RESUMEN

The hypothalamus is comprised of heterogenous cell populations and includes highly complex neural circuits that regulate the autonomic nerve system. Its dysfunction therefore results in severe endocrine disorders. Although recent experiments have been conducted for in vitro organogenesis of hypothalamic neurons from embryonic stem (ES) or induced pluripotent stem (iPS) cells, whether these stem cell-derived hypothalamic neurons can be useful for regenerative medicine remains unclear. We therefore performed orthotopic transplantation of mouse ES cell (mESC)-derived hypothalamic neurons into adult mouse brains. We generated electrophysiologically functional hypothalamic neurons from mESCs and transplanted them into the supraoptic nucleus of mice. Grafts extended their axons along hypothalamic nerve bundles in host brain, and some of them even projected into the posterior pituitary (PPit), which consists of distal axons of the magnocellular neurons located in hypothalamic supraoptic and paraventricular nuclei. The axonal projections to the PPit were not observed when the mESC-derived hypothalamic neurons were ectopically transplanted into the substantia nigra reticular part. These findings suggest that our stem cell-based orthotopic transplantation approach might contribute to the establishment of regenerative medicine for hypothalamic and pituitary disorders.

Asunto(s)

Hipotálamo , Células Madre Embrionarias de Ratones , Animales , Ratones , Hipotálamo/fisiología , Axones/fisiología , Neuronas/fisiología , Núcleo Supraóptico , Núcleo Hipotalámico Paraventricular

RESUMEN

Hypothalamic melanin-concentrating hormone (MCH) neurons are important regulators of multiple physiological processes, such as sleep, feeding, and memory. Despite the increasing interest in their neuronal functions, the molecular mechanism underlying MCH neuron development remains poorly understood. We report that a three-dimensional culture of mouse embryonic stem cells (mESCs) can generate hypothalamic-like tissues containing MCH-positive neurons, which reproduce morphologic maturation, neuronal connectivity, and neuropeptide/neurotransmitter phenotype of native MCH neurons. Using this in vitro system, we demonstrate that Hedgehog (Hh) signaling serves to produce major neurochemical subtypes of MCH neurons characterized by the presence or absence of cocaine- and amphetamine-regulated transcript (CART). Without exogenous Hh signals, mESCs initially differentiated into dorsal hypothalamic/prethalamic progenitors and finally into MCH+CART+ neurons through a specific intermediate progenitor state. Conversely, activation of the Hh pathway specified ventral hypothalamic progenitors that generate both MCH+CART- and MCH+CART+ neurons. These results suggest that in vivo MCH neurons may originate from multiple cell lineages that arise through early dorsoventral patterning of the hypothalamus. Additionally, we found that Hh signaling supports the differentiation of mESCs into orexin/hypocretin neurons, a well-defined cell group intermingled with MCH neurons in the lateral hypothalamic area (LHA). The present study highlights and improves the utility of mESC culture in the analysis of the developmental programs of specific hypothalamic cell types.

Asunto(s)

Hormonas Hipotalámicas , Células Madre Embrionarias de Ratones , Animales , Proteínas Hedgehog/metabolismo , Hormonas Hipotalámicas/metabolismo , Hipotálamo/metabolismo , Melaninas/metabolismo , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Orexinas/metabolismo , Hormonas Hipofisarias/metabolismo

RESUMEN

5'-Nucleotidase domain-containing protein 2 (NT5DC2) has been revealed by genome-wide association studies (GWAS) as a gene implicated in neuropsychiatric disorders related to the abnormality of dopamine (DA) activity in the brain. Based on its amino acid sequence, NT5DC2 is assumed to be a member of the family of haloacid dehalogenase-type phosphatases; although there is no information about its function and structural conformation. We recently reported that NT5DC2 binds to tyrosine hydroxylase (TH) and that the down-regulation of NT5DC2 tended to increase DA synthesis. In this study, we investigated whether NT5DC2 could regulate the catalytic activity of TH, which converts tyrosine to DOPA, because the phosphorylation level of TH, controlled by protein kinases and phosphatases, is well known to regulate its catalytic activity. The down-regulation of NT5DC2 by siRNA increased mainly DOPA synthesis by TH in PC12D cells, although this down-regulation tended to increase the conversion of DOPA to DA by aromatic L-amino acid decarboxylase. The increased DOPA synthesis should be attributed to the catalytic activity of TH controlled by its phosphorylation, because Western blot analysis revealed that the down-regulation of NT5DC2 tended to increase the level of TH phosphorylated at its Ser residues, but not that of the TH protein. Moreover, the induction of kinase activity by forskolin markedly potentiated the phosphorylation of TH at its Ser40 in PC12D cells having down-regulated NT5DC2. Immunocytochemical analysis of PC12D cells demonstrated that NT5DC2, TH protein, and TH phosphorylated at its Ser40 were predominantly localized in the cytoplasm and that the localization of NT5DC2 and TH proteins partially overlapped. Collectively, our results indicate that NT5DC2 could work to inhibit the DOPA synthesis by decreasing the phosphorylation of TH at its Ser40. We propose that NT5DC2 might decrease this phosphorylation of TH by promoting dephosphorylation or by inhibiting kinase activity.

