RESUMO
Cholinergic striatal interneurons (ChIs) express the vesicular glutamate transporter 3 (VGLUT3) which allows them to regulate the striatal network with glutamate and acetylcholine (ACh). In addition, VGLUT3-dependent glutamate increases ACh vesicular stores through vesicular synergy. A missense polymorphism, VGLUT3-p.T8I, was identified in patients with substance use disorders (SUDs) and eating disorders (EDs). A mouse line was generated to understand the neurochemical and behavioral impact of the p.T8I variant. In VGLUT3T8I/T8I male mice, glutamate signaling was unchanged but vesicular synergy and ACh release were blunted. Mutant male mice exhibited a reduced DA release in the dorsomedial striatum but not in the dorsolateral striatum, facilitating habit formation and exacerbating maladaptive use of drug or food. Increasing ACh tone with donepezil reversed the self-starvation phenotype observed in VGLUT3T8I/T8I male mice. Our study suggests that unbalanced dopaminergic transmission in the dorsal striatum could be a common mechanism between SUDs and EDs.
Assuntos
Corpo Estriado , Dopamina , Animais , Masculino , Dopamina/metabolismo , Camundongos , Corpo Estriado/metabolismo , Humanos , Acetilcolina/metabolismo , Transtornos Relacionados ao Uso de Substâncias/metabolismo , Transtornos Relacionados ao Uso de Substâncias/genética , Transdução de Sinais/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Interneurônios/metabolismo , Interneurônios/efeitos dos fármacos , Transtornos da Alimentação e da Ingestão de Alimentos/metabolismo , Transtornos da Alimentação e da Ingestão de Alimentos/genética , Transtornos da Alimentação e da Ingestão de Alimentos/fisiopatologia , Camundongos Endogâmicos C57BL , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Mutação , Mutação de Sentido Incorreto , Proteínas Vesiculares de Transporte de AcetilcolinaRESUMO
Changes in the amount of daylight (photoperiod) alter physiology and behaviour1,2. Adaptive responses to seasonal photoperiods are vital to all organisms-dysregulation associates with disease, including affective disorders3 and metabolic syndromes4. The circadian rhythm circuitry is implicated in such responses5,6, yet little is known about the precise cellular substrates that underlie phase synchronization to photoperiod change. Here we identify a brain circuit and system of axon branch-specific and reversible neurotransmitter deployment that are critical for behavioural and sleep adaptation to photoperiod. A type of neuron called mrEn1-Pet17 in the mouse brainstem median raphe nucleus segregates serotonin from VGLUT3 (also known as SLC17A8, a proxy for glutamate) to different axonal branches that innervate specific brain regions involved in circadian rhythm and sleep-wake timing8,9. This branch-specific neurotransmitter deployment did not distinguish between daylight and dark phase; however, it reorganized with change in photoperiod. Axonal boutons, but not cell soma, changed neurochemical phenotype upon a shift away from equinox light/dark conditions, and these changes were reversed upon return to equinox conditions. When we genetically disabled Vglut3 in mrEn1-Pet1 neurons, sleep-wake periods, voluntary activity and clock gene expression did not synchronize to the new photoperiod or were delayed. Combining intersectional rabies virus tracing and projection-specific neuronal silencing, we delineated a preoptic area-to-mrEn1Pet1 connection that was responsible for decoding the photoperiodic inputs, driving the neurotransmitter reorganization and promoting behavioural synchronization. Our results reveal a brain circuit and periodic, branch-specific neurotransmitter deployment that regulates organismal adaptation to photoperiod change.
Assuntos
Adaptação Fisiológica , Axônios , Ritmo Circadiano , Neurotransmissores , Fotoperíodo , Animais , Feminino , Camundongos , Adaptação Fisiológica/fisiologia , Sistemas de Transporte de Aminoácidos Acídicos/deficiência , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Axônios/metabolismo , Axônios/fisiologia , Ritmo Circadiano/fisiologia , Proteínas CLOCK/genética , Escuridão , Núcleo Dorsal da Rafe/citologia , Núcleo Dorsal da Rafe/metabolismo , Vias Neurais/fisiologia , Neurotransmissores/metabolismo , Área Pré-Óptica/citologia , Área Pré-Óptica/metabolismo , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/fisiologia , Vírus da Raiva , Serotonina/metabolismo , Sono/fisiologia , Vigília/fisiologiaRESUMO
Social behavior is important for our well-being, and its dysfunctions impact several pathological conditions. Although the involvement of glutamate is undeniable, the relevance of vesicular glutamate transporter type 3 (VGluT3), a specific vesicular transporter, in the control of social behavior is not sufficiently explored. Since midbrain median raphe region (MRR) is implicated in social behavior and the nucleus contains high amount of VGluT3+ neurons, we compared the behavior of male VGluT3 knock-out (KO) and VGluT3-Cre mice, the latter after chemogenetic MRR-VGluT3 manipulation. Appropriate control groups were included. Behavioral test battery was used for social behavior (sociability, social discrimination, social interaction, resident intruder test) and possible confounding factors (open field, elevated plus maze, Y-maze tests). Neuronal activation was studied by c-Fos immunohistochemistry. Human relevance was confirmed by VGluT3 gene expression in relevant human brainstem areas. VGluT3 KO mice exhibited increased anxiety, social interest, but also aggressive behavior in anxiogenic environment and impaired social memory. For KO animals, social interaction induced lower cell activation in the anterior cingulate, infralimbic cortex, and medial septum. In turn, excitation of MRR-VGluT3+ neurons was anxiolytic. Inhibition increased social interest 24â h later but decreased mobility and social behavior in aggressive context. Chemogenetic activation increased the number of c-Fos+ neurons only in the MRR. We confirmed the increased anxiety-like behavior and impaired memory of VGluT3 KO strain and revealed increased, but inadequate, social behavior. MRR-VGluT3 neurons regulated mobility and social and anxiety-like behavior in a context-dependent manner. The presence of VGluT3 mRNA on corresponding human brain areas suggests clinical relevance.
Assuntos
Ansiedade , Camundongos Knockout , Comportamento Social , Animais , Masculino , Humanos , Ansiedade/metabolismo , Núcleos da Rafe/metabolismo , Camundongos , Neurônios/metabolismo , Camundongos Endogâmicos C57BL , Comportamento Animal/fisiologia , Camundongos Transgênicos , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Agressão/fisiologiaRESUMO
The malate aspartate shuttle (MAS) plays a pivotal role in transporting cytosolic reducing equivalents - electrons - into the mitochondria for energy conversion at the electron transport chain (ETC) and in the process of oxidative phosphorylation. The MAS consists of two pairs of cytosolic and mitochondrial isoenzymes (malate dehydrogenases 1 and 2; and glutamate oxaloacetate transaminases 1 and 2) and two transporters (malate-2-oxoglutarate carrier and aspartate glutamate carrier (AGC), the latter of which has two tissue-dependent isoforms AGC1 and AGC2). While the inner mitochondrial membrane is impermeable to NADH, the MAS forms one of the main routes for mitochondrial electron uptake by promoting uptake of malate. Inherited bi-allelic pathogenic variants in five of the seven components of the MAS have been described hitherto and cause a wide spectrum of symptoms including early-onset epileptic encephalopathy. This review provides an overview of reported patients suffering from MAS deficiencies. In addition, we give an overview of diagnostic procedures and research performed on patient-derived cellular models and tissues. Current cellular models are briefly discussed and novel ways to achieve a better understanding of MAS deficiencies are highlighted.
Assuntos
Ácido Aspártico , Malato Desidrogenase , Malatos , Mitocôndrias , Humanos , Malatos/metabolismo , Malato Desidrogenase/metabolismo , Malato Desidrogenase/genética , Mitocôndrias/metabolismo , Mitocôndrias/genética , Mitocôndrias/patologia , Ácido Aspártico/metabolismo , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/patologia , Erros Inatos do Metabolismo/metabolismo , Erros Inatos do Metabolismo/diagnóstico , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/deficiência , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Fosforilação Oxidativa , AntiportersRESUMO
Exposure to stressors has profound effects on sleep that have been linked to serotonin (5-HT) neurons of the dorsal raphe nucleus (DR). However, the DR also comprises glutamatergic neurons expressing vesicular glutamate transporter type 3 (DRVGLUT3), leading us to examine their role. Cell-type-specific tracing revealed that DRVGLUT3 neurons project to brain areas regulating arousal and stress. We found that chemogenetic activation of DRVGLUT3 neurons mimics stress-induced sleep perturbations. Furthermore, deleting VGLUT3 in the DR attenuated stress-induced sleep perturbations, especially after social defeat stress. In the DR, VGLUT3 is found in subsets of 5-HT and non-5-HT neurons. We observed that both populations are activated by acute stress, including those projecting to the ventral tegmental area. However, deleting VGLUT3 in 5-HT neurons minimally affected sleep regulation. These findings suggest that VGLUT3 expression in the DR drives stress-induced sleep perturbations, possibly involving non-5-HT DRVGLUT3 neurons.
Assuntos
Núcleo Dorsal da Rafe , Neurônios , Sono , Estresse Psicológico , Animais , Masculino , Núcleo Dorsal da Rafe/metabolismo , Camundongos , Estresse Psicológico/metabolismo , Neurônios/metabolismo , Sono/fisiologia , Serotonina/metabolismo , Camundongos Endogâmicos C57BL , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genéticaRESUMO
Alcohol dependence results in long-lasting neuroadaptive changes in meso-corticolimbic system, especially in the nucleus accumbens (NAc), which drives relapse-like ethanol drinking upon abstinence or withdrawal. Within NAc, altered glutamate homeostasis is one of the neuroadaptive changes caused by alcohol dependence. Accumbal glutamate homeostasis is tightly maintained through glutamate transporter 1 (GLT-1) and cystine-glutamate antiporter (xCT). But the role of GLT-1 and xCT in relapse-like ethanol drinking is poorly understood. Here, we used alcohol-preferring (P) rats in relapse-like ethanol drinking paradigm to (a) determine the effect of relapse-like ethanol drinking on gene and protein expression of GLT-1 and xCT in NAc, measured by quantitative polymerase chain reaction (qPCR) and Western blot, respectively; (b) examine if glutamate uptake is affected by relapse-like ethanol drinking in NAc, measured by radioactive glutamate uptake assay; (c) elucidate if upregulation of either/both GLT-1 or/and xCT through ceftriaxone is/are required to attenuate relapse-like ethanol drinking. The GLT-1 or xCT protein expression was suppressed during ceftriaxone treatments through microinjection of GLT-1/xCT anti-sense vivo-morpholinos. We found that relapse-like ethanol drinking did not affect the gene and protein expression of GLT-1 and xCT in NAc. The glutamate uptake was also unaltered. Ceftriaxone (200 mg/kg body weight, i.p.) treatments during the last 5 days of abstinence attenuated relapse-like ethanol drinking. The suppression of GLT-1 or xCT expression prevented the ceftriaxone-induced attenuation of relapse-like ethanol drinking. These findings confirm that upregulation of both GLT-1 and xCT within NAc is crucial for ceftriaxone-mediated attenuation of relapse-like ethanol drinking.
Assuntos
Alcoolismo , Ceftriaxona , Consumo de Bebidas Alcoólicas/metabolismo , Alcoolismo/genética , Alcoolismo/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Ceftriaxona/metabolismo , Ceftriaxona/farmacologia , Etanol/farmacologia , Transportador 2 de Aminoácido Excitatório/genética , Ácido Glutâmico/metabolismo , Núcleo Accumbens , Ratos , RecidivaRESUMO
BACKGROUND: Myelin-associated glycoprotein (MAG) is a key molecule involved in the nurturing effect of myelin on ensheathed axons. MAG also inhibits axon outgrowth after injury. In preclinical stroke models, administration of a function-blocking anti-MAG monoclonal antibody (mAb) aimed to improve axon regeneration demonstrated reduced lesion volumes and a rapid clinical improvement, suggesting a mechanism of immediate neuroprotection rather than enhanced axon regeneration. In addition, it has been reported that antibody-mediated crosslinking of MAG can protect oligodendrocytes (OLs) against glutamate (Glu) overload by unknown mechanisms. PURPOSE: To unravel the molecular mechanisms underlying the protective effect of anti-MAG therapy with a focus on neuroprotection against Glu toxicity. RESULTS: MAG activation (via antibody crosslinking) triggered the clearance of extracellular Glu by its uptake into OLs via high affinity excitatory amino acid transporters. This resulted not only in protection of OLs but also nearby neurons. MAG activation led to a PKC-dependent activation of factor Nrf2 (nuclear-erythroid related factor-2) leading to antioxidant responses including increased mRNA expression of metabolic enzymes from the glutathione biosynthetic pathway and the regulatory chain of cystine/Glu antiporter system xc- increasing reduced glutathione (GSH), the main antioxidant in cells. The efficacy of early anti-MAG mAb administration was demonstrated in a preclinical model of excitotoxicity induced by intrastriatal Glu administration and extended to a model of Experimental Autoimmune Encephalitis showing axonal damage secondary to demyelination. CONCLUSIONS: MAG activation triggers Glu uptake into OLs under conditions of Glu overload and induces a robust protective antioxidant response.
Assuntos
Anticorpos Monoclonais/imunologia , Ácido Glutâmico/metabolismo , Glicoproteína Associada a Mielina/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Axônios/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Ácido Glutâmico/administração & dosagem , Ácido Glutâmico/farmacologia , Glutationa/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína Associada a Mielina/imunologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Neurônios/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteína Quinase C/metabolismo , Ratos , Receptores de Glutamato/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Mutations in TUBB4A are associated with a spectrum of neurologic disorders categorized as TUBB4A-related leukoencephalopathy. Affected children can present with global developmental delay or normal early development, followed by a variable loss of skills over time. Further research is needed to characterize the factors associated with the divergent developmental trajectories in this rare monogenic disorder because this phenotypic spectrum is not fully explained by genotype alone.To characterize early psychomotor features, developmental milestones and age of disease onset were collected from medical records (n=54 individuals). Three subcohorts were identified: individuals with the common p.Asp249Asn variant vs all other genotypes with either early (<12 months of age) or late onset of presentation. Individuals with the p.Asp249Asn variant or those with non-p.Asp249Asn genotypes with later disease onset attained key milestones, including head control, sitting, and independent walking. Subjects with early-onset, non-p.Asp249Asn-associated disease were less likely to achieve developmental milestones. Next, we defined the developmental severity as the percentage of milestones attained by age 2 years. The mild form was defined as attaining at least 75% of key developmental milestones. Among cohort categorized as mild, individuals with p.Asp249Asn variant were more likely to lose acquired abilities when compared with non-p.Asp249Asn individuals.Our results suggest multiple influences on developmental trajectory, including a strong contribution from genotype and age of onset. Further studies are needed to identify additional factors that influence overall outcomes to better counsel families and to design clinical trials with appropriate clinical endpoints.
Assuntos
Gânglios da Base/patologia , Cerebelo/patologia , Deficiências do Desenvolvimento/complicações , Deficiências do Desenvolvimento/genética , Leucoencefalopatias/complicações , Leucoencefalopatias/genética , Adolescente , Sistemas de Transporte de Aminoácidos Acídicos/deficiência , Sistemas de Transporte de Aminoácidos Acídicos/genética , Antiporters/deficiência , Antiporters/genética , Atrofia , Criança , Desenvolvimento Infantil , Pré-Escolar , Estudos de Coortes , Deficiências do Desenvolvimento/patologia , Feminino , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/complicações , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Humanos , Lactente , Recém-Nascido , Leucoencefalopatias/patologia , Masculino , Doenças Mitocondriais/complicações , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Mutação , Transtornos Psicomotores/complicações , Transtornos Psicomotores/genética , Transtornos Psicomotores/patologia , Estudos Retrospectivos , Tubulina (Proteína)/genéticaRESUMO
PYCR2 pathogenic variants lead to an autosomal recessive hypomyelinating leukodystrophy 10 (HLD10), characterized by global developmental delay, microcephaly, facial dysmorphism, movement disorder, and hypomyelination. This study identified the first two unrelated Thai patients with HLD10. Patient 1 harbored the novel compound heterozygous variants, c.257T>G (p.Val86Gly) and c.400G>A (p.Val134Met), whereas patient 2 possessed the homozygous variant, c.400G>A (p.Val134Met), in PYCR2. Haplotype analysis revealed that the two families' members shared a 2.3 Mb region covering the c.400G>A variant, indicating a common ancestry. The variant was estimated to age 1450 years ago. Since the c.400G>A was detected in three out of four mutant alleles and with a common ancestry, this variant might be common in Thai patients. We also reviewed the phenotype and genotype of all 35 previously reported PYCR2 patients and found that majorities of cases were homozygous with a consanguineous family history, except patient 1 and another reported case who were compound heterozygous. All patients had microcephaly and developmental delay. Hypotonia and peripheral spasticity were common. Hypomyelination or delayed myelination was a typical radiographic feature. Here, we report the first two Thai patients with HLD10 with the novel PYCR2 variants expanding the genotypic spectrum and suggest that the c.400G>A might be a common mutation in Thai patients.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/deficiência , Antiporters/deficiência , Deficiências do Desenvolvimento/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Microcefalia/genética , Doenças Mitocondriais/genética , Transtornos dos Movimentos/genética , Transtornos Psicomotores/genética , Pirrolina Carboxilato Redutases/genética , Adolescente , Alelos , Sistemas de Transporte de Aminoácidos Acídicos/genética , Antiporters/genética , Criança , Pré-Escolar , Deficiências do Desenvolvimento/complicações , Deficiências do Desenvolvimento/patologia , Feminino , Genótipo , Haplótipos/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/complicações , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Homozigoto , Humanos , Masculino , Microcefalia/complicações , Microcefalia/patologia , Doenças Mitocondriais/complicações , Doenças Mitocondriais/patologia , Transtornos dos Movimentos/complicações , Transtornos dos Movimentos/patologia , Mutação , Linhagem , Fenótipo , Transtornos Psicomotores/complicações , Transtornos Psicomotores/patologia , Adulto JovemRESUMO
The mammalian cochlea cannot regenerate functional hair cells (HCs) spontaneously. Atoh1 overexpression as well as other strategies are unable to generate functional HCs. Here, we simultaneously upregulated the expression of Gfi1, Pou4f3, and Atoh1 in postnatal cochlear supporting cells (SCs) in vivo, which efficiently converted SCs into HCs. The newly regenerated HCs expressed HC markers Myo7a, Calbindin, Parvalbumin, and Ctbp2 and were innervated by neurites. Importantly, many new HCs expressed the mature and terminal marker Prestin or vesicular glutamate transporter 3 (vGlut3), depending on the subtypes of the source SCs. Finally, our patch-clamp analysis showed that the new HCs in the medial region acquired a large K+ current, fired spikes transiently, and exhibited signature refinement of ribbon synapse functions, in close resemblance to native wild-type inner HCs. We demonstrated that co-upregulating Gfi1, Pou4f3, and Atoh1 enhances the efficiency of HC generation and promotes the functional maturation of new HCs.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ligação a DNA/genética , Células Ciliadas Auditivas/metabolismo , Proteínas de Homeodomínio/genética , Células Labirínticas de Suporte/metabolismo , Organogênese/genética , Fator de Transcrição Brn-3C/genética , Fatores de Transcrição/genética , Potenciais de Ação/fisiologia , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Calbindinas/genética , Calbindinas/metabolismo , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/citologia , Proteínas de Homeodomínio/metabolismo , Transporte de Íons , Células Labirínticas de Suporte/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/metabolismo , Miosina VIIa/genética , Miosina VIIa/metabolismo , Neuritos/metabolismo , Neuritos/ultraestrutura , Parvalbuminas/genética , Parvalbuminas/metabolismo , Técnicas de Patch-Clamp , Potássio/metabolismo , Transdução de Sinais , Fator de Transcrição Brn-3C/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Cochlear outer hair cells (OHCs) are known to uniquely participate in auditory processing through their electromotility, and like inner hair cells, are also capable of releasing vesicular glutamate onto spiral ganglion (SG) neurons: in this case, onto the sparse Type II SG neurons. However, unlike glutamate signaling at the inner hair cell-Type I SG neuron synapse, which is robust across a wide spectrum of sound intensities, glutamate signaling at the OHC-Type II SG neuron synapse is weaker and has been hypothesized to occur only at intense, possibly damaging sound levels. Here, we tested the ability of the OHC-Type II SG pathway to signal to the brain in response to moderate, nondamaging sound (80 dB SPL) as well as to intense sound (115 dB SPL). First, we determined the VGluTs associated with OHC signaling and then confirmed the loss of glutamatergic synaptic transmission from OHCs to Type II SG neurons in KO mice using dendritic patch-clamp recordings. Next, we generated genetic mouse lines in which vesicular glutamate release occurs selectively from OHCs, and then assessed c-Fos expression in the cochlear nucleus in response to sound. From these analyses, we show, for the first time, that glutamatergic signaling at the OHC-Type II SG neuron synapse is capable of activating cochlear nucleus neurons, even at moderate sound levels.SIGNIFICANCE STATEMENT Evidence suggests that cochlear outer hair cells (OHCs) release glutamate onto Type II spiral ganglion neurons only when exposed to loud sound, and that Type II neurons are activated by tissue damage. Knowing whether moderate level sound, without tissue damage, activates this pathway has functional implications for this fundamental auditory pathway. We first determined that OHCs rely largely on VGluT3 for synaptic glutamate release. We then used a genetically modified mouse line in which OHCs, but not inner hair cells, release vesicular glutamate to demonstrate that moderate sound exposure activates cochlear nucleus neurons via the OHC-Type II spiral ganglion pathway. Together, these data indicate that glutamate signaling at the OHC-Type II afferent synapse participates in auditory function at moderate sound levels.
Assuntos
Estimulação Acústica/métodos , Núcleo Coclear/metabolismo , Ácido Glutâmico/metabolismo , Células Ciliadas Auditivas Externas/metabolismo , Neurônios/metabolismo , Gânglio Espiral da Cóclea/metabolismo , Vias Aferentes/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Vias Auditivas/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Amino acids are delivered into developing wheat grains to support the accumulation of storage proteins in the starchy endosperm, and transporters play important roles in regulating this process. RNA-seq, RT-qPCR, and promoter-GUS assays showed that three amino acid transporters are differentially expressed in the endosperm transfer cells (TaAAP2), starchy endosperm cells (TaAAP13), and aleurone cells and embryo of the developing grain (TaAAP21), respectively. Yeast complementation revealed that all three transporters can transport a broad spectrum of amino acids. RNAi-mediated suppression of TaAAP13 expression in the starchy endosperm did not reduce the total nitrogen content of the whole grain, but significantly altered the composition and distribution of metabolites in the starchy endosperm, with increasing concentrations of some amino acids (notably glutamine and glycine) from the outer to inner starchy endosperm cells compared with wild type. Overexpression of TaAAP13 under the endosperm-specific HMW-GS (high molecular weight glutenin subunit) promoter significantly increased grain size, grain nitrogen concentration, and thousand grain weight, indicating that the sink strength for nitrogen transport was increased by manipulation of amino acid transporters. However, the total grain number was reduced, suggesting that source nitrogen remobilized from leaves is a limiting factor for productivity. Therefore, simultaneously increasing loading of amino acids into the phloem and delivery to the spike would be required to increase protein content while maintaining grain yield.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Grão Comestível/metabolismo , Triticum/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Cromatografia Líquida de Alta Pressão , Grão Comestível/crescimento & desenvolvimento , Endosperma/metabolismo , Regulação da Expressão Gênica de Plantas , Glutens/genética , Glutens/metabolismo , Espectroscopia de Ressonância Magnética , Nitrogênio/metabolismo , Floema/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Interferência de RNA , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real , Triticum/genética , Regulação para CimaRESUMO
BACKGROUND: In man two mitochondrial aspartate/glutamate carrier (AGC) isoforms, known as aralar and citrin, are required to accomplish several metabolic pathways. In order to fill the existing gap of knowledge in Drosophila melanogaster, we have studied aralar1 gene, orthologue of human AGC-encoding genes in this organism. METHODS: The blastp algorithm and the "reciprocal best hit" approach have been used to identify the human orthologue of AGCs in Drosophilidae and non-Drosophilidae. Aralar1 proteins have been overexpressed in Escherichia coli and functionally reconstituted into liposomes for transport assays. RESULTS: The transcriptional organization of aralar1 comprises six isoforms, three constitutively expressed (aralar1-RA, RD and RF), and the remaining three distributed during the development or in different tissues (aralar1-RB, RC and RE). Aralar1-PA and Aralar1-PE, representative of all isoforms, have been biochemically characterized. Recombinant Aralar1-PA and Aralar1-PE proteins share similar efficiency to exchange glutamate against aspartate, and same substrate affinities than the human isoforms. Interestingly, although Aralar1-PA and Aralar1-PE diverge only in their EF-hand 8, they greatly differ in their specific activities and substrate specificity. CONCLUSIONS: The tight regulation of aralar1 transcripts expression and the high request of aspartate and glutamate during early embryogenesis suggest a crucial role of Aralar1 in this Drosophila developmental stage. Furthermore, biochemical characterization and calcium sensitivity have identified Aralar1-PA and Aralar1-PE as the human aralar and citrin counterparts, respectively. GENERAL SIGNIFICANCE: The functional characterization of the fruit fly mitochondrial AGC transporter represents a crucial step toward a complete understanding of the metabolic events acting during early embryogenesis.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/genética , Antiporters/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Sistemas de Transporte de Aminoácidos Acídicos/química , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Antiporters/química , Antiporters/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/química , Drosophila melanogaster/metabolismo , Evolução Molecular , Humanos , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Amacrine cells of the retina are conspicuously variable in their morphologies, their population demographics, and their ensuing functions. Vesicular glutamate transporter 3 (VGluT3) amacrine cells are a recently characterized type of amacrine cell exhibiting local dendritic autonomy. The present analysis has examined three features of this VGluT3 population, including their density, local distribution, and dendritic spread, to discern the extent to which these are interrelated, using male and female mice. We first demonstrate that Bax-mediated cell death transforms the mosaic of VGluT3 cells from a random distribution into a regular mosaic. We subsequently examine the relationship between cell density and mosaic regularity across recombinant inbred strains of mice, finding that, although both traits vary across the strains, they exhibit minimal covariation. Other genetic determinants must therefore contribute independently to final cell number and to mosaic order. Using a conditional KO approach, we further demonstrate that Bax acts via the bipolar cell population, rather than cell-intrinsically, to control VGluT3 cell number. Finally, we consider the relationship between the dendritic arbors of single VGluT3 cells and the distribution of their homotypic neighbors. Dendritic field area was found to be independent of Voronoi domain area, while dendritic coverage of single cells was not conserved, simply increasing with the size of the dendritic field. Bax-KO retinas exhibited a threefold increase in dendritic coverage. Each cell, however, contributed less dendrites at each depth within the plexus, intermingling their processes with those of neighboring cells to approximate a constant volumetric density, yielding a uniformity in process coverage across the population.SIGNIFICANCE STATEMENT Different types of retinal neuron spread their processes across the surface of the retina to achieve a degree of dendritic coverage that is characteristic of each type. Many of these types achieve a constant coverage by varying their dendritic field area inversely with the local density of like-type neighbors. Here we report a population of retinal amacrine cells that do not develop dendritic arbors in relation to the spatial positioning of such homotypic neighbors; rather, this cell type modulates the extent of its dendritic branching when faced with a variable number of overlapping dendritic fields to approximate a uniformity in dendritic density across the retina.
Assuntos
Células Amácrinas/fisiologia , Sistemas de Transporte de Aminoácidos Acídicos/fisiologia , Dendritos/fisiologia , Retina/citologia , Retina/fisiologia , Sistemas de Transporte de Aminoácidos Acídicos/genética , Animais , Apoptose/fisiologia , Contagem de Células , Morte Celular , Mapeamento Cromossômico , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Aferentes/fisiologia , Locos de Características Quantitativas , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/fisiologiaRESUMO
The cell surface glycoprotein CD44 has various types of splicing variants, which contribute to its multiple distinct cellular functions. Recently, it was reported that the CD44v8-10 isoform interacts with the system Xc(-) transporter-related protein (xCT), and inhibits the accumulation of reactive oxygen species by promoting the synthesis of the antioxidant glutathione in human tumour cells. In this study, we investigated the expression and function of CD44 variants and xCT in canine tumours. From semi-quantitative reverse transcription polymerase chain reaction analysis, the mRNA expression of the CD44v8-10 isoform was observed in canine tumour tissues as well as human cases. The overexpression of CD44v8-10 may promote the synthesis of glutathione and enhance the resistance to radiation of canine breast tumour cells. Furthermore, canine xCT mRNA expression was significantly upregulated in the canine breast tumour tissues as compared to the normal tissues surrounding the tumours. To investigate the function of canine xCT, we treated canine tumour cells with the xCT inhibitor sulfasalazine. Consequently, the sulfasalazine-treated cells were more sensitive to oxidative stress than the non-treated cells. Taken together, these results suggested that CD44v8-10 and xCT play important roles in the therapy resistance of canine tumours as well as human tumours.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/genética , Doenças do Cão/genética , Regulação Neoplásica da Expressão Gênica , Receptores de Hialuronatos/genética , Sistemas de Transporte de Aminoácidos Acídicos/antagonistas & inibidores , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama , Doenças do Cão/metabolismo , Cães , Feminino , Glutationa/metabolismo , Receptores de Hialuronatos/metabolismo , Isoformas de Proteínas , Espécies Reativas de Oxigênio/metabolismo , Sulfassalazina/farmacologia , Regulação para CimaRESUMO
Neurological diseases were often characterized by progressive neuronal death, and emerging evidences suggested that ferroptosis may be an active driver of multiple neurodegenerative diseases. However, the mechanisms underlying ferroptosis in neuron cells are unclear. Here, we demonstrated that ferroptotic stimuli caused injury in neuron-like PC12 cells by modulating the expression of proteins involved in iron metabolism and lipid peroxidation at multiple levels, such as altering iron import/export, activating ferritinophagy, and decreasing glutathione (GSH) level. Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates multiple genes involved in ferroptosis, however, its exact role remain elusive. Our mechanistic inquiry revealed that Nrf2 expression enhanced iron storage capacity by increasing ferritin heavy chain 1 (FTH1) expression in PC12 cells. Moreover, Nrf2 alleviated the decrease in GSH level by promoting the expression of genes related to GSH synthesis, including solute carrier family 7 member 11 (SLC7A11) and cysteine ligase (GCL). The contribution of Nrf2 on ferroptosis resistance was further verified by increasing cell tolerance to oxidative stress. Furthermore, Nfe2l2 (Nrf2) knockdown sensitized cells to ferroptotic cell death. Taken together, our findings suggested that iron accumulation caused by altering iron metabolism and the decrease of GSH content are key factors in determining ferroptosis in PC12 cells, and Nrf2 inhibits ferroptosis by combating iron-induced oxidative stress. Our present study provided new clues for the intervention and prevention against ferroptosis-associated neurological diseases.
Assuntos
Ferroptose/efeitos dos fármacos , Glutationa/biossíntese , Sobrecarga de Ferro/tratamento farmacológico , Fator 2 Relacionado a NF-E2/biossíntese , Neurônios/efeitos dos fármacos , Sistemas de Transporte de Aminoácidos Acídicos/biossíntese , Sistemas de Transporte de Aminoácidos Acídicos/genética , Animais , Técnicas de Silenciamento de Genes , Glutationa/deficiência , Ferro/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Células PC12 , RNA Interferente Pequeno , RatosRESUMO
Innocuous mechanical stimuli, such as rubbing or stroking the skin, relieve itch through the activation of low-threshold mechanoreceptors. However, the mechanisms behind this inhibition remain unknown. We presently investigated whether stroking the skin reduces the responses of superficial dorsal horn neurons to pruritogens in male C57BL/6J mice. Single-unit recordings revealed that neuronal responses to chloroquine were enhanced during skin stroking, and this was followed by suppression of firing below baseline levels after the termination of stroking. Most of these neurons additionally responded to capsaicin. Stroking did not suppress neuronal responses to capsaicin, indicating state-dependent inhibition. Vesicular glutamate transporter 3 (VGLUT3)-lineage sensory nerves compose a subset of low-threshold mechanoreceptors. Stroking-related inhibition of neuronal responses to chloroquine was diminished by optogenetic inhibition of VGLUT3-lineage sensory nerves in male and female Vglut3-cre/NpHR-EYFP mice. Conversely, in male and female Vglut3-cre/ChR2-EYFP mice, optogenetic stimulation of VGLUT3-lineage sensory nerves inhibited firing responses of spinal neurons to pruritogens after the termination of stimulation. This inhibition was nearly abolished by spinal delivery of the κ-opioid receptor antagonist nor-binaltorphimine dihydrochloride, but not the neuropeptide Y receptor Y1 antagonist BMS193885. Optogenetic stimulation of VGLUT3-lineage sensory nerves inhibited pruritogen-evoked scratching without affecting mechanical and thermal pain behaviors. Therefore, VGLUT3-lineage sensory nerves appear to mediate inhibition of itch by tactile stimuli.SIGNIFICANCE STATEMENT Rubbing or stroking the skin is known to relieve itch. We investigated the mechanisms behind touch-evoked inhibition of itch in mice. Stroking the skin reduced the activity of itch-responsive spinal neurons. Optogenetic inhibition of VGLUT3-lineage sensory nerves diminished stroking-evoked inhibition, and optogenetic stimulation of VGLUT3-lineage nerves inhibited pruritogen-evoked firing. Together, our results provide a mechanistic understanding of touch-evoked inhibition of itch.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Mecanorreceptores/metabolismo , Prurido/metabolismo , Limiar Sensorial , Tato , Potenciais de Ação , Sistemas de Transporte de Aminoácidos Acídicos/genética , Animais , Capsaicina/farmacologia , Di-Hidropiridinas/farmacologia , Feminino , Masculino , Mecanorreceptores/efeitos dos fármacos , Mecanorreceptores/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Inibição Neural , Compostos de Fenilureia/farmacologia , Fármacos do Sistema Sensorial/farmacologiaRESUMO
Sphingolipids are abundant and essential molecules in eukaryotes that have crucial functions as signaling molecules and as membrane components. Sphingolipid biosynthesis starts in the endoplasmic reticulum with the condensation of serine and palmitoyl-CoA. Sphingolipid biosynthesis is highly regulated to maintain sphingolipid homeostasis. Even though, serine is an essential component of the sphingolipid biosynthesis pathway, its role in maintaining sphingolipid homeostasis has not been precisely studied. Here we show that serine uptake is an important factor for the regulation of sphingolipid biosynthesis in Saccharomyces cerevisiae. Using genetic experiments, we find the broad-specificity amino acid permease Gnp1 to be important for serine uptake. We confirm these results with serine uptake assays in gnp1Δ cells. We further show that uptake of exogenous serine by Gnp1 is important to maintain cellular serine levels and observe a specific connection between serine uptake and the first step of sphingolipid biosynthesis. Using mass spectrometry-based flux analysis, we further observed imported serine as the main source for de novo sphingolipid biosynthesis. Our results demonstrate that yeast cells preferentially use the uptake of exogenous serine to regulate sphingolipid biosynthesis. Our study can also be a starting point to analyze the role of serine uptake in mammalian sphingolipid metabolism.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismo , Esfingolipídeos/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Homeostase , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Esfingolipídeos/biossínteseRESUMO
The retina possesses few types of neurons so; it is considered an excellent model for understanding the neural mechanisms underlying basic neural information processing in the brain. Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system and retina. The present study was carried out to characterize the expression pattern of vesicular glutamate transporter 2 (Vglut2) and 3 (Vglut3) and glutamate receptor 1 (GluR1) and 2 (GluR2) mRNAs in the retina of adult laughing dove (Streptopelia senegalensis) by RT-PCR and in situ hybridization histochemistry. The cerebellum of adult laughing dove was used as a positive control in this study. Vglut2 mRNA was highly expressed only in the granular layer of the cerebellum while Vglut3 mRNA was weakly expressed only in the Purkinje cells layer. In the retina, Vglut2 mRNA was highly expressed in the ganglion cell layer and moderately expressed in the inner nuclear layer while Vglut3 mRNA was moderately expressed only in the inner nuclear layer. GluR1 mRNA was intensely expressed in the Purkinje cells layer while GluR2 mRNA signals were highly detectable in both granular and Purkinje cells layers of the cerebellum. In the retina, moderate expression of GluR1 and intense expression of GluR2 was found in both ganglion cell layer and the internal half of inner nuclear layer mostly amacrine cells. These results suggest that some retinal neuronal cells in the adult laughing dove are glutamatergic. Therefore, GluR1 and 2 are suggested as useful markers for glutamatergic retinal neuronal cells in the adult laughing doves.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , RNA Mensageiro/metabolismo , Receptores de Glutamato/metabolismo , Retina/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Animais , Columbidae , Hibridização In Situ , RNA Mensageiro/genética , Receptores de Glutamato/genética , Proteína Vesicular 2 de Transporte de Glutamato/genéticaRESUMO
CASE: We are reporting the third unrelated case of cerebral aspartate-glutamate carrier isoform 1 (AGC1) deficiency. Patient is a 21-month-old Yemeni male who presented with refractory seizure disorder and developmental arrest. Neuroimaging showed cerebral volume loss and diminished N-acetylaspartate (NAA) peak. Whole exome sequencing revealed a homozygous novel missense variant in the SLC25A12 gene. Patient's seizure frequency abated drastically following initiation of ketogenic diet. DISCUSSION AND CONCLUSION: Cerebral AGC1 deficiency results in dysfunction of mitochondrial malate aspartate shuttle, thereby prohibiting myelin synthesis. There are significant phenotypic commonalities between our patient and previously reported cases including intractable epilepsy, psychomotor delay, cerebral atrophy, and diminished NAA peak. Our report also provides evidence regarding beneficial effect of ketogenic diet in this rare neurometabolic epilepsy.