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Proc Natl Acad Sci U S A ; 118(38)2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34479991


COVID-19 induces a robust, extended inflammatory "cytokine storm" that contributes to an increased morbidity and mortality, particularly in patients with type 2 diabetes (T2D). Macrophages are a key innate immune cell population responsible for the cytokine storm that has been shown, in T2D, to promote excess inflammation in response to infection. Using peripheral monocytes and sera from human patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and a murine hepatitis coronavirus (MHV-A59) (an established murine model of SARS), we identified that coronavirus induces an increased Mφ-mediated inflammatory response due to a coronavirus-induced decrease in the histone methyltransferase, SETDB2. This decrease in SETDB2 upon coronavirus infection results in a decrease of the repressive trimethylation of histone 3 lysine 9 (H3K9me3) at NFkB binding sites on inflammatory gene promoters, effectively increasing inflammation. Mφs isolated from mice with a myeloid-specific deletion of SETDB2 displayed increased pathologic inflammation following coronavirus infection. Further, IFNß directly regulates SETDB2 in Mφs via JaK1/STAT3 signaling, as blockade of this pathway altered SETDB2 and the inflammatory response to coronavirus infection. Importantly, we also found that loss of SETDB2 mediates an increased inflammatory response in diabetic Mϕs in response to coronavirus infection. Treatment of coronavirus-infected diabetic Mφs with IFNß reversed the inflammatory cytokine production via up-regulation of SETDB2/H3K9me3 on inflammatory gene promoters. Together, these results describe a potential mechanism for the increased Mφ-mediated cytokine storm in patients with T2D in response to COVID-19 and suggest that therapeutic targeting of the IFNß/SETDB2 axis in T2D patients may decrease pathologic inflammation associated with COVID-19.

Coronavirus/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/virologia , Macrófagos/metabolismo , Animais , COVID-19/imunologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Síndrome da Liberação de Citocina , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/genética , Feminino , Histona-Lisina N-Metiltransferase/genética , Humanos , Inflamação/metabolismo , Inflamação/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , SARS-CoV-2/metabolismo , Transdução de Sinais
Stem Cells Dev ; 29(17): 1145-1159, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32438891


Bipolar disorder (BP) is a complex psychiatric condition characterized by severe fluctuations in mood for which underlying pathological mechanisms remain unclear. Family and twin studies have identified a hereditary component to the disorder, but a single causative gene (or set of genes) has not been identified. MicroRNAs (miRNAs) are small, noncoding RNAs ∼20 nucleotides in length, that are responsible for the posttranslational regulation of multiple genes. They have been shown to play important roles in neural development as well as in the adult brain, and several miRNAs have been reported to be dysregulated in postmortem brain tissue isolated from bipolar patients. Because there are no viable cellular models to study BP, we have taken advantage of the recent discovery that somatic cells can be reprogrammed to pluripotency then directed to form the full complement of neural cells. Analysis of RNAs extracted from Control and BP patient-derived neurons identified 58 miRNAs that were differentially expressed between the two groups. Using quantitative polymerase chain reaction we validated six miRNAs that were elevated and two miRNAs that were expressed at lower levels in BP-derived neurons. Analysis of the targets of the miRNAs indicate that they may regulate a number of cellular pathways, including axon guidance, Mapk, Ras, Hippo, Neurotrophin, and Wnt signaling. Many are involved in processes previously implicated in BP, such as cell migration, axon guidance, dendrite and synapse development, and function. We have validated targets of several different miRNAs, including AXIN2, BDNF, RELN, and ANK3 as direct targets of differentially expressed miRNAs using luciferase assays. Identification of pathways altered in patient-derived neurons suggests that disruption of these regulatory networks that may contribute to the complex phenotypes in BP.

Biochim Biophys Acta ; 1842(1): 79-87, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24129262


The development of therapies for Amyotrophic Lateral Sclerosis (ALS) has been hindered by the lack of biomarkers for both identifying early disease and for monitoring the effectiveness of drugs. The identification of ALS biomarkers in presymptomatic individuals might also provide clues to the earliest biochemical correlates of the disease. Previous attempts to use plasma metabolites as biomarkers have led to contradictory results, presumably because of heterogeneity in both the underlying genetics and the disease stage in the clinical population. To eliminate these two sources of heterogeneity we have characterized plasma amino acids and other metabolites in the SOD1(G93A) transgenic mouse model for ALS. Presymptomatic SOD1(G93A) mice have significant differences in concentrations of several plasma metabolites compared to wild type animals, most notably in the concentrations of aspartate, cystine/cysteine, and phosphoethanolamine, and in changes indicative of methylation defects. There are significant changes in amino acid compositions between 50 and 70days of age in both the SOD1(G93A) and wild type mice, and several of the age-related and disease-related differences in metabolite concentration were also gender-specific. Many of the SOD1(G93A)-related differences could be altered by treatment of mice with methionine sulfoximine, which extends the lifespan of this mouse, inhibits glutamine synthetase, and modifies brain methylation reactions. These studies show that assaying plasma metabolites can effectively distinguish transgenic mice from wild type, suggesting that one or more plasma metabolites might be useful biomarkers for the disease in humans, especially if genetic and longitudinal analysis is used to reduce population heterogeneity.

Aminoácidos/sangue , Esclerose Amiotrófica Lateral/sangue , Mutação , Superóxido Dismutase/sangue , Fatores Etários , Esclerose Amiotrófica Lateral/tratamento farmacológico , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Animais , Biomarcadores/sangue , Modelos Animais de Doenças , Feminino , Glutamato-Amônia Ligase/antagonistas & inibidores , Glutamato-Amônia Ligase/sangue , Humanos , Longevidade/efeitos dos fármacos , Masculino , Metaboloma/efeitos dos fármacos , Metionina Sulfoximina/farmacologia , Metilação , Camundongos , Camundongos Transgênicos , Fatores Sexuais , Superóxido Dismutase/genética , Superóxido Dismutase-1
Gend Med ; 9(6): 524-35, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23217569


OBJECTIVE: To investigate the role of sex and the role of ammonia and amino acid metabolism, specifically the activity of glutamine synthetase, in survival and disease progression in amyotrophic lateral sclerosis. METHODS: We tested treatment with methionine sulfoximine (MSO) on the lifespan and neuromuscular ability of male and female SOD1 mice as measured by their ability to maintain their grip on an inverted wire grid. We also tested the effects of castration and ovariectomization on those measurements. RESULTS: MSO treatment improves the survival of both male and female mice, but the effects are significantly greater on female mice. Saline-treated (control) female mice have delayed neuromuscular degeneration compared with saline-treated male mice, and MSO further delays disease progression in females, to a greater extent than in males. Ovariectomization or castration completely eliminates the effect of the drug on either survival or neuromuscular deterioration. CONCLUSIONS: Sex is an important factor in disease progression and the response of SOD1 mice to a drug targeting a central enzyme in nitrogen metabolism, with female sex hormones playing a greater role than male sex hormones. Glutamine synthetase, or its reactants and products, therefore plays a role in this disease, and the sex specificity of treatments aimed at this or other metabolic targets may therefore be an important factor in the development of therapies to treat amyotrophic lateral sclerosis.

Esclerose Amiotrófica Lateral/tratamento farmacológico , Esclerose Amiotrófica Lateral/enzimologia , Inibidores Enzimáticos/uso terapêutico , Glutamato-Amônia Ligase/metabolismo , Metionina Sulfoximina/uso terapêutico , Esclerose Amiotrófica Lateral/fisiopatologia , Animais , Modelos Animais de Doenças , Progressão da Doença , Feminino , Glutamato-Amônia Ligase/antagonistas & inibidores , Estimativa de Kaplan-Meier , Longevidade , Masculino , Camundongos , Camundongos Transgênicos , Músculo Esquelético/fisiopatologia , Orquiectomia , Ovariectomia , Fatores Sexuais , Superóxido Dismutase/genética , Fatores de Tempo
J Neurol Sci ; 290(1-2): 41-7, 2010 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-20060132


In an effort to alter the levels of neurochemicals involved in excitotoxicity, we treated mice with methionine sulfoximine (MSO), an inhibitor of glutamine synthetase. Since glutamate toxicity has been proposed as a mechanism for the degeneration of motor neurons in a variety of neurodegenerative diseases, we tested the effects of MSO on the transgenic mouse that overexpresses the mutant human SOD1(G93A) gene, an animal model for the primary inherited form of the human neurodegenerative disease amyotrophic lateral sclerosis (ALS). This treatment in vivo reduced glutamine synthetase activity measured in vitro by 85%. Proton magnetic resonance spectroscopy, with magic angle spinning of intact samples of brain tissue, showed that MSO treatment reduced brain levels of glutamine by 60% and of glutamate by 30% in both the motor cortex and the anterior striatum, while also affecting levels of GABA and glutathione. Kaplan-Meyer survival analysis revealed that MSO treatment significantly extended the lifespan of these mice by 8% (p<0.01). These results show that in the SOD1(G93A) model of neurodegenerative diseases, the concentration of brain glutamate (determined with (1)H-MRS) can be lowered by inhibiting in vivo the synthesis of glutamine with non-toxic doses of MSO.

Esclerose Amiotrófica Lateral/tratamento farmacológico , Esclerose Amiotrófica Lateral/metabolismo , Antagonistas de Aminoácidos Excitatórios/farmacologia , Metionina Sulfoximina/farmacologia , Esclerose Amiotrófica Lateral/fisiopatologia , Animais , Biomarcadores/análise , Biomarcadores/sangue , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Glutamato-Amônia Ligase/antagonistas & inibidores , Glutamato-Amônia Ligase/metabolismo , Ácido Glutâmico/metabolismo , Glutamina/antagonistas & inibidores , Glutamina/metabolismo , Glutationa/metabolismo , Humanos , Estimativa de Kaplan-Meier , Espectroscopia de Ressonância Magnética , Metionina Sulfoximina/uso terapêutico , Camundongos , Camundongos Transgênicos , Córtex Motor/efeitos dos fármacos , Córtex Motor/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Neurotoxinas/antagonistas & inibidores , Neurotoxinas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Resultado do Tratamento , Ácido gama-Aminobutírico/metabolismo
Front Biosci ; 12: 3892-8, 2007 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-17485345


The multifunctional protein CAD initiates de novo pyrimidine biosynthesis in mammalian cells. CAD is activated by MAP kinase (Erk1/2) just prior to the S phase of the cell cycle, when the demand for pyrimidine nucleotides is greatest, and down-regulated as the cells emerge from S phase by protein kinase A (PKA) phosphorylation. MAP kinase phosphorylates Thr456, while PKA phosphorylates Ser1406 and Ser1859, although only Ser1406 is involved in regulation. LC/mass spectrometry showed that Ser1873, a residue that lies within a putative protein kinase C (PKC) consensus sequence is also phosphorylated. Purified CAD was reacted with ATP and a panel of eight PKC isozymes. Most isozymes resulted in limited CAD phosphorylation, but the delta and epsilon isozymes were most effective. While the level of Thr456 phosphorylation is very low in confluent cells, exposure of stationary BHK 165-23 cells to the PKC activator, phorbol 12-myristate-13-acetate (PMA) resulted in a 3-fold increase in the modification of this residue. The stimulation of Thr456 phosphorylation was blocked by PKC inhibitors. The PKA inhibitor, H-89, also stimulated PMA-induced Thr456 modification probably because PKA mediated phosphorylation of CAD Ser1406 antagonizes the MAP kinase phosphorylation. Thus, the extent of Thr456 phosphorylation and the activation of pyrimidine biosynthesis depend on the synergistic and antagonistic interactions of three signaling pathways, MAP kinase, PKC and PKA. Deletions mutants lacking the putative PKC site, Ser1873 do not exhibit PMA induced Thr456 phosphorylation. We conclude that the activating MAP kinase phosphorylation of CAD proceeds through a PKC dependent pathway.

Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Pirimidinas/metabolismo , Regulação para Cima , Animais , Linhagem Celular , Cricetinae , Ativação Enzimática , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia