RESUMO
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.
Assuntos
Transportador 2 de Aminoácido Excitatório , Presenilina-1 , Animais , Humanos , Camundongos , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Sítios de Ligação , Transportador 2 de Aminoácido Excitatório/química , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Neurônios/metabolismo , Presenilina-1/química , Presenilina-1/genética , Presenilina-1/metabolismo , Ligação Proteica , Peptídeos/metabolismoRESUMO
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.
Assuntos
Aquaporina 4 , Astrócitos , Transportador 2 de Aminoácido Excitatório , Camundongos Transgênicos , Tauopatias , Proteínas tau , Animais , Humanos , Masculino , Camundongos , Aquaporina 4/metabolismo , Aquaporina 4/genética , Astrócitos/metabolismo , Astrócitos/patologia , Concussão Encefálica/metabolismo , Concussão Encefálica/patologia , Transportador 2 de Aminoácido Excitatório/metabolismo , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/biossíntese , Camundongos Endogâmicos C57BL , Fenótipo , Proteínas tau/metabolismo , Proteínas tau/genética , Tauopatias/metabolismo , Tauopatias/patologia , Tauopatias/genéticaRESUMO
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.
Assuntos
Ansiedade , Hipotireoidismo Congênito , Transportador 2 de Aminoácido Excitatório , Hipocampo , Ratos Wistar , Animais , Masculino , Hipocampo/metabolismo , Ansiedade/metabolismo , Ansiedade/etiologia , Ratos , Feminino , Hipotireoidismo Congênito/metabolismo , Gravidez , Transportador 2 de Aminoácido Excitatório/metabolismo , Transportador 2 de Aminoácido Excitatório/genética , Hormônios Tireóideos/metabolismo , Transportador 1 de Aminoácido Excitatório/metabolismo , Transportador 1 de Aminoácido Excitatório/genética , Transportador 3 de Aminoácido Excitatório/metabolismo , Transportador 3 de Aminoácido Excitatório/genética , Comportamento Animal/fisiologia , Propiltiouracila , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Sistema X-AG de Transporte de Aminoácidos/genética , Efeitos Tardios da Exposição Pré-Natal/metabolismoRESUMO
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.
Assuntos
Esclerose Lateral Amiotrófica , Modelos Animais de Doenças , Bainha de Mielina , Testosterona , Animais , Camundongos , Bainha de Mielina/metabolismo , Bainha de Mielina/efeitos dos fármacos , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Masculino , Testosterona/farmacologia , Medula Espinal/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Transportador 2 de Aminoácido Excitatório/metabolismo , Transportador 2 de Aminoácido Excitatório/genética , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologiaRESUMO
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.
Assuntos
Homólogo AlkB 5 da RNA Desmetilase , Astrócitos , Transtorno Depressivo Maior , Camundongos Knockout , Animais , Astrócitos/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Camundongos , Humanos , Transtorno Depressivo Maior/metabolismo , Transtorno Depressivo Maior/genética , Transtorno Depressivo Maior/patologia , Masculino , Feminino , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Estresse Psicológico/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Transportador 2 de Aminoácido Excitatório/genética , Comportamento Animal , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/patologia , Depressão/metabolismo , Depressão/genética , Adulto , Transmissão Sináptica , Pessoa de Meia-IdadeRESUMO
Excitatory amino acid transporters (EAATs) are responsible for excitatory amino acid transportation and are associated with auto-immune diseases in the central nervous system and peripheral tissues. However, the subcellular location and function of EAAT2 in macrophages are still obscure. In this study, we demonstrated that LPS stimulation increases expression of EAAT2 (coded by Slc1a2) via NF-κB signaling. EAAT2 is necessary for inflammatory macrophage polarization through sustaining mTORC1 activation. Mechanistically, lysosomal EAAT2 mediates lysosomal glutamate and aspartate efflux to maintain V-ATPase activation, which sustains macropinocytosis and mTORC1. We also found that mice with myeloid depletion of Slc1a2 show alleviated inflammatory responses in LPS-induced systemic inflammation and high-fat diet induced obesity. Notably, patients with type II diabetes (T2D) have a higher level of expression of lysosomal EAAT2 and activation of mTORC1 in blood macrophages. Taken together, our study links the subcellular location of amino acid transporters with the fate decision of immune cells, which provides potential therapeutic targets for the treatment of inflammatory diseases.
Assuntos
Transportador 2 de Aminoácido Excitatório , Inflamação , Lisossomos , Macrófagos , Alvo Mecanístico do Complexo 1 de Rapamicina , Animais , Macrófagos/metabolismo , Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Transportador 2 de Aminoácido Excitatório/metabolismo , Transportador 2 de Aminoácido Excitatório/genética , Lisossomos/metabolismo , Inflamação/metabolismo , Inflamação/imunologia , Humanos , Lipopolissacarídeos/farmacologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/imunologia , Ácido Glutâmico/metabolismo , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Ácido Aspártico/metabolismo , Masculino , Obesidade/metabolismo , Obesidade/imunologia , Transdução de Sinais , Dieta Hiperlipídica/efeitos adversos , Pinocitose/efeitos dos fármacosRESUMO
Astrocyte-elevated gene-1 (AEG-1/MTDH/LYRIC) has garnered signficant attention in cancer research, yet, its role in inflammation-associated astrogliosis remains underexplored. This study aims to elucidate the effects of AEG-1 on reactive astrogliosis, including proliferation, migration, and glutamate uptake in primary astrocytes derived from rats. We first confirmed the effect of AEG-1 on these parameters. Subsequently, we investigated whether AEG-1 plays a role in the process of pro-inflammation factors such as tumor necrosis factor-alpha (TNF-α) induced astrogliosis. Our findings revealed that AEG-1-lentivirus infection led to hypertrophic cell bodies and enhanced expression of astrogliosis markers, including glial fibrillary acidic protein (GFAP) and vimentin. Additionally, AEG-1 was found to upregulate the mRNA and protein expression levels of EAAT2, a major glutamate transporter in the brain predominantly expressed by astrocytes and responsible for 90% of glutamate clearance. Furthermore, TNF-α was shown to promote astrogliosis, as well as astrocyte proliferation and migration, by upregulating AEG-1 expression through the NF-κB pathway. Collectively, these results suggest a potential role for AEG-1 in inflammation-related astrogliosis.
Assuntos
Astrócitos , Gliose , Proteínas de Membrana , NF-kappa B , Proteínas de Ligação a RNA , Fator de Necrose Tumoral alfa , Regulação para Cima , Animais , Astrócitos/metabolismo , Astrócitos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , NF-kappa B/metabolismo , Gliose/metabolismo , Gliose/patologia , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Ratos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ratos Sprague-Dawley , Transdução de Sinais , Células Cultivadas , Transportador 2 de Aminoácido Excitatório/metabolismo , Transportador 2 de Aminoácido Excitatório/genética , Proteína Glial Fibrilar Ácida/metabolismo , Ácido Glutâmico/metabolismoRESUMO
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.