RESUMO
N-methyl-D-aspartate receptor (NMDAR) modulators have recently received increased attention as potential therapeutics for posttraumatic stress disorder (PTSD). Here, we tested a novel NMDAR-positive modulator, NYX-783, in the following two rodent models of PTSD: an auditory fear-conditioning model and a single-prolonged stress (SPS) model. We examined the ability of NYX-783 to reduce subsequent fear-based behaviors by measuring enhanced fear extinction and reduced spontaneous recovery (spontaneous return of fear) in male mice. NYX-783 administration significantly reduced spontaneous recovery in both PTSD models and enhanced fear extinction in the SPS model. Furthermore, NYX-783 increased the NMDA-induced inward currents of excitatory and inhibitory neurons in the infralimbic medial prefrontal cortex (IL mPFC) and that the GluN2B subunit of NMDARs on pyramidal neurons in the IL mPFC is required for its effect on spontaneous recovery. The downstream expression of brain-derived neurotrophic factor was required for NYX-783 to achieve its behavioral effect. These results elucidate the cellular targets of NYX-783 and the molecular mechanisms underlying the inhibition of spontaneous recovery. These preclinical findings support the hypothesis that NYX-783 may have therapeutic potential for PTSD treatment and may be particularly useful for inhibiting spontaneous recovery.
Assuntos
Medo , Receptores de N-Metil-D-Aspartato , Animais , Extinção Psicológica/fisiologia , Medo/fisiologia , Masculino , Camundongos , Córtex Pré-Frontal/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Calsyntenin-3 (Clstn3) is a postsynaptic adhesion molecule that induces presynaptic differentiation via presynaptic neurexins (Nrxns), but whether Nrxns directly bind to Clstn3 has been a matter of debate. Here, using LC-MS/MS-based protein analysis, confocal microscopy, RNAscope assays, and electrophysiological recordings, we show that ß-Nrxns directly interact via their LNS domain with Clstn3 and Clstn3 cadherin domains. Expression of splice site 4 (SS4) insert-positive ß-Nrxn variants, but not insert-negative variants, reversed the impaired Clstn3 synaptogenic activity observed in Nrxn-deficient neurons. Consistently, Clstn3 selectively formed complexes with SS4-positive Nrxns in vivo Neuron-specific Clstn3 deletion caused significant reductions in number of excitatory synaptic inputs. Moreover, expression of Clstn3 cadherin domains in CA1 neurons of Clstn3 conditional knockout mice rescued structural deficits in excitatory synapses, especially within the stratum radiatum layer. Collectively, our results suggest that Clstn3 links to SS4-positive Nrxns to induce presynaptic differentiation and orchestrate excitatory synapse development in specific hippocampal neural circuits, including Schaffer collateral afferents.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Animais , Caderinas/metabolismo , Proteínas de Ligação ao Cálcio/fisiologia , Cromatografia Líquida/métodos , Hipocampo/metabolismo , Proteínas de Membrana/fisiologia , Camundongos , Proteínas do Tecido Nervoso/fisiologia , Moléculas de Adesão de Célula Nervosa/fisiologia , Neurônios/metabolismo , Sinapses/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Gephyrin is a central scaffold protein that mediates development, function, and plasticity of mammalian inhibitory synapses by interacting with various inhibitory synaptic proteins. Here, we show that IQSEC3, a guanine nucleotide exchange factor for ARF6, directly interacts with gephyrin, an interaction that is critical for the inhibitory synapse localization of IQSEC3. Overexpression of IQSEC3 increases inhibitory, but not excitatory, synapse density in a guanine nucleotide exchange factor activity-dependent manner. Conversely, knockdown of IQSEC3 decreases size of gephyrin cluster without altering gephyrin puncta density. Collectively, these data reveal that IQSEC3 acts together with gephyrin to regulate inhibitory synapse development.
Assuntos
Proteínas de Transporte , Fatores de Troca do Nucleotídeo Guanina , Proteínas de Membrana , Sinapses , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ratos , Sinapses/genética , Sinapses/metabolismoRESUMO
Glycosylation, the addition of glycans or carbohydrates to proteins, lipids, or other glycans, is a complex post-translational modification that plays a crucial role in cellular function. It is estimated that at least half of all mammalian proteins undergo glycosylation, underscoring its importance in the functioning of cells. This is reflected in the fact that a significant portion of the human genome, around 2%, is devoted to encoding enzymes involved in glycosylation. Changes in glycosylation have been linked to various neurological disorders, including Alzheimer's disease, Parkinson's disease, autism spectrum disorder, and schizophrenia. Despite its widespread occurrence, the role of glycosylation in the central nervous system remains largely unknown, particularly with regard to its impact on behavioral abnormalities in brain diseases. This review focuses on examining the role of three types of glycosylation: N-glycosylation, O-glycosylation, and O-GlcNAcylation, in the manifestation of behavioral and neurological symptoms in neurodevelopmental, neurodegenerative, and neuropsychiatric disorders.