Asunto(s)

Estudio de Asociación del Genoma Completo , Tirosina 3-Monooxigenasa , Dopamina , Fosforilación , Tirosina , Tirosina 3-Monooxigenasa/metabolismo

RESUMEN

Tyrosine hydroxylase (TH), which catalyzes the conversion of l-tyrosine to l-DOPA, is the rate-limiting enzyme in the biosynthesis of catecholamines. It is well known that both α-synuclein and 14-3-3 protein family members bind to the TH molecule and regulate phosphorylation of its N-terminus by kinases to control the catalytic activity. In this present study we investigated whether other proteins aside from these 2 proteins might also bind to TH molecules. Nano-LC-MS/MS analysis revealed that 5'-nucleotidase domain-containing protein 2 (NT5DC2), belonging to a family of haloacid dehalogenase-type (HAD) phosphatases, was detected in the immunoprecipitate of PC12D cell lysates that had been reacted with Dynabeads protein G-anti-TH antibody conjugate. Surprisingly, NT5DC2 had already been revealed by Genome-Wide Association Studies (GWAS) as a gene implicated in neuropsychiatric disorders such as schizophrenia, bipolar disorder, which are diseases related to the abnormality of dopamine activity in the brain, although the role that NT5DC2 plays in these diseases remains unknown. Therefore, we investigated the effect of NT5DC2 on the TH molecule. The down-regulation of NT5DC2 by siRNA increased the synthesis of catecholamines (dopamine, noradrenaline, and adrenaline) in PC12D cells. These increases might be attributed to the catalytic activity of TH and not to the intracellular stability of TH, because the intracellular content of TH assessed by Western blotting was not changed by the down-regulation of NT5DC2. Collectively, our results indicate that NT5DC2 inhibited the synthesis of dopamine by decreasing the enzymatic activity of TH.

Asunto(s)

5'-Nucleotidasa/metabolismo , Catecolaminas/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , 5'-Nucleotidasa/genética , Animales , Línea Celular , Cromatografía Liquida , Regulación hacia Abajo , Células PC12 , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/genética , Ratas , Espectrometría de Masas en Tándem

RESUMEN

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its stability is a fundamental factor to maintain the level of the catecholamines in cells. However, the intracellular stability of TH determined by the degradation remains unknown; although the TH molecule phosphorylated at its Ser19 was observed in the nucleus, and the phosphorylation suspected to trigger its proteasome-mediated degradation. Computer-assisted analysis using the cNLS Mapper program predicted that two sequences of nuclear localization signals (NLS) exist in the N-terminus of TH molecule containing the phosphorylation sites Ser19, Ser31, and Ser40 (Pro9-Arg38 and Lys12-Ile42): the NLS scores indicated that TH could become localized in both nucleus and cytoplasm. Moreover, inhibition of the importin α/ß-mediated nuclear import pathway increased the level of TH phosphorylated at its Ser19 in PC12D cells. The results suggest that TH might be imported to nucleus from cytoplasm to be degraded. Recent studies revealed that proteasomes predominantly exist in the nucleus rather than in the cytoplasm to degrade the nuclear proteins related to cell-cycle progression, gene expression, DNA damage, and DNA repair. Therefore, these studies suggest that the relationship between the phosphorylation and the nuclear localization of the TH molecule should be a matter of focus to understand the mechanism of proteasome-mediated degradation of the enzyme as a first priority.

