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1.
J Vis Exp ; (207)2024 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-38884489

RESUMEN

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry. In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1+ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodo Red-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.


Asunto(s)
Citometría de Flujo , Microglía , Sinapsis , Animales , Microglía/citología , Microglía/metabolismo , Ratones , Sinapsis/fisiología , Sinapsis/química , Citometría de Flujo/métodos , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/análisis , Sinaptosomas/metabolismo , Encéfalo/citología
2.
J Mol Neurosci ; 74(3): 60, 2024 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-38904846

RESUMEN

Our former studies have identified the alleviating effect of Calycosin (CA) on spinal cord injury (SCI). In this study, our purpose is to explore the influence of CA on SCI from the perspective of promoting axon growth. The SCI animal model was constructed by spinal cord compression, wherein rat primary cortex neuronal isolation was performed, and the axonal growth restriction cell model was established via chondroitin sulfate proteoglycan (CSPG) treatment. The expressions of axon regeneration markers were measured via immunofluorescent staining and western blot, and the direct target of CA was examined using silver staining. Finally, the expression of the protein tyrosine phosphatase receptor type S (PTPRS) was assessed using western blot. CA treatment increased neuronal process outgrowth and the expressions of axon regeneration markers, such as neurofilament H (NF-H), vesicular glutamate transporter 1 (vGlut1), and synaptophysin (Syn) in both SCI model rats and CSPG-treated primary cortical neurons, and PTPRS levels were elevated after SCI induction. In addition, PTPRS was the direct target of CA, and according to in vivo findings, exposure to CA reduced the PTPRS content. Furthermore, PTPRS overexpression inhibited CA's enhancement of axon regeneration marker content and neuronal axon lengths. CA improves SCI by increasing axon development through regulating PTPRS expression.


Asunto(s)
Axones , Isoflavonas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal , Sinaptofisina , Animales , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/tratamiento farmacológico , Ratas , Isoflavonas/farmacología , Isoflavonas/uso terapéutico , Axones/efectos de los fármacos , Axones/metabolismo , Células Cultivadas , Sinaptofisina/metabolismo , Sinaptofisina/genética , Proteínas de Neurofilamentos/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/genética , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/citología , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Masculino , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Proyección Neuronal/efectos de los fármacos , Femenino , Proteína 2 de Transporte Vesicular de Glutamato
3.
Acta Histochem ; 126(4): 152170, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38936136

RESUMEN

We previously reported the presence of P2X3 purinoceptors (P2X3)-expressing subserosal afferent nerve endings consisting of net- and basket-like nerve endings in the rat gastric antrum. These nerve endings may morphologically be vagal mechanoreceptors activated by antral peristalsis. The present study investigated immunoreactivities for vesicular glutamate transporter (VGLUT) 1 and VGLUT2 as well as exocytosis-related proteins, i.e., core components of the SNARE complex (SNAP25, Stx1, and VAMP2) and synaptotagmin-1 (Syt1), in whole-mount preparations of the rat gastric antrum using double immunofluorescence. VGLUT1 immunoreactivity was not detected, whereas VGLUT2 immunoreactivity was observed in P2X3-immunoreactive subserosal nerve endings composed of both net- and basket-like endings. In net-like nerve endings, intense VGLUT2 immunoreactivity was localized in polygonal bulges of reticular nerve fibers and peripheral axon terminals. Furthermore, intense immunoreactivities for SNAP25, Stx1, and VAMP2 were localized in net-like nerve endings. Intense immunoreactivities for VAMP2 and Syt1 were observed in VGLUT2-immunoreactive net-like nerve endings. In basket-like nerve endings, VGLUT2 immunoreactivity was localized in pleomorphic terminal structures and small bulges surrounding the subserosal ganglion, whereas immunoreactivities for SNAP25, Stx1, and VAMP2 were weak in these nerve endings. VGLUT2-immunoreactive basket-like nerve endings were weakly immunoreactive for VAMP2 and Syt1. These results suggest that subserosal afferent nerve endings release glutamate by exocytosis mainly from net-like nerve endings to modulate their mechanoreceptor function.


