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1.
Sheng Wu Gong Cheng Xue Bao ; 36(10): 2113-2125, 2020 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-33169576

RESUMO

Glutamic acid is an important amino acid with wide range of applications and huge market demand. Therefore, by performing transcriptome sequencing and re-sequencing analysis on Corynebacterium glutamicum E01 and high glutamate-producing strain C. glutamicum G01, we identified and selected genes with significant differences in transcription and gene levels in the central metabolic pathway that may have greatly influenced glutamate synthesis and further increased glutamic acid yield. The oxaloacetate node and α-ketoglutarate node play an important role in glutamate synthesis. The oxaloacetate node and α-ketoglutarate node were studied to explore effect on glutamate production. Based on the integrated strain constructed from the above experimental results, the growth rate in a 5-L fermenter was slightly lower than that of the original strain, but the glutamic acid yield after 48 h reached (136.1±5.53) g/L, higher than the original strain (93.53±4.52) g/L, an increase by 45.5%; sugar-acid conversion rate reached 58.9%, an increase of 13.7% compared to 45.2% of the original strain. The application of the above experimental strategy improved the glutamic acid yield and the sugar-acid conversion rate, and provided a theoretical basis for the metabolic engineering of Corynebacterium glutamicum.


Assuntos
Ciclo do Ácido Cítrico , Ácido Glutâmico , Redes e Vias Metabólicas , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Ácido Glutâmico/metabolismo , Engenharia Metabólica , Redes e Vias Metabólicas/genética
2.
Nat Commun ; 11(1): 5038, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33028830

RESUMO

Epilepsy is one of the most common neurological disorders, yet its pathophysiology is poorly understood due to the high complexity of affected neuronal circuits. To identify dysfunctional neuronal subtypes underlying seizure activity in the human brain, we have performed single-nucleus transcriptomics analysis of >110,000 neuronal transcriptomes derived from temporal cortex samples of multiple temporal lobe epilepsy and non-epileptic subjects. We found that the largest transcriptomic changes occur in distinct neuronal subtypes from several families of principal neurons (L5-6_Fezf2 and L2-3_Cux2) and GABAergic interneurons (Sst and Pvalb), whereas other subtypes in the same families were less affected. Furthermore, the subtypes with the largest epilepsy-related transcriptomic changes may belong to the same circuit, since we observed coordinated transcriptomic shifts across these subtypes. Glutamate signaling exhibited one of the strongest dysregulations in epilepsy, highlighted by layer-wise transcriptional changes in multiple glutamate receptor genes and strong upregulation of genes coding for AMPA receptor auxiliary subunits. Overall, our data reveal a neuronal subtype-specific molecular phenotype of epilepsy.


Assuntos
Epilepsia Resistente a Medicamentos/genética , Epilepsia do Lobo Temporal/genética , Neurônios/patologia , Lobo Temporal/patologia , Transcriptoma/genética , Adolescente , Adulto , Biópsia , Estudos de Casos e Controles , Núcleo Celular/genética , Núcleo Celular/metabolismo , Conjuntos de Dados como Assunto , Epilepsia Resistente a Medicamentos/diagnóstico , Epilepsia Resistente a Medicamentos/patologia , Epilepsia Resistente a Medicamentos/cirurgia , Epilepsia do Lobo Temporal/diagnóstico , Epilepsia do Lobo Temporal/patologia , Epilepsia do Lobo Temporal/cirurgia , Feminino , Ácido Glutâmico/metabolismo , Humanos , Imagem por Ressonância Magnética , Masculino , Microdissecção , Pessoa de Meia-Idade , Modelos Genéticos , Rede Nervosa/metabolismo , Rede Nervosa/patologia , Neurônios/citologia , Neurônios/metabolismo , RNA-Seq , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Transdução de Sinais/genética , Análise de Célula Única , Lobo Temporal/citologia , Lobo Temporal/diagnóstico por imagem , Lobo Temporal/cirurgia , Transcrição Genética , Regulação para Cima , Adulto Jovem
3.
Science ; 370(6514): 364-368, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33060364

RESUMO

The heart consumes circulating nutrients to fuel lifelong contraction, but a comprehensive mapping of human cardiac fuel use is lacking. We used metabolomics on blood from artery, coronary sinus, and femoral vein in 110 patients with or without heart failure to quantify the uptake and release of 277 metabolites, including all major nutrients, by the human heart and leg. The heart primarily consumed fatty acids and, unexpectedly, little glucose; secreted glutamine and other nitrogen-rich amino acids, indicating active protein breakdown, at a rate ~10 times that of the leg; and released intermediates of the tricarboxylic acid cycle, balancing anaplerosis from amino acid breakdown. Both heart and leg consumed ketones, glutamate, and acetate in direct proportionality to circulating levels, indicating that availability is a key driver for consumption of these substrates. The failing heart consumed more ketones and lactate and had higher rates of proteolysis. These data provide a comprehensive and quantitative picture of human cardiac fuel use.


