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1.
Gels ; 10(5)2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38786256

RESUMO

To determine the evolution of microbial community and microbial shift under anaerobic processes, this study investigates the use of denaturing gradient gel electrophoresis (DGGE). In the DGGE, short- and medium-sized DNA fragments are separated based on their melting characteristics, and this technique is used in this study to understand the dominant bacterial community in mesophilic and thermophilic anaerobic digestion processes. Dairy manure is known for emitting greenhouse gases (GHGs) such as methane, and GHG emissions from manure is a biological process that is largely dependent on the manure conditions, microbial community presence in manure, and their functions. Additional efforts are needed to understand the GHG emissions from manure and develop control strategies to minimize the biological GHG emissions from manure. To study the microbial shift during anaerobic processes responsible for GHG emission, we conducted a series of manure anaerobic digestion experiments, and these experiments were conducted in lab-scale reactors operated under various temperature conditions (28 °C, 36 °C, 44 °C, and 52 °C). We examined the third variable region (V3) of the 16S rRNA gene fingerprints of bacterial presence in anaerobic environment by PCR amplification and DGGE separation. Results showed that bacterial community was affected by the temperature conditions and anaerobic incubation time of manure. The microbial community structure of the original manure changed over time during anaerobic processes, and the community composition changed substantially with the temperature of the anaerobic process. At Day 0, the sequence similarity confirmed that most of the bacteria were similar (>95%) to Acinetobacter sp. (strain: ATCC 31012), a Gram-negative bacteria, regardless of temperature conditions. At day 7, the sequence similarity of DNA fragments of reactors (28 °C) was similar to Acinetobacter sp.; however, the DNA fragments of effluent of reactors at 44 °C and 52 °C were similar to Coprothermobacter proteolyticus (strain: DSM 5265) (similarity: 97%) and Tepidimicrobium ferriphilum (strain: DSM 16624) (similarity: 100%), respectively. At day 60, the analysis showed that DNA fragments of effluent of 28 °C reactor were similar to Galbibacter mesophilus (strain: NBRC 10162) (similarity: 87%), and DNA fragments of effluent of 36 °C reactors were similar to Syntrophomonas curvata (strain: GB8-1) (similarity: 91%). In reactors with a relatively higher temperature, the DNA fragments of effluent of 44 °C reactor were similar to Dielma fastidiosa (strain: JC13) (similarity: 86%), and the DNA fragments of effluent of 52 °C reactor were similar to Coprothermobacter proteolyticus (strain: DSM 5265) (similarity: 99%). To authors' knowledge, this is one of the few studies where DGGE-based approach is utilized to study and compare microbial shifts under mesophilic and thermophilic anaerobic digestions of manure simultaneously. While there were challenges in identifying the bands during gradient gel electrophoresis, the joint use of DGGE and sequencing tool can be potentially useful for illustrating and comparing the change in microbial community structure under complex anaerobic processes and functionality of microbes for understanding the consequential GHG emissions from manure.

2.
Proc Natl Acad Sci U S A ; 120(30): e2221413120, 2023 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-37433002

RESUMO

Effects of micronutrients on brain connectivity are incompletely understood. Analyzing human milk samples across global populations, we identified the carbocyclic sugar myo-inositol as a component that promotes brain development. We determined that it is most abundant in human milk during early lactation when neuronal connections rapidly form in the infant brain. Myo-inositol promoted synapse abundance in human excitatory neurons as well as cultured rat neurons and acted in a dose-dependent manner. Mechanistically, myo-inositol enhanced the ability of neurons to respond to transsynaptic interactions that induce synapses. Effects of myo-inositol in the developing brain were tested in mice, and its dietary supplementation enlarged excitatory postsynaptic sites in the maturing cortex. Utilizing an organotypic slice culture system, we additionally determined that myo-inositol is bioactive in mature brain tissue, and treatment of organotypic slices with this carbocyclic sugar increased the number and size of postsynaptic specializations and excitatory synapse density. This study advances our understanding of the impact of human milk on the infant brain and identifies myo-inositol as a breast milk component that promotes the formation of neuronal connections.