Asunto(s)
Exocitosis , Ácido Glutámico , Inmunohistoquímica , Terminaciones Nerviosas , Antro Pilórico , Receptores Purinérgicos P2X3 , Proteína 2 de Transporte Vesicular de Glutamato , Animales , Masculino , Ratas , Receptores Purinérgicos P2X3/metabolismo , Ácido Glutámico/metabolismo , Antro Pilórico/inervación , Antro Pilórico/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Terminaciones Nerviosas/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Ratas Wistar , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sinaptotagmina I/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Sintaxina 1/metabolismo
4.
Cereb Cortex ; 34(13): 121-128, 2024 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-38696601

RESUMEN

Previous studies in autism spectrum disorder demonstrated an increased number of excitatory pyramidal cells and a decreased number of inhibitory parvalbumin+ chandelier interneurons in the prefrontal cortex of postmortem brains. How these changes in cellular composition affect the overall abundance of excitatory and inhibitory synapses in the cortex is not known. Herein, we quantified the number of excitatory and inhibitory synapses in the prefrontal cortex of 10 postmortem autism spectrum disorder brains and 10 control cases. To identify excitatory synapses, we used VGlut1 as a marker of the presynaptic component and postsynaptic density protein-95 as marker of the postsynaptic component. To identify inhibitory synapses, we used the vesicular gamma-aminobutyric acid transporter as a marker of the presynaptic component and gephyrin as a marker of the postsynaptic component. We used Puncta Analyzer to quantify the number of co-localized pre- and postsynaptic synaptic components in each area of interest. We found an increase in the number of excitatory synapses in upper cortical layers and a decrease in inhibitory synapses in all cortical layers in autism spectrum disorder brains compared with control cases. The alteration in the number of excitatory and inhibitory synapses could lead to neuronal dysfunction and disturbed network connectivity in the prefrontal cortex in autism spectrum disorder.


Asunto(s)
Proteínas de la Membrana , Corteza Prefrontal , Sinapsis , Corteza Prefrontal/metabolismo , Corteza Prefrontal/patología , Humanos , Masculino , Femenino , Sinapsis/patología , Sinapsis/metabolismo , Adulto , Persona de Mediana Edad , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/patología , Adulto Joven , Adolescente , Niño , Trastorno Autístico/metabolismo , Trastorno Autístico/patología , Inhibición Neural/fisiología , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo
5.
Neuron ; 112(12): 1978-1996.e6, 2024 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-38599212

RESUMEN

Interactions among neuronal, glial, and vascular components are crucial for retinal angiogenesis and blood-retinal barrier (BRB) maturation. Although synaptic dysfunction precedes vascular abnormalities in many retinal pathologies, how neuronal activity, specifically glutamatergic activity, regulates retinal angiogenesis and BRB maturation remains unclear. Using in vivo genetic studies in mice, single-cell RNA sequencing (scRNA-seq), and functional validation, we show that deep plexus angiogenesis and paracellular BRB maturation are delayed in Vglut1-/- retinas where neurons fail to release glutamate. By contrast, deep plexus angiogenesis and paracellular BRB maturation are accelerated in Gnat1-/- retinas, where constitutively depolarized rods release excessive glutamate. Norrin expression and endothelial Norrin/ß-catenin signaling are downregulated in Vglut1-/- retinas and upregulated in Gnat1-/- retinas. Pharmacological activation of endothelial Norrin/ß-catenin signaling in Vglut1-/- retinas rescues defects in deep plexus angiogenesis and paracellular BRB maturation. Our findings demonstrate that glutamatergic neuronal activity regulates retinal angiogenesis and BRB maturation by modulating endothelial Norrin/ß-catenin signaling.


Asunto(s)
Barrera Hematorretinal , Proteínas del Ojo , Ácido Glutámico , Proteínas del Tejido Nervioso , Transducción de Señal , beta Catenina , Animales , Barrera Hematorretinal/metabolismo , beta Catenina/metabolismo , Ratones , Ácido Glutámico/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Ojo/metabolismo , Proteínas del Ojo/genética , Transducción de Señal/fisiología , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Neuronas/metabolismo , Ratones Noqueados , Neovascularización Retiniana/metabolismo , Retina/metabolismo , Ratones Endogámicos C57BL , Angiogénesis
6.
Neuroscience ; 546: 75-87, 2024 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-38552733

RESUMEN

Alzheimer's disease (AD) is a progressive neurodegenerative disorder for which there are very limited treatment options. Dysfunction of the excitatory neurotransmitter system is thought to play a major role in the pathogenesis of this condition. Vesicular glutamate transporters (VGLUTs) are key to controlling the quantal release of glutamate. Thus, expressional changes in disease can have implications for aberrant neuronal activity, raising the possibility of a therapeutic target. There is no information regarding the expression of VGLUTs in the human medial temporal lobe in AD, one of the earliest and most severely affected brain regions. This study aimed to quantify and compare the layer-specific expression of VGLUT1 and VGLUT2 between control and AD cases in the hippocampus, subiculum, entorhinal cortex, and superior temporal gyrus. Free-floating fluorescent immunohistochemistry was used to label VGLUT1 and VGLUT2 in the hippocampus, subiculum, entorhinal cortex, and superior temporal gyrus. Sections were imaged using laser-scanning confocal microscopy and transporter densitometric analysis was performed. VGLUT1 density was not significantly different in AD tissue, except lower staining density observed in the dentate gyrus stratum moleculare (p = 0.0051). VGLUT2 expression was not altered in the hippocampus and entorhinal cortex of AD cases but was significantly lower in the subiculum (p = 0.015) and superior temporal gyrus (p = 0.0023). This study indicates a regionally specific vulnerability of VGLUT1 and VGLUT2 expression in the medial temporal lobe and superior temporal gyrus in AD. However, the causes and functional consequences of these disturbances need to be further explored to assess VGLUT1 and VGLUT2 as viable therapeutic targets.


Asunto(s)
Enfermedad de Alzheimer , Lóbulo Temporal , Proteína 1 de Transporte Vesicular de Glutamato , Proteína 2 de Transporte Vesicular de Glutamato , Humanos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Lóbulo Temporal/metabolismo , Lóbulo Temporal/patología , Masculino , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Anciano , Femenino , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Anciano de 80 o más Años , Persona de Mediana Edad , Inmunohistoquímica
7.
Nature ; 622(7981): 120-129, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37674083

RESUMEN

Multimodal astrocyte-neuron communications govern brain circuitry assembly and function1. For example, through rapid glutamate release, astrocytes can control excitability, plasticity and synchronous activity2,3 of synaptic networks, while also contributing to their dysregulation in neuropsychiatric conditions4-7. For astrocytes to communicate through fast focal glutamate release, they should possess an apparatus for Ca2+-dependent exocytosis similar to neurons8-10. However, the existence of this mechanism has been questioned11-13 owing to inconsistent data14-17 and a lack of direct supporting evidence. Here we revisited the astrocyte glutamate exocytosis hypothesis by considering the emerging molecular heterogeneity of astrocytes18-21 and using molecular, bioinformatic and imaging approaches, together with cell-specific genetic tools that interfere with glutamate exocytosis in vivo. By analysing existing single-cell RNA-sequencing databases and our patch-seq data, we identified nine molecularly distinct clusters of hippocampal astrocytes, among which we found a notable subpopulation that selectively expressed synaptic-like glutamate-release machinery and localized to discrete hippocampal sites. Using GluSnFR-based glutamate imaging22 in situ and in vivo, we identified a corresponding astrocyte subgroup that responds reliably to astrocyte-selective stimulations with subsecond glutamate release events at spatially precise hotspots, which were suppressed by astrocyte-targeted deletion of vesicular glutamate transporter 1 (VGLUT1). Furthermore, deletion of this transporter or its isoform VGLUT2 revealed specific contributions of glutamatergic astrocytes in cortico-hippocampal and nigrostriatal circuits during normal behaviour and pathological processes. By uncovering this atypical subpopulation of specialized astrocytes in the adult brain, we provide insights into the complex roles of astrocytes in central nervous system (CNS) physiology and diseases, and identify a potential therapeutic target.


Asunto(s)
Astrocitos , Sistema Nervioso Central , Ácido Glutámico , Transducción de Señal , Adulto , Humanos , Astrocitos/clasificación , Astrocitos/citología , Astrocitos/metabolismo , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Neuronas/metabolismo , Transmisión Sináptica , Calcio/metabolismo , Exocitosis , Análisis de Expresión Génica de una Sola Célula , Proteína 1 de Transporte Vesicular de Glutamato/deficiencia , Proteína 1 de Transporte Vesicular de Glutamato/genética , Eliminación de Gen , Corteza Cerebral/citología , Corteza Cerebral/metabolismo
8.
J Alzheimers Dis ; 94(1): 227-246, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37212097

RESUMEN

BACKGROUND: Altered glutamatergic neurotransmission may contribute to impaired default mode network (DMN) function in Alzheimer's disease (AD). Among the DMN hub regions, frontal cortex (FC) was suggested to undergo a glutamatergic plasticity response in prodromal AD, while the status of glutamatergic synapses in the precuneus (PreC) during clinical-neuropathological AD progression is not known. OBJECTIVE: To quantify vesicular glutamate transporter VGluT1- and VGluT2-containing synaptic terminals in PreC and FC across clinical stages of AD. METHODS: Unbiased sampling and quantitative confocal immunofluorescence of cortical VGluT1- and VGluT2-immunoreactive profiles and spinophilin-labeled dendritic spines were performed in cases with no cognitive impairment (NCI), mild cognitive impairment (MCI), mild-moderate AD (mAD), or moderate-severe AD (sAD). RESULTS: In both regions, loss of VGluT1-positive profile density was seen in sAD compared to NCI, MCI, and mAD. VGluT1-positive profile intensity in PreC did not differ across groups, while in FC it was greater in MCI, mAD, and sAD compared to NCI. VGluT2 measures were stable in PreC while FC had greater VGluT2-positive profile density in MCI compared to sAD, but not NCI or mAD. Spinophilin measures in PreC were lower in mAD and sAD compared to NCI, while in FC they were stable across groups. Lower VGluT1 and spinophilin measures in PreC, but not FC, correlated with greater neuropathology. CONCLUSION: Frank loss of VGluT1 in advanced AD relative to NCI occurs in both DMN regions. In FC, an upregulation of VGluT1 protein content in remaining glutamatergic terminals may contribute to this region's plasticity response in AD.


Asunto(s)
Enfermedad de Alzheimer , Humanos , Enfermedad de Alzheimer/patología , Proteínas de Transporte Vesicular de Glutamato/metabolismo , Red en Modo Predeterminado , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Terminales Presinápticos/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo
9.
Nat Commun ; 14(1): 2723, 2023 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-37169755

RESUMEN

Vesicular glutamate transporters accumulate glutamate in synaptic vesicles, where they also function as a major Cl- efflux pathway. Here we combine heterologous expression and cellular electrophysiology with mathematical modeling to understand the mechanisms underlying this dual function of rat VGLUT1. When glutamate is the main cytoplasmic anion, VGLUT1 functions as H+-glutamate exchanger, with a transport rate of around 600 s-1 at -160 mV. Transport of other large anions, including aspartate, is not stoichiometrically coupled to H+ transport, and Cl- permeates VGLUT1 through an aqueous anion channel with unitary transport rates of 1.5 × 105 s-1 at -160 mV. Mathematical modeling reveals that H+ coupling is sufficient for selective glutamate accumulation in model vesicles and that VGLUT Cl- channel function increases the transport efficiency by accelerating glutamate accumulation and reducing ATP-driven H+ transport. In summary, we provide evidence that VGLUT1 functions as H+-glutamate exchanger that is partially or fully uncoupled by other anions.


Asunto(s)
Vesículas Sinápticas , Proteínas de Transporte Vesicular de Glutamato , Ratas , Animales , Proteínas de Transporte Vesicular de Glutamato/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Vesículas Sinápticas/metabolismo , Aniones/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Ácido Glutámico/metabolismo
10.
Cells ; 12(3)2023 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-36766715

RESUMEN

Glioblastoma (GB) is the most prevalent primary brain cancer and the most aggressive form of glioma because of its poor prognosis and high recurrence. To confirm the importance of epigenetics in glioma, we explored The Cancer Gene Atlas (TCGA) database and we found that several histone/DNA modifications and chromatin remodeling factors were affected at transcriptional and genetic levels in GB compared to lower-grade gliomas. We associated these alterations in our own cohort of study with a significant reduction in the bulk levels of acetylated lysines 9 and 14 of histone H3 in high-grade compared to low-grade tumors. Within GB, we performed an RNA-seq analysis between samples exhibiting the lowest and highest levels of acetylated H3 in the cohort; these results are in general concordance with the transcriptional changes obtained after histone deacetylase (HDAC) inhibition of GB-derived cultures that affected relevant genes in glioma biology and treatment (e.g., A2ML1, CD83, SLC17A7, TNFSF18). Overall, we identified a transcriptional signature linked to histone acetylation that was potentially associated with good prognosis, i.e., high overall survival and low rate of somatic mutations in epigenetically related genes in GB. Our study identifies lysine acetylation as a key defective histone modification in adult high-grade glioma, and offers novel insights regarding the use of HDAC inhibitors in therapy.


Asunto(s)
Glioblastoma , Glioma , Humanos , Adulto , Histonas/metabolismo , Glioblastoma/genética , Acetilación , Inhibidores de Histona Desacetilasas/farmacología , Glioma/genética , Proteína 1 de Transporte Vesicular de Glutamato
11.
Artículo en Inglés | MEDLINE | ID: mdl-36209771

RESUMEN

The NMDA antagonist ketamine demonstrated a fast antidepressant activity in treatment-resistant depression. Pre-clinical studies suggest that de novo synthesis of the brain-derived neurotrophic factor (BDNF) in the PFC might be involved in the rapid antidepressant action of ketamine. Applying a genetic model of impaired glutamate release, this study aims to further identify the molecular mechanisms that could modulate antidepressant action and resistance to treatment. To that end, mice knocked-down for the vesicular glutamate transporter 1 (VGLUT1+/-) were used. We analyzed anhedonia and helpless behavior as well as the expression of the proteins linked to glutamate transmission in the PFC of mice treated with ketamine or the reference antidepressant reboxetine. Moreover, we analyzed the acute effects of ketamine in VGLUT1+/- mice pretreated with chronic reboxetine or those that received a PFC rescue expression of VGLUT1. Chronic reboxetine rescued the depressive-like phenotype of the VGLUT1+/- mice. In addition, it enhanced the expression of the proteins linked to the AMPA signaling pathway as well as the immature form of BDNF (pro-BDNF). Unlike WT mice, ketamine had no effect on anhedonia or pro-BDNF expression in VGLUT1+/- mice; it also failed to decrease phosphorylated eukaryote elongation factor 2 (p-eEF2). Nevertheless, we found that reboxetine administered as pretreatment or PFC overexpression of VGLUT1 did rescue the antidepressant-like activity of acute ketamine in the mice. Our results strongly suggest that not only do PFC VGLUT1 levels modulate the rapid-antidepressant action of ketamine, but also highlight a possible mechanism for antidepressant resistance in some patients.


Asunto(s)
Ketamina , Proteína 1 de Transporte Vesicular de Glutamato , Animales , Ratones , Anhedonia , Antidepresivos/uso terapéutico , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Depresión/metabolismo , Modelos Animales de Enfermedad , Ácido Glutámico/metabolismo , Ketamina/farmacología , Neuronas/metabolismo , Corteza Prefrontal/metabolismo , Reboxetina/farmacología , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo
12.
Behav Brain Res ; 436: 114072, 2023 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-36030906

RESUMEN

D-galactose (d-gal) is broadly used in animal aging studies as its chronic administration mimics learning and memory impairments related to aging in humans. However, within the few studies that utilize chronic oral d-gal intake, none of them is focused on alteration in synaptic structure and function. We examined the effects of 6-weeks oral d-gal intake (200 mg/kg and 500 mg/kg, dissolved in tap water) on age-related changes, with emphasis on the prefrontal cortex (PFC) and hippocampus (HIP) of adult male Wistar rats. Memory assessment was followed by histological examination of the PFC and HIP (Nissl staining and Iba-1 immunostaining), while in crude synaptosomal fractions the state of oxidative stress and the expression of proteins involved in glutamatergic signaling was determined. Although applied dosages compromised memory, alterations such as impaired sensory-motor function and aberrant morphology were not detected. In the PFC, analysis of microglia revealed reduction of branching pattern following d-gal intake, in parallel with increased oxidative damage of proteins, lipids and disturbed pro-oxidant antioxidant balance. These changes in the PFC were further accompanied with decreased levels of vesicular glutamate transporter 1, syntaxin-1 and NMDA receptor 2B subunit in both treated groups. Simultaneously, the increased hippocampal oxidative damage of lipids was detected. Results indicate successful provocation of age-related changes following oral d-gal intake, and suggest greater sensitivity of the PFC to d-gal treatment than HIP.


Asunto(s)
Antioxidantes , Galactosa , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Galactosa/farmacología , Hipocampo/metabolismo , Humanos , Lípidos , Masculino , Estrés Oxidativo , Corteza Prefrontal/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/farmacología , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Agua/metabolismo , Agua/farmacología
13.
Int J Mol Sci ; 23(23)2022 Dec 03.
Artículo en Inglés | MEDLINE | ID: mdl-36499607

RESUMEN

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease; its pathogenesis is still being intensively studied to explain the reasons for the significant genetic and phenotypic heterogeneity of the disease. To search for new genes involved in HCM development, we analyzed gene expression profiles coupled with DNA methylation profiles in the hypertrophied myocardia of HCM patients. The transcriptome analysis identified significant differences in the levels of 193 genes, most of which were underexpressed in HCM. The methylome analysis revealed 1755 nominally significant differentially methylated positions (DMPs), mostly hypomethylated in HCM. Based on gene ontology enrichment analysis, the majority of biological processes, overrepresented by both differentially expressed genes (DEGs) and DMP-containing genes, are involved in the regulation of locomotion and muscle structure development. The intersection of 193 DEGs and 978 DMP-containing genes pinpointed eight common genes, the expressions of which correlated with the methylation levels of the neighboring DMPs. Half of these genes (AUTS2, BRSK2, PRRT1, and SLC17A7), regulated by the mechanism of DNA methylation, were underexpressed in HCM and were involved in neurogenesis and synapse functioning. Our data, suggesting the involvement of innervation-associated genes in HCM, provide additional insights into disease pathogenesis and expand the field of further research.


Asunto(s)
Cardiomiopatía Hipertrófica , Transcriptoma , Humanos , Cardiomiopatía Hipertrófica/metabolismo , Perfilación de la Expresión Génica , Metilación de ADN , Ontología de Genes , Proteína 1 de Transporte Vesicular de Glutamato/genética
14.
J Neurosci ; 42(43): 8078-8094, 2022 10 26.
Artículo en Inglés | MEDLINE | ID: mdl-36104279

RESUMEN

The most caudal part of the striatum in rodents, the tail of the striatum (TS), has many features that distinguish it from the rostral striatum, such as its biased distributions of dopamine receptor subtypes, lack of striosomes and matrix compartmentalization, and involvement in sound-driven behaviors. However, information regarding the TS is still limited. We demonstrate in this article that the TS of the male mouse contains GABAergic neurons of a novel type that were detected immunohistochemically with the neurofilament marker SMI-32. Their somata were larger than cholinergic giant aspiny neurons, were located in a narrow space adjacent to the globus pallidus (GP), and extended long dendrites laterally toward the intermediate division (ID) of the trilaminar part of the TS, the region targeted by axons from the primary auditory cortex (A1). Although vesicular glutamate transporter 1-positive cortical axon terminals rarely contacted these TS large (TSL) neurons, glutamic acid decarboxylase-immunoreactive and enkephalin-immunoreactive boutons densely covered somata and dendrites of TSL neurons, forming symmetrical synapses. Analyses of GAD67-CrePR knock-in mice revealed that these axonal boutons originated from nearby medium spiny neurons (MSNs) in the ID. All MSNs examined in the ID in turn received inputs from the A1. Retrograde tracers injected into the rostral zona incerta and ventral medial nucleus of the thalamus labeled somata of TSL neurons. TSL neurons share many morphological features with GP neurons, but their strategically located dendrites receive inputs from closely located MSNs in the ID, suggesting faster responses than distant GP neurons to facilitate auditory-evoked, prompt disinhibition in their targets.SIGNIFICANCE STATEMENT This study describes a newly found population of neurons in the mouse striatum, the brain region responsible for appropriate behaviors. They are large GABAergic neurons located in the most caudal part of the striatum [tail of the striatum (TS)]. These TS large (TSL) neurons extended dendrites toward a particular region of the TS where axons from the primary auditory cortex (A1) terminated. These dendrites received direct synaptic inputs heavily from nearby GABAergic neurons of the striatum that in turn received inputs from the A1. TSL neurons sent axons to two subcortical regions outside basal ganglia, one of which is related to arousal. Specialized connectivity of TSL neurons suggests prompt disinhibitory actions on their targets to facilitate sound-evoked characteristic behaviors.


Asunto(s)
Dendritas , Glutamato Descarboxilasa , Masculino , Animales , Ratones , Dendritas/metabolismo , Glutamato Descarboxilasa/metabolismo , Neuronas GABAérgicas/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Sinapsis/metabolismo , Cuerpo Estriado/metabolismo , Axones/metabolismo , Encefalinas/metabolismo , Receptores Dopaminérgicos/metabolismo , Colinérgicos
15.
Biol Pharm Bull ; 45(9): 1385-1388, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36047209

RESUMEN

Docosahexaenoic acid (DHA; 22:6n-3), which is enriched in the neuronal membrane, plays a variety of roles in the brain. Vesicular glutamate transporters (VGLUTs) are responsible for incorporating glutamine into synaptic vesicles. We investigated the influence of DHA on the fatty acid profile and the levels of VGLUT1 and VGLUT2 proteins in differentiated NG108-15 cells, a neuroblastoma-glioma hybrid cell line. NG108-15 cells were plated and 24 h later the medium was replaced with Dulbecco's modified Eagle's medium supplemented with 1% fetal bovine serum, 0.2 mM dibutyryl cAMP, and 100 nM dexamethasone, which was added to induce differentiation. After 6 d, the amount of DHA in the cells was increased by addition of DHA to the medium. VGLUT2 levels were increased by the addition of DHA. These data indicate that DHA affected the levels of VGLUT2 in NG108-15 cells under differentiation-promoting conditions, suggesting that DHA affects brain functions involving VGLUT2.


Asunto(s)
Ácidos Docosahexaenoicos , Vesículas Sinápticas , Ácidos Docosahexaenoicos/farmacología , Ácido Glutámico/metabolismo , Neuronas/metabolismo , Vesículas Sinápticas/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo
16.
Mol Psychiatry ; 27(12): 5213-5226, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36028572

RESUMEN

The excitatory neurotransmitter glutamate shapes learning and memory, but the underlying epigenetic mechanism of glutamate regulation in neuron remains poorly understood. Here, we showed that lysine demethylase KDM6B was expressed in excitatory neurons and declined in hippocampus with age. Conditional knockout of KDM6B in excitatory neurons reduced spine density, synaptic vesicle number and synaptic activity, and impaired learning and memory without obvious effect on brain morphology in mice. Mechanistically, KDM6B upregulated vesicular glutamate transporter 1 and 2 (VGLUT1/2) in neurons through demethylating H3K27me3 at their promoters. Tau interacted and recruited KDM6B to the promoters of Slc17a7 and Slc17a6, leading to a decrease in local H3K27me3 levels and induction of VGLUT1/2 expression in neurons, which could be prevented by loss of Tau. Ectopic expression of KDM6B, VGLUT1, or VGLUT2 restored spine density and synaptic activity in KDM6B-deficient cortical neurons. Collectively, these findings unravel a fundamental mechanism underlying epigenetic regulation of synaptic plasticity and cognition.


Asunto(s)
Epigénesis Genética , Histona Demetilasas con Dominio de Jumonji , Plasticidad Neuronal , Proteínas tau , Animales , Ratones , Cognición/fisiología , Ácido Glutámico/metabolismo , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Plasticidad Neuronal/genética , Plasticidad Neuronal/fisiología , Sinapsis/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/genética , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/genética , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Proteínas tau/metabolismo
17.
Neuro Endocrinol Lett ; 43(2): 88-98, 2022 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-35933616

RESUMEN

OBJECTIVE: To investigate the effects of estrogen on the threshold and temperature of orofacial pain and explore the influence on the function of glutamate and GABA neurons in the orofacial pain temperature perception pathway by observing the expression of vesicular glutamate transporter 2 (Vglut2) and vesicular GABA transporter 1 (Vgat1). METHODS: A total of 24 adult female Sprague-Dawley rats were divided into three groups: sham operation (SHAM), ovariectomized (OVX) and ovariectomized plus estrogen intervention (OVX+E) (n = 8 per group). The threshold of mechanical pain of the orofacial region was assessed with von Frey filaments, and the temperature of the rat orofacial region was monitored by infrared thermography. Changes in the expression of Vglut2 and Vgat1 in glutamatergic and GABAergic neurons in the trigeminal ganglion (TG), spinal trigeminal nucleus (Sp5C), lateral parabrachial nucleus (LPB) and ventral posteromedial nucleus of the thalamus (VPM) were assessed by immunostaining and Western blotting. RESULTS: Under low-estrogen conditions, the mechanical pain threshold of the orofacial region of rats decreased significantly, and the temperature of the orofacial region increased significantly. The expression of Vglut2 and Vgat1 in the TG and Sp5C showed a downward trend, and the decline in Vgat1 was greater than that in Vglut2. Conversely, both proteins were upregulated in the LPB and VPM, and the magnitude of the changes in Vglut2 was greater than that in Vgat1. Estrogen therapy reversed these changes. CONCLUSION: Under low-estrogen conditions, the proportion of glutamate and GABA neurons in the orofacial pain and temperature sensation pathway changes, which leads to the imbalance of neurotransmission function and the enhancement of excitatory transmission of these two kinds of neurons and finally leads to a decrease in the orofacial pain threshold and an increase in temperature.


Asunto(s)
Dolor Facial , Sensación , Animales , Femenino , Ratas , Estrógenos/farmacología , Glutamatos , Ratas Sprague-Dawley , Temperatura , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores
18.
Neuron ; 110(9): 1483-1497.e7, 2022 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-35263617

RESUMEN

Vesicular transporters (VTs) define the type of neurotransmitter that synaptic vesicles (SVs) store and release. While certain mammalian neurons release multiple transmitters, it is not clear whether the release occurs from the same or distinct vesicle pools at the synapse. Using quantitative single-vesicle imaging, we show that a vast majority of SVs in the rodent brain contain only one type of VT, indicating specificity for a single neurotransmitter. Interestingly, SVs containing dual transporters are highly diverse (27 types) but small in proportion (2% of all SVs), excluding the largest pool that carries VGLUT1 and ZnT3 (34%). Using VGLUT1-ZnT3 SVs, we demonstrate that the transporter colocalization influences the SV content and synaptic quantal size. Thus, the presence of diverse transporters on the same vesicle is bona fide, and depending on the VT types, this may act to regulate neurotransmitter type, content, and release in space and time.


Asunto(s)
Proteínas de Transporte de Neurotransmisores , Vesículas Sinápticas , Animales , Mamíferos , Proteínas de Transporte de Membrana , Neurotransmisores , Sinapsis , Vesículas Sinápticas/fisiología , Proteína 1 de Transporte Vesicular de Glutamato
19.
Exp Biol Med (Maywood) ; 247(11): 910-920, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35285286

RESUMEN

Platinum-based chemotherapy, especially carboplatin, is the primary measure to treat patients with ovarian cancer (OC). However, OC patients still have an adverse prognosis due to emergency of chemotherapy resistance. Ovarian serous cystadenocarcinoma (OSC) is the most common histological subtype of OC. Therefore, identifying the key factors that affect chemotherapy resistance and searching novel treatments had become a top priority. In this study, we analyzed carboplatin response-related mRNA, miRNA, DNA methylation, and alternative splicing (AS) and established a drug-resistant signature for carboplatin in OSC. This drug-resistant signature was obviously higher in resistant group than in non-resistant group and had accuracy predictive performance, which demonstrated that this signature could be considered as a superior indicator for OSC patients with carboplatin resistance. Furthermore, we selected three potential small molecule drugs including liranaftate, siguazodan, and tramiprostate to inhibit carboplatin resistance of OSC. In addition, we also identified ZINC00000205417, ZINC00000140928, and ZINC00021908260 were potential small molecule compounds for SLC17A7 based on Molecular Operating Environment (MOE) virtual screening. Finally, we confirmed the drug-like properties of these small molecule drugs via evaluating absorption, distribution, metabolism, elimination, and toxicity (ADMET) property. In summary, the signature could be used as biomarker for carboplatin resistance and small molecule drugs targeting these genes could improve clinical treatment for OSC in the future.


Asunto(s)
Cistadenocarcinoma Seroso , Neoplasias Ováricas , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carboplatino/farmacología , Carboplatino/uso terapéutico , Carcinoma Epitelial de Ovario/tratamiento farmacológico , Carcinoma Epitelial de Ovario/genética , Cistadenocarcinoma Seroso/tratamiento farmacológico , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Proteína 1 de Transporte Vesicular de Glutamato
20.
J Comp Neurol ; 530(7): 1112-1125, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-34468980

RESUMEN

Glutamate is packaged in vesicles via two main vesicular transporter (VGLUT) proteins, VGLUT1 and VGLUT2, which regulate its storage and release from synapses of excitatory neurons. Studies in rodents, primates, ferrets, and tree shrews suggest that these transporters may identify distinct subsets of excitatory projections in visual structures, particularly in thalamocortical pathways where they tend to correlate with modulatory and driver projections, respectively. Despite being a well-studied model of thalamocortical connectivity, little is known about their expression pattern in the cat visual system. To expand current knowledge on their distribution and how they correlated with known driver and modulator projecting sites, we examined the protein expression patterns of VGLUT1 and VGLUT2 in the visual thalamus of the cat (lateral geniculate nucleus and the pulvinar complex). We also studied their expression pattern in relevant visual structures projecting to or receiving significant thalamic projections, such as the primary visual cortex and the superior colliculus. Our results indicate that both VGLUTs are consistently present throughout the cat visual system and show laminar or nuclei specificity in their distribution, which suggests, as in other species, that VGLUT1 and VGLUT2 represent distinct populations of glutamatergic projections.


Asunto(s)
Hurones , Tálamo , Animales , Hurones/metabolismo , Hibridación in Situ , Tálamo/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo
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