RESUMEN
Regulatory T cells (T(reg) cells) develop from progenitor thymocytes after the engagement of T cell antigen receptors (TCRs) with high-affinity ligands, but the underlying molecular mechanisms are still unclear. Here we show that the Nr4a nuclear receptors, which are encoded by immediate-early genes upregulated by TCR stimulation in thymocytes, have essential roles in T(reg) cell development. Mice that lacked all Nr4a factors could not produce T(reg) cells and died early owing to systemic autoimmunity. Nr4a receptors directly activated the promoter of the gene encoding the transcription factor Foxp3, and forced activation of Nr4a receptors bypassed low-strength TCR signaling to drive the T(reg) cell developmental program. Our results suggest that Nr4a receptors have key roles in determining CD4(+) T cell fates in the thymus and thus contribute to immune homeostasis.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas del Tejido Nervioso/fisiología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/fisiología , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/fisiología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Esteroides/fisiología , Receptores de Hormona Tiroidea/fisiología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Animales , Autoinmunidad/genética , Diferenciación Celular , Células Cultivadas , Proteínas de Unión al ADN/genética , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Genes Inmediatos-Precoces , Homeostasis , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genética , Transducción de Señal , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/citología , Timocitos/metabolismoRESUMEN
The gene encoding 5'-nucleotidase domain-containing protein 2 (NT5DC2) has been associated with neuropsychiatric disorders related to the abnormality of dopamine activity in the brain. However, its physiological functions remain unclear. In this study, we analyzed the features of NT5DC2 that influence its binding with tyrosine hydroxylase (TH) and its effects on dihydroxyphenylalanine (DOPA) synthesis, using NT5DC2 overexpressed in PC12D cells by the pCMV vector. Western blot analysis revealed that the purified NT5DC2-DYKDDDDK-tag (NT5DC2-tag) protein can bind with the phosphorylated form of recombinant human TH type 1 (rhTH1), apart from the endogenous TH in PC12D cells. Proteomic analysis by mass spectrometry revealed that the purified NT5DC2-tag protein has the potential to bind to 41 proteins with multiple phosphorylation sites in PC12D cells (NT5DC2 binding proteins: positive, 391 sites/41 proteins; and negative, 85 sites/27 proteins). Overexpression of NT5DC2 in PC12D cells decreased DOPA levels in the medium. When the lysate of PC12D cells overexpressing NT5DC2 was incubated at 37 °C, the phosphorylated form of endogenous TH in PC12D cells decreased. This decrease was also detected when phosphorylated rhTH1 was incubated with purified NT5DC2-tag. Overall, our results suggest that NT5DC2 regulates DOPA synthesis by promoting the dephosphorylation of TH, similar to a phosphatase. Therefore, our study provides useful information for understanding various disorders associated with abnormalities in dopamine levels in the brain.
Asunto(s)
Oxigenasas de Función Mixta , Tirosina 3-Monooxigenasa , Humanos , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismo , Fosforilación , Oxigenasas de Función Mixta/metabolismo , Dopamina , Proteínas Portadoras/metabolismo , Proteómica , Dihidroxifenilalanina/metabolismoRESUMEN
Tetrahydrobiopterin (BH4) is an essential cofactor for dopamine and serotonin synthesis in monoaminergic neurons, phenylalanine metabolism in hepatocytes, and nitric oxide synthesis in endothelial and immune cells. BH4 is consumed as a cofactor or is readily oxidized by autooxidation. Quinonoid dihydropteridine reductase (QDPR) is an enzyme that reduces quinonoid dihydrobiopterin (qBH2) back to BH4, and we have previously demonstrated the significance of QDPR in maintaining BH4 in vivo using Qdpr-KO mice. In addition to the levels of BH4 in the cells, the ratios of oxidized to reduced forms of BH4 are supposed to be important for regulating nitric oxide synthase (NOS) via the so-called uncoupling of NOS. However, previous studies were limited due to the absence of specific and high-affinity inhibitors against QDPR. Here, we performed a high-throughput screening for a QDPR inhibitor and identified Compound 9b with an IC50 of 0.72 µM. To understand the inhibition mechanism, we performed kinetic analyses and molecular dynamics simulations. Treatment with 9b combined with methotrexate (MTX), an inhibitor of another BH4-reducing enzyme, dihydrofolate reductase (DHFR), significantly oxidized intracellular redox states in HepG2, Jurkat, SH-SY5Y, and PC12D cells. Collectively, these findings suggest that 9b may enhance the anticancer and anti-autoimmune effects of MTX.