Asunto(s)

Núcleo Celular/metabolismo , Citoplasma/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Secuencia de Aminoácidos , Animales , Núcleo Celular/química , Citoplasma/química , Humanos , Líquido Intracelular/química , Líquido Intracelular/metabolismo , Fosforilación/fisiología , Complejo de la Endopetidasa Proteasomal/análisis , Tirosina 3-Monooxigenasa/análisis

RESUMEN

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its stability is a fundamental factor to maintain the level of the catecholamines in cells. However, the intracellular stability determined by the degradation pathway remains unknown. In this study, we investigated the mechanism by which phosphorylation of TH affected the proteasome pathway. The inhibition of proteasomes by MG-132 increased the percentage of TH molecules phosphorylated at their Ser19, Ser31 and/or Ser40 among the total TH proteins to about 70% in PC12D cells over a 24-hr period; although the percentage of phosphorylated TH molecules was about 20% under basal conditions. Moreover, the inhibition of proteasomes by epoxomicin with high specificity increased primarily the quantity of TH molecules phosphorylated at their Ser19. The phosphorylation of Ser19 potentiated Ser40 phosphorylation in cells by a process known as hierarchical phosphorylation. Therefore, the proteasome inhibition might result in an increase in the levels of all 3 phosphorylated TH forms, thus complicating interpretation of data. Conversely, activation of proteasome degradation by IU-1, which is an inhibitor for the deubiquitinating activity of USP14, decreased only the quantity of TH molecules phosphorylated at their Ser19, although it did not decrease that of TH phosphorylated at its Ser31 and Ser40 or that of TH molecules. These results suggest that the phosphorylation of Ser19 in the N-terminal portion of TH is critical as a trigger for the degradation of this enzyme by the ubiquitin-proteasome pathway.

Asunto(s)

Complejo de la Endopetidasa Proteasomal/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Animales , Células PC12 , Fosforilación , Proteolisis , Ratas , Transducción de Señal , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitinación

RESUMEN

We previously showed that aripiprazole increases intracellular NADPH and glucose-6-phosphate dehydrogenase mRNA in PC12 cells. Aripiprazole presumably activates a system that concurrently detoxifies reactive oxygen species and replenishes NADPH. Nrf2, a master transcriptional regulator of redox homeostasis genes, also activates the pentose phosphate pathway, including NADPH production. Therefore, our aim was to determine whether aripiprazole activates Nrf2 in PC12 cells. Aripiprazole increased mRNA expression of Nrf2-dependent genes (NAD(P)H-quinone oxidoreductase-1, Nqo1; heme oxygenase-1, HO1; and glutamate-cysteine ligase catalytic subunit) and protein expression of Nqo1 and HO1 in these cells (p < 0.05). To maintain increased Nrf2 activity, it is necessary to inhibit Nrf2 degradation; this is done by causing Nrf2 to dissociate from Keap1 or ß-TrCP. However, in aripiprazole-treated cells, the relative amount of Nrf2 anchored to Keap1 or ß-TrCP was unaffected and Nrf2 in the nuclear fraction decreased (p < 0.05). Aripiprazole did not affect phosphorylation of Nrf2 at Ser40 and decreased the relative amount of acetylated Nrf2 (p < 0.05). The increase in Nqo1 and HO1 in aripiprazole-treated cells cannot be explained by the canonical Nrf2-degrading pathways. Further experiments are needed to determine the biochemical mechanisms underlying the aripiprazole-induced increase in these enzymes.

Asunto(s)

Antipsicóticos/farmacología , Aripiprazol/farmacología , Hemo Oxigenasa (Desciclizante)/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Acetilación/efectos de los fármacos , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Supervivencia Celular/efectos de los fármacos , Citosol/efectos de los fármacos , Citosol/enzimología , Glutamato-Cisteína Ligasa/metabolismo , Peróxido de Hidrógeno/toxicidad , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch , Células PC12 , Fosforilación/efectos de los fármacos , Ratas , Proteínas con Repetición de beta-Transducina/metabolismo

RESUMEN

We previously reported that an optimal dose of lipopolysaccharide (LPS) markedly extends the lifespan of murine primary-cultured microglia by suppressing cell death pathways. In this study, we investigated the effects of LPS pretreatment on UV light-induced apoptosis of cells from the microglial cell line BV-2. More than half of BV-2 cells were apoptotic, and procaspase-3 was cleaved into its active form at 3 h of UV irradiation. In contrast, in BV-2 cells treated with LPS for 24 h, UV irradiation caused neither apoptosis nor procaspase-3 cleavage. LPS treatment arrested the cell cycle in G1 phase and upregulated cyclin-dependent kinase inhibitor p21(Waf1/Cip1) and growth arrest and DNA damage-inducible (GADD) 45α in BV-2 cells. When p21(Waf1/Cip1) and GADD45α were knocked down by small interfering RNA, procaspase-3 was cleaved into its active form to induce apoptosis. Our findings suggest that LPS inhibits UV-induced apoptosis in BV-2 cells through arrest of the cell cycle in G1 phase by upregulation of p21(Waf1/Cip1) and GADD45α. Excessive activation of microglia may play a critical role in the exacerbation of neurodegeneration, therefore, normalizing the precise regulation of apoptosis may be a new strategy to prevent the deterioration caused by neurodegenerative disorders.

Asunto(s)

Apoptosis/efectos de los fármacos , Fase G1/efectos de los fármacos , Lipopolisacáridos/farmacología , Microglía/efectos de los fármacos , Rayos Ultravioleta , Animales , Apoptosis/efectos de la radiación , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Citometría de Flujo , Fase G1/efectos de la radiación , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de la radiación , Ratones , Microglía/efectos de la radiación , Análisis de Secuencia por Matrices de Oligonucleótidos , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , ARN Mensajero , ARN Interferente Pequeño/farmacología , Factores de Tiempo

RESUMEN

In aripiprazole-treated PC12 cells, we previously showed that the mitochondrial membrane potential (Δψm) was rather increased in spite of lowered cytochrome c oxidase activity. To address these inconsistent results, we focused the NADPH generation by glucose-6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme of the pentose phosphate pathway (PPP), to titrate reactive oxygen species (ROS) that results in the Δψm maintenance. G6PD may be also involved in another inconsistent result of lowered intracellular lactate level in aripiprazole-treated PC12 cells, because PPP competes glucose-6-phosphate with the glycolytic pathway, resulting in the downregulation of glycolysis. Therefore, we assayed intracellular amounts of NADPH, ROS, and the activities of the enzymes generating or consuming NADPH (G6PD, NADP(+)-dependent isocitrate dehydrogenase, NADP(+)-dependent malic enzyme, glutathione reductase, and NADPH oxidase [NOX]) and estimated glycolysis in 50 µM aripiprazole-, clozapine-, and haloperidol-treated PC12 cells. NADPH levels were enhanced only in aripiprazole-treated ones. Only haloperidol increased ROS. However, the enzyme activities did not show significant changes toward enhancing NADPH level except for the aripiprazole-induced decrease in NOX activity. Thus, the lowered NOX activity could have contributed to the aripiprazole-induced increase in the NADPH level by lowering ROS generation, resulting in maintained Δψm. Although the aforementioned assumption was invalid, the ratio of fructose-1,6-bisphosphate to fructose-6-phosphate was decreased by all antipsychotics examined. Pyruvate kinase activity was enhanced only by aripiprazole. In summary, these observations indicate that aripiprazole possibly possesses the pharmacological superiority to clozapine and haloperidol in the ROS generation and the adjustment of glycolytic pathway.

Asunto(s)

Antipsicóticos/farmacología , NADPH Oxidasas/metabolismo , NADP/metabolismo , Neuronas/efectos de los fármacos , Piperazinas/farmacología , Quinolonas/farmacología , Animales , Aripiprazol , Neuronas/metabolismo , Células PC12 , Ratas , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines, is a key protein involved in the pathogenesis of neurodegenerative diseases such as Parkinson's disease. Elucidation of the mechanisms regulating the synthesis, degradation, and activity of TH should be a first target in order to understand the role of this enzyme in pathogenesis. Recently, several reports suggest that the ubiquitin-proteasome pathway is a prerequisite for the degradation of TH and that the N-terminal part of TH plays a critical role in the degradation. In this report, we propose the mechanism by which the N-terminal part of TH regulates the degradation of this enzyme. Moreover, we integrate our findings with recent progress in other areas of TH regulation.