Assuntos
Transtorno do Espectro Autista , Doenças do Sistema Nervoso , Animais , Humanos , Glicosilação , Proteínas , Processamento de Proteína Pós-Traducional , Polissacarídeos/metabolismo , Sintomas Comportamentais , Mamíferos/metabolismoRESUMO
Animals tend to alternate between different choices, which requires the ability to remember recent choices. The Y-maze spontaneous alternation test is widely used in various animal models for assessing short-term memory, and its precise evaluation depends upon the accurate determination of the arm visit sequence. However, an objective method for defining arm visits is lacking owing to uncertainty regarding the extent to which an animal must go into the arm to be considered visited. Here, we conducted quantitative analyses on mice behavior in the Y-maze while systematically varying the arm visit threshold and assessed the effect of acute social isolation on spatial working memory. Our results revealed that 24-h social isolation significantly reduced spontaneous alternation rate when the arm threshold was set at the distal part of the arm. Furthermore, the memory of the recently visited arms faded away faster in the socially isolated mice. However, other behavioral factors were comparable to those of the group-housed mice, indicating a specific impairment of short-term memory. Our findings suggest that the location of arm visit threshold is critical for the precise evaluation of short-term memory, and our study provides a method for comprehensively and systematically assessing spontaneous alternation behavior in the Y-maze.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Memória de Curto Prazo , Animais , Camundongos , Isolamento Social , Aprendizagem em Labirinto , Rememoração MentalRESUMO
Gangliosides abundant in the nervous system have been implicated in a broad range of biological functions, including the regulation of cell proliferation and death. Glutamate-induced cell death, which is accompanied by an accumulation of reactive oxygen species (ROS), is a major contributor to pathological cell death within the nervous system. However, the mechanism underlying this neuronal cell death has not been fully elucidated. In this study, we report that ganglioside GM3 is involved in neuronal cell death. GM3 was up-regulated in the mouse hippocampal cell line HT22 death caused by glutamate. Increment in GM3 levels by both the exogenous addition of GM3 and the overexpression of the GM3 synthase gene induced neuronal cell death. Overexpression of GM3 synthase by microinjecting mRNA into zebrafish embryos resulted in neuronal cell death in the central nervous system (CNS). Conversely, RNA interference-mediated silencing of GM3 synthase rescued glutamate-induced neuronal death, as evidenced by the inhibition of massive ROS production and intracellular calcium ion influx. 12-lipoxygenase (12-lipoxygenase) (12-LOX) was recruited to glycosphingolipid-enriched microdomains (GEM) in a GM3-dependent manner during oxidative glutamate toxicity. Our findings suggest that GM3 acts as not only a mediator of oxidative HT22 death by glutamate but also a modulator of in vivo neuronal cell death.
Assuntos
Apoptose , Gangliosídeo G(M3)/fisiologia , Neurônios/metabolismo , Sequência de Aminoácidos , Animais , Araquidonato 12-Lipoxigenase/metabolismo , Cálcio/metabolismo , Linhagem Celular , Gangliosídeo G(M3)/antagonistas & inibidores , Gangliosídeo G(M3)/toxicidade , Ácido Glutâmico/toxicidade , Hipocampo/citologia , Microdomínios da Membrana/enzimologia , Camundongos , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/efeitos dos fármacos , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Alinhamento de Sequência , Sialiltransferases/antagonistas & inibidores , Sialiltransferases/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Slit- and Trk-like (Slitrks) are a six-member family of synapse organizers that control excitatory and inhibitory synapse formation by forming trans-synaptic adhesions with LAR receptor protein tyrosine phosphatases (PTPs). Intriguingly, genetic mutations of Slitrks have been associated with a multitude of neuropsychiatric disorders. However, nothing is known about the neuronal and synaptic consequences of these mutations. Here, we report the structural and functional effects on synapses of various rare de novo mutations identified in patients with schizophrenia or Tourette syndrome. A number of single amino acid substitutions in Slitrk1 (N400I or T418S) or Slitrk4 (V206I or I578V) reduced their surface expression levels. These substitutions impaired glycosylation of Slitrks expressed in HEK293T cells, caused retention of Slitrks in the endoplasmic reticulum and cis-Golgi compartment in COS-7 cells and neurons, and abolished Slitrk binding to PTPδ. Furthermore, these substitutions eliminated the synapse-inducing activity of Slitrks, abolishing their functional effects on synapse density in cultured neurons. Strikingly, a valine-to-methionine mutation in Slitrk2 (V89M) compromised synapse formation activity in cultured neuron, without affecting surface transport, expression, or synapse-inducing activity in coculture assays. Similar deleterious effects were observed upon introduction of the corresponding valine-to-methionine mutation into Slitrk1 (V85M), suggesting that this conserved valine residue plays a key role in maintaining the synaptic functions of Slitrks. Collectively, these data indicate that inactivation of distinct cellular mechanisms caused by specific Slitrk dysfunctions may underlie Slitrk-associated neuropsychiatric disorders in humans, and provide a robust cellular readout for the development of knowledge-based therapies.
RESUMO
The four members of the LRRTM family (LRRTM1-4) are postsynaptic adhesion molecules essential for excitatory synapse development. They have also been implicated in neuropsychiatric diseases. Here, we focus on LRRTM3, showing that two distinct LRRTM3 variants generated by alternative splicing regulate LRRTM3 interaction with PSD-95, but not its excitatory synapse-promoting activity. Overexpression of either LRRTM3 variant increased excitatory synapse density in dentate gyrus (DG) granule neurons, whereas LRRTM3 knockdown decreased it. LRRTM3 also controlled activity-regulated AMPA receptor surface expression in an alternative splicing-dependent manner. Furthermore, Lrrtm3-knockout mice displayed specific alterations in excitatory synapse density, excitatory synaptic transmission and excitability in DG granule neurons but not in CA1 pyramidal neurons. Lastly, LRRTM3 required only specific splice variants of presynaptic neurexins for their synaptogenic activity. Collectively, our data highlight alternative splicing and differential presynaptic ligand utilization in the regulation of LRRTMs, revealing key regulatory mechanisms for excitatory synapse development.
Assuntos
Processamento Alternativo , Moléculas de Adesão Celular Neuronais/metabolismo , Potenciais Pós-Sinápticos Excitadores , Animais , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/crescimento & desenvolvimento , Região CA1 Hipocampal/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Células Cultivadas , Giro Denteado/citologia , Giro Denteado/crescimento & desenvolvimento , Giro Denteado/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurogênese , Transporte Proteico , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Ratos , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Sinapses/fisiologiaRESUMO
Synaptic adhesion molecules regulate various aspects of synapse development, function and plasticity. These functions mainly involve trans-synaptic interactions and positive regulations, whereas cis-interactions and negative regulation are less understood. Here we report that SALM4, a member of the SALM/Lrfn family of synaptic adhesion molecules, suppresses excitatory synapse development through cis inhibition of SALM3, another SALM family protein with synaptogenic activity. Salm4-mutant (Salm4(-/-)) mice show increased excitatory synapse numbers in the hippocampus. SALM4 cis-interacts with SALM3, inhibits trans-synaptic SALM3 interaction with presynaptic LAR family receptor tyrosine phosphatases and suppresses SALM3-dependent presynaptic differentiation. Importantly, deletion of Salm3 in Salm4(-/-) mice (Salm3(-/-); Salm4(-/-)) normalizes the increased excitatory synapse number. These results suggest that SALM4 negatively regulates excitatory synapses via cis inhibition of the trans-synaptic SALM3-LAR adhesion.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Diferenciação Celular/fisiologia , Moléculas de Adesão de Célula Nervosa/metabolismo , Terminações Pré-Sinápticas/fisiologia , Células Piramidais/fisiologia , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Animais , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/fisiologia , Adesão Celular/fisiologia , Moléculas de Adesão Celular Neuronais/genética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Masculino , Glicoproteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Proteínas do Tecido Nervoso , Moléculas de Adesão de Célula Nervosa/genética , Transmissão Sináptica/fisiologiaRESUMO
Although it is known that evaporated metals can penetrate into films of various organic molecules that are a few nanometers thick, there has been little work aimed at exploring the interaction of the common electrode metals used in devices with fullerene derivatives, such as organic photovoltaics (OPVs) or perovskite solar cells that use fullerenes as electron transport layers. In this paper, we show that when commonly used electrode metals (e.g., Au, Ag, Al, Ca, etc.) are evaporated onto films of fullerene derivatives (such as [6,6]-phenyl-C61-butyric acid methyl ester (PCBM)), the metal penetrates many tens of nanometers into the fullerene layer. This penetration decreases the effective electrical thickness of fullerene-based sandwich structure devices, as measured by the device's geometric capacitance, and thus significantly alters the device physics. For the case of Au/PCBM, the metal penetrates a remarkable 70 nm into the fullerene, and we see penetration of similar magnitude in a wide variety of fullerene derivative/evaporated metal combinations. Moreover, using transmission electron microscopy to observed cross-sections of the films, we show that when gold is evaporated onto poly(3-hexylthiophene) (P3HT)/PCBM sequentially processed OPV quasi-bilayers, Au nanoparticles with diameters of â¼3-20 nm are formed and are dispersed entirely throughout the fullerene-rich overlayer. The plasmonic absorption and scattering from these nanoparticles are readily evident in the optical transmission spectrum, demonstrating that the interpenetrated metal significantly alters the optical properties of fullerene-rich active layers. This opens a number of possibilities in terms of contact engineering and light management so that metal penetration in devices that use fullerene derivatives could be used to advantage, making it critical that researchers are aware of the electronic and optical consequences of exposing fullerene-derivative films to evaporated electrode metals.
Assuntos
Eletroquímica/instrumentação , Fulerenos/química , Metais/química , Nanoestruturas/química , Eletricidade , Eletrodos , Ésteres/química , Nanoestruturas/ultraestrutura , Semicondutores , VolatilizaçãoRESUMO
Ginseng, the root of Panax ginseng Meyer, has been used frequently in traditional oriental medicine and is popular globally. Ginsenosides, which are the saponins in ginseng, are the major components having pharmacological and biological activities, including anti-diabetic and anti-tumor activities. In this study, we investigated the effects of total saponin from Korean red ginseng (TSKRG) on thrombin-produced thromboxane A2 (TXA2), an aggregating thrombogenic molecule, and its associated microsomal enzymes cyclooxygenase (COX)-1 and TXA2 synthase (TXAS). Thrombin (0.5 U/mL) increased TXA2 production up to 169 ng/10(8) platelets as compared with control (0.2 ng/10(8) platelets). However, TSKRG inhibited potently TXA2 production to the control level in a dose-dependent manner, which was associated with the strong inhibition of COX-1 and TXAS activities in platelet microsomes having cytochrome c reductase activity. The results demonstrate TSKRG is a beneficial traditional oriental medicine in platelet-mediated thrombotic diseases via suppression of COX-1 and TXAS to inhibit production of TXA2.
RESUMO
AIM: In this study, we investigated the effect of (-)-epigallocatechin-3-gallate (EGCG) on cyclic nucleotide production and vasodilator-stimulated phosphoprotein (VASP) phosphorylation in collagen (10 µg/mL)-stimulated platelet aggregation. METHODS: Washed platelets (10(8)/mL) from Sprague-Dawley rats (6-7 weeks old, male) were preincubated for 3 min at 37°C in the presence of 2 mM exogenous CaCl(2) with or without EGCG or other materials, stimulated with collagen (10 µg/mL) for 5 min, and then used for the determination of intracellular cytosolic Ca(2+) ([Ca(2+)](i)), thromboxane A(2) (TXA(2)), adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP), and VASP phosphorylation. RESULTS: EGCG dose-dependently inhibited collagen-induced platelet aggregation by inhibiting both [Ca(2+)](i) mobilization and TXA(2) production. Of two aggregation-inhibiting molecules, cAMP and cGMP, EGCG significantly increased intracellular levels of cAMP, but not cGMP. EGCG-elevated cAMP level was decreased by SQ22536, an adenylate cyclase inhibitor, but not by etazolate, a cAMPspecific phosphodiesterase inhibitor. In addition, EGCG elevated the phosphorylation of VASP-Ser(157), a cAMP-dependent protein kinase (A-kinase) substrate, but not the phosphorylation of VASP-Ser(239), a cGMP-dependent protein kinase substrate, in intact platelets and collagen-induced platelets, and VASP-Ser(157) phosphorylation by EGCG was inhibited by both an adenylate cyclase inhibitor SQ22536 and an A-kinase inhibitor Rp-8-Br-cAMPS. We have demonstrated that EGCG increases cAMP via adenylate cyclase activation and subsequently phosphorylates VASP-Ser(157) through A-kinase activation to inhibit [Ca(2+)](i) mobilization and TXA(2) production on collagen-induced platelet aggregation. CONCLUSIONS: These results strongly indicate that EGCG is a beneficial compound elevating cAMP level in collagen-platelet interaction, which may result in the prevention of platelet aggregation-mediated thrombotic diseases.