Asunto(s)
Biopterinas , Dihidropteridina Reductasa , Sinergismo Farmacológico , Metotrexato , Metotrexato/farmacología , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Humanos , Dihidropteridina Reductasa/metabolismo , Inhibidores Enzimáticos/farmacología , Oxidación-Reducción/efectos de los fármacos , Animales , Simulación de Dinámica MolecularRESUMEN
Dopamine (DA) exerts well-known functions in the brain as a neurotransmitter. In addition, it plays important physiological roles in peripheral organs, but it is largely unknown how and where peripheral DA is synthesized and regulated. Catecholamines in peripheral tissues are either produced within the tissue itself and/or derived from sympathetic neurons, which release neurotransmitters for uptake by peripheral tissues. To evaluate DA-producing ability of each peripheral tissue, we generated conditional KO mice (cKO mice) in which the tyrosine hydroxylase (TH) gene is ablated in the sympathoadrenal system, thus eliminating sympathetic neurons as a DA source. We then examined the alterations in the noradrenaline (NA), DA, and 3,4-dihydroxyphenylalanine (DOPA) contents in peripheral organs and performed immunohistochemical analyses of TH-expressing cells. In the heart and pancreas of cKO mice, both the TH protein and NA levels were significantly decreased, and the DA contents were decreased in parallel with NA contents, indicating that the DA supply originated from sympathetic neurons. We found TH-immunoreactive cells in the stomach and lung, where the TH protein showed a decreasing trend, but the DA levels were not decreased in cKO mice. Moreover, we found a significant correlation between the DA content in the kidney and the plasma DOPA concentration, suggesting that the kidney takes up DOPA from blood to make DA. The aforementioned data unravel differences in the DA biosynthetic pathway among tissues and support the role of sympathetic neurons as a DA supplier.
Asunto(s)
Glándulas Suprarrenales/metabolismo , Vías Biosintéticas , Catecolaminas/metabolismo , Dopamina/biosíntesis , Neuronas/metabolismo , Sistema Nervioso Simpático/metabolismo , Tirosina 3-Monooxigenasa/fisiología , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de ÓrganosRESUMEN
Increasing evidence suggests the involvement of peripheral amino acid metabolism in the pathophysiology of neuropsychiatric disorders, whereas the molecular mechanisms are largely unknown. Tetrahydrobiopterin (BH4) is a cofactor for enzymes that catalyze phenylalanine metabolism, monoamine synthesis, nitric oxide production, and lipid metabolism. BH4 is synthesized from guanosine triphosphate and regenerated by quinonoid dihydropteridine reductase (QDPR), which catalyzes the reduction of quinonoid dihydrobiopterin. We analyzed Qdpr-/- mice to elucidate the physiological significance of the regeneration of BH4. We found that the Qdpr-/- mice exhibited mild hyperphenylalaninemia and monoamine deficiency in the brain, despite the presence of substantial amounts of BH4 in the liver and brain. Hyperphenylalaninemia was ameliorated by exogenously administered BH4, and dietary phenylalanine restriction was effective for restoring the decreased monoamine contents in the brain of the Qdpr-/- mice, suggesting that monoamine deficiency was caused by the secondary effect of hyperphenylalaninemia. Immunohistochemical analysis showed that QDPR was primarily distributed in oligodendrocytes but hardly detectable in monoaminergic neurons in the brain. Finally, we performed a behavioral assessment using a test battery. The Qdpr-/- mice exhibited enhanced fear responses after electrical foot shock. Taken together, our data suggest that the perturbation of BH4 metabolism should affect brain monoamine levels through alterations in peripheral amino acid metabolism, and might contribute to the development of anxiety-related psychiatric disorders. Cover Image for this issue: https://doi.org/10.1111/jnc.15398.
Asunto(s)
Biopterinas , Fenilcetonurias , Animales , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Dihidropteridina Reductasa , Miedo , Humanos , Ratones , Fenilalanina , Fenilcetonurias/genética , Fenilcetonurias/metabolismoRESUMEN
Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of dopamine (DA), and the regulation of its activity is important for DA homeostasis. In this study, we focused on the modification of TH through a cysteine residue. We found that incubation with N-ethylmaleimide (NEM), a cysteine modification reagent, inactivated TH. The responsible cysteine was identified as Cys176 of human TH with recombinant mutant proteins. We further examined how NEM modification was affected by the states of TH. DA binding, a feedback inhibition mechanism of TH, delayed the modification and inactivation of TH by NEM. In contrast, the S40E mutant, which mimics the phosphorylation of Ser40 that suppresses DA binding and is thus considered as an active state of TH, did not affect modification and inactivation. These results suggest that the modification of Cys176 can inhibit even phosphorylated active TH. In addition, we found that DA oxides, which are generated by oxidative stress in dopaminergic neurons, reacted with TH through Cys176 and inhibited its activity, similar to NEM. These results suggest that the modification of Cys176 of TH could be involved in the mechanisms of neurotoxicity caused by DA oxides.
Asunto(s)
Cisteína/metabolismo , Tirosina 3-Monooxigenasa/química , Tirosina 3-Monooxigenasa/metabolismo , Dopamina/metabolismo , Activación Enzimática , Etilmaleimida , Humanos , Mutación/genética , Fosforilación , Relación Estructura-Actividad , Tirosina 3-Monooxigenasa/genéticaRESUMEN
6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor for aromatic L-amino acid hydroxylases, including tyrosine hydroxylase (TH), alkylglycerol monooxygenase, and three types of nitric oxide (NO) synthases (NOS). Sepiapterin reductase (SPR) catalyzes the third step of BH4 biosynthesis. SPR gene-disrupted (Spr-/- ) mice exhibit a dystonic posture, low body weight, hyperphenylalaninemia, and unstable hypertension with endothelial dysfunction. In this study, we found that Spr-/- mice suffered from a high incidence of severe priapism. Their erections persisted for months. The biopterin, BH4, and norepinephrine contents, and TH protein levels in the penile tissue of Spr-/- mice without and with priapism were significantly reduced compared to those of Spr+/+ mice. In contrast, their neural NOS (nNOS) protein levels were increased, and the cyclic guanosine monophosphate (cGMP) levels were remarkably elevated in the penises of Spr-/- mice with priapism. The symptoms were relieved by repeated administration of BH4. The biopterin, BH4, and norepinephrine contents were increased in penile homogenates from BH4-supplemented Spr-/- mice, and the TH protein levels tended to increase, and their nitrite plus nitrate levels were significantly lower than those of vehicle-treated Spr-/- mice and were approximately the same as vehicle- and BH4-supplemented Spr+/+ mice. Thus, we deduced that the priapism of Spr-/- mice is primarily caused by hypofunction of the sympathetic neurons due to cofactor depletion and the loss of TH protein and, further, dysregulation of the NO/cGMP signaling pathway, which would be caused by disinhibition of nNOS-containing neurons and/or abnormal catabolism of cyclic nucleotides is suggested.
Asunto(s)
Priapismo , Oxidorreductasas de Alcohol , Animales , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Humanos , Masculino , Ratones , Neuronas/metabolismo , Norepinefrina/metabolismo , Priapismo/etiología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Quinonoid dihydropteridine reductase (QDPR) regenerates tetrahydrobiopterin (BH4), which is an essential cofactor for catecholamine and serotonin (5-hydroxytryptamine, 5-HT) biosynthesis. Serotonin is known as an important platelet agonist, but its role under BH4-synthesizing or recycling enzymes deficiency is unknown. In the present study, we evaluated the effect of Qdpr gene disruption on platelet aggregation using knockout (Qdpr-/-) mice. Platelet aggregation was monitored by light transmission aggregometry using adenosine diphosphate (ADP) and collagen as agonists. We also assessed how platelet aggregation was modified by 5-HT recovery through supplementation with 5-hydroxytryptophan (5-HTP), a 5-HT precursor, or by blocking the serotonin 5-HT2A receptor. Platelet aggregation in the Qdpr-/- mice was significantly suppressed in comparison with that in wild-type (Qdpr+/+) mice, particularly at the maintenance phase of aggregation. 5-HT storage was decreased in Qdpr-/- platelets, and 5-HTP supplementation recovered not only the intraplatelet 5-HT levels but also platelet aggregation. In addition, 5-HT signal blockade using sarpogrelate suppressed platelet aggregation in Qdpr+/+ mice, and platelets in Qdpr-/- mice were hardly affected. Our results indicate that QDPR deficiency suppresses platelet aggregation by impairing 5-HT biosynthesis in mice.