Assuntos
Ácidos Graxos/metabolismo , Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Acetatos/metabolismo , Idoso , Glicemia/metabolismo , Ciclo do Ácido Cítrico , Feminino , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Humanos , Cetonas/metabolismo , Perna (Membro)/irrigação sanguínea , Masculino , Metabolômica , Pessoa de Meia-Idade , Contração Miocárdica , Proteólise
4.
Yakugaku Zasshi ; 140(10): 1235-1242, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32999202

RESUMO

The central nervous system (CNS) is segregated from the circulating blood and peripheral tissues by endothelial and epithelial barriers. To overcome refractory CNS diseases, it is important to understand the membrane transport systems of drugs and the endogenous compounds that relate to the pathogenesis of CNS diseases at these barriers. The endothelial barrier in the brain is the blood-brain barrier (BBB). Our studies clarified the efflux transport of prostaglandin E2 (PGE2), a modulator of neural excitation and inflammatory responses, across the BBB via plasma membrane transporters such as organic anion transporter 3 (Oat3) and multidrug resistance-associated protein 4 (Mrp4). This efflux transport was attenuated by peripheral inflammation or cerebral treatment with neuroexcitatory l-glutamate, suggesting that BBB-mediated PGE2 elimination was altered under several pathological conditions. We also examined excitatory amino acid transporter (EAAT) 1 and 3 as l-glutamate efflux transporters of the inner blood-retinal barrier (BRB) and blood-cerebrospinal barrier. It was considered that these efflux membrane transporters participated in the homeostasis of neuroexcitatory and neuroinflammatory responses in the brain and retina. Moreover, we identified connexin 43 (Cx43) hemichannels as a new membrane transport system that is activated under pathological conditions and recognizes several monocarboxylate drugs, such as valproate. As it is expected that the action of these membrane transporters across the CNS barriers is of great importance in understanding the pathology of various neuroexcitatory diseases, our studies should contribute to the establishment of therapeutic strategies for refractory CNS diseases.


Assuntos
Transporte Biológico , Barreira Hematoencefálica/metabolismo , Barreira Hematorretiniana/metabolismo , Encéfalo/metabolismo , Doenças do Sistema Nervoso Central/etiologia , Doenças do Sistema Nervoso Central/metabolismo , Desenvolvimento de Medicamentos , Proteínas de Membrana Transportadoras/metabolismo , Retina/metabolismo , Animais , Doenças do Sistema Nervoso Central/tratamento farmacológico , Conexina 43/metabolismo , Dinoprostona/metabolismo , Transportador 1 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Camundongos , Terapia de Alvo Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo
5.
J Vis Exp ; (163)2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32986031

RESUMO

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.


Assuntos
Ácido Glutâmico/metabolismo , Hipocampo/fisiologia , Terminações Pré-Sinápticas/fisiologia , Área Tegmentar Ventral/anatomia & histologia , Área Tegmentar Ventral/fisiologia , Potenciais de Ação , Amplificadores Eletrônicos , Animais , Dependovirus/metabolismo , Fluorescência , Imageamento Tridimensional , Masculino , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Fibras Ópticas , Optogenética
6.
PLoS Biol ; 18(8): e3000820, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866173

RESUMO

Mutations in the gene encoding the microtubule-severing protein spastin (spastic paraplegia 4 [SPG4]) cause hereditary spastic paraplegia (HSP), associated with neurodegeneration, spasticity, and motor impairment. Complicated forms (complicated HSP [cHSP]) further include cognitive deficits and dementia; however, the etiology and dysfunctional mechanisms of cHSP have remained unknown. Here, we report specific working and associative memory deficits upon spastin depletion in mice. Loss of spastin-mediated severing leads to reduced synapse numbers, accompanied by lower miniature excitatory postsynaptic current (mEPSC) frequencies. At the subcellular level, mutant neurons are characterized by longer microtubules with increased tubulin polyglutamylation levels. Notably, these conditions reduce kinesin-microtubule binding, impair the processivity of kinesin family protein (KIF) 5, and reduce the delivery of presynaptic vesicles and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Rescue experiments confirm the specificity of these results by showing that wild-type spastin, but not the severing-deficient and disease-associated K388R mutant, normalizes the effects at the synaptic, microtubule, and transport levels. In addition, short hairpin RNA (shRNA)-mediated reduction of tubulin polyglutamylation on spastin knockout background normalizes KIF5 transport deficits and attenuates the loss of excitatory synapses. Our data provide a mechanism that connects spastin dysfunction with the regulation of kinesin-mediated cargo transport, synapse integrity, and cognition.