Assuntos
Catequina/análogos & derivados , AMP Cíclico/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Inibidores de Adenilil Ciclases , Animais , Catequina/farmacologia , Colágeno/farmacologia , AMP Cíclico/biossíntese , GMP Cíclico/farmacologia , Masculino , Fosforilação , Ratos , Ratos Sprague-DawleyRESUMO
N-Acetylglucosaminyltransferase-V (GnT-V) has been reported to be up-regulated in invasive/metastatic cancer cells, but a comprehensive understanding of how the transferase correlates with the invasive/metastatic potential is not currently available. Through a glycomics approach, we identified 30 proteins, including tissue inhibitor of metalloproteinase-1 (TIMP-1), as a target protein for GnT-V in human colon cancer cell WiDr. TIMP-1 was aberrantly glycosylated as characterized by the addition of beta1,6-N-acetylglucosamine, polylactosaminylation, and sialylation in GnT-V-overexpressing WiDr cells. Compared with normal TIMP-1, the aberrantly glycosylated TIMP-1 showed the weaker inhibition on both matrix metalloproteinase (MMP)-2 and MMP-9, and this aberrancy was closely associated with cancer cell invasion and metastasis in vivo as well as in vitro. Integrated data, both of TIMP-1 expression level and aberrant glycosylation, could provide important information to aid to improve the clinical outcome of colon cancer patients.
Assuntos
Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , N-Acetilglucosaminiltransferases/metabolismo , Proteômica/métodos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Movimento Celular/efeitos dos fármacos , Progressão da Doença , Eletroforese em Gel Bidimensional , Inibidores Enzimáticos/farmacologia , Gelatinases/antagonistas & inibidores , Glicosilação/efeitos dos fármacos , Células HT29 , Humanos , Cinética , Espectrometria de Massas , Proteínas Mutantes/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Ligação Proteica/efeitos dos fármacos , TransfecçãoRESUMO
To gain a better understanding of the mechanism underlying colon cancer and to search for potential markers of colon cancer prognosis, a comparative proteomic analysis of colon cancer WiDr cells was conducted using 2-DE and lectin blot, followed by identification based on ESI-MS. Through these approaches 14 proteins were identified as candidate target proteins for N-acetylglucosaminyl transferase V (GnT-V) that would be expected to be implicated in the progression of colon cancer. We selected protein tyrosine phosphatase kappa (PTPkappa) as a model protein to validate this approach to the discovery of novel biomarkers in colon cancer. PTPkappa underwent an aberrant glycosylation in GnT-V-overexpressing WiDr cells, and the aberrantly glycosylated PTPkappa was vulnerable to proteolytic cleavage. The enhanced cleavage of PTPkappa in GnT-V-overexpressing cells was responsible for the mitigation of the homophilic binding capacity, resulting in an increase in cancer cell migration.
Assuntos
Movimento Celular , Neoplasias do Colo/enzimologia , N-Acetilglucosaminiltransferases/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Eletroforese em Gel Bidimensional , Glicosilação , Humanos , Lectinas/metabolismo , Fragmentos de Peptídeos/imunologia , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores , Espectrometria de Massas por Ionização por Electrospray , Células Tumorais CultivadasRESUMO
To understand better the mechanism underlying gastric cancer and search for potential markers for gastric cancer prognosis, the proteomic analysis of gastric cancer tissues was conducted using two-dimensional gel electrophoresis and lectin blot, followed by electrospray ionization-mass spectrometry. These approaches permitted identification of glyco- or putative glycosylated proteins which may be associated with tumorigenesis. The proteins identified include molecules involved in sugar metabolism, signal transduction, proteolysis, and stress, as well as several unknown proteins, which were aberrantly glycosylated as evidenced by the L-phytohemagglutinin blot.