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1.
Mol Brain ; 17(1): 20, 2024 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-38685046

RESUMO

While the excessive inflammation in cancer cachexia is well-known to be induced by the overproduction of inflammatory mediators in the periphery, microflora disruption and brain dysfunction are also considered to contribute to the induction of cancer cachexia. Hypothalamic microglia play a crucial role in brain inflammation and central-peripheral immune circuits via the production of inflammatory mediators. In the present study, we evaluated possible changes in excessive secretion of gut microbiota-derived endotoxin and the expression timeline of several inflammation-regulatory mediators and their inhibiting modulators in hypothalamic microglia of a mouse model of cancer cachexia following transplantation of pancreatic cancer cells. We demonstrated that the plasma level of lipopolysaccharide (LPS) was significantly increased with an increase in anaerobic bacteria, especially Firmicutes, in the gut at the late stage of tumor-bearing mice that exhibited dramatic appetite loss, sarcopenia and severe peripheral immune suppression. At the early stage, in which tumor-bearing mice had not yet displayed "cachexia symptoms", the mRNA expression of pro-inflammatory cytokines, but not of the neurodegenerative and severe inflammatory modulator lipocalin-2 (LCN2), was significantly increased, whereas at the late "cachexia stage", the level of LCN2 mRNA was significantly increased along with significant decreases in levels of inhibitory immune checkpoint receptors programmed death receptor-1 (PD-1) and CD112R in hypothalamic microglia. In addition, a high density of activated neurons in the paraventricular nucleus (PVN) of the hypothalamus region and a significant increase in corticosterone secretion were found in cachexia model mice. Related to the cachexia state, released corticosterone was clearly increased in normal mice with specific activation of PVN neurons. A marked decrease in the natural killer cell population was also observed in the spleen of mice with robust activation of PVN neurons as well as mice with cancer cachexia. On the other hand, in vivo administration of LPS in normal mice induced hypothalamic microglia with low expression of inhibitory immune checkpoint receptors. These findings suggest that the induction of cancer cachexia may parallel exacerbation of the hypothalamic inflammatory status with polarization to microglia expressed with low levels of inhibitory immune checkpoint receptors following LPS release from the gut microflora.


Assuntos
Caquexia , Hipotálamo , Lipocalina-2 , Lipopolissacarídeos , Microglia , Animais , Caquexia/complicações , Caquexia/patologia , Microglia/metabolismo , Hipotálamo/metabolismo , Lipocalina-2/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Linhagem Celular Tumoral , Camundongos , Receptor de Morte Celular Programada 1/metabolismo , Microbioma Gastrointestinal , Citocinas/metabolismo , Neoplasias/complicações , Camundongos Endogâmicos C57BL , Mediadores da Inflamação/metabolismo , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico
2.
Biochem