Assuntos
Aleitamento Materno , Leite Humano , Feminino , Lactente , Humanos , Animais , Camundongos , Ratos , Neurônios , Inositol/farmacologia , Açúcares
3.
Nat Commun ; 14(1): 459, 2023 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-36709330

RESUMO

Multiple trans-synaptic complexes organize synapse development, yet their roles in the mature brain and cooperation remain unclear. We analyzed the postsynaptic adhesion protein LRRTM1 in the prefrontal cortex (PFC), a region relevant to cognition and disorders. LRRTM1 knockout (KO) mice had fewer synapses, and we asked whether other synapse organizers counteract further loss. This determined that the immunoglobulin family member SynCAM 1 controls synapse number in PFC and was upregulated upon LRRTM1 loss. Combined LRRTM1 and SynCAM 1 deletion substantially lowered dendritic spine number in PFC, but not hippocampus, more than the sum of single KO impairments. Their cooperation extended presynaptically, and puncta of Neurexins, LRRTM1 partners, were less abundant in double KO (DKO) PFC. Electrophysiology and fMRI demonstrated aberrant neuronal activity in DKO mice. Further, DKO mice were impaired in social interactions and cognitive tasks. Our results reveal concerted roles of LRRTM1 and SynCAM 1 across synaptic, network, and behavioral domains.


Assuntos
Molécula 1 de Adesão Celular , Proteínas de Membrana , Proteínas do Tecido Nervoso , Sinapses , Animais , Camundongos , Cognição , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Córtex Pré-Frontal/metabolismo , Sinapses/metabolismo , Molécula 1 de Adesão Celular/genética , Molécula 1 de Adesão Celular/metabolismo
4.
Materials (Basel) ; 15(23)2022 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-36499941

RESUMO

The present work investigates the friction reduction capability of two types of micro-textures (grooves and dimples) created on steel surfaces using a vertical milling machine. The wear studies were conducted using a pin-on-disc tribometer, with the results indicating a better friction reduction capacity in the case of the dimple texture as compared to the grooved texture. The microscopic images of the pin surface revealed deep furrows and significant damage on the pin surfaces of the groove-textured disc. An optimization of the textured surfaces was performed using an artificial neural network (ANN) model, predicting the influence of the surface texture as a function of the load, depth of cut and distance between the micro-textures.

5.
Curr Top Dev Biol ; 142: 319-370, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33706921

RESUMO

Synaptic connectivity patterns underlie brain functions. How recognition molecules control where and when neurons form synapses with each other, therefore, is a fundamental question of cellular neuroscience. This chapter delineates adhesion and signaling complexes as well as secreted factors that contribute to synaptic partner recognition in the vertebrate brain. The sections follow a developmental perspective and discuss how recognition molecules (1) guide initial synaptic wiring, (2) provide for the rejection of incorrect partner choices, (3) contribute to synapse specification, and (4) support the removal of inappropriate synapses once formed. These processes involve a rich repertoire of molecular players and key protein families are described, notably the Cadherin and immunoglobulin superfamilies, Semaphorins/Plexins, Leucine-rich repeat containing proteins, and Neurexins and their binding partners. Molecular themes that diversify these recognition systems are defined and highlighted throughout the text, including the neuron-type specific expression and combinatorial action of recognition factors, alternative splicing, and post-translational modifications. Methodological innovations advancing the field such as proteomic approaches and single cell expression studies are additionally described. Further, the chapter highlights the importance of choosing an appropriate brain region to analyze synaptic recognition factors and the advantages offered by laminated structures like the hippocampus or retina. In a concluding section, the profound disease relevance of aberrant synaptic recognition for neurodevelopmental and psychiatric disorders is discussed. Based on the current progress, an outlook is presented on research goals that can further advance insights into how recognition molecules provide for the astounding precision and diversity of synaptic connections.


Assuntos
Encéfalo/fisiologia , Sinapses , Humanos , Proteômica
6.
Structure ; 28(3): 274-276, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32130888

RESUMO

In this issue of Structure, Cai et al. (2020) describe crystal structures of the postsynaptic protein Shank3, a homolog of cortactin binding protein 1 (CBP1), in complex with small G proteins Rap1 and H-Ras. Functional studies suggest that binding of Ras and Rap to Shank3 is modulated by synaptic plasticity.