Asunto(s)

Complejo de la Endopetidasa Proteasomal/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Animales , Catecolaminas/metabolismo , Humanos , Fosforilación

RESUMEN

Postmortem brain biochemistry has revealed that the main symptom of movement disorder in Parkinson's disease (PD) is caused by a deficiency in dopamine (DA) at the nerve terminals of degenerating nigro-striatal DA neurons in the striatum. Since tyrosine hydroxylase (TH) is the rate-limiting enzyme for the biosynthesis of DA, TH may play an important role in the disease process of PD. DA regulated by TH activity is thought to interact with α-synuclein protein, which results in intracellular aggregates called Lewy bodies and causes apoptotic cell death during the aging process. Human TH has several isoforms produced by alternative mRNA splicing, which may affect activation by phosphorylation of serine residues in the N-terminus of TH. The activity and protein level of TH are decreased to cause DA deficiency in the striatum in PD. However, the homo-specific activity (activity/enzyme protein) of TH is increased. This increase in TH homo-specific activity suggests activation by increased phosphorylation at the N-terminus of the TH protein for a compensatory increase in DA synthesis. We recently found that phosphorylation of the N-terminal portion of TH triggers proteasomal degradation of the enzyme to increase TH turnover. We propose a hypothesis that this compensatory activation of TH by phosphorylation in the remaining DA neurons may contribute to a further decrease in TH protein and activity in DA neurons in PD, causing a vicious circle of decreasing TH activity, protein level and DA contents. Furthermore, increased TH homo-specific activity leading to an increase in DA may cause toxic reactive oxygen species in the neurons to promote neurodegeneration.

Asunto(s)

Encéfalo/enzimología , Enfermedad de Parkinson/patología , Tirosina 3-Monooxigenasa/metabolismo , Encéfalo/patología , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Humanos , Cambios Post Mortem

RESUMEN

1. Previously, we reported that an optimal dose of lipopolysaccharide (LPS) markedly extends the life span of mouse primary-cultured microglia by suppressing apoptotic and autophagic cell death pathways. The aim of the present study was to assess how these cells protect themselves against reactive oxygen species (ROS) generated by LPS treatment. 2. The study was conducted in microglia obtained from murine neonate brain, which are destined to die within a few days under ordinary culture conditions. 3. The generation of ROS was maximal after 15 h LPS treatment (1 ng/mL LPS and 100 ng/mL LPS). The expression of inducible nitric oxide (NO) synthase protein was significantly increased by Day 1 of LPS treatment and was followed by the production of NO. The expression of either Cu/Zn- or Mn-superoxide dismutase protein (SOD) was also increased by 16 h and Day 1 of LPS treatment. LPS did not affect the expression of Cu/Zn- and Mn-SOD proteins, nor did it extend the life span of microglia that had mutated Toll-like receptor (TLR) 4. 4. The findings of the present study suggest that SODs function as a potent barrier to overcome ROS generated in primary-cultured microglia following LPS treatment and that TLR4 may be significantly involved in inducing these proteins. The microglia may be able to protect themselves against oxidative stress, allowing them to live for more than 1 month. Because long-lived microglia may play a critical role in the exacerbation of neurodegeneration, bringing activated microglia back to their resting stage could be a new and promising strategy to inhibit the deterioration underlying neurodegenerative disorders.

Asunto(s)

Radicales Libres/metabolismo , Microglía/metabolismo , Estrés Oxidativo , Superóxido Dismutasa/metabolismo , Animales , Catalasa/metabolismo , Separación Celular , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C3H , Microglía/citología , Microglía/efectos de los fármacos , Enfermedades Neurodegenerativas/inducido químicamente , Enfermedades Neurodegenerativas/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa-1 , Receptor Toll-Like 4/metabolismo

RESUMEN

Aripiprazole is the only atypical antipsychotic drug known to cause the phosphorylation of AMP-activated protein kinase (AMPK) in PC12 cells. However, the molecular mechanisms underlying this phosphorylation in aripiprazole-treated PC12 cells have not yet been clarified. Here, using PC12 cells, we show that these cells incubated for 24 h with aripiprazole at 50 µM and 25 mM glucose underwent a decrease in their NAD⁺/NADH ratio. Aripiprazole suppressed cytochrome c oxidase (COX) activity but enhanced the activities of pyruvate dehydrogenase (PDH), citrate synthase and Complex I. The changes in enzyme activities coincided well with those in NADH, NAD⁺, and NAD⁺/NADH ratio. However, the bioenergetic peril judged by the lowered COX activity might not be accompanied by excessive occurrence of apoptotic cell death in aripiprazole-treated cells, because the mitochondrial membrane potential was not decreased, but rather increased. On the other hand, when PC12 cells were incubated for 24 h with clozapine at 50 µM and 25 mM glucose, the NAD⁺/NADH ratio did not change. Also, the COX activity was decreased; and the PDH activity was enhanced. These results suggest that aripiprazole-treated PC12 cells responded to the bioenergetic peril more effectively than the clozapine-treated ones to return the ATP biosynthesis back toward its ordinary level. This finding might be related to the fact that aripiprazole alone causes phosphorylation of AMPK in PC12 cells.