Asunto(s)
Dihidropteridina Reductasa , Agregación Plaquetaria , 5-Hidroxitriptófano/farmacología , Adenosina Difosfato/farmacología , Animales , Biopterinas/análogos & derivados , Catecolaminas , Colágeno , Dihidropteridina Reductasa/genética , Dihidropteridina Reductasa/farmacología , Ratones , Receptor de Serotonina 5-HT2A , Serotonina/farmacologíaRESUMEN
Some bipolar cells in the human retina are known to express voltage-gated Na+ channels. However, it is unclear which types of channels are expressed, and whether Na+ channel expression is limited to specific types of bipolar cells. In the present study, we examined the types of voltage-gated Na+ channels expressed in human bipolar cells and the morphology of bipolar cells with voltage-gated Na+ currents. To investigate the expression of voltage-gated Na+ channels in human bipolar cells, we examined whether Na+ channel transcripts could be detected in single bipolar cells using the reverse transcription polymerase chain reaction (RT-PCR) technique. The voltage-gated Na+ current was recorded from isolated bipolar cells using the patch-clamp recording technique. Types of bipolar cells that have the Na+ currents were investigated by analyzing their morphology after staining with Lucifer yellow. Using RT-PCR, the SCN2A Na+ channel was detected in 5 of 6 isolated bipolar cells. This suggests that a subset of human bipolar cells expresses the SCN2A Na+ channel. Under voltage-clamp conditions, depolarizing voltage steps induced a fast transient inward current in cone bipolar cells with axon terminal boutons that stratified at the ON layer, which includes the stratum 3, 4, and 5 of the inner plexiform layer (IPL, n = 2/11 cells). The fast transient inward current of isolated bipolar cells was blocked by 1 µM of tetrodotoxin (TTX), a voltage-gated Na+ channel blocker. No fast transient inward current was recorded with axon terminals that stratify at the OFF layer, which includes stratum 1 and 2 of the IPL (n = 4). Thus, a subset of ON cone bipolar cells at least expresses the putative voltage-gated Na+ channel SCN2A in the human retina. The Na+ channels in the bipolar cells may serve to amplify the release of neurotransmitter, glutamate, when membrane potential is rapidly depolarized and thereby selectively accelerating light responses.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Canal de Sodio Activado por Voltaje NAV1.2/genética , Células Bipolares de la Retina/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Adulto , Anciano , Electrofisiología , Femenino , Humanos , Masculino , Potenciales de la Membrana , Persona de Mediana Edad , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacologíaRESUMEN
6-Pyruvoyl tetrahydropterin synthase (PTS) converts 7,8-dihydroneopterin triphosphate into 6-pyruvoyltetrahydropterin and is a critical enzyme for the de novo synthesis of tetrahydrobiopterin, an essential cofactor for aromatic amino acid hydroxylases and nitric-oxide synthases. Neopterin derived from 7,8-dihydroneopterin triphosphate is secreted by monocytes/macrophages, and is a well-known biomarker for cellular immunity. Because PTS activity in the cell can be a determinant of neopterin production, here we used recombinant human PTS protein to investigate how its activity is regulated, especially depending on redox conditions. Human PTS has two cysteines: Cys-43 at the catalytic site and Cys-10 at the N terminus. PTS can be oxidized and consequently inactivated by H2O2 treatment, oxidized GSH, or S-nitrosoglutathione, and determining the oxidized modifications of PTS induced by each oxidant by MALDI-TOF MS, we show that PTS is S-glutathionylated in the presence of GSH and H2O2S-Glutathionylation at Cys-43 protected PTS from H2O2-induced irreversible sulfinylation and sulfonylation. We also found that PTS expressed in HeLa and THP-1 cells is reversibly modified under oxidative stress conditions. Our findings suggest that PTS activity and S-glutathionylation is regulated by the cellular redox environment and that reversible S-glutathionylation protects PTS against oxidative stress.