Assuntos
Proteínas rap1 de Ligação ao GTP , Proteínas ras , Proteínas rap de Ligação ao GTP
7.
Artigo em Inglês | MEDLINE | ID: mdl-30914943

RESUMO

AMPA-type glutamate receptors (AMPARs) are clustered into functional nanodomains at postsynaptic sites through anchorage by the scaffolding protein, postsynaptic density protein-95 (PSD-95). The synaptic abundance of AMPARs is dynamically controlled in various forms of synaptic plasticity. Removal of AMPARs from the synapse in long-term depression (LTD) requires mobilization of PSD-95 away from the synapse. The molecular mechanisms underlying PSD-95 dispersal from the synapse during LTD are not completely understood. Here we show that, following Ca2+ influx, binding of Ca2+/calmodulin (CaM) to PSD-95 triggers loss of synaptic PSD-95 as well as surface AMPARs during chemically induced LTD in cultured rat neurons. Our data suggest that a reduction in PSD-95 palmitoylation mediates the effect of Ca2+/CaM on PSD-95 synaptic levels during LTD. These findings reveal a novel molecular mechanism for synaptic AMPAR regulation in LTD.

8.
Front Mol Neurosci ; 12: 8, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30766476

RESUMO

Many postsynaptic proteins undergo palmitoylation, the reversible attachment of the fatty acid palmitate to cysteine residues, which influences trafficking, localization, and protein interaction dynamics. Both palmitoylation by palmitoyl acyl transferases (PAT) and depalmitoylation by palmitoyl-protein thioesterases (PPT) is regulated in an activity-dependent, localized fashion. Recently, palmitoylation has received attention for its pivotal contribution to various forms of synaptic plasticity, the dynamic modulation of synaptic strength in response to neuronal activity. For instance, palmitoylation and depalmitoylation of the central postsynaptic scaffold protein postsynaptic density-95 (PSD-95) is important for synaptic plasticity. Here, we provide a comprehensive review of studies linking palmitoylation of postsynaptic proteins to synaptic plasticity.

9.
F1000Res ; 7: 234, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29560257

RESUMO

The ability of neurons and circuits to maintain their excitability and activity levels within the appropriate dynamic range by homeostatic mechanisms is fundamental for brain function. Neuronal hyperactivity, for instance, could cause seizures.  One such homeostatic process is synaptic scaling, also known as synaptic homeostasis. It involves a negative feedback process by which neurons adjust (scale) their postsynaptic strength over their whole synapse population to compensate for increased or decreased overall input thereby preventing neuronal hyper- or hypoactivity that could otherwise result in neuronal network dysfunction. While synaptic scaling is well-established and critical, our understanding of the underlying molecular mechanisms is still in its infancy. Homeostatic adaptation of synaptic strength is achieved through upregulation (upscaling) or downregulation (downscaling) of the functional availability of AMPA-type glutamate receptors (AMPARs) at postsynaptic sites.  Understanding how synaptic AMPARs are modulated in response to alterations in overall neuronal activity is essential to gain valuable insights into how neuronal networks adapt to changes in their environment, as well as the genesis of an array of neurological disorders. Here we discuss the key molecular mechanisms that have been implicated in tuning the synaptic abundance of postsynaptic AMPARs in order to maintain synaptic homeostasis.

10.
Nat Commun ; 9(1): 1050, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29535304

RESUMO

G protein-coupled receptors (GPCRs) transduce pleiotropic intracellular signals in a broad range of physiological responses and disease states. Activated GPCRs can undergo agonist-induced phosphorylation by G protein receptor kinases (GRKs) and second messenger-dependent protein kinases such as protein kinase A (PKA). Here, we characterize spatially segregated subpopulations of ß2-adrenergic receptor (ß2AR) undergoing selective phosphorylation by GRKs or PKA in a single cell. GRKs primarily label monomeric ß2ARs that undergo endocytosis, whereas PKA modifies dimeric ß2ARs that remain at the cell surface. In hippocampal neurons, PKA-phosphorylated ß2ARs are enriched in dendrites, whereas GRK-phosphorylated ß2ARs accumulate in soma, being excluded from dendrites in a neuron maturation-dependent manner. Moreover, we show that PKA-phosphorylated ß2ARs are necessary to augment the activity of L-type calcium channel. Collectively, these findings provide evidence that functionally distinct subpopulations of this prototypical GPCR exist in a single cell.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinases de Receptores Acoplados a Proteína G/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Animais , Canais de Cálcio Tipo L/metabolismo , Células HEK293 , Hipocampo/metabolismo , Humanos , Camundongos , Neurônios/metabolismo , Fosforilação , Imagem Individual de Molécula , Análise de Célula Única
11.
Neuron ; 97(5): 1094-1109.e9, 2018 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-29429936

RESUMO

Despite the central role PSD-95 plays in anchoring postsynaptic AMPARs, how PSD-95 itself is tethered to postsynaptic sites is not well understood. Here we show that the F-actin binding protein α-actinin binds to the very N terminus of PSD-95. Knockdown (KD) of α-actinin phenocopies KD of PSD-95. Mutating lysine at position 10 or lysine at position 11 of PSD-95 to glutamate, or glutamate at position 53 or glutamate and aspartate at positions 213 and 217 of α-actinin, respectively, to lysine impairs, in parallel, PSD-95 binding to α-actinin and postsynaptic localization of PSD-95 and AMPARs. These experiments identify α-actinin as a critical PSD-95 anchor tethering the AMPAR-PSD-95 complex to postsynaptic sites.


Assuntos
Actinina/metabolismo , Proteína 4 Homóloga a Disks-Large/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/metabolismo , Actinina/química , Actinina/genética , Sequência de Aminoácidos , Animais , Células Cultivadas , Proteína 4 Homóloga a Disks-Large/química , Proteína 4 Homóloga a Disks-Large/genética , Feminino , Células HEK293 , Humanos , Masculino , Estrutura Secundária de Proteína , Ratos
12.
EMBO J ; 37(1): 122-138, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29118000

RESUMO

Postsynaptic density protein-95 (PSD-95) localizes AMPA-type glutamate receptors (AMPARs) to postsynaptic sites of glutamatergic synapses. Its postsynaptic displacement is necessary for loss of AMPARs during homeostatic scaling down of synapses. Here, we demonstrate that upon Ca2+ influx, Ca2+/calmodulin (Ca2+/CaM) binding to the N-terminus of PSD-95 mediates postsynaptic loss of PSD-95 and AMPARs during homeostatic scaling down. Our NMR structural analysis identified E17 within the PSD-95 N-terminus as important for binding to Ca2+/CaM by interacting with R126 on CaM. Mutating E17 to R prevented homeostatic scaling down in primary hippocampal neurons, which is rescued via charge inversion by ectopic expression of CaMR126E, as determined by analysis of miniature excitatory postsynaptic currents. Accordingly, increased binding of Ca2+/CaM to PSD-95 induced by a chronic increase in Ca2+ influx is a critical molecular event in homeostatic downscaling of glutamatergic synaptic transmission.


Assuntos
Sinalização do Cálcio , Calmodulina/metabolismo , Proteína 4 Homóloga a Disks-Large/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Sinapses/fisiologia , Animais , Calmodulina/química , Calmodulina/genética , Células Cultivadas , Proteína 4 Homóloga a Disks-Large/química , Proteína 4 Homóloga a Disks-Large/genética , Ácido Glutâmico/metabolismo , Hipocampo/citologia , Lipoilação , Modelos Moleculares , Neurônios/citologia , Ligação Proteica , Conformação Proteica , Ratos , Receptores de Glutamato/metabolismo , Transmissão Sináptica , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismo
13.
Sci Signal ; 10(463)2017 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-28119465

RESUMO

The L-type Ca2+ channel Cav1.2 controls multiple functions throughout the body including heart rate and neuronal excitability. It is a key mediator of fight-or-flight stress responses triggered by a signaling pathway involving ß-adrenergic receptors (ßARs), cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA). PKA readily phosphorylates Ser1928 in Cav1.2 in vitro and in vivo, including in rodents and humans. However, S1928A knock-in (KI) mice have normal PKA-mediated L-type channel regulation in the heart, indicating that Ser1928 is not required for regulation of cardiac Cav1.2 by PKA in this tissue. We report that augmentation of L-type currents by PKA in neurons was absent in S1928A KI mice. Furthermore, S1928A KI mice failed to induce long-term potentiation in response to prolonged theta-tetanus (PTT-LTP), a form of synaptic plasticity that requires Cav1.2 and enhancement of its activity by the ß2-adrenergic receptor (ß2AR)-cAMP-PKA cascade. Thus, there is an unexpected dichotomy in the control of Cav1.2 by PKA in cardiomyocytes and hippocampal neurons.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Neurônios/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Serina/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/fisiologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Imidazóis/farmacologia , Isoproterenol/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Fosforilação/efeitos dos fármacos , Propanolaminas/farmacologia , Ratos Sprague-Dawley , Receptores Adrenérgicos beta 2/genética , Serina/genética , Transdução de Sinais/efeitos dos fármacos
14.
EMBO J ; 35(12): 1330-45, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27103070

RESUMO

Agonist-triggered downregulation of ß-adrenergic receptors (ARs) constitutes vital negative feedback to prevent cellular overexcitation. Here, we report a novel downregulation of ß2AR signaling highly specific for Cav1.2. We find that ß2-AR binding to Cav1.2 residues 1923-1942 is required for ß-adrenergic regulation of Cav1.2. Despite the prominence of PKA-mediated phosphorylation of Cav1.2 S1928 within the newly identified ß2AR binding site, its physiological function has so far escaped identification. We show that phosphorylation of S1928 displaces the ß2AR from Cav1.2 upon ß-adrenergic stimulation rendering Cav1.2 refractory for several minutes from further ß-adrenergic stimulation. This effect is lost in S1928A knock-in mice. Although AMPARs are clustered at postsynaptic sites like Cav1.2, ß2AR association with and regulation of AMPARs do not show such dissociation. Accordingly, displacement of the ß2AR from Cav1.2 is a uniquely specific desensitization mechanism of Cav1.2 regulation by highly localized ß2AR/cAMP/PKA/S1928 signaling. The physiological implications of this mechanism are underscored by our finding that LTP induced by prolonged theta tetanus (PTT-LTP) depends on Cav1.2 and its regulation by channel-associated ß2AR.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Processamento de Proteína Pós-Traducional , Receptores Adrenérgicos beta 2/metabolismo , Animais , Camundongos , Fosforilação
15.
EMBO J ; 33(12): 1341-53, 2014 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-24705785

RESUMO

Postsynaptic density protein-95 (PSD-95) is a central element of the postsynaptic architecture of glutamatergic synapses. PSD-95 mediates postsynaptic localization of AMPA receptors and NMDA receptors and plays an important role in synaptic plasticity. PSD-95 is released from postsynaptic membranes in response to Ca(2+) influx via NMDA receptors. Here, we show that Ca(2+)/calmodulin (CaM) binds at the N-terminus of PSD-95. Our NMR structure reveals that both lobes of CaM collapse onto a helical structure of PSD-95 formed at its N-terminus (residues 1-16). This N-terminal capping of PSD-95 by CaM blocks palmitoylation of C3 and C5, which is required for postsynaptic PSD-95 targeting and the binding of CDKL5, a kinase important for synapse stability. CaM forms extensive hydrophobic contacts with Y12 of PSD-95. The PSD-95 mutant Y12E strongly impairs binding to CaM and Ca(2+)-induced release of PSD-95 from the postsynaptic membrane in dendritic spines. Our data indicate that CaM binding to PSD-95 serves to block palmitoylation of PSD-95, which in turn promotes Ca(2+)-induced dissociation of PSD-95 from the postsynaptic membrane.


Assuntos
Calmodulina/metabolismo , Hipocampo/citologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Modelos Neurológicos , Neurônios/metabolismo , Densidade Pós-Sináptica/metabolismo , Animais , Células Cultivadas , Proteína 4 Homóloga a Disks-Large , Fluorescência , Técnicas Histológicas , Immunoblotting , Imunoprecipitação , Espectroscopia de Ressonância Magnética , Conformação Proteica , Ratos
16.
J Neurosci ; 33(9): 4151-64, 2013 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-23447623

RESUMO

Selective control of receptor trafficking provides a mechanism for remodeling the receptor composition of excitatory synapses, and thus supports synaptic transmission, plasticity, and development. GluN3A (formerly NR3A) is a nonconventional member of the NMDA receptor (NMDAR) subunit family, which endows NMDAR channels with low calcium permeability and reduced magnesium sensitivity compared with NMDARs comprising only GluN1 and GluN2 subunits. Because of these special properties, GluN3A subunits act as a molecular brake to limit the plasticity and maturation of excitatory synapses, pointing toward GluN3A removal as a critical step in the development of neuronal circuitry. However, the molecular signals mediating GluN3A endocytic removal remain unclear. Here we define a novel endocytic motif (YWL), which is located within the cytoplasmic C-terminal tail of GluN3A and mediates its binding to the clathrin adaptor AP2. Alanine mutations within the GluN3A endocytic motif inhibited clathrin-dependent internalization and led to accumulation of GluN3A-containing NMDARs at the cell surface, whereas mimicking phosphorylation of the tyrosine residue promoted internalization and reduced cell-surface expression as shown by immunocytochemical and electrophysiological approaches in recombinant systems and rat neurons in primary culture. We further demonstrate that the tyrosine residue is phosphorylated by Src family kinases, and that Src-activation limits surface GluN3A expression in neurons. Together, our results identify a new molecular signal for GluN3A internalization that couples the functional surface expression of GluN3A-containing receptors to the phosphorylation state of GluN3A subunits, and provides a molecular framework for the regulation of NMDAR subunit composition with implications for synaptic plasticity and neurodevelopment.


Assuntos
Endocitose/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Tirosina/metabolismo , Trifosfato de Adenosina/farmacocinética , Motivos de Aminoácidos/efeitos dos fármacos , Motivos de Aminoácidos/genética , Análise de Variância , Animais , Biofísica , Biotinilação , Células Cultivadas , Córtex Cerebral/citologia , Chlorocebus aethiops , Clatrina/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Estimulação Elétrica , Embrião de Mamíferos , Endocitose/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Ácido Glutâmico/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Humanos , Imunoprecipitação , Mutagênese/fisiologia , Mutação/fisiologia , Neurônios/efeitos dos fármacos , Neurotransmissores/farmacologia , Técnicas de Patch-Clamp , Isótopos de Fósforo/farmacocinética , Fosforilação/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Conformação Proteica , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/genética , Transfecção , Transferrina/metabolismo
17.
Neurobiol Dis ; 48(3): 290-8, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22801082

RESUMO

Excitotoxicity due to excessive activation of glutamate receptors is a primary mediator of cell death in acute and chronic neurological disorders, and NMDA-type glutamate receptors (NMDARs) are thought to be involved. NMDARs assemble from heteromeric combinations of GluN1, GluN2 and GluN3 subunits, yielding a variety of receptor subtypes that differ in biophysical properties, signaling, and synaptic targeting. Inclusion of inhibitory GluN3 subunits reduces Ca2+ influx via NMDAR channels and alters their synaptic targeting, thus modifying the two hallmarks of NMDARs that are critical for their roles on neuronal death and survival. Here we evaluated the neuroprotective potential of GluN3A subunits by analyzing the susceptibility to striatal excitotoxic damage of transgenic mice overexpressing GluN3A. We found that mild GluN3A overexpression protected susceptible striatal neurons from lesions induced by the neurotoxin 3-nitropropionic acid (3-NP), an inhibitor of mitochondrial complex II/succinate dehydrogenase. GluN3A-mediated neuroprotection was dose-dependent, and correlated with the levels of transgenic GluN3A expressed by two different mice strains. Neuroprotection was associated with a potent reduction of the activation of calpain, a Ca2+-dependent protease, which was measured as a decrease in 3-NP-induced fodrin and STEP cleavage in GluN3A transgenic mice relative to controls. We further show that transgenic GluN3A subunits incorporate into extrasynaptic compartments in mouse striatum, suggesting that reductions of toxic calpain activation might be linked to inhibition by GluN3A of pathological extrasynaptic NMDAR activity.


Assuntos
Calpaína/metabolismo , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Western Blotting , Convulsivantes/toxicidade , Corpo Estriado/patologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Camundongos Transgênicos , Nitrocompostos/toxicidade , Propionatos/toxicidade , Subunidades Proteicas/metabolismo
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