Assuntos
Ácido Glutâmico/metabolismo , Cinesina/metabolismo , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Memória de Curto Prazo , Neurônios/metabolismo , Espastina/deficiência , Tubulina (Proteína)/metabolismo , Potenciais de Ação , Animais , Membrana Celular/metabolismo , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Potenciais Pós-Sinápticos Excitadores , Hipocampo/patologia , Hipocampo/fisiopatologia , Camundongos Knockout , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Atividade Motora , Neurônios/patologia , Neurônios/ultraestrutura , Transporte Proteico , Espastina/metabolismo , Sinapses/metabolismo , Sinapses/ultraestrutura , Vesículas Sinápticas/metabolismo
7.
Nat Commun ; 11(1): 4388, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32873805

RESUMO

Presynaptic spike timing-dependent long-term depression (t-LTD) at hippocampal CA3-CA1 synapses is evident until the 3rd postnatal week in mice, disappearing during the 4th week. At more mature stages, we found that the protocol that induced t-LTD induced t-LTP. We characterized this form of t-LTP and the mechanisms involved in its induction, as well as that driving this switch from t-LTD to t-LTP. We found that this t-LTP is expressed presynaptically at CA3-CA1 synapses, as witnessed by coefficient of variation, number of failures, paired-pulse ratio and miniature responses analysis. Additionally, this form of presynaptic t-LTP does not require NMDARs but the activation of mGluRs and the entry of Ca2+ into the postsynaptic neuron through L-type voltage-dependent Ca2+ channels and the release of Ca2+ from intracellular stores. Nitric oxide is also required as a messenger from the postsynaptic neuron. Crucially, the release of adenosine and glutamate by astrocytes is required for t-LTP induction and for the switch from t-LTD to t-LTP. Thus, we have discovered a developmental switch of synaptic transmission from t-LTD to t-LTP at hippocampal CA3-CA1 synapses in which astrocytes play a central role and revealed a form of presynaptic LTP and the rules for its induction.


Assuntos
Astrócitos/metabolismo , Hipocampo/crescimento & desenvolvimento , Potenciação de Longa Duração/fisiologia , Transmissão Sináptica/fisiologia , Adenosina/metabolismo , Animais , Feminino , Ácido Glutâmico/metabolismo , Hipocampo/citologia , Masculino , Camundongos , Técnicas de Patch-Clamp , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo
8.
Nat Commun ; 11(1): 4614, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929069

RESUMO

The suprachiasmatic nucleus (SCN) is a complex structure dependent upon multiple mechanisms to ensure rhythmic electrical activity that varies between day and night, to determine circadian adaptation and behaviours. SCN neurons are exposed to glutamate from multiple sources including from the retino-hypothalamic tract and from astrocytes. However, the mechanism preventing inappropriate post-synaptic glutamatergic effects is unexplored and unknown. Unexpectedly we discovered that TRESK, a calcium regulated two-pore potassium channel, plays a crucial role in this system. We propose that glutamate activates TRESK through NMDA and AMPA mediated calcium influx and calcineurin activation to then oppose further membrane depolarisation and rising intracellular calcium. Hence, in the absence of TRESK, glutamatergic activity is unregulated leading to membrane depolarisation, increased nocturnal SCN firing, inverted basal calcium levels and impaired sensitivity in light induced phase delays. Our data reveals TRESK plays an essential part in SCN regulatory mechanisms and light induced adaptive behaviours.


