N-methyl-D-aspartate receptors (NMDARs) are a subtype of ionotropic glutamate receptors critical for synaptic transmission and plasticity, and for the development of neural circuits. Rare or de-novo variants in GRIN genes encoding NMDAR subunits have been associated with neurodevelopmental disorders characterized by intellectual disability, developmental delay, autism, schizophrenia, or epilepsy. In recent years, some disease-associated variants in GRIN genes have been characterized using recombinant receptors expressed in non-neuronal cells, and a few variants have also been studied in neuronal preparations or animal models. Here we review the current literature on the functional evaluation of human disease-associated variants in GRIN1, GRIN2A and GRIN2B genes at all levels of analysis. Focusing on the impact of different patient variants at the level of receptor function, we discuss effects on receptor agonist and co-agonist affinity, channel open probability, and receptor cell surface expression. We consider how such receptor-level functional information may be used to classify variants as gain-of-function or loss-of-function, and discuss the limitations of this classification at the synaptic, cellular, or system level. Together this work by many laboratories worldwide yields valuable insights into NMDAR structure and function, and represents significant progress in the effort to understand and treat GRIN disorders. Keywords: NMDA receptor , GRIN genes, Genetic variants, Electrophysiology, Synapse, Animal models.

• MeSH
• Genetic Predisposition to Disease MeSH
• Genetic Variation MeSH
• Humans MeSH
• Neurodevelopmental Disorders genetics   MeSH
• Nerve Tissue Proteins genetics   metabolism   MeSH
• Receptors, N-Methyl-D-Aspartate * genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• GRIN1 protein, human MeSH Browser
• N-methyl D-aspartate receptor subtype 2A MeSH Browser
• NR2B NMDA receptor MeSH Browser
• Nerve Tissue Proteins MeSH
• Receptors, N-Methyl-D-Aspartate * MeSH

NMDA receptors (NMDARs) are ionotropic glutamate receptors that play a key role in excitatory neurotransmission. The number and subtype of surface NMDARs are regulated at several levels, including their externalization, internalization, and lateral diffusion between the synaptic and extrasynaptic regions. Here, we used novel anti-GFP (green fluorescent protein) nanobodies conjugated to either the smallest commercially available quantum dot 525 (QD525) or the several nanometer larger (and thus brighter) QD605 (referred to as nanoGFP-QD525 and nanoGFP-QD605, respectively). Targeting the yellow fluorescent protein-tagged GluN1 subunit in rat hippocampal neurons, we compared these two probes to a previously established larger probe, a rabbit anti-GFP IgG together with a secondary IgG conjugated to QD605 (referred to as antiGFP-QD605). The nanoGFP-based probes allowed faster lateral diffusion of the NMDARs, with several-fold increased median values of the diffusion coefficient (D). Using thresholded tdTomato-Homer1c signals to mark synaptic regions, we found that the nanoprobe-based D values sharply increased at distances over 100 nm from the synaptic edge, while D values for antiGFP-QD605 probe remained unchanged up to a 400 nm distance. Using the nanoGFP-QD605 probe in hippocampal neurons expressing the GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits, we detected subunit-dependent differences in the synaptic localization of NMDARs, D value, synaptic residence time, and synaptic-extrasynaptic exchange rate. Finally, we confirmed the applicability of the nanoGFP-QD605 probe to study differences in the distribution of synaptic NMDARs by comparing to data obtained with nanoGFPs conjugated to organic fluorophores, using universal point accumulation imaging in nanoscale topography and direct stochastic optical reconstruction microscopy.SIGNIFICANCE STATEMENT Our study systematically compared the localization and mobility of surface NMDARs containing GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits expressed in rodent hippocampal neurons, using anti-green fluorescent protein (GFP) nanobodies conjugated to the quantum dot 605 (nanoGFP-QD605), as well as nanoGFP probes conjugated with small organic fluorophores. Our comprehensive analysis showed that the method used to delineate the synaptic region plays an important role in the study of synaptic and extrasynaptic pools of NMDARs. In addition, we showed that the nanoGFP-QD605 probe has optimal parameters for studying the mobility of NMDARs because of its high localization accuracy comparable to direct stochastic optical reconstruction microscopy and longer scan time compared with universal point accumulation imaging in nanoscale topography. The developed approaches are readily applicable to the study of any GFP-labeled membrane receptors expressed in mammalian neurons.

• Keywords
• GluN subunit, excitatory synapse, glutamate receptor, lateral diffusion, live microscopy, mammalian neuron,
• MeSH
• Hippocampus metabolism   MeSH
• Immunoglobulin G metabolism   MeSH
• Single-Domain Antibodies * metabolism   MeSH
• Rabbits MeSH
• Rats MeSH
• Neurons metabolism   MeSH
• Receptors, N-Methyl-D-Aspartate * metabolism   MeSH
• Mammals MeSH
• Synapses physiology   MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Immunoglobulin G MeSH
• Single-Domain Antibodies * MeSH
• Receptors, N-Methyl-D-Aspartate * MeSH

Alzheimer's disease (AD) is a complex disease with an unknown etiology. Available treatments, limited to cholinesterase inhibitors and N-methyl-d-aspartate receptor (NMDAR) antagonists, provide symptomatic relief only. As single-target therapies have not proven effective, rational specific-targeted combination into a single molecule represents a more promising approach for treating AD, and is expected to yield greater benefits in alleviating symptoms and slowing disease progression. In the present study, we designed, synthesized, and biologically evaluated 24 novel N-methylpropargylamino-quinazoline derivatives. Initially, compounds were thoroughly inspected by in silico techniques determining their oral and CNS availabilities. We tested, in vitro, the compounds' effects on cholinesterases and monoamine oxidase A/B (MAO-A/B), as well as their impacts on NMDAR antagonism, dehydrogenase activity, and glutathione levels. In addition, we inspected selected compounds for their cytotoxicity on undifferentiated and differentiated neuroblastoma SH-SY5Y cells. We collectively highlighted II-6h as the best candidate endowed with a selective MAO-B inhibition profile, NMDAR antagonism, an acceptable cytotoxicity profile, and the potential to permeate through BBB. The structure-guided drug design strategy applied in this study imposed a novel concept for rational drug discovery and enhances our understanding on the development of novel therapeutic agents for treating AD.

• Keywords
• Alzheimer’s disease, N-methyl-d-aspartate receptor, acetylcholinesterase, enzyme inhibition, monoamine oxidase A/B, multi-target directed ligands,
• MeSH
• Acetylcholinesterase metabolism   MeSH
• Alzheimer Disease * drug therapy   MeSH
• Cholinesterase Inhibitors therapeutic use   MeSH
• Monoamine Oxidase Inhibitors therapeutic use   MeSH
• Humans MeSH
• Monoamine Oxidase metabolism   MeSH
• Neuroblastoma * drug therapy   MeSH
• Drug Design MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Acetylcholinesterase MeSH
• Cholinesterase Inhibitors MeSH
• Monoamine Oxidase Inhibitors MeSH
• Monoamine Oxidase MeSH