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1.
J Neuroinflammation ; 21(1): 130, 2024 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-38750510

RESUMEN

Epidemiological studies have unveiled a robust link between exposure to repetitive mild traumatic brain injury (r-mTBI) and elevated susceptibility to develop neurodegenerative disorders, notably chronic traumatic encephalopathy (CTE). The pathogenic lesion in CTE cases is characterized by the accumulation of hyperphosphorylated tau in neurons around small cerebral blood vessels which can be accompanied by astrocytes that contain phosphorylated tau, the latter termed tau astrogliopathy. However, the contribution of tau astrogliopathy to the pathobiology and functional consequences of r-mTBI/CTE or whether it is merely a consequence of aging remains unclear. We addressed these pivotal questions by utilizing a mouse model harboring tau-bearing astrocytes, GFAPP301L mice, subjected to our r-mTBI paradigm. Despite the fact that r-mTBI did not exacerbate tau astrogliopathy or general tauopathy, it increased phosphorylated tau in the area underneath the impact site. Additionally, gene ontology analysis of tau-bearing astrocytes following r-mTBI revealed profound alterations in key biological processes including immunological and mitochondrial bioenergetics. Moreover, gene array analysis of microdissected astrocytes accrued from stage IV CTE human brains revealed an immunosuppressed astroglial phenotype similar to tau-bearing astrocytes in the GFAPP301L model. Additionally, hippocampal reduction of proteins involved in water transport (AQP4) and glutamate homeostasis (GLT1) was found in the mouse model of tau astrogliopathy. Collectively, these findings reveal the importance of understanding tau astrogliopathy and its role in astroglial pathobiology under normal circumstances and following r-mTBI. The identified mechanisms using this GFAPP301L model may suggest targets for therapeutic interventions in r-mTBI pathogenesis in the context of CTE.


Asunto(s)
Acuaporina 4 , Astrocitos , Transportador 2 de Aminoácidos Excitadores , Ratones Transgénicos , Tauopatías , Proteínas tau , Astrocitos/metabolismo , Astrocitos/patología , Animales , Ratones , Proteínas tau/metabolismo , Proteínas tau/genética , Acuaporina 4/metabolismo , Acuaporina 4/genética , Tauopatías/metabolismo , Tauopatías/patología , Tauopatías/genética , Humanos , Transportador 2 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/biosíntesis , Conmoción Encefálica/metabolismo , Conmoción Encefálica/patología , Masculino , Fenotipo , Ratones Endogámicos C57BL
2.
Nat Commun ; 15(1): 4347, 2024 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-38773146

RESUMEN

Epigenetic mechanisms bridge genetic and environmental factors that contribute to the pathogenesis of major depression disorder (MDD). However, the cellular specificity and sensitivity of environmental stress on brain epitranscriptomics and its impact on depression remain unclear. Here, we found that ALKBH5, an RNA demethylase of N6-methyladenosine (m6A), was increased in MDD patients' blood and depression models. ALKBH5 in astrocytes was more sensitive to stress than that in neurons and endothelial cells. Selective deletion of ALKBH5 in astrocytes, but not in neurons and endothelial cells, produced antidepressant-like behaviors. Astrocytic ALKBH5 in the mPFC regulated depression-related behaviors bidirectionally. Meanwhile, ALKBH5 modulated glutamate transporter-1 (GLT-1) m6A modification and increased the expression of GLT-1 in astrocytes. ALKBH5 astrocyte-specific knockout preserved stress-induced disruption of glutamatergic synaptic transmission, neuronal atrophy and defective Ca2+ activity. Moreover, enhanced m6A modification with S-adenosylmethionine (SAMe) produced antidepressant-like effects. Our findings indicate that astrocytic epitranscriptomics contribute to depressive-like behaviors and that astrocytic ALKBH5 may be a therapeutic target for depression.


Asunto(s)
Desmetilasa de ARN, Homólogo 5 de AlkB , Astrocitos , Trastorno Depresivo Mayor , Ratones Noqueados , Animales , Astrocitos/metabolismo , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Ratones , Humanos , Trastorno Depresivo Mayor/metabolismo , Trastorno Depresivo Mayor/genética , Trastorno Depresivo Mayor/patología , Masculino , Femenino , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Neuronas/metabolismo , Estrés Psicológico/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/genética , Conducta Animal , Corteza Prefrontal/metabolismo , Corteza Prefrontal/patología , Depresión/metabolismo , Depresión/genética , Adulto , Transmisión Sináptica , Persona de Mediana Edad
3.
Nat Commun ; 15(1): 4549, 2024 May 29.
Artículo en Inglés | MEDLINE | ID: mdl-38811525

RESUMEN

Breast cancer metastasis to the brain is a clinical challenge rising in prevalence. However, the underlying mechanisms, especially how cancer cells adapt a distant brain niche to facilitate colonization, remain poorly understood. A unique metabolic feature of the brain is the coupling between neurons and astrocytes through glutamate, glutamine, and lactate. Here we show that extracellular vesicles from breast cancer cells with a high potential to develop brain metastases carry high levels of miR-199b-5p, which shows higher levels in the blood of breast cancer patients with brain metastases comparing to those with metastatic cancer in other organs. miR-199b-5p targets solute carrier transporters (SLC1A2/EAAT2 in astrocytes and SLC38A2/SNAT2 and SLC16A7/MCT2 in neurons) to hijack the neuron-astrocyte metabolic coupling, leading to extracellular retention of these metabolites and promoting cancer cell growth. Our findings reveal a mechanism through which cancer cells of a non-brain origin reprogram neural metabolism to fuel brain metastases.


Asunto(s)
Astrocitos , Neoplasias Encefálicas , Neoplasias de la Mama , MicroARNs , Neuronas , Humanos , MicroARNs/metabolismo , MicroARNs/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias Encefálicas/secundario , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Femenino , Animales , Línea Celular Tumoral , Astrocitos/metabolismo , Astrocitos/patología , Neuronas/metabolismo , Neuronas/patología , Ratones , Transportador 2 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/genética , Vesículas Extracelulares/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Regulación Neoplásica de la Expresión Génica , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Ácido Láctico/metabolismo , Proliferación Celular
4.
Horm Behav ; 162: 105548, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38636205

RESUMEN

Thyroid hormones are crucial for brain development and their deficiency during fetal and postnatal periods can lead to mood and cognitive disorders. We aimed to examine the consequences of thyroid hormone deficiency on anxiety-related behaviors and protein expression of hippocampal glutamate transporters in congenital hypothyroid male offspring rats. Possible beneficial effects of treadmill exercise have also been examined. Congenital hypothyroidism was induced by adding propylthiouracil (PTU) to drinking water of pregnant Wistar rats from gestational day 6 until the end of the weaning period (postnatal day 28). Next, following 4 weeks of treadmill exercise (5 days per week), anxiety-related behaviors were examined using elevated plus maze (EPM) and light/dark box tests. Thereafter, protein expression of astrocytic (GLAST and GLT-1) and neuronal (EAAC1) glutamate transporters were measured in the hippocampus by immunoblotting. Hypothyroid rats showed decreased anxiety-like behavior, as measured by longer time spent in the open arms of the EPM and in the light area of the light/dark box, compared to control rats. Hypothyroid rats had significantly higher GLAST and GLT-1 and lower EAAC1 protein levels in the hippocampus than did the euthyroid rats. Following exercise, anxiety levels decreased in the euthyroid group while protein expression of EAAC1 increased and returned to normal levels in the hypothyroid group. Our findings indicate that thyroid hormone deficiency was associated with alterations in protein expression of glutamate transporters in the hippocampus. Up-regulation of hippocampal GLAST and GLT-1 could be at least one of the mechanisms associated with the anxiolytic effects of congenital hypothyroidism.


Asunto(s)
Ansiedad , Hipotiroidismo Congénito , Transportador 2 de Aminoácidos Excitadores , Hipocampo , Ratas Wistar , Animales , Masculino , Hipocampo/metabolismo , Ansiedad/metabolismo , Ansiedad/etiología , Ratas , Femenino , Hipotiroidismo Congénito/metabolismo , Embarazo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/genética , Hormonas Tiroideas/metabolismo , Transportador 1 de Aminoácidos Excitadores/metabolismo , Transportador 1 de Aminoácidos Excitadores/genética , Transportador 3 de Aminoácidos Excitadores/metabolismo , Transportador 3 de Aminoácidos Excitadores/genética , Conducta Animal/fisiología , Propiltiouracilo , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Sistema de Transporte de Aminoácidos X-AG/genética , Efectos Tardíos de la Exposición Prenatal/metabolismo
5.
Biomolecules ; 14(4)2024 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-38672445

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a fatal motoneuron degenerative disease that is associated with demyelination. The Wobbler (WR) mouse exhibits motoneuron degeneration, gliosis and myelin deterioration in the cervical spinal cord. Since male WRs display low testosterone (T) levels in the nervous system, we investigated if T modified myelin-relative parameters in WRs in the absence or presence of the aromatase inhibitor, anastrozole (A). We studied myelin by using luxol-fast-blue (LFB) staining, semithin sections, electron microscopy and myelin protein expression, density of IBA1+ microglia and mRNA expression of inflammatory factors, and the glutamatergic parameters glutamine synthetase (GS) and the transporter GLT1. Controls and WR + T showed higher LFB, MBP and PLP staining, lower g-ratios and compact myelin than WRs and WR + T + A, and groups showing the rupture of myelin lamellae. WRs showed increased IBA1+ cells and mRNA for CD11b and inflammatory factors (IL-18, TLR4, TNFαR1 and P2Y12R) vs. controls or WR + T. IBA1+ cells, and CD11b were not reduced in WR + T + A, but inflammatory factors' mRNA remained low. A reduction of GS+ cells and GLT-1 immunoreactivity was observed in WRs and WR + T + A vs. controls and WR + T. Clinically, WR + T but not WR + T + A showed enhanced muscle mass, grip strength and reduced paw abnormalities. Therefore, T effects involve myelin protection, a finding of potential clinical translation.


Asunto(s)
Esclerosis Amiotrófica Lateral , Modelos Animales de Enfermedad , Vaina de Mielina , Testosterona , Animales , Ratones , Vaina de Mielina/metabolismo , Vaina de Mielina/efectos de los fármacos , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Masculino , Testosterona/farmacología , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Transportador 2 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/genética , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología
6.
J Biol Chem ; 300(4): 107172, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38499151

RESUMEN

The recently discovered interaction between Presenilin 1 (PS1), a catalytic subunit of γ-secretase responsible for generating amyloid-ß peptides, and GLT-1, a major glutamate transporter in the brain (EAAT2), provides a mechanistic link between these two key factors involved in Alzheimer's disease (AD) pathology. Modulating this interaction can be crucial to understand the consequence of such crosstalk in AD context and beyond. However, the interaction sites between these two proteins are unknown. Herein, we utilized an alanine scanning approach coupled with FRET-based fluorescence lifetime imaging microscopy to identify the interaction sites between PS1 and GLT-1 in their native environment within intact cells. We found that GLT-1 residues at position 276 to 279 (TM5) and PS1 residues at position 249 to 252 (TM6) are crucial for GLT-1-PS1 interaction. These results have been cross validated using AlphaFold Multimer prediction. To further investigate whether this interaction of endogenously expressed GLT-1 and PS1 can be prevented in primary neurons, we designed PS1/GLT-1 cell-permeable peptides (CPPs) targeting the PS1 or GLT-1 binding site. We used HIV TAT domain to allow for cell penetration which was assayed in neurons. First, we assessed the toxicity and penetration of CPPs by confocal microscopy. Next, to ensure the efficiency of CPPs, we monitored the modulation of GLT-1-PS1 interaction in intact neurons by fluorescence lifetime imaging microscopy. We saw significantly less interaction between PS1 and GLT-1 with both CPPs. Our study establishes a new tool to study the functional aspect of GLT-1-PS1 interaction and its relevance in normal physiology and AD models.


Asunto(s)
Transportador 2 de Aminoácidos Excitadores , Presenilina-1 , Animales , Humanos , Ratones , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Sitios de Unión , Transportador 2 de Aminoácidos Excitadores/química , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Neuronas/metabolismo , Presenilina-1/química , Presenilina-1/genética , Presenilina-1/metabolismo , Unión Proteica , Péptidos/metabolismo
7.
Ann Clin Transl Neurol ; 10(9): 1695-1699, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37452008

RESUMEN

Dravet syndrome (DS) is a monogenic, often refractory, epilepsy resultant from SCN1A haploinsufficiency in humans. A novel therapeutic target in DS that can be engaged in isolation or as adjunctive therapy is highly desirable. Here, we demonstrate reduced expression of the rodent glutamate transporter type 1 (GLT-1) in a DS mouse model, and in wild type mouse strains where Scn1a haploinsufficiency is most likely to cause epilepsy, indicating that GLT-1 depression may play a role in DS seizures. As GLT-1 can be upregulated by common and safe FDA-approved medications, this strategy may be an attractive, viable, and novel avenue for DS treatment.


Asunto(s)
Epilepsias Mioclónicas , Epilepsia , Transportador 2 de Aminoácidos Excitadores , Animales , Humanos , Ratones , Sistema de Transporte de Aminoácidos X-AG , Epilepsias Mioclónicas/genética , Canal de Sodio Activado por Voltaje NAV1.1/genética , Convulsiones , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo
8.
Neurochem Res ; 48(9): 2847-2856, 2023 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-37178383

RESUMEN

Glial cells give rise to glioblastoma multiform as a primary brain tumor. In glioblastomas, neurons are destroyed via excitotoxicity which is the accumulation of excess glutamate in synaptic cavity. Glutamate Transporter 1 (GLT-1) is the main transporter that absorbs the excessive glutamate. Sirtuin 4 (SIRT4) was shown to have a potential protective role against excitotoxicity in previous studies. In this study, the regulation of dynamic GLT-1 expression by SIRT4 was analyzed in glia (immortalized human astrocytes) and glioblastoma (U87) cells. The expression of GLT-1 dimers and trimers were reduced and the ubiquitination of GLT-1 was increased in glioblastoma cells when SIRT4 was silenced; however GLT-1 monomer was not affected. In glia cells, SIRT4 reduction did not affect GLT-1 monomer, dimer, trimer expression or the ubiquitination of GLT-1. The phosphorylation of Nedd4-2 and the expression of PKC did not change in glioblastoma cells when SIRT4 was silenced but increased in glia cells. We also showed that SIRT4 deacetylates PKC in glia cells. In addition, GLT-1 was shown to be deacetylated by SIRT4 which might be a priority for ubiquitination. Therefore, we conclude that GLT-1 expression is regulated differently in glia and glioblastoma cells. SIRT4 activators or inhibitors of ubiquitination may be used to prevent excitotoxicity in glioblastomas.


Asunto(s)
Transportador 2 de Aminoácidos Excitadores , Glioblastoma , Sirtuinas , Humanos , Astrocitos/metabolismo , Transportador 2 de Aminoácidos Excitadores/genética , Glioblastoma/metabolismo , Ácido Glutámico/metabolismo , Proteínas Mitocondriales/metabolismo , Neuronas/metabolismo , Sirtuinas/metabolismo , Ubiquitinación , Proteolisis
9.
Psychopharmacology (Berl) ; 240(4): 837-851, 2023 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-36725696

RESUMEN

RATIONALE AND OBJECTIVE: Post-traumatic stress disorder (PTSD) is a prevalent and debilitating psychiatric disorder. However, its specific etiological mechanism remains unclear. Previous studies have shown that traumatic stress changes metabotropic glutamate receptor 5 (mGluR5) expression in the hippocampus (HIP) and prefrontal cortex (PFC). More importantly, mGluR5 expression is often accompanied by alterations in brain-derived neurotrophic factor (BDNF). Furthermore, BDNF/tropomyosin-associated kinase B (TrkB) signaling plays multiple roles, including roles in neuroplasticity and antidepressant activity, by regulating glutamate transporter-1 (GLT-1) expression. This study aims to explore the effects of inhibiting mGluR5 on PTSD-like behaviors and BDNF, TrkB, and GLT-1 expression in the HIP and PFC of inevitable foot shock (IFS)-treated rats. METHODS: Seven-day IFS was used to establish a PTSD rat model, and 2-methyl-6-(phenylethynyl)-pyridine (MPEP) (10 mg/kg, intraperitoneal injection) was used to inhibit the activity of mGluR5 during IFS in rats. After modeling, behavioral changes and mGluR5, BDNF, TrkB, and GLT-1 expression in the PFC and HIP were examined. RESULTS: First, the IFS procedure induced PTSD-like behavior. Second, IFS increased the expression of mGluR5 and decreased BDNF, TrkB, and GLT-1 expression in the PFC and HIP. Third, the mGluR5 antagonist blocked the above behavioral and molecular alterations. CONCLUSIONS: mGluR5 was involved in IFS-induced PTSD-like behavior by changing BDNF, TrkB, and GLT-1 expression.


Asunto(s)
Factor Neurotrófico Derivado del Encéfalo , Transportador 2 de Aminoácidos Excitadores , Receptor del Glutamato Metabotropico 5 , Receptor trkB , Trastornos por Estrés Postraumático , Animales , Ratas , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Hipocampo/metabolismo , Corteza Prefrontal/metabolismo , Receptor del Glutamato Metabotropico 5/genética , Receptor del Glutamato Metabotropico 5/metabolismo , Receptor trkB/genética , Receptor trkB/metabolismo , Trastornos por Estrés Postraumático/genética , Trastornos por Estrés Postraumático/metabolismo , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo
10.
J Biol Chem ; 299(1): 102793, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36509140

RESUMEN

Astrocytic excitatory amino acid transporter 2 (EAAT2) plays a major role in removing the excitatory neurotransmitter L-glutamate (L-Glu) from synaptic clefts in the forebrain to prevent excitotoxicity. Polyunsaturated fatty acids such as docosahexaenoic acid (DHA, 22:6 n-3) enhance synaptic transmission, and their target molecules include EAATs. Here, we aimed to investigate the effect of DHA on EAAT2 and identify the key amino acid for DHA/EAAT2 interaction by electrophysiological recording of L-Glu-induced current in Xenopus oocytes transfected with EAATs, their chimeras, and single mutants. DHA transiently increased the amplitude of EAAT2 but tended to decrease that of excitatory amino acid transporter subtype 1 (EAAT1), another astrocytic EAAT. Single mutation of leucine (Leu) 434 to alanine (Ala) completely suppressed the augmentation by DHA, while mutation of EAAT1 Ala 435 (corresponding to EAAT2 Leu434) to Leu changed the effect from suppression to augmentation. Other polyunsaturated fatty acids (docosapentaenoic acid, eicosapentaenoic acid, arachidonic acid, and α-linolenic acid) similarly augmented the EAAT2 current and suppressed the EAAT1 current. Finally, our docking analysis suggested the most stable docking site is the lipid crevice of EAAT2, in close proximity to the L-Glu and sodium binding sites, suggesting that the DHA/Leu434 interaction might affect the elevator-like slide and/or the shapes of the other binding sites. Collectively, our results highlight a key molecular detail in the DHA-induced regulation of synaptic transmission involving EAATs.


Asunto(s)
Ácidos Docosahexaenoicos , Transportador 2 de Aminoácidos Excitadores , Transmisión Sináptica , Xenopus laevis , Ácidos Docosahexaenoicos/metabolismo , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , Ácido Glutámico/metabolismo , Leucina , Mutación , Xenopus laevis/metabolismo
11.
Metab Brain Dis ; 38(1): 1-16, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36173507

RESUMEN

Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS). Excitatory amino acid transporters (EAATs) have important roles in the uptake of glutamate and termination of glutamatergic transmission. Up to now, five EAAT isoforms (EAAT1-5) have been identified in mammals. The main focus of this review is EAAT2. This protein has an important role in the pathoetiology of epilepsy. De novo dominant mutations, as well as inherited recessive mutation in this gene, have been associated with epilepsy. Moreover, dysregulation of this protein is implicated in a range of neurological diseases, namely amyotrophic lateral sclerosis, alzheimer's disease, parkinson's disease, schizophrenia, epilepsy, and autism. In this review, we summarize the role of EAAT2 in epilepsy and other neurological disorders, then provide an overview of the therapeutic modulation of this protein.


Asunto(s)
Epilepsia , Esquizofrenia , Animales , Humanos , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , Epilepsia/genética , Esquizofrenia/metabolismo , Transporte Biológico , Ácido Glutámico/metabolismo , Mamíferos/metabolismo
12.
Nat Commun ; 13(1): 4714, 2022 08 11.
Artículo en Inglés | MEDLINE | ID: mdl-35953475

RESUMEN

Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states at 3.2 Å and 2.8 Å, respectively. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. We provide the characterization of the molecular features of EAAT2 and its selective inhibition mechanism that may facilitate structure-based drug design for EAAT2.


Asunto(s)
Transportador 2 de Aminoácidos Excitadores/química , Ácido Glutámico , Animales , Sitios de Unión , Encéfalo/metabolismo , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , Transportador 3 de Aminoácidos Excitadores/genética , Transportador 3 de Aminoácidos Excitadores/metabolismo , Ácido Glutámico/metabolismo , Humanos , Mamíferos/metabolismo , Neuroglía/metabolismo
13.
Artículo en Inglés | MEDLINE | ID: mdl-35840289

RESUMEN

OBJECTIVE: Recently, we reported on a new MDD-like mouse model based on a regionally selective knockdown of astroglial glutamate transporters, GLAST/GLT-1, in infralimbic cortex (IL) which evokes widespread changes in mouse brain associated with the typical alterations found in MDD patients. To further characterize this new MDD-like mouse model, here we examine some transcriptional elements of glutamatergic/GABAergic neurotransmission and neuroplasticity in forebrain regions in the GLT-1 knockdown mice. Furthermore, we assess the acute ketamine effects on these transcriptional processes. MATERIAL AND METHODS: We used a small interfering RNA (siRNA) pool targeting GLT-1 mRNA to disrupt the GLT-1 transcription in mouse IL. Histological assays were performed to examine postsynaptic density protein-95 (PSD95), neuritin (NRN), glutamine acid descarboxilase-65 (GAD65), and GLT-1 mRNA expression in IL and hippocampus. RESULTS: Knockdown of GLT-1 in mouse IL leads to decreased expression of PSD95 and NRN neuroplasticity mRNAs in IL and hippocampus, which was reversed by an acute dose of ketamine antidepressant. Likewise, a single dose of ketamine also increased the mRNA levels of GAD65 and GLT-1 in IL of GLT-1 knockdown mice, reaching the basal values of control mice. CONCLUSIONS: The glutamatergic neuronal hyperactivity and deficits in the GABA system resulting from siRNA-induced astroglial glutamate transporter knockdown in IL can compromise the integrity/plasticity of neurocircuits affected in MDD. Suitable depressive-like animal models to address the neurobiological changes in MDD are an unmet need and the development of the GLAST/GLT-1 knockdown mouse model may represent a better option to understand the rapid-acting antidepressant effects of ketamine.


Asunto(s)
Astrocitos , Ketamina , Plasticidad Neuronal , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Animales , Antidepresivos/farmacología , Antidepresivos/uso terapéutico , Astrocitos/metabolismo , Depresión/genética , Depresión/metabolismo , Transportador 2 de Aminoácidos Excitadores/efectos de los fármacos , Transportador 2 de Aminoácidos Excitadores/genética , Humanos , Ketamina/metabolismo , Ketamina/farmacología , Ketamina/uso terapéutico , Ratones , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo
14.
Addict Biol ; 27(4): e13178, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35754102

RESUMEN

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.


Asunto(s)
Alcoholismo , Ceftriaxona , Consumo de Bebidas Alcohólicas/metabolismo , Alcoholismo/genética , Alcoholismo/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animales , Ceftriaxona/metabolismo , Ceftriaxona/farmacología , Etanol/farmacología , Transportador 2 de Aminoácidos Excitadores/genética , Ácido Glutámico/metabolismo , Núcleo Accumbens , Ratas , Recurrencia
15.
Neurosci Lett ; 780: 136637, 2022 05 29.
Artículo en Inglés | MEDLINE | ID: mdl-35439550

RESUMEN

Glutamate transporter 1 is the principal transporter that mediates glutamate clearance in the mammalian brain. In rodents, it is referred to as GLT-1, whereas in humans it is referred to as EAAT2. We have cloned a novel and abundantly expressed carboxyl-terminal splice variant of this transporter in both rodents and humans, which we denote as GLT-1d/EAAT2d. The novel splice variant results from usage of internal splice sites and the splicing event leads to novel extra sequence spliced in after exon 10. The open reading frames of GLT-1d and EAAT2d encode proteins of 572 and 566 amino acids respectively; both contain a C-terminal PDZ motif. When expressed in COS7 cells, the proteins function as glutamate transporters that are inhibited by dihydrokainate (a GLT-1/EAAT2 transporter inhibitor). RT-PCR amplification using GLT-1d specific primers confirmed expression of message in all brain regions examined (forebrain, midbrain, hindbrain and cerebellum) as well as spinal cord, astrocyte cultures, retina and peripheral tissues (liver, testis, small intestine and lung). Quantitative RT-PCR analysis showed that expression of GLT-1d is developmentally regulated. In adult human brain, EAAT2d message is âˆ¼ 30% of the level of EAAT2a message (the most abundant form), potentially making it the second most abundantly expressed form of EAAT2 in the brain. The amino terminal region of GLT-1d is also alternately spliced; the brain and testis forms contain a sequence corresponding to the amino acid sequence MASTEG whereas the corresponding liver sequence is MVS. In summary, we have cloned a novel EAAT2/GLT-1 splice variant from human and rodent brains. The splice variant is abundantly expressed in the brain, spinal cord, retina, liver and testis; it is a functional glutamate transporter; therefore, we conclude that it will likely have a functional role in glutamate homeostasis in the rodent and human nervous system, during development, adulthood, and plausibly in pathological states.


Asunto(s)
Transportador 2 de Aminoácidos Excitadores , Roedores , Adulto , Animales , Encéfalo/metabolismo , Clonación Molecular , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , Ácido Glutámico/metabolismo , Humanos , Masculino , Roedores/metabolismo
16.
Mol Neurobiol ; 59(7): 3996-4014, 2022 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-35451738

RESUMEN

Long noncoding RNAs (lncRNAs) play an important regulatory role in various diseases. However, the role of lncRNAs in brain ischemic tolerance (BIT) induced by cerebral ischemic preconditioning (CIPC) is still unknown. The lncRNA profile of rat cortical astrocytes pretreated with ischemic preconditioning was analyzed by high-throughput sequencing. The results of Cell-Counting Kit-8 (CCK-8) assay showed that a novel lncRNA, NONRATT009133.2, which we referred to as brain ischemia-related factor (BIRF), was highly correlated with BIT. Through bioinformatics analysis, we predicted that BIRF, miR-330-5p, and GLT-1 (also named Slc1a2) might constitute a ceRNA regulatory network in the induction of BIT. We found that BIRF was upregulated by CIPC, which promoted GLT-1 expression and BIT induction. BIRF could directly bind to miR-330-5p. Furthermore, miR-330-5p directly targeted GLT-1, and miR-330-5p inhibited both GLT-1 expression and BIT induction in vitro and in vivo. Moreover, BIRF acts as a molecular sponge to competitively bind to miR-330-5p with GLT-1 mRNA, while the miR-330-5p inhibitor reversed all the effects of BIRF siRNA on GLT-1 expression and neuronal vitality. Taken together, our results demonstrate the important roles of the BIRF/miR-330-5p/GLT-1 axis in the induction of BIT by CIPC. BIRF may be a potentially effective therapeutic strategy against stroke injury.


Asunto(s)
Isquemia Encefálica , Transportador 2 de Aminoácidos Excitadores , Precondicionamiento Isquémico , MicroARNs , ARN Largo no Codificante , Animales , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Clorprofam , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Ratas
17.
BMC Psychiatry ; 22(1): 171, 2022 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-35260124

RESUMEN

BACKGROUND: Recent studies have shown that the excitatory amino acid transporters (EAATs) are associated with schizophrenia. The aim of this study was to investigate the relationship between the polymorphism of EAAT1 and EAAT2 genes and schizophrenia in Chinese Han population. METHODS: A total of 233 patients with schizophrenia and 342 healthy controls were enrolled. Two SNPs in EAAT1 gene (rs2269272, rs2731880) and four SNPs in EAAT2 gene (rs12360706, rs3088168, rs12294045, rs10836387) were genotyped by SNaPshot. Clinical features were collected using a self-made questionnaire. Psychotic symptoms of patients were measured by the Positive and Negative Syndrome Scale (PANSS), and patients' cognitive function was assessed by Matrics Consensus Cognitive Battery (MCCB). RESULTS: Significant difference in allelic distributions between cases and controls was confirmed at locus rs12294045 (Ρ = 0.004) of EAAT2 gene. Different genotypes of rs12294045 were associated with family history (P = 0.046), in which patients with CT genotype had higher proportion of family history of psychosis. The polymorphism of rs12294045 was related to working operational memory (LNS: P = 0.016) and verbal learning function (HVLT-R: P = 0.042) in patients in which CT genotype had lower scores. However, these differences were no longer significant after Bonferroni correction. CONCLUSIONS: Our study showed that the polymorphism of rs12294045 in EAAT2 gene may be associated with schizophrenia in Chinese Han population. CT genotype may be one of the risk factors for family history and cognitive deficits of patients.


Asunto(s)
Disfunción Cognitiva , Transportador 2 de Aminoácidos Excitadores/genética , Esquizofrenia , Pueblo Asiatico/genética , China , Disfunción Cognitiva/diagnóstico , Humanos , Polimorfismo de Nucleótido Simple , Esquizofrenia/diagnóstico
19.
ACS Chem Neurosci ; 13(5): 676-687, 2022 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-35148069

RESUMEN

Excitatory neurotoxicity caused by the accumulation of glutamate in the synaptic cleft is an important cause of Parkinson's disease (PD). Astrocyte glutamate transporter 1 (GLT-1) is the main transporter responsible for transporting glutamate, and investigations toward the regulation of GLT-1 in astrocytes can reveal important insights. Vitamin C (VC) has important protective effects on the brain, but its effect on the regulation of GLT-1 expression is unclear. The purpose of this study was to explore any regulatory effect of VC on GLT-1 expression in astrocytes and to clarify the possible mechanism of such regulation. We found that GLT-1 expression was impaired in 1-methyl-4-phenylpyridinium iodide (MPP+)-treated astrocytes, and the transport capacity for glutamate was significantly reduced. Pretreatment with VC restored the GLT-1 expression in the MPP+-treated astrocytes. Intraperitoneal VC administration in a PD murine model confirmed that GLT-1 expression was restored in midbrain tissue. The VC-dependent rescue of GLT-1 expression in the MPP+-treated astrocytes was shown to be due to inhibition of GLT-1 ubiquitination. Transcriptome sequence analysis revealed a number of differentially expressed genes as a result of VC treatment on MPP+-treated astrocytes, including the downregulation of HECT Domain E3 ubiquitin protein ligase 1 (Hectd1). After knocking down Hectd1, the impaired GLT-1 expression caused by MPP+ was alleviated, while overexpression of Hectd1 significantly reduced the expression of GLT-1. After overexpression of Hectd1, VC could no longer increase GLT-1 expression of MPP+-treated astrocytes, indicating that HECTD1 is essential for VC regulation of GLT-1. Thus, VC reduces the ubiquitination of GLT-1 in astrocytes by inhibiting the expression of HECTD1. Our findings have identified a novel mechanism by which VC regulates the expression of GLT-1 in astrocytes.


Asunto(s)
Ácido Ascórbico/farmacología , Astrocitos , Transportador 2 de Aminoácidos Excitadores , Ubiquitina-Proteína Ligasas , Animales , Astrocitos/metabolismo , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , Ácido Glutámico/metabolismo , Ratones , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación
20.
Epilepsia ; 63(2): 388-401, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34961934

RESUMEN

OBJECTIVE: Mutations in the gene solute carrier family member 1A2 (SLC1A2) encoding the excitatory amino acid transporter 2 (EAAT2) are associated with severe forms of epileptic encephalopathy. EAAT2 is expressed in glial cells and presynaptic nerve terminals and represents the main l-glutamate uptake carrier in the mammalian brain. It does not only function as a secondary active glutamate transporter, but also as an anion channel. How naturally occurring mutations affect these two transport functions of EAAT2 and how such alterations cause epilepsy is insufficiently understood. METHODS: Here we studied the functional consequences of three disease-associated mutations, which predict amino acid exchanges p.Gly82Arg (G82R), p.Leu85Pro (L85P), and p.Pro289Arg (P289R), by heterologous expression in mammalian cells, biochemistry, confocal imaging, and whole-cell patch-clamp recordings of EAAT2 l-glutamate transport and anion current. RESULTS: G82R and L85P exchange amino acid residues that contribute to the formation of the EAAT anion pore. They enlarge the pore diameter sufficiently to permit the passage of l-glutamate and thus function as l-glutamate efflux pathways. The mutation P289R decreases l-glutamate uptake, but increases anion currents despite a lower membrane expression. SIGNIFICANCE: l-glutamate permeability of the EAAT anion pore is an unexpected functional consequence of naturally occurring single amino acid substitutions. l-glutamate efflux through mutant EAAT2 anion channels will cause glutamate excitotoxicity and neuronal hyperexcitability in affected patients. Antagonists that selectively suppress the EAAT anion channel function could serve as therapeutic agents in the future.


Asunto(s)
Encefalopatías , Transportador 2 de Aminoácidos Excitadores , Aminoácidos/metabolismo , Animales , Aniones/metabolismo , Transportador 2 de Aminoácidos Excitadores/química , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Humanos , Mamíferos/metabolismo , Mutación/genética
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