Asunto(s)

Antipsicóticos/farmacología , Carbono/metabolismo , Clozapina/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glucólisis/efectos de los fármacos , Piperazinas/farmacología , Quinolonas/farmacología , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis/efectos de los fármacos , Aripiprazol , Supervivencia Celular/efectos de los fármacos , Dihidrolipoamida Deshidrogenasa/genética , Dihidrolipoamida Deshidrogenasa/metabolismo , Relación Dosis-Respuesta a Droga , Complejo IV de Transporte de Electrones/metabolismo , Líquido Extracelular/efectos de los fármacos , Glucosa/farmacología , Hipoxantina Fosforribosiltransferasa/genética , Hipoxantina Fosforribosiltransferasa/metabolismo , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Cetona Oxidorreductasas/genética , Cetona Oxidorreductasas/metabolismo , Ácido Láctico/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , NAD/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Células PC12/efectos de los fármacos , Células PC12/enzimología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Ácido Pirúvico/metabolismo , ARN Mensajero/metabolismo , Ratas , Factores de Tiempo

RESUMEN

BACKGROUND: Activated microglia secrete inflammatory cytokines and may play roles in the progression of neurodegenerative diseases. However, the mechanism underlying microglial activation remains unclear. OBJECTIVE: Our aim was to examine the regulation of activated microglia through their cell death and survival pathways. METHODS: We used mouse primary-cultured microglia, which are destined to die within a few days under ordinary culture conditions. The microglia live for longer than 1 month, without any measurable increase in apoptotic or necrotic cell death, when kept activated by sublethal concentrations of lipopolysaccharide (LPS). RESULTS: LPS-treated microglia showed changes in shape. LPS treatment had no effect on the level of the proapoptotic Bcl-2-associated X protein but increased the level of the antiapoptotic protein Bcl-xL at day 1. Furthermore, the level of microtubule-associated light chain 3-II, a marker protein for autophagy, was decreased 3 h after exposure to LPS. CONCLUSION: An increase in Bcl-xL seems to inhibit both apoptosis and autophagy. Our results suggest that long-lived microglia resulting from exposure to the optimal dose of LPS may play critical roles in the progression of neurodegeneration.

Asunto(s)

Apoptosis/fisiología , Citocinas/metabolismo , Microglía/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Encéfalo/citología , Caspasa 3/metabolismo , Células Cultivadas , Lipopolisacáridos/farmacología , Ratones , Transducción de Señal/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismo , Proteína bcl-X/metabolismo

RESUMEN

This review summarizes the effects of neuroinflammatory stress on the subventricular zone (SVZ), where new neurons are constitutively produced in the adult brain, especially focusing on the relation with Parkinson's disease (PD), because the SVZ is under the control of dopaminergic afferents from the substantia nigra (SN). In Lewy bodies-positive-PD, microglia is known to phagocytoze aggregated α-synuclein, resulting in the release of inflammatory cytokines. The neurogenesis in the SVZ should be affected in PD brain by the neuroinflammatory process. The administration of lipopolysaccaharide is available as an alternative model for microglia-induced loss of dopaminergic neurons and also the impairment of stem cell maintenance. Therefore, the research on the neuroinflammatory process in the SVZ gives us a hint to prevent the outbreak of PD or at least slow the disease process.

Asunto(s)

Ventrículos Cerebrales/patología , Inflamación/patología , Sistema Nervioso/patología , Enfermedad de Parkinson/patología , Estrés Fisiológico , Animales , Humanos , Transducción de Señal

RESUMEN

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its N-terminus plays a critical role in the intracellular stability of the enzyme. In the present study, we investigated the mechanism by which the N-terminal region of TH affects this stability. TH molecules phosphorylated at their Ser31 and Ser40 were localized predominantly in the cytoplasm of PC12D cells. However, those molecules phosphorylated at Ser19 were found mainly in the nucleus, whereas they seemed to be negligible in the cytoplasm. The inhibition of proteasomes increased the quantity of TH molecules phosphorylated at their Ser19 and Ser40, although it did not increase that of TH molecules or that of TH phosphorylated at its Ser31. The inhibition of autophagy did not affect the amount of the TH molecule or that of its three phosphorylated forms. Deletion mutants of human TH type-1 lacking the N-terminal region containing the three phosphorylation sites possessed high stability of the enzyme in PC12D cells. These results suggest that the phosphorylation of the N-terminal portion of TH regulates the degradation of this enzyme by the ubiquitin-proteasome pathway.

Asunto(s)

Complejo de la Endopetidasa Proteasomal/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Ubiquitina/metabolismo , Animales , Autofagia , Humanos , Lisosomas/enzimología , Células PC12 , Fosforilación , Estructura Terciaria de Proteína/genética , Ratas , Eliminación de Secuencia , Tirosina 3-Monooxigenasa/genética

RESUMEN

By converting changes in intracellular energy status to changes in cell membrane polarization, ATP-sensitive K(+) (K(ATP)) channels in hypothalamic appetite-regulating neurons play a critical role in linking neuronal electrochemical function, metabolic and energy status, and feeding behavior. Most atypical antipsychotics (AAPs) increase the appetite of patients with schizophrenia and thus cause obesity. This study aimed to explain the mechanism underlying AAP-induced appetite stimulation, based on the fact that the efficiency of fatty acid uptake into mitochondria generating ATP through ß-oxidation is determined by the rate of fatty acid synthesis. Using PC12 cells exposed to clozapine, olanzapine, risperidone, quetiapine, ziprasidone, aripiprazole, and haloperidol, we measured intracellular ATP and mRNA and protein expression of enzymes and related substances involved in fatty acid synthesis and K(ATP) channel function. Forty-eight-hour treatment of cells with 50 µM aripiprazole in 5.6 mM glucose decreased intracellular ATP. Only 50 µM aripiprazole phosphorylated AMP-activated protein kinase (AMPK); none of the other antipsychotics did so to a detectable level. Expression of carnitine palmitoyltransferase 1a, uncoupling protein 2, and sulfonylurea receptor 1 was unaffected by the antipsychotics, although expression of their mRNA was affected by AAPs. Pyrilamine (H(1) receptor antagonist), ketanserin (5HT(2) receptor antagonist), and raclopride (D(2) receptor antagonist) alone or in combination had no effect on expression of the aforementioned proteins. Therefore, although this study did not differentiate orexigenic and non-orexigenic AAPs, it suggests that aripiprazole is unique in its ability to activate AMPK.

Asunto(s)

Proteínas Quinasas Activadas por AMP/metabolismo , Antipsicóticos/farmacología , Haloperidol/farmacología , Piperazinas/farmacología , Quinolonas/farmacología , Animales , Regulación del Apetito/efectos de los fármacos , Regulación del Apetito/fisiología , Aripiprazol , Ácidos Grasos/biosíntesis , Fosforilación Oxidativa/efectos de los fármacos , Células PC12 , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , ARN Mensajero/análisis , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas

RESUMEN

Because the subventricular zone (SVZ) constantly supplies newly generated neurons to the olfactory bulb (OB) along the rostral migratory stream (RMS) in adult brain, SVZ-RMS-OB axis has been thought to work as a unit. We previously reported that peripherally injected lipopolysaccharide (LPS) induces apoptosis in the OB in young adult mice. Therefore, this study was undertaken to examine whether peripherally injected LPS induces apoptotic cell death also in the SVZ. Two mouse strains were used: C3H/HeN and Toll-like receptor 4-mutated C3H/HeJ, and wild-type C57BL/6 and TNFR1(-/-)-2(-/-), in which the genes tumor necrosis factor receptor (TNFR)1 and TNFR2 are knocked out. Immunohistochemical study and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay done on the SVZ-RMS pathway of young adult male mice showed that peripherally injected LPS switches on the apoptotic signal by cleaving pro-caspase-3, thus possibly increasing the number of cells dying from apoptosis in these areas in adult mice. Activation of the tumor necrosis factor (TNF)-alpha-TNFR system played a critical role in fully inducing apoptosis in this pathway. We suggest that TNF-alpha was probably released not from microglia but from astrocytes in the SVZ and RMS.

Asunto(s)

Apoptosis/efectos de los fármacos , Ventrículos Cerebrales/citología , Ventrículos Cerebrales/efectos de los fármacos , Lipopolisacáridos/farmacología , Animales , Caspasa 3/metabolismo , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proteínas de Dominio Doblecortina , Proteína Ácida Fibrilar de la Glía/metabolismo , Etiquetado Corte-Fin in Situ/métodos , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/fisiología , Neuropéptidos/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Receptores Tipo II del Factor de Necrosis Tumoral/deficiencia , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Microglial activation has been implicated in the recognition and phagocytic removal of degenerating neurons; however, this process must be tightly regulated in the central nervous system, because prolonged activation could damage normal neurons. We report that mouse primary-cultured microglia, which are destined to die within a few days under ordinary culture conditions, can live for more than 1 month when kept activated by lipopolysaccharide (LPS) treatment. Primary-cultured microglia treated with sublethal doses of LPS remained viable, without any measurable increase in apoptotic or necrotic cell death. LPS-treated microglia had an arborescent shape, with enlarged somata and thickened cell bodies. Although the amount of intracellular ATP in these microglia was reduced by 2 h after the start of LPS treatment, this had no effect on the viability of the cells. LPS treatment of microglia increased the antiapoptotic factor Bcl-xL protein level at day 1, although the level of the proapoptotic Bcl-associated X-protein was unaffected. Furthermore, the level of microtubule-associated light chain 3, a marker protein for autophagy, decreased at 3 h after exposure to LPS. These data show that the optimal dose of LPS suppresses the induction of both apoptosis and autophagy in primary-cultured microglia, allowing the cells to stay alive for more than 1 month. Because long-lived microglia may play critical roles in the exacerbation of neurodegeneration, our findings suggest that inducing a resting stage in active microglia could be a new and promising strategy to inhibit the deterioration of neurodegenerative disease.

Asunto(s)

Lipopolisacáridos/farmacología , Microglía/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Adenosina Trifosfato/metabolismo , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Microglía/citología , Proteínas Asociadas a Microtúbulos/metabolismo , Necrosis/tratamiento farmacológico , ARN Mensajero/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Proteína bcl-X/metabolismo

RESUMEN

Peripheral administration of lipopolysaccharide (LPS) in an amount that produces acute stress has been found to affect the catecholamine systems in the brain. Acute peripheral LPS administration activated norepinephrine (NE) metabolism in the locus ceruleus (LC). Approximately 40% of murine LC neurons project to the olfactory bulb (OB) and the anterior olfactory nucleus (AON). Thus, we investigated the effects of a single intra-peritoneal (i.p.) LPS injection on catecholamine biosynthesis in the OB and AON in 8-week-old C3H/HeN male mice. In the AON, the content of dopamine (DA), but not that of NE, was highly increased 6 h after LPS injection. In the OB, the contents of DA and NE did not change; but within 2 h after a single i.p. LPS injection, the mRNA levels of IkappaB, TNF-alpha, and TNF-alpha receptor type 1 were significantly enhanced. Almost all TNF-alpha-immunoreactive cells in the OB of the LPS-injected mice were located in the granule cell layer, and unexpectedly, they were not microglia but astroglia. The number of TUNEL-positive cells identified exclusively in the granule cell layer was significantly increased at 24 h after LPS injection. Therefore, our data suggest that astroglia activated by peripherally injected LPS may release TNF-alpha, which may trigger apoptosis in the granule cell layer in the OB. The increase in DA content in the AON and the production of TNF-alpha and apoptotic cells in the OB by acute peripheral LPS administration are not likely to be related.

Asunto(s)

Dopamina/metabolismo , Lipopolisacáridos/farmacología , Bulbo Olfatorio/efectos de los fármacos , Bulbo Olfatorio/metabolismo , Animales , Apoptosis/fisiología , Catecolaminas/metabolismo , Citocinas/genética , Citocinas/metabolismo , Locus Coeruleus/citología , Locus Coeruleus/metabolismo , Masculino , Ratones , Ratones Endogámicos C3H , Bulbo Olfatorio/citología , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo