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1.
Methods Mol Biol ; 2799: 1-11, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38727899

RESUMO

N-methyl-D-aspartate receptors (NMDAR) are ligand-gated ion channels mediating excitatory neurotransmission and are important for normal brain development, cognitive abilities, and motor functions. Pathogenic variants in the Glutamate receptor Ionotropic N-methyl-D-aspartate (GRIN) genes (GRIN1, GRIN2A-D) encoding NMDAR subunits have been associated with a wide spectrum of neurodevelopmental disorders and epilepsies ranging from treatable focal epilepsies to devastating early-onset developmental and epileptic encephalopathies. Genetic variants in NMDA receptor genes can cause a range of complex alterations to receptor properties resulting in various degrees of loss-of-function, gain-of-function, or mixtures thereof. Understanding how genetic variants affect the function of the receptors, therefore, represents an important first step in the ongoing development towards targeted therapies. Currently, targeted treatment options for GRIN-related diseases are limited. However, treatment with memantine has been reported to significantly reduce seizure frequency in a few individuals with developmental and epileptic encephalopathies harboring de novo gain-of-function GRIN2A missense variants, and supplementary treatment with L-serine has been associated with improved motor and cognitive performance as well as reduced seizure frequency in patients with GRIN2B loss-of-function missense variants as well as GRIN2A and GRIN2B null variants.


Assuntos
Epilepsia , Transtornos do Neurodesenvolvimento , Receptores de N-Metil-D-Aspartato , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Humanos , Transtornos do Neurodesenvolvimento/genética , Epilepsia/genética , Epilepsia/tratamento farmacológico , Predisposição Genética para Doença , Variação Genética , Memantina/uso terapêutico , Memantina/farmacologia
2.
Methods Mol Biol ; 2799: 29-46, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38727901

RESUMO

The expression and activity of ionotropic glutamate receptors control signal transduction at the excitatory synapses in the CNS. The NMDAR comprises two obligatory GluN1 subunits and two GluN2 or GluN3 subunits in different combinations. Each GluN subunit consists of four domains: the extracellular amino-terminal and agonist-binding domains, the transmembrane domain, and the intracellular C-terminal domain (CTD). The CTD interaction with various classes of intracellular proteins is critical for trafficking and synaptic localization of NMDARs. Amino acid mutations or the inclusion of premature stop codons in the CTD could contribute to the emergence of neurodevelopmental and neuropsychiatric disorders. Here, we describe the method of preparing primary hippocampal neurons and lentiviral particles expressing GluN subunits that can be used as a model to study cell surface expression and synaptic localization of NMDARs. We also show a simple method of fluorescence immunostaining of eGFP-tagged GluN2 subunits and subsequent microscopy technique and image analysis to study the effects of disease-associated mutations in the CTDs of GluN2A and GluN2B subunits.


Assuntos
Hipocampo , Neurônios , Receptores de N-Metil-D-Aspartato , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Hipocampo/metabolismo , Hipocampo/citologia , Neurônios/metabolismo , Animais , Subunidades Proteicas/metabolismo , Subunidades Proteicas/genética , Células Cultivadas , Ratos , Humanos , Lentivirus/genética , Cultura Primária de Células/métodos , Expressão Gênica
3.
Methods Mol Biol ; 2799: 13-27, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38727900

RESUMO

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.


Assuntos
Baculoviridae , Cromatografia de Afinidade , Cromatografia em Gel , Receptores de N-Metil-D-Aspartato , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/isolamento & purificação , Receptores de N-Metil-D-Aspartato/química , Animais , Baculoviridae/genética , Cromatografia de Afinidade/métodos , Humanos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Expressão Gênica , Células Sf9
4.
Methods Mol Biol ; 2799: 79-105, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38727904

RESUMO

The analysis of rare NMDAR gene variants in mice, coupled with a fundamental understanding of NMDAR function, plays a crucial role in achieving therapeutic success when addressing NMDAR dysfunctions in human patients. For the generation of such NMDAR mouse models, a basic knowledge of receptor structure, along with skills in database sequence analysis, cloning in E. coli, genetic manipulation of embryonic stem (ES) cells, and ultimately the genetic modification of mouse embryos, is essential. Primarily, this chapter will focus on the design and synthesis of NMDAR gene-targeting vectors that can be used successfully for the genetic manipulation of mice. We will outline the core principles of the design and synthesis of a gene targeting vector that facilitates the introduction of single-point mutations in NMDAR-encoding genes in mice. The transformation of ES cells, selection of positive ES cell colonies, manipulation of mouse embryos, and genotyping strategies will be described briefly.


Assuntos
Receptores de N-Metil-D-Aspartato , Animais , Camundongos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Humanos , Células-Tronco Embrionárias/metabolismo , Marcação de Genes/métodos , Vetores Genéticos/genética
5.
Methods Mol Biol ; 2799: 139-150, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38727906

RESUMO

Epilepsy is one of the most represented neurological diseases worldwide. However, in many cases, the precise molecular mechanisms of epileptogenesis and ictiogenesis are unknown. Because of their important role in synaptic function and neuronal excitability, NMDA receptors are implicated in various epileptogenic mechanisms. Most of these are subunit specific and require a precise analysis of the subunit composition of the NMDARs implicated. Here, we describe an express electrophysiological method to analyze the contribution of NMDAR subunits to spontaneous postsynaptic activity in identified cells in brain slices using patch clamp whole cell recordings.


Assuntos
Técnicas de Patch-Clamp , Receptores de N-Metil-D-Aspartato , Sinapses , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Técnicas de Patch-Clamp/métodos , Sinapses/metabolismo , Sinapses/fisiologia , Encéfalo/metabolismo , Encéfalo/citologia , Neurônios/metabolismo , Camundongos , Ratos , Subunidades Proteicas/metabolismo
6.
Methods Mol Biol ; 2799: 47-54, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38727902

RESUMO

Transfection allows the introduction of foreign nucleic acid into eukaryotic cells. It is an important tool in understanding the roles of NMDARs in neurons. Here we describe using lipofection-mediated transfection to introduce cDNA encoding NMDAR subunits into postmitotic rodent primary cortical neurons maintained in culture.


Assuntos
Neurônios , Transfecção , Neurônios/metabolismo , Neurônios/citologia , Animais , Transfecção/métodos , Células Cultivadas , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Camundongos , Cultura Primária de Células/métodos , DNA Complementar/genética
7.
Methods Mol Biol ; 2799: 55-77, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38727903

RESUMO

NMDA-type ionotropic glutamate receptors are critically involved in many brain functions and are implicated in a variety of brain disorders. Seven NMDA receptor subunits exist (GluN1, GluN2A-D, and GluN3A-B) that assemble into tetrameric receptor subtypes with distinct functional properties and physiological roles. The majority NMDA receptors are composed of two GluN1 and two GluN2 subunits, which can assemble into four diheteromeric receptors subtypes composed of GluN1 and one type of GluN2 subunit (e.g., GluN1/2A), and presumably also six triheteromeric receptor subtypes composed of GluN1 and two different GluN2 subunits (e.g., GluN1/2A/2B). Furthermore, the GluN1 subunit exists as eight splice variants (e.g., GluN1-1a and GluN1-1b isoforms), and two different GluN1 isoforms can co-assemble to also form triheteromeric NMDA receptors (e.g., GluN1-1a/1b/2A). Here, we describe a method to faithfully express triheteromeric NMDA receptors in heterologous expression systems by controlling the identity of two of the four subunits. This method overcomes the problem that co-expression of three different NMDA receptor subunits generates two distinct diheteromeric receptor subtypes as well as one triheteromeric receptor subtype, thereby confounding studies that require a homogenous population of triheteromeric NMDA receptors. The method has been applied to selectively express recombinant triheteromeric GluN1/2A/2B, GluN1/2A/2C, GluN1/2B/2D, GluN1-1a/GluN1-1b/2A, GluN1-1a/GluN1-1b/2B receptors with negligible co-expression of the respective diheteromeric receptor subtypes. This method therefore enables quantitative evaluation of functional and pharmacological properties of triheteromeric NMDA receptors, some of which are abundant NMDA receptor subtypes in the adult brain.


Assuntos
Isoformas de Proteínas , Subunidades Proteicas , Receptores de N-Metil-D-Aspartato , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Humanos , Subunidades Proteicas/metabolismo , Subunidades Proteicas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Células HEK293 , Animais , Membrana Celular/metabolismo , Expressão Gênica
8.
Methods Mol Biol ; 2799: 151-175, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38727907

RESUMO

In vertebrate central neurons, NMDA receptors are glutamate- and glycine-gated ion channels that allow the passage of Na+ and Ca2+ ions into the cell when these neurotransmitters are simultaneously present. The passage of Ca2+ is critical for initiating the cellular processes underlying various forms of synaptic plasticity. These Ca2+ ions can autoregulate the NMDA receptor signal through multiple distinct mechanisms to reduce the total flux of cations. One such mechanism is the ability of Ca2+ ions to exclude the passage of Na+ ions resulting in a reduced unitary current conductance. In contrast to the well-characterized Mg2+ block, this "channel block" mechanism is voltage-independent. In this chapter, we discuss theoretical and experimental considerations for the study of channel block by Ca2+ using single-channel patch-clamp electrophysiology. We focus on two classic methodologies to quantify the dependence of unitary channel conductance on external concentrations of Ca2+ as the basis for quantifying Ca2+ block.


Assuntos
Cálcio , Técnicas de Patch-Clamp , Receptores de N-Metil-D-Aspartato , Receptores de N-Metil-D-Aspartato/metabolismo , Cálcio/metabolismo , Técnicas de Patch-Clamp/métodos , Animais , Ativação do Canal Iônico , Humanos , Sódio/metabolismo
9.
Methods Mol Biol ; 2799: 107-138, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38727905

RESUMO

NMDAR-dependent forms of synaptic plasticity in brain regions like the hippocampus are widely believed to provide the neural substrate for long-term associative memory formation. However, the experimental data are equivocal at best and may suggest a more nuanced role for NMDARs and synaptic plasticity in memory. Much of the experimental data available comes from studies in genetically modified mice in which NMDAR subunits have been deleted or mutated in order to disrupt NMDAR function. Behavioral assessment of long-term memory in these mice has involved tests like the Morris watermaze and the radial arm maze. Here we describe these behavioral tests and some of the different testing protocols that can be used to assess memory performance. We discuss the importance of distinguishing selective effects on learning and memory processes from nonspecific effects on sensorimotor or motivational aspects of performance.


Assuntos
Aprendizagem em Labirinto , Memória de Longo Prazo , Receptores de N-Metil-D-Aspartato , Memória Espacial , Animais , Receptores de N-Metil-D-Aspartato/metabolismo , Camundongos , Memória de Longo Prazo/fisiologia , Aprendizagem em Labirinto/fisiologia , Memória Espacial/fisiologia , Hipocampo/fisiologia , Hipocampo/metabolismo , Comportamento Animal/fisiologia , Plasticidade Neuronal/fisiologia
10.
Methods Mol Biol ; 2799: 177-200, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38727908

RESUMO

In the mammalian central nervous system (CNS), fast excitatory transmission relies primarily on the ionic fluxes generated by ionotropic glutamate receptors (iGluRs). Among iGluRs, NMDA receptors (NMDARs) are unique in their ability to pass large, Ca2+-rich currents. Importantly, their high Ca2+ permeability is essential for normal CNS function and is under physiological control. For this reason, the accurate measurement of NMDA receptor Ca2+ permeability represents a valuable experimental step in evaluating the mechanism by which these receptors contribute to a variety of physiological and pathological conditions. In this chapter, we provide a theoretical and practical overview of the common methods used to estimate the Ca2+ permeability of ion channels as they apply to NMDA receptors. Specifically, we describe the principles and methodology used to calculate relative permeability (PCa/PNa) and fractional permeability (Pf), along with the relationship between these two metrics. With increasing knowledge about the structural dynamics of ion channels and of the ongoing environmental fluctuations in which channels operate in vivo, the ability to quantify the Ca2+ entering cells through specific ion channels remains a tool essential to delineating the molecular mechanisms that support health and cause disease.


Assuntos
Cálcio , Técnicas de Patch-Clamp , Receptores de N-Metil-D-Aspartato , Receptores de N-Metil-D-Aspartato/metabolismo , Cálcio/metabolismo , Técnicas de Patch-Clamp/métodos , Animais , Humanos , Permeabilidade , Permeabilidade da Membrana Celular
11.
Methods Mol Biol ; 2799: 257-267, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38727912

RESUMO

The NMDAR is a heterotetramer composed of two GluN1 subunits and two GluN2 and/or GluN3 subunits, with the GluN2 subunits exhibiting significant diversity in their structure and function. Recent studies have highlighted the importance of characterizing the specific roles of each GluN2 subunit across central nervous system regions and developmental stages, as well as their unique contributions to NMDAR-mediated signaling and plasticity. Understanding the distinct functions of GluN2 subunits is critical for the development of targeted therapeutic strategies for NMDAR-related disorders. However, measuring the functional contribution of individual GluN2 subtypes in ex vivo slices is challenging. Conventionally, pharmacological or genetic approaches are used, but, in many cases, this is not possible or is restricted to population-level NMDAR responses. Here, we describe a technique for using biophysical properties of miniature synaptic NMDAR responses as a proxy to measure the functional contribution of specific GluN2-NMDAR subunits to individual synapses within a neuron.


Assuntos
Subunidades Proteicas , Receptores de N-Metil-D-Aspartato , Sinapses , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Animais , Sinapses/metabolismo , Subunidades Proteicas/metabolismo , Camundongos , Neurônios/metabolismo , Ratos , Técnicas de Patch-Clamp/métodos , Transmissão Sináptica
12.
Methods Mol Biol ; 2799: 201-223, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38727909

RESUMO

Neuronal N-methyl-D-aspartate (NMDA) receptors are well known for their pivotal role in memory formation. Originally, they were thought to be exclusive to neurons. However, numerous studies revealed their functional expression also on various types of glial cells in the nervous system. Here, the methodology on how to study the physiology of NMDA receptors selectively on astrocytes will be described in detail. Astrocytes are the main class of neuroglia that control transmitter and ion homeostasis, which link cerebral blood flow and neuronal energy demands, but also affect synaptic transmission directly.


Assuntos
Astrócitos , Receptores de N-Metil-D-Aspartato , Astrócitos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Camundongos , Técnicas de Patch-Clamp/métodos , Células Cultivadas , Neurônios/metabolismo , Ratos
13.
Methods Mol Biol ; 2799: 281-290, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38727914

RESUMO

Artificial intelligence underwent remarkable advancement in the past decade, revolutionizing our way of thinking and unlocking unprecedented opportunities across various fields, including drug development. The emergence of large pretrained models, such as ChatGPT, has even begun to demonstrate human-level performance in certain tasks.However, the difficulties of deploying and utilizing AI and pretrained model for nonexpert limited its practical use. To overcome this challenge, here we presented three highly accessible online tools based on a large pretrained model for chemistry, the Uni-Mol, for drug development against CNS diseases, including those targeting NMDA receptor: the blood-brain barrier (BBB) permeability prediction, the quantitative structure-activity relationship (QSAR) analysis system, and a versatile interface of the AI-based molecule generation model named VD-gen. We believe that these resources will effectively bridge the gap between cutting-edge AI technology and NMDAR experts, facilitating rapid and rational drug development.


Assuntos
Barreira Hematoencefálica , Aprendizado Profundo , Relação Quantitativa Estrutura-Atividade , Receptores de N-Metil-D-Aspartato , Receptores de N-Metil-D-Aspartato/metabolismo , Humanos , Barreira Hematoencefálica/metabolismo , Desenvolvimento de Medicamentos/métodos
14.
Methods Mol Biol ; 2799: 243-255, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38727911

RESUMO

Zebrafish are a powerful system to study brain development and to dissect the activity of complex circuits. One advantage is that they display complex behaviors, including prey capture, learning, responses to photic and acoustic stimuli, and social interaction (Dreosti et al., Front Neural Circuits 9:39, 2015; Bruckner et al., PLoS Biol 20:e3001838, 2022; Zoodsma et al., Mol Autism 13:38, 2022) that can be probed to assess brain function. Many of these behaviors are easily assayed at early larval stages, offering a noninvasive and high-throughput readout of nervous system function. Additionally, larval zebrafish readily uptake small molecules dissolved in water making them ideal for behavioral-based drug screens. Together, larval zebrafish and their behavioral repertoire offer a means to rapidly dissect brain circuitry and can serve as a template for high-throughput small molecule screens.NMDA receptor subunits are highly conserved in zebrafish compared to mammals (Zoodsma et al., Mol Autism 13:38, 2022; Cox et al., Dev Dyn 234:756-766, 2005; Zoodsma et al., J Neurosci 40:3631-3645, 2020). High amino acid and domain structure homology between humans and zebrafish underlie conserved functional similarities. Here we describe a set of behavioral assays that are useful to study the NMDA receptor activity in brain function.


Assuntos
Comportamento Animal , Receptores de N-Metil-D-Aspartato , Peixe-Zebra , Animais , Peixe-Zebra/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Comportamento Animal/efeitos dos fármacos , Larva/metabolismo , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos
15.
Methods Mol Biol ; 2799: 269-280, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38727913

RESUMO

N-Methyl-D-aspartate (NMDA) receptors are glutamate-gated excitatory channels that play essential roles in brain functions. While high-resolution structures were solved for an allosterically inhibited form of functional NMDA receptor, other key functional states (particularly the active open-channel state) have not yet been resolved at atomic resolutions. To decrypt the molecular mechanism of the NMDA receptor activation, structural modeling and simulation are instrumental in providing detailed information about the dynamics and energetics of the receptor in various functional states. In this chapter, we describe coarse-grained modeling of the NMDA receptor using an elastic network model and related modeling/analysis tools (e.g., normal mode analysis, flexibility and hotspot analysis, cryo-EM flexible fitting, and transition pathway modeling) based on available structures. Additionally, we show how to build an atomistic model of the active-state receptor with targeted molecular dynamics (MD) simulation and explore its energetics and dynamics with conventional MD simulation. Taken together, these modeling and simulation can offer rich structural and dynamic information which will guide experimental studies of the activation of this key receptor.


Assuntos
Simulação de Dinâmica Molecular , Receptores de N-Metil-D-Aspartato , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/química , Conformação Proteica , Humanos , Microscopia Crioeletrônica/métodos , Modelos Moleculares
16.
Addict Biol ; 29(5): e13401, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38782631

RESUMO

Addictive properties of propofol have been demonstrated in both humans and animals. The nucleus accumbens (NAc) shell (NAsh) in the brain, along with the interactions between N-methyl-D-aspartate receptor (NMDAR) and the dopamine D1 receptor (D1R), as well as their downstream ERK/CREB signalling pathway in the NAc, are integral in regulating reward-seeking behaviour. Nevertheless, it remains unclear whether NMDARs and the NMDAR-D1R/ERK/CREB signalling pathway in the NAsh are involved in mediating propofol addiction. To investigate it, we conducted experiments with adult male Sprague-Dawley rats to establish a model of propofol self-administration behaviour. Subsequently, we microinjected D-AP5 (a competitive antagonist of NMDARs, 1.0-4.0 µg/0.3 µL/site) or vehicle into bilateral NAsh in rats that had previously self-administered propofol to examine the impact of NMDARs within the NAsh on propofol self-administration behaviour. Additionally, we examined the protein expressions of NR2A and NR2B subunits, and the D1R/ERK/CREB signalling pathways within the NAc. The results revealed that propofol administration behaviour was enhanced by D-AP5 pretreatment in NAsh, accompanied by elevated expressions of phosphorylation of NR2A (Tyr1246) and NR2B (Tyr1472) subunits. There were statistically significant increases in the expressions of D1Rs, as well as in the phosphorylated ERK1/2 (p-ERK1/2) and CREB (p-CREB). This evidence substantiates a pivotal role of NMDARs in the NAsh, with a particular emphasis on the NR2A and NR2B subunits, in mediating propofol self-administration behaviour. Furthermore, it suggests that this central reward processing mechanism may operate through the NMDAR-D1R/ERK/CREB signal transduction pathway.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Núcleo Accumbens , Propofol , Ratos Sprague-Dawley , Receptores de Dopamina D1 , Receptores de N-Metil-D-Aspartato , Autoadministração , Transdução de Sinais , Animais , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Propofol/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Masculino , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D1/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos
18.
Int J Mol Sci ; 25(9)2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38731978

RESUMO

Alzheimer's disease (AD) is the main cause of dementia worldwide. Given that learning and memory are impaired in this pathology, NMDA receptors (NMDARs) appear as key players in the onset and progression of the disease. NMDARs are glutamate receptors, mainly located at the post-synapse, which regulate voltage-dependent influx of calcium into the neurons. They are heterotetramers, and there are different subunits that can be part of the receptors, which are usually composed of two obligatory GluN1 subunits plus either two NR2A or two NR2B subunits. NR2A are mostly located at the synapse, and their activation is involved in the expression of pro-survival genes. Conversely, NR2B are mainly extrasynaptic, and their activation has been related to cell death and neurodegeneration. Thus, activation of NR2A and/or inactivation of NR2B-containing NMDARS has been proposed as a therapeutic strategy to treat AD. Here, we wanted to investigate the main differences between both subunits signalling in neuronal primary cultures of the cortex and hippocampus. It has been observed that Aß induces a significant increase in calcium release and also in MAPK phosphorylation signalling in NR2B-containing NMDAR in cortical and hippocampal neurons. However, while NR2A-containing NMDAR decreases neuronal death and favours cell viability after Aß treatment, NR2B-containing NMDAR shows higher levels of cytotoxicity and low levels of neuronal survival. Finally, it has been detected that NMDAR has no effect on pTau axonal transport. The present results demonstrate a different role between GluNA and GluNB subunits in neurodegenerative diseases such as Alzheimer's.


Assuntos
Doença de Alzheimer , Neurônios , Receptores de N-Metil-D-Aspartato , Receptores de N-Metil-D-Aspartato/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Neurônios/metabolismo , Hipocampo/metabolismo , Peptídeos beta-Amiloides/metabolismo , Cálcio/metabolismo , Humanos , Camundongos , Fosforilação , Células Cultivadas , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Ratos
19.
Exp Brain Res ; 242(6): 1507-1515, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38719948

RESUMO

Alzheimer's disease is a progressive neurodegenerative disorder characterized by impairments in synaptic plasticity and cognitive performance. Current treatments are unable to achieve satisfactory therapeutic effects or reverse the progression of the disease. Calcineurin has been implicated as part of a critical signaling pathway for learning and memory, and neuronal calcineurin may be hyperactivated in AD. To investigate the effects and underlying mechanisms of FK506, a calcineurin inhibitor, on Alzheimer-like behavior and synaptic dysfunction in the 3 × Tg-AD transgenic mouse model of Alzheimer's disease, we investigated the effect of FK506 on cognitive function and synaptic plasticity in the 3 × Tg-AD transgenic mouse model of Alzheimer's disease. The results showed that FK506 treatment ameliorated cognitive deficits, as indicated by the decreased latency in the water maze, and attenuated tau hyperphosphorylation in 3 × Tg-AD mice. Treatment with FK506 also reduced the levels of certain markers of postsynaptic deficits, including PSD-95 and NR2B, and reversed the long-term potentiation deficiency and dendritic spine impairments in 3 × Tg-AD mice. These findings suggest that treatment with calcineurin inhibitors such as FK506 can be an effective therapeutic strategy to rescue synaptic deficit and cognitive impairment in familial Alzheimer's disease and related tauopathies.


Assuntos
Doença de Alzheimer , Inibidores de Calcineurina , Modelos Animais de Doenças , Camundongos Transgênicos , Tacrolimo , Animais , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Tacrolimo/farmacologia , Inibidores de Calcineurina/farmacologia , Camundongos , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Calcineurina/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Proteínas tau/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Masculino , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Proteína 4 Homóloga a Disks-Large/metabolismo
20.
Life Sci Alliance ; 7(8)2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38749544

RESUMO

Calcium signaling is integral for neuronal activity and synaptic plasticity. We demonstrate that the calcium response generated by different sources modulates neuronal activity-mediated protein synthesis, another process essential for synaptic plasticity. Stimulation of NMDARs generates a protein synthesis response involving three phases-increased translation inhibition, followed by a decrease in translation inhibition, and increased translation activation. We show that these phases are linked to NMDAR-mediated calcium response. Calcium influx through NMDARs elicits increased translation inhibition, which is necessary for the successive phases. Calcium through L-VGCCs acts as a switch from translation inhibition to the activation phase. NMDAR-mediated translation activation requires the contribution of L-VGCCs, RyRs, and SOCE. Furthermore, we show that IP3-mediated calcium release and SOCE are essential for mGluR-mediated translation up-regulation. Finally, we signify the relevance of our findings in the context of Alzheimer's disease. Using neurons derived from human fAD iPSCs and transgenic AD mice, we demonstrate the dysregulation of NMDAR-mediated calcium and translation response. Our study highlights the complex interplay between calcium signaling and protein synthesis, and its implications in neurodegeneration.


Assuntos
Sinalização do Cálcio , Cálcio , Neurônios , Biossíntese de Proteínas , Receptores de Glutamato Metabotrópico , Receptores de N-Metil-D-Aspartato , Animais , Receptores de N-Metil-D-Aspartato/metabolismo , Camundongos , Cálcio/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Humanos , Neurônios/metabolismo , Camundongos Transgênicos , Doença de Alzheimer/metabolismo , Plasticidade Neuronal , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia
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