Assuntos
Adaptação Ocular , Escuridão , Canais de Potássio/metabolismo , Núcleo Supraquiasmático/fisiologia , Animais , Comportamento Animal , Cálcio/metabolismo , Ácido Glutâmico/metabolismo , Luz , Potenciais da Membrana/efeitos da radiação , Camundongos Endogâmicos C57BL , Canais de Potássio/deficiência , Transdução de Sinais/efeitos da radiação , Núcleo Supraquiasmático/efeitos da radiação
9.
J Headache Pain ; 21(1): 105, 2020 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-32842964

RESUMO

BACKGROUND: Different pain syndromes may be characterized by different profiles of mediators reflecting pathophysiological differences, and these alterations may be measured in a simple saliva sample. The aims of the current study were to compare concentration of glutamate, serotonin (5-HT), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and substance P (SP) in saliva and plasma from a well-defined group of patients with chronic temporomandibular disorders myalgia (TMD-myalgia) with a group of pain-free controls, and further investigate the relationship between these markers and clinical characteristics. METHODS: Patients diagnosed according to the diagnostic criteria for TMD (n = 39), and matched healthy pain-free controls (n = 39) were included. Stimulated whole saliva and plasma samples were collected in the morning. Glutamate was analysed using a colorimetric assay, and 5-HT and SP were analysed by commercially available ELISA. Levels of NGF and BDNF were determined using multiplex electrochemiluminescence assay panel. RESULTS: Patients expressed higher salivary and plasma levels of glutamate (saliva: 40.22 ± 13.23 µmol/L; plasma: 30.31 ± 18.73 µmol/L) than controls (saliva: 33.24 ± 11.27 µmol/L; plasma: 20.41 ± 15.96 µmol/L) (p < 0.05). Salivary NGF (0.319 ± 0.261 pg/ml) and BDNF (3.57 ± 1.47 pg/ml) were lower in patients compared to controls (NGF: 0.528 ± 0.477 pg/ml; BDNF 4.62 ± 2.51 pg/ml)(p's < 0.05). Contrary, plasma BDNF, was higher in patients (263.33 ± 245.13 pg/ml) than controls (151.81 ± 125.90 pg/ml) (p < 0.05). 5-HT was undetectable in saliva. Neither plasma 5-HT, nor SP levels differed between groups. BDNF and NGF concentrations correlated to levels of psychological distress (p < 0.0005). CONCLUSION: The higher levels of salivary and plasma glutamate in patients with TMD-myalgia compared to controls strengthens its importance in the pathophysiology of TMD-myalgia. However, the lack of correlation to pain levels question its role as a putative biomarker. Patients with TMD-myalgia further had lower levels of salivary NGF and BDNF, but higher plasma BDNF. These results and their correlations to psychological distress warrant further investigations.


Assuntos
Saliva/metabolismo , Transtornos da Articulação Temporomandibular/sangue , Transtornos da Articulação Temporomandibular/metabolismo , Adulto , Biomarcadores/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Feminino , Ácido Glutâmico/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Mialgia/metabolismo , Fator de Crescimento Neural/metabolismo , Substância P/metabolismo , Adulto Jovem
10.
Biol Res ; 53(1): 36, 2020 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-32843088

RESUMO

BACKGROUND: To investigate the thalamic neurotransmitters and functional connections in the development of chronic constriction injury (CCI)-induced neuropathic pain. METHODS: The paw withdrawal threshold was measured by mechanical stimulation the right hind paw with the von frey hair in the rats of CCI-induced neuropathic pain. The N-acetylaspartate (NAA) and Glutamate (Glu) in thalamus were detected by magnetic resonance spectrum (MRS) process. The thalamic functional connectivity with other brain regions was scanned by functional magnetic resonance image (fMRI). RESULTS: The paw withdrawal threshold of the ipsilateral side showed a noticeable decline during the pathological process. Increased concentrations of Glu and decreased levels of NAA in the thalamus were significantly correlated with mechanical allodynia in the neuropathic pain states. The thalamic regional homogeneity (ReHo) decreased during the process of neuropathic pain. The functional connectivity among the thalamus with the insula and somatosensory cortex were significantly increased at different time points (7, 14, 21 days) after CCI surgery. CONCLUSION: Our study suggests that dynamic changes in thalamic NAA and Glu levels contribute to the thalamic functional connection hyper-excitation during CCI-induced neuropathic pain. Enhanced thalamus-insula functional connection might have a significant effect on the occurrence of neuropathic pain.


Assuntos
Neuralgia , Neurotransmissores/metabolismo , Tálamo/metabolismo , Ferimentos e Lesões/fisiopatologia , Animais , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Constrição , Ácido Glutâmico/metabolismo , Hiperalgesia , Ratos , Tálamo/fisiopatologia
11.
J Pharmacol Sci ; 144(3): 151-164, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32807662

RESUMO

Glutamate is the major excitatory neurotransmitter in the central nervous system. Glutamate transmission efficiency depends on the correct functionality and expression of a plethora of receptors and transporters, located both on neurons and glial cells. Of note, glutamate reuptake by dedicated transporters prevents its accumulation at the synapse as well as non-physiological spillover. Indeed, extracellular glutamate increase causes aberrant synaptic signaling leading to neuronal excitotoxicity and death. Moreover, extrasynaptic glutamate diffusion is strongly associated with glia reaction and neuroinflammation. Glutamate-induced excitotoxicity is mainly linked to an impaired ability of glial cells to reuptake and respond to glutamate, then this is considered a common hallmark in many neurodegenerative diseases, including Parkinson's disease (PD). In this review, we discuss the function of astrocytes and microglia in glutamate homeostasis, focusing on how glial dysfunction causes glutamate-induced excitotoxicity leading to neurodegeneration in PD.


Assuntos
Ácido Glutâmico/metabolismo , Ácido Glutâmico/toxicidade , Neuroglia/metabolismo , Neurotransmissores/metabolismo , Neurotransmissores/toxicidade , Doença de Parkinson/etiologia , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Homeostase , Humanos , Inflamação , Doença de Parkinson/metabolismo , Receptores de Glutamato/metabolismo
12.
Am J Physiol Cell Physiol ; 319(3): C605-C610, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32783655

RESUMO

Epileptic seizures are the manifestation of hypersynchronous and excessive neuronal excitation. While the glutamatergic and GABAergic neurons play major roles in shaping fast neuronal excitation/inhibition homeostasis, it is well illustrated that astrocytes profoundly regulate neuronal excitation by controlling glutamate, GABA, cannabinoids, adenosine, and concentration of K+ around neurons. However, little is known about whether microglia take part in the regulation of acute neuronal excitation and ongoing epileptic behaviors. We proposed that if microglia are innately ready to respond to epileptic overexcitation, depletion of microglia might alter neuronal excitability and severity of acute epileptic seizures. We found that microglia depletion by plx3397, an inhibitor of CSF1R, exacerbates seizure severity and excitotoxicity-induced neuronal degeneration, indicating that microglia are rapidly responsive to the change of excitation/inhibition homeostasis and participate in the protection of neurons from overexcitation.


Assuntos
Astrócitos/metabolismo , Hipocampo/fisiologia , Microglia/fisiologia , Convulsões/fisiopatologia , Animais , Ácido Glutâmico/metabolismo , Camundongos , Neurônios/fisiologia , Convulsões/metabolismo
13.
Nat Commun ; 11(1): 4218, 2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32839452

RESUMO

Exposure to social stress and dysregulated serotonergic neurotransmission have both been implicated in the etiology of psychiatric disorders. However, the serotonergic circuit involved in stress vulnerability is still unknown. Here, we explored whether a serotonergic input from the dorsal raphe (DR) to ventral tegmental area (VTA) influences vulnerability to social stress. We identified a distinct, anatomically and functionally defined serotonergic subpopulation in the DR that projects to the VTA (5-HTDR→VTA neurons). Moreover, we found that susceptibility to social stress decreased the firing activity of 5-HTDR→VTA neurons. Importantly, the bidirectional manipulation of 5-HTDR→VTA neurons could modulate susceptibility to social stress. Our findings reveal that the activity of 5-HTDR→VTA neurons may be an essential factor in determining individual levels of susceptibility to social stress and suggest that targeting specific serotonergic circuits may aid the development of therapies for the treatment of stress-related disorders.


Assuntos
Núcleo Dorsal da Rafe/fisiologia , Vias Neurais/fisiologia , Neurônios Serotoninérgicos/fisiologia , Estresse Psicológico/fisiopatologia , Transmissão Sináptica/fisiologia , Área Tegmentar Ventral/fisiologia , Animais , Núcleo Dorsal da Rafe/citologia , Núcleo Dorsal da Rafe/metabolismo , Ácido Glutâmico/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Neurônios Serotoninérgicos/citologia , Neurônios Serotoninérgicos/metabolismo , Serotonina/metabolismo , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/metabolismo
14.
Neuron ; 107(5): 864-873.e4, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32610039

RESUMO

Like ventral tegmental area (VTA) dopamine (DA) neurons, VTA glutamate neuron activity can support positive reinforcement. However, a subset of VTA neurons co-release DA and glutamate, and DA release might be responsible for behavioral reinforcement induced by VTA glutamate neuron activity. To test this, we used optogenetics to stimulate VTA glutamate neurons in which tyrosine hydroxylase (TH), and thus DA biosynthesis, was conditionally ablated using either floxed Th mice or viral-based CRISPR/Cas9. Both approaches led to loss of TH expression in VTA glutamate neurons and loss of DA release from their distal terminals in nucleus accumbens (NAc). Despite loss of the DA signal, optogenetic activation of VTA glutamate cell bodies or axon terminals in NAc was sufficient to support reinforcement. These results suggest that glutamate release from VTA is sufficient to promote reinforcement independent of concomitant DA co-release, establishing a non-DA mechanism by which VTA activity can support reward-seeking behaviors.


Assuntos
Ácido Glutâmico/metabolismo , Motivação/fisiologia , Neurônios/metabolismo , Área Tegmentar Ventral/metabolismo , Animais , Comportamento Animal/fisiologia , Dopamina/metabolismo , Camundongos , Optogenética , Reforço Psicológico , Recompensa
15.
J Biosci Bioeng ; 130(5): 464-470, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32713813

RESUMO

Cyanobacteria can grow photoautotrophically, producing a range of substances by absorbing sunlight and utilizing carbon dioxide, and can potentially be used as industrial microbes that have minimal sugar requirements. To evaluate this potential, we explored the possibility of l-glutamate production using the Synechocystis sp. PCC6803. The ybjL gene encoding the putative l-glutamate exporter from Escherichia coli was introduced, and l-glutamate production reached 2.3 g/L in 143 h (34°C, 100 µmol m-2 s-1). Then, we attempted to produce two flavor substances, (S)-linalool, a monoterpene alcohol, and the sesquiterpene (+)-valencene. The Synechocystis sp. PCC6803 strain in which the linalool synthase gene (LINS) from Actinidia arguta (AaLINS) was expressed under control of the tac promoter (GT0846K-Ptac-AaLINS) produced 11.4 mg/L (S)-linalool in 160 h (30°C, 50 µmol m-2 s-1). The strain in which AaLINS2 and the mutated farnesyl diphosphate synthase gene ispA∗ (S80F) from E. coli (GT0846K-PpsbA2-AaLINS-ispA∗) were expressed from the PpsbA2 promoter accumulated 11.6 mg/L (S)-linalool in 160 h. Genome analysis revealed that both strains had mutations in slr1270, suggesting that loss of Slr1270 function was necessary for high linalool accumulation. For sesquiterpene production, the valencene synthase gene from Callitropsis nootkatensis and the fernesyl diphosphate synthase (ispA) gene from E. coli were introduced, and the resultant strain produced 9.6 mg/L of (+)-valencene in 166 h (30°C, 50 µmol m-2 s-1). This study highlights the production efficiency of engineered cyanobacteria, providing insight into potential industrial applications.


Assuntos
Monoterpenos Acíclicos/química , Monoterpenos Acíclicos/metabolismo , Ácido Glutâmico/metabolismo , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Synechocystis/metabolismo , Escherichia coli/genética , Aromatizantes/química , Aromatizantes/metabolismo , Engenharia Genética , Ácido Glutâmico/química , Estereoisomerismo , Synechocystis/genética
16.
Life Sci ; 257: 118049, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32634430

RESUMO

AIMS: Mild traumatic brain injury (mTBI) is an important risk factor for cognitive impairment. Despite intense efforts to develop efficient treatments, the current therapies are not often effective and far from satisfactory. Silymarin has been suggested as a therapeutic agent in the treatment of traumatic brain injury. This study aimed to determine whether silymarin can exert neuroprotective effects on memory impairment following mTBI in mice. MAIN METHODS: After mTBI induction, mice were treated with silymarin once daily for 20 consecutive days by oral gavage. To investigate cognitive functions, animals were subjected to Y-maze, novel-object recognition, and Morris-water maze. Levels of tumor necrosis factor (TNF)-α, glutamate, and brain derived neurotrophic factor (BDNF) were measured in the hippocampus. KEY FINDINGS: Our findings showed that mTBI resulted in a significant decline in memory in the Y-maze and Morris-water maze in both sexes, whereas only impaired cognitive function in males in the novel-object recognition. We found notable increases in TNF-α and glutamate levels in the hippocampus of both sexes, while there was only a significant decrease in hippocampal BDNF in mTBI-induced females. In addition, silymarin treatment improved cognitive impairments in mTBI-induced males but not in females. Silymarin significantly reduced TNF-α and glutamate levels, and increased BDNF levels in the hippocampus of mTBI-induced male but not in female mice. SIGNIFICANCE: This study demonstrates that silymarin treatment sex-dependently improves cognitive impairment in mTBI-induced mice, and suggests that silymarin may be a therapeutic agent for cognitive decline following mTBI in males. Further studies are needed to establish the validity of these findings in humans.


Assuntos
Concussão Encefálica/tratamento farmacológico , Cognição/efeitos dos fármacos , Silimarina/uso terapêutico , Animais , Animais não Endogâmicos , Concussão Encefálica/metabolismo , Lesões Encefálicas/tratamento farmacológico , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Cognição/fisiologia , Disfunção Cognitiva/tratamento farmacológico , Modelos Animais de Doenças , Feminino , Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Memória/efeitos dos fármacos , Camundongos , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fatores Sexuais , Silimarina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
17.
Mol Pharmacol ; 98(4): 454-461, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32606204

RESUMO

Endogenous opioid peptides in the amygdala regulate many of our behaviors and emotional responses. In particular, the endogenous opioid enkephalin plays a significant role in regulating amygdala activity, but its action is strongly limited by peptidases, which degrade enkephalin into inactive fragments. Inhibiting peptidases may be an attractive method to enhance endogenous opioid signaling; however, we do not know which specific peptidase(s) to target. Using inhibition of glutamate release onto the intercalated cells of the amygdala as an assay for enkephalin activity, we applied specific peptidase inhibitors to determine which peptidase(s) regulate enkephalin signaling in this region. Thiorphan (10 µM), captopril (1 µM), or bestatin (10 µM) were used to inhibit the activity of neprilysin, angiotensin-converting enzyme, or aminopeptidase N, respectively. In rat brain slices containing the intercalated cells, we found that inhibition of glutamate release by a submaximal concentration of enkephalin was doubled by application of all three peptidase inhibitors combined. Then, we tested inhibitors individually and found that inhibition of neprilysin alone could enhance enkephalin responses to the same extent as inhibitors of all three peptidases combined. This indicates neprilysin is the predominant peptidase responsible for degrading enkephalins in the intercalated cells of the amygdala. This differs from the striatum, locus coeruleus, and spinal cord, where multiple peptidases metabolize enkephalin. These data highlight the importance of knowing which specific peptidase(s) control opioid actions in the relevant neural circuit and how they change in disease states to allow rational choices of drugs targeting the specific peptidase of interest. SIGNIFICANCE STATEMENT: Endogenous opioids modulate many of our emotional and behavioral responses. In the amygdala, they modulate our pain, fear, and addictive behaviors. Their actions are terminated when they are catabolized into inactive fragments by at least three different peptidases. In this study, we found that neprilysin selectively controls endogenous opioid concentrations at synapses in the intercalated cells of the amygdala. This peptidase may be a target for regulation of endogenous opioid modulation of amygdala-mediated emotional and behavioral responses.


Assuntos
Tonsila do Cerebelo/metabolismo , Encefalinas/metabolismo , Neprilisina/metabolismo , Inibidores de Proteases/farmacologia , Animais , Captopril/farmacologia , Sinapses Elétricas/efeitos dos fármacos , Sinapses Elétricas/metabolismo , Ácido Glutâmico/metabolismo , Leucina/análogos & derivados , Leucina/farmacologia , Masculino , Neprilisina/antagonistas & inibidores , Proteólise/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Tiorfano/farmacologia
18.
Biochim Biophys Acta Proteins Proteom ; 1868(9): 140461, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32474108

RESUMO

d-Amino acids are physiologically important components of peptidoglycan in the bacterial cell wall, maintaining cell structure and aiding adaptation to environmental changes through peptidoglycan remodelling. Therefore, the biosynthesis of d-amino acids is essential for bacteria to adapt to different environmental conditions. The peptidoglycan of the extremely thermophilic bacterium Thermus thermophilus contains d-alanine (d-Ala) and d-glutamate (d-Glu), but its d-amino acid metabolism remains poorly understood. Here, we investigated the enzyme activity and function of the product of the TTHA1643 gene, which is annotated to be a Glu racemase in the T. thermophilus HB8 genome. Among 21 amino acids tested, TTHA1643 showed highly specific activity toward Glu as the substrate. The catalytic efficiency (kcat/Km) of TTHA1643 toward d- and l-Glu was comparable; however, the kcat value was 18-fold higher for l-Glu than for d-Glu. Temperature and pH profiles showed that the racemase activity of TTHA1643 is high under physiological conditions for T. thermophilus growth. To assess physiological relevance, we constructed a TTHA1643-deficient strain (∆TTHA1643) by replacing the TTHA1643 gene with the thermostable hygromycin resistance gene. Growth of the ∆TTHA1643 strain in synthetic medium without d-Glu was clearly diminished relative to wild type, although the TTHA1643 deletion was not lethal, suggesting that alternative d-Glu biosynthetic pathways may exist. The deterioration in growth was restored by adding d-Glu to the culture medium, showing that d-Glu is required for normal growth of T. thermophilus. Collectively, our findings show that TTHA1643 is a Glu racemase and has the physiological function of d-Glu production in T. thermophilus.


Assuntos
Isomerases de Aminoácido/química , Isomerases de Aminoácido/genética , Isomerases de Aminoácido/metabolismo , Thermus thermophilus/enzimologia , Sequência de Aminoácidos , Aminoácidos/metabolismo , Parede Celular/química , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/metabolismo , Deleção de Genes , Genoma Bacteriano , Ácido Glutâmico/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Proteínas Recombinantes , Especificidade por Substrato , Temperatura , Thermus thermophilus/genética , Thermus thermophilus/crescimento & desenvolvimento , Thermus thermophilus/fisiologia , Transcriptoma
19.
Chem Biol Interact ; 327: 109164, 2020 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-32524992

RESUMO

General anaesthetics are some of the most widely used and essential therapeutic agents. However, despite over a century of research, the molecular mechanisms of general anaesthesia in the central nervous system remain elusive. Ketamine (ketamine hydrochloride) has been approved for use in general anaesthesia either alone or in combination with other medications. It is a superb drug for use in short-term medical procedures that do not require skeletal muscle relaxation, and it has approval for the induction of general anaesthesia as a pre-anaesthetic to other general anaesthetic agents. However, Several questions remain unsolved, including the exact identification of the neural substrate of consciousness and its components, the pharmacodynamic interactions between anaesthetic agents, the mechanisms of cognitive alterations that follow an anaesthetic procedure, the identification of an eventual unitary mechanism of anaesthesia-induced alteration of consciousness, the relationship between network effects and the biochemical targets of anaesthetic agents, leading to difficulties in between-studies comparisons. Thus, the glutamate and dopamine systems play distinct roles in terms of neuronal signalling, yet both have proposed to contribute significantly to the pathophysiology of neuropsychiatric diseases. Imaging of the glutamate system and other aspects of research on the dopamine system have produced less consistent findings, potentially due to methodological limitations and the heterogeneity of the disorder. In this review, we discuss the neural circuits through which the two systems interact and how their disruption may cause psychotic symptoms. We also summarize from a molecular perspective of mechanisms of action of ketamine as general anaesthetics on ligand-gated ion channels mediated modulation of dopamine in the brain region.


Assuntos
Anestésicos Dissociativos/farmacologia , Dopamina/metabolismo , Ácido Glutâmico/metabolismo , Ketamina/farmacologia , Anestesia/efeitos adversos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Humanos , Transtornos Psicóticos/fisiopatologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/fisiopatologia
20.
Neuron ; 107(3): 436-453.e12, 2020 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-32485136

RESUMO

New methods for investigating human astrocytes are urgently needed, given their critical role in the central nervous system. Here we show that CD49f is a novel marker for human astrocytes, expressed in fetal and adult brains from healthy and diseased individuals. CD49f can be used to purify fetal astrocytes and human induced pluripotent stem cell (hiPSC)-derived astrocytes. We provide single-cell and bulk transcriptome analyses of CD49f+ hiPSC-astrocytes and demonstrate that they perform key astrocytic functions in vitro, including trophic support of neurons, glutamate uptake, and phagocytosis. Notably, CD49f+ hiPSC-astrocytes respond to inflammatory stimuli, acquiring an A1-like reactive state, in which they display impaired phagocytosis and glutamate uptake and fail to support neuronal maturation. Most importantly, we show that conditioned medium from human reactive A1-like astrocytes is toxic to human and rodent neurons. CD49f+ hiPSC-astrocytes are thus a valuable resource for investigating human astrocyte function and dysfunction in health and disease.


Assuntos
Astrócitos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Integrina alfa6/metabolismo , Doença de Alzheimer/metabolismo , Animais , Astrócitos/fisiologia , Biomarcadores/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Ácido Glutâmico/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/fisiopatologia , Camundongos , Técnicas de Patch-Clamp , Fagocitose/fisiologia , RNA-Seq , Análise de Célula Única
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