Asunto(s)
Cisteína/química , Regulación Enzimológica de la Expresión Génica , Glutatión/química , Liasas de Fósforo-Oxígeno/metabolismo , Pterinas/metabolismo , Células HeLa , Humanos , Oxidación-Reducción , Estrés Oxidativo , Liasas de Fósforo-Oxígeno/química , Sustancias ProtectorasRESUMEN
BACKGROUND: Nuclear receptor subfamily 4 group A member 1 (NR4A1), an orphan nuclear receptor, has been implicated in several biological events such as metabolism, apoptosis, and inflammation. Recent studies indicate a potential role for NR4A1 in mast cells, yet its role in allergic responses remains largely unknown. OBJECTIVES: The aim of this study was to clarify the role of NR4A1 in mast cell activation and anaphylaxis. METHODS: To evaluate the function of NR4A1 in mast cells, the impacts of siRNA knockdown, gene knockout, adenoviral overexpression, and pharmacological inhibition of NR4A1 on FcεRI signaling and effector functions in mouse bone marrow-derived mast cells (BMMCs) in vitro and on anaphylactic responses in vivo were evaluated. RESULTS: Knockdown or knockout of NR4A1 markedly suppressed degranulation and lipid mediator production by FcεRI-crosslinked BMMCs, while its overexpression augmented these responses. Treatment with a NR4A1 antagonist also blocked mast cell activation to a similar extent as NR4A1 knockdown or knockout. Moreover, mast cell-specific NR4A1-deficient mice displayed dampened anaphylactic responses in vivo. Mechanistically, NR4A1 promoted FcεRI signaling by counteracting the liver kinase B1 (LKB1)/adenosine monophosphate-activated protein kinase (AMPK) axis. Following FcεRI crosslinking, NR4A1 bound to the LKB1/AMPK complex and sequestered it in the nucleus, thereby promoting FcεRI downstream signaling pathways. Silencing or knockout of LKB1/AMPK largely abrogated the effect of NR4A1 on mast cell activation. Additionally, NR4A1 facilitated spleen tyrosine kinase activation independently of LKB1/AMPK. CONCLUSIONS: Nuclear receptor subfamily 4 group A member 1 positively regulates mast cell activation by antagonizing the LKB1-AMPK-dependent negative regulatory axis. This finding may provide a novel therapeutic strategy for the development of anti-allergic compounds.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Anafilaxia/metabolismo , Mastocitos/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de IgE/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Animales , Basófilos/metabolismo , Células de la Médula Ósea/metabolismo , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/antagonistas & inhibidores , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Anafilaxis Cutánea Pasiva , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridinas/farmacologíaRESUMEN
Parkinson's disease (PD) is an aging-related movement disorder mainly caused by a deficiency of neurotransmitter dopamine (DA) in the striatum of the brain and is considered to be due to progressive degeneration of nigro-striatal DA neurons. Most PD is sporadic without family history (sPD), and there are only a few percent of cases of young-onset familial PD (fPD, PARKs) with the chromosomal locations and the genes identified. Tyrosine hydroxylase (TH), tetrahydrobiopterin (BH4)-dependent and iron-containing monooxygenase, catalyzes the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), which is the initial and rate-limiting step in the biosynthesis of catecholamines (DA, noradrenaline, and adrenaline). PD affects specifically TH-containing catecholamine neurons. The most marked neurodegeneration in patients with DA deficiency is observed in the nigro-striatal DA neurons, which contain abundant TH. Accordingly, TH has been speculated to play some important roles in the pathophysiology in PD. However, this decrease in TH is thought to be secondary due to neurodegeneration of DA neurons caused by some as yet unidentified genetic and environmental factors, and thus, TH deficiency may not play a direct role in PD. This manuscript provides an overview of the role of human TH in the pathophysiology of PD, covering the following aspects: (1) structures of the gene and protein of human TH in relation to PD; (2) similarity and dissimilarity between the phenotypes of aging-related sPD and those of young-onset fPD or DOPA-responsive dystonia due to DA deficiency in the striatum with decreased TH activity caused by mutations in either the TH gene or GTP cyclohydrolase I (GCH1) gene; and (3) genetic variants of the TH gene (polymorphisms, rare variants, and mutations) in PD, as discovered recently by advanced genome analysis.
Asunto(s)
Enfermedad de Parkinson/enzimología , Tirosina 3-Monooxigenasa/metabolismo , Humanos , Enfermedades Neurodegenerativas/enzimología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/fisiopatología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/fisiopatología , Tirosina 3-Monooxigenasa/genéticaRESUMEN
Microglia cells fulfill key homeostatic functions and essentially contribute to host defense within the CNS. Altered activation of microglia, in turn, has been implicated in neuroinflammatory and neurodegenerative diseases. In this study, we identify the nuclear receptor (NR) Nr4a1 as key rheostat controlling the activation threshold and polarization of microglia. In steady-state microglia, ubiquitous neuronal-derived stress signals such as ATP induced expression of this NR, which contributed to the maintenance of a resting and noninflammatory microglia phenotype. Global and microglia-specific deletion of Nr4a1 triggered the spontaneous and overwhelming activation of microglia and resulted in increased cytokine and NO production as well as in an accelerated and exacerbated form of experimental autoimmune encephalomyelitis. Ligand-induced activation of Nr4a1 accordingly ameliorated the course of this disease. Our current data thus identify Nr4a1 as regulator of microglia activation and potentially new target for the treatment of inflammatory CNS diseases such as multiple sclerosis.
Asunto(s)
Sistema Nervioso Central/inmunología , Microglía/fisiología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Adenosina Trifosfato/farmacología , Animales , Células Cultivadas , Enfermedades del Sistema Nervioso Central/terapia , Citocinas/biosíntesis , Citocinas/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/fisiopatología , Encefalomielitis Autoinmune Experimental/prevención & control , Regulación de la Expresión Génica , Humanos , Inflamación , Activación de Macrófagos , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/inmunología , Esclerosis Múltiple/terapia , Enfermedades Neurodegenerativas/terapia , Óxido Nítrico/biosíntesis , Óxido Nítrico/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/deficienciaRESUMEN
Dopamine (DA) is a neurotransmitter in the brain, playing a central role in several disease conditions, including tetrahydrobiopterin (BH4) metabolism disorders and Parkinson's disease (PD). BH4 metabolism disorders present a variety of clinical manifestations including motor disturbance via altered DA metabolism, since BH4 is a cofactor for tyrosine hydroxylase (TH), a rate-limiting enzyme for DA synthesis. Genetically, BH4 metabolism disorders are, in an autosomal recessive pattern, caused by a variant in genes encoding enzymes for BH4 synthesis or recycling, including 6-pyruvoyltetrahydropterin synthase (PTPS) or dihydropteridine reductase (DHPR), respectively. Although BH4 metabolism disorders and its metabolisms have been studied, it is unclear how gene variants cause aberrant DA synthesis in patient neurons. Here, we generated induced pluripotent stem cells (iPSCs) from BH4 metabolism disorder patients with PTPS or DHPR variants, corrected the gene variant in the iPSCs using the CRISPR/Cas9 system, and differentiated the BH4 metabolism disorder patient- and isogenic control iPSCs into midbrain DA neurons. We found that by the gene correction, the BH4 amount, TH protein level and extracellular DA level were restored in DA neuronal culture using PTPS deficiency iPSCs. Furthermore, the pharmacological correction by BH4 precursor sepiapterin treatment also improved the phenotypes of PTPS deficiency. These results suggest that patient iPSCs with BH4 metabolism disorders provide an opportunity for screening substances for treating aberrant DA synthesis-related disorders.
Asunto(s)
Biopterinas/análogos & derivados , Dopamina/genética , Células Madre Pluripotentes Inducidas/metabolismo , Enfermedades Metabólicas/genética , Enfermedad de Parkinson/genética , Biopterinas/metabolismo , Diferenciación Celular/genética , Dopamina/biosíntesis , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Genotipo , Humanos , Cariotipo , Enfermedades Metabólicas/metabolismo , Enfermedades Metabólicas/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Pterinas/metabolismo , Tirosina 3-Monooxigenasa/genéticaRESUMEN
Tetrahydrobiopterin (BH4) is a cofactor for tyrosine hydroxylase that is essential for the biosynthesis of dopamine. Parkinson's disease (PD) is characterized by a progressive degeneration of nigrostriatal dopaminergic neurons, and biomarkers reflecting the degree of neurodegeneration are important not only for basic research but also for clinical diagnosis and the treatment of the disease. Although the total neopterin and biopterin levels in the cerebrospinal fluids (CSF) of the patients with PD were reported, alterations in the composition of reduced and oxidized forms of pteridine compounds have not been examined. In this study, we first examined the time-dependent alterations in BH4 and other reduced pteridine compounds in the CSF of an MPTP-treated monkey as a primate PD model. We found that the CSF levels of BH4 and dihydroneopterin, an intermittent metabolite of BH4-biosynthesis, altered inversely with progression of neurodegeneration, whereas those of dihydrobiopterin and neopterin were relatively low and constant. Next, we assayed the amounts of reduced pteridine compounds in the CSF of 36 pre-symptomatic LRRK2-mutation (N1437H or G2019S) carriers (LRRK2-carrier), 13 patients with PD symptoms (LRRK2-PD), 46 patients with sporadic PD (sPD), and 26 non-PD individuals. The BH4 levels were significantly lower in both the LRRK2-PD and sPD patients, and the LRRK2-carriers exhibited higher BH4 levels compared with the sPD patients. The total neopterin levels in the CSF of the LRRK2-PD were significantly higher than those in the sPD and non-PD individuals, which indicated greater inflammatory responses in the brains of LRRK2-PD patients. The present results suggest that detailed analyses of pteridine levels in the CSF might be useful for understanding the pathophysiology of familial PD and for monitoring PD progression.
Asunto(s)
Heterocigoto , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/líquido cefalorraquídeo , Mutación/genética , Enfermedad de Parkinson/líquido cefalorraquídeo , Enfermedad de Parkinson/genética , Pteridinas/líquido cefalorraquídeo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores/líquido cefalorraquídeo , Femenino , Humanos , Intoxicación por MPTP/líquido cefalorraquídeo , Intoxicación por MPTP/genética , Macaca , Masculino , Persona de Mediana EdadRESUMEN
OBJECTIVE: The hemodialysis (HD) diet, which is a high-calorie and high-fat regimen, may inadvertently lead to an inadequate dietary intake, resulting in undernutrition among elderly HD patients. Therefore, an attempt was made to improve the dietary intake by implementing a modified diet regimen in eligible elderly HD patients. SUBJECTS: Elderly HD patients who had ingested < 50% of the meals provided and were diagnosed with undernutrition among all elderly patients institutionalized at the special elderly nursing home annexed to Nagasaki Kidney Hospital between June and November 2012. RESULTS: Of the elderly HD patients in the nursing home (n = 27), the study included a total of 7 consecutive patients (male/female, 1/6; mean age, 84.1±6.4 years old; duration of HD, 4.3±3.8 years; geriatric nutritional index [GNRI], 83.5±8.3; normalized protein catabolic ratio [nPCR], 0.78±0.14). The modified diet regimen, which involved reducing food portion sizes and incorporating a liquid diet, led to a significant increase in their dietary intake from 48.1% at baseline to 97.1% of the meals provided 3 months after the start of the modified HD diet regimen. Their GNRI also significantly increased from 83.5±8.3 to 86.1±10.2, and their serum albumin levels significantly increased from 3.2±0.2 g/dL to 3.4±0.4 g/dL, suggesting improvements in their nutritional status. CONCLUSIONS: The attempted dietary approach for elderly HD patients was shown to potentially increase their dietary intake and improve their nutritional status without affecting the efficiency of HD being implemented.
Asunto(s)
Ingestión de Energía , Anciano , Anciano de 80 o más Años , Dieta , Femenino , Humanos , Masculino , Estado Nutricional , Proyectos Piloto , Diálisis RenalRESUMEN
Tetrahydrobiopterin (BH4) is an essential cofactor for the aromatic amino acid hydroxylases, alkylglycerol monooxygenase, and nitric oxide synthases (NOS). Inborn errors of BH4 metabolism lead to severe insufficiency of brain monoamine neurotransmitters while augmentation of BH4 by supplementation or stimulation of its biosynthesis is thought to ameliorate endothelial NOS (eNOS) dysfunction, to protect from (cardio-) vascular disease and/or prevent obesity and development of the metabolic syndrome. We have previously reported that homozygous knock-out mice for the 6-pyruvolytetrahydropterin synthase (PTPS; Pts-ko/ko) mice with no BH4 biosynthesis die after birth. Here we generated a Pts-knock-in (Pts-ki) allele expressing the murine PTPS-p.Arg15Cys with low residual activity (15% of wild-type in vitro) and investigated homozygous (Pts-ki/ki) and compound heterozygous (Pts-ki/ko) mutants. All mice showed normal viability and depending on the severity of the Pts alleles exhibited up to 90% reduction of PTPS activity concomitant with neopterin elevation and mild reduction of total biopterin while blood L-phenylalanine and brain monoamine neurotransmitters were unaffected. Yet, adult mutant mice with compromised PTPS activity (i.e., Pts-ki/ko, Pts-ki/ki or Pts-ko/wt) had increased body weight and elevated intra-abdominal fat. Comprehensive phenotyping of Pts-ki/ki mice revealed alterations in energy metabolism with proportionally higher fat content but lower lean mass, and increased blood glucose and cholesterol. Transcriptome analysis indicated changes in glucose and lipid metabolism. Furthermore, differentially expressed genes associated with obesity, weight loss, hepatic steatosis, and insulin sensitivity were consistent with the observed phenotypic alterations. We conclude that reduced PTPS activity concomitant with mildly compromised BH4-biosynthesis leads to abnormal body fat distribution and abdominal obesity at least in mice. This study associates a novel single gene mutation with monogenic forms of obesity.
Asunto(s)
Tejido Adiposo/metabolismo , Biopterinas/análogos & derivados , Distribución de la Grasa Corporal , Obesidad Abdominal/genética , Liasas de Fósforo-Oxígeno/genética , Alelos , Animales , Biopterinas/biosíntesis , Biopterinas/genética , Peso Corporal/genética , Colesterol/genética , Femenino , Genotipo , Glucosa/genética , Heterocigoto , Homocigoto , Metabolismo de los Lípidos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación/genética , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo III/genética , Fenilalanina/genética , Transcriptoma/genéticaRESUMEN
The protein amount of tyrosine hydroxylase (TH), that is the rate-limiting enzyme for the biosynthesis of dopamine (DA), should be tightly regulated, whereas its degradation pathway is largely unknown. In this study, we analyzed how the TH protein is chemically modified and subsequently degraded under deficiencies of DA and tetrahydrobiopterin (BH4), a cofactor for TH, by using pharmacological agents in PC12D cells and cultured mesencephalic neurons. When inhibition of DA- or BH4-synthesizing enzymes greatly reduced the DA contents in PC12D cells, a marked and persistent increase in phosphorylated TH at (40)Ser (p40-TH) was concomitantly observed. This phosphorylation was mediated by D2 dopamine auto-receptor and cAMP-dependent protein kinase (PKA). Our immunoprecipitation experiments showed that the increase in the p40-TH level was accompanied with its poly-ubiquitination. Treatment of PC12D cells with cycloheximide showed that total-TH protein level was reduced by the DA- or BH4-depletion. Notably, this reduction in the total-TH protein level was sensitive not only to a 26S proteasomal inhibitor, MG-132, but also to a PKA inhibitor, H-89. These data demonstrated that DA deficiency should induce compensatory activation of TH via phosphorylation at (40)Ser through D2-autoreceptor and PKA-mediated pathways, which in turn give a rise to its degradation through an ubiquitin-proteasome pathway, resulting in a negative spiral of DA production when DA deficiency persists.
Asunto(s)
Biopterinas/análogos & derivados , Dopamina/deficiencia , Neuronas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Ubiquitina/metabolismo , Animales , Biopterinas/deficiencia , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cicloheximida/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Regulación de la Expresión Génica , Isoquinolinas/farmacología , Leupeptinas/farmacología , Mesencéfalo/citología , Mesencéfalo/efectos de los fármacos , Mesencéfalo/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Células PC12 , Fosforilación/efectos de los fármacos , Cultivo Primario de Células , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Proteolisis/efectos de los fármacos , Ratas , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Serina/metabolismo , Transducción de Señal , Sulfonamidas/farmacología , Tirosina 3-Monooxigenasa/genética , Ubiquitina/genética , Ubiquitinación/efectos de los fármacosRESUMEN
Intracellular redox state is a critical factor for fundamental cellular functions, including regulation of the activities of various metabolic enzymes as well as ROS production and elimination. Genetically-encoded fluorescent redox sensors, such as roGFP (Hanson, G. T., et al. (2004)) and Redoxfluor (Yano, T., et al. (2010)), have been developed to investigate the redox state of living cells. However, these sensors are not useful in cells that contain, for example, other colored pigments. We therefore intended to obtain simpler redox sensor proteins, and have developed oxidation-sensitive fluorescent proteins called Oba-Q (oxidation balance sensed quenching) proteins. Our sensor proteins derived from CFP and Sirius can be used to monitor the intracellular redox state as their fluorescence is drastically quenched upon oxidation. These blue-shifted spectra of the Oba-Q proteins enable us to monitor various redox states in conjunction with other sensor proteins.
Asunto(s)
Proteínas Fluorescentes Verdes/metabolismo , Líquido Intracelular/metabolismo , Técnicas Biosensibles/métodos , Glutatión/metabolismo , Disulfuro de Glutatión/metabolismo , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Mutagénesis , Oxidación-Reducción , Teoría Cuántica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de FluorescenciaRESUMEN
OBJECTIVES: Aicardi-Goutières syndrome (AGS) is a rare, genetically determined, early onset progressive encephalopathy associated with autoimmune manifestations. AGS is usually inherited in an autosomal recessive manner. The disease is rare, therefore the clinical manifestations and genotype-phenotype correlations, particularly with regard to autoimmune diseases, are still unclear. Here we performed a nationwide survey of AGS patients in Japan and analysed the genetic and clinical data. METHODS: Patients were recruited via questionnaires sent to paediatric or adult neurologists in Japanese hospitals and institutions. Genetic analysis was performed and clinical data were collected. RESULTS: Fourteen AGS patients were identified from 13 families; 10 harboured genetic mutations. Three patients harboured dominant-type TREX1 mutations. These included two de novo cases: one caused by a novel heterozygous p.His195Tyr mutation and the other by a novel somatic mosaicism resulting in a p.Asp200Asn mutation. Chilblain lesions were observed in all patients harbouring dominant-type TREX1 mutations. All three patients harbouring SAMHD1 mutations were diagnosed with autoimmune diseases, two with SLE and one with SS. The latter is the first reported case. CONCLUSION: This study is the first to report a nationwide AGS survey, which identified more patients with sporadic AGS carrying de novo dominant-type TREX1 mutations than expected. There was a strong association between the dominant-type TREX1 mutations and chilblain lesions, and between SAMHD1 mutations and autoimmunity. These findings suggest that rheumatologists should pay attention to possible sporadic AGS cases presenting with neurological disorders and autoimmune manifestations.