMDARs, which typically comprise GluN1 and GluN2 subunits, have different biophysical properties than GluN3-containing NMDARs: GluN3-containing NMDARs have smaller unitary conductance, less Ca2+-permeability and lower Mg2+-sensitivity than those of conventional NMDARs. However, there are very few specific modulators for GluN3-containing NMDARs. Here, we developed a cell-based high-throughput calcium assay and identified 3-fluoro-1,2-phenylene bis (3-hydroxybenzoate) (WZB117) as a relatively selective inhibitor of GluN1/GluN3 receptors. The IC50 value of WZB117 on GluN1/GluN3A receptors expressed in HEK-293 cells was 1.15 ± 0.34 µM. Consistently, WZB117 exhibited strong inhibitory activity against glycine-induced currents in the presence of CGP-78608 but only slightly affected the NMDA-, KA- and AMPA-induced currents in the acutely isolated rat hippocampal neurons. Among the four types of endogenous currents, only the first one is primarily mediated by GluN1/GluN3 receptors. Mechanistic studies showed that WZB117 inhibited the GluN1/GluN3A receptors in a glycine-, voltage- and pH-independent manner, suggesting it is an allosteric modulator. Site-directed mutagenesis and chimera construction further revealed that WZB117 may act on the GluN3A pre-M1 region with key determinants different from those of previously identified modulators. Together, our study developed an efficient method to discover modulators of GluN3-containing NMDARs and characterized WZB117 as a novel allosteric inhibitor of GluN1/GluN3 receptors.

Structural basis of ketamine action on human NMDA receptors.

Ketamine is a non-competitive channel blocker of N-methyl-D-aspartate (NMDA) receptors1. A single sub-anaesthetic dose of ketamine produces rapid (within hours) and long-lasting antidepressant effects in patients who are resistant to other antidepressants2,3. Ketamine is a racemic mixture of S- and R-ketamine enantiomers, with S-ketamine isomer being the more active antidepressant4. Here we describe the cryo-electron microscope structures of human GluN1-GluN2A and GluN1-GluN2B NMDA receptors in complex with S-ketamine, glycine and glutamate. Both electron density maps uncovered the binding pocket for S-ketamine in the central vestibule between the channel gate and selectivity filter. Molecular dynamics simulation showed that S-ketamine moves between two distinct locations within the binding pocket. Two amino acids-leucine 642 on GluN2A (homologous to leucine 643 on GluN2B) and asparagine 616 on GluN1-were identified as key residues that form hydrophobic and hydrogen-bond interactions with ketamine, and mutations at these residues reduced the potency of ketamine in blocking NMDA receptor channel activity. These findings show structurally how ketamine binds to and acts on human NMDA receptors, and pave the way for the future development of ketamine-based antidepressants.

Gating mechanism and a modulatory niche of human GluN1-GluN2A NMDA receptors.

N-methyl-D-aspartate (NMDA) receptors are glutamate-gated calcium-permeable ion channels that are widely implicated in synaptic transmission and plasticity. Here, we report a gallery of cryo-electron microscopy (cryo-EM) structures of the human GluN1-GluN2A NMDA receptor at an overall resolution of 4 Å in complex with distinct ligands or modulators. In the full-length context of GluN1-GluN2A receptors, we visualize the competitive antagonists bound to the ligand-binding domains (LBDs) of GluN1 and GluN2A subunits, respectively. We reveal that the binding of positive allosteric modulator shortens the distance between LBDs and the transmembrane domain (TMD), which further stretches the opening of the gate. In addition, we unexpectedly visualize the binding cavity of the "foot-in-the-door" blocker 9-aminoacridine within the LBD-TMD linker region, differing from the conventional "trapping" blocker binding site at the vestibule within the TMD. Our study provides molecular insights into the crosstalk between LBDs and TMD during channel activation, inhibition, and allosteric transition.

Homozygous variants in PANX1 cause human oocyte death and female infertility.

PANX1, one of the members of the pannexin family, is a highly glycosylated channel-forming protein. Recently, we identified heterozygous variants in PANX1 that follow an autosomal dominant inheritance pattern and cause female infertility characterized by oocyte death. In this study, we screened for novel PANX1 variants in patients with the phenotype of oocyte death and discovered a new type of inheritance pattern accompanying PANX1 variants. We identified two novel homozygous missense variants in PANX1 [NM_015368.4 c.712T>C (p.(Ser238Pro) and c.899G>A (p.(Arg300Gln))] associated with the oocyte death phenotype in two families. Both of the homozygous variants altered the PANX1 glycosylation pattern in cultured cells, led to aberrant PANX1 channel activation, and resulted in mouse oocyte death after fertilization in vitro. It is worth noting that the destructive effect of the two homozygous variants on PANX1 function was weaker than that caused by the recently reported heterozygous variants. Our findings enrich the variational spectrum of PANX1 and expand the inheritance pattern of PANX1 variants to an autosomal recessive mode. This highlights the critical role of PANX1 in human oocyte development and helps us to better understand the genetic basis of female infertility due to oocyte death.

Specific Hypothalamic Neurons Required for Sensing Conspecific Male Cues Relevant to Inter-male Aggression.

The hypothalamus regulates innate social interactions, but how hypothalamic neurons transduce sex-related sensory signals emitted by conspecifics to trigger appropriate behaviors remains unclear. Here, we addressed this issue by identifying specific hypothalamic neurons required for sensing conspecific male cues relevant to inter-male aggression. By in vivo recording of neuronal activities in behaving mice, we showed that neurons expressing dopamine transporter (DAT+) in the ventral premammillary nucleus (PMv) of the hypothalamus responded to male urine cues in a vomeronasal organ (VNO)-dependent manner in naive males. Retrograde trans-synaptic tracing further revealed a specific group of neurons in the bed nucleus of the stria terminalis (BNST) that convey male-relevant signals from VNO to PMv. Inhibition of PMvDAT+ neurons abolished the preference for male urine cues and reduced inter-male attacks, while activation of these neurons promoted urine marking and aggression. Thus, PMvDAT+ neurons exemplify a hypothalamic node that transforms sex-related chemo-signals into recognition and behaviors.

A pannexin 1 channelopathy causes human oocyte death.

Connexins and pannexins are two protein families that play an important role in cellular communication. Pannexin 1 (PANX1), one of the members of pannexin family, is a channel protein. It is glycosylated and forms three species, GLY0, GLY1, and GLY2. Here, we describe four independent families in which mutations in PANX1 cause familial or sporadic female infertility via a phenotype that we term "oocyte death." The mutations, which are associated with oocyte death, alter the PANX1 glycosylation pattern, influence the subcellular localization of PANX1 in cultured cells, and result in aberrant PANX1 channel activity, ATP release in oocytes, and mutant PANX1 GLY1. Overexpression of a patient-derived mutation in mice causes infertility, recapitulating the human oocyte death phenotype. Our findings demonstrate the critical role of PANX1 in human oocyte development, provide a genetic explanation for a subtype of infertility, and suggest a potential target for therapeutic intervention for this disease.

Prediction of ligand modulation patterns on membrane receptors via lysine reactivity profiling.

Herein we provide a mass spectrometry-based lysine reactivity profiling strategy to monitor the ligand modulation of protein receptors, which is achieved by active dimethyl labeling of lysine residues and comparison of the alterations of labeling reactivity during ligand binding. The small-molecule ligand modulation patterns on the catechol-O-methyltransferase and the N-methyl-d-aspartate receptors have been predicted, including both binding regions and related conformational changes.

Structural Basis of the Proton Sensitivity of Human GluN1-GluN2A NMDA Receptors.

N-methyl-D-aspartate (NMDA) receptors are critical for synaptic development and plasticity. While glutamate is the primary agonist, protons can modulate NMDA receptor activity at synapses during vesicle exocytosis by mechanisms that are unknown. We used cryo-electron microscopy to solve the structures of the human GluN1-GluN2A NMDA receptor at pH 7.8 and pH 6.3. Our structures demonstrate that the proton sensor predominantly resides in the N-terminal domain (NTD) of the GluN2A subunit and reveal the allosteric coupling mechanism between the proton sensor and the channel gate. Under high-pH conditions, the GluN2A-NTD adopts an "open-and-twisted" conformation. However, upon protonation at the lower pH, the GluN2A-NTD transits from an open- to closed-cleft conformation, causing rearrangements between the tetrameric NTDs and agonist-binding domains. The conformational mobility observed in our structures (presumably from protonation) is supported by molecular dynamics simulation. Our findings reveal the structural mechanisms by which protons allosterically inhibit human GluN1-GluN2A receptor activity.

Structure and symmetry inform gating principles of ionotropic glutamate receptors.

Ionotropic glutamate receptors (iGluRs) transduce signals derived from release of the excitatory neurotransmitter glutamate from pre-synaptic neurons into excitation of post-synaptic neurons on a millisecond time-scale. In recent years, the elucidation of full-length iGluR structures of NMDA, AMPA and kainate receptors by X-ray crystallography and single particle cryo-electron microscopy has greatly enhanced our understanding of the interrelationships between receptor architecture and gating mechanism. Here we briefly review full-length iGluR structures and discuss the similarities and differences between NMDA receptors and non-NMDA iGluRs. We focus on distinct conformations, including ligand-free, agonist-bound active, agonist-bound desensitized and antagonist-bound conformations as well as modulator and auxiliary protein-bound states. These findings provide insights into structure-based mechanisms of iGluR gating and modulation which together shape the amplitude and time course of the excitatory postsynaptic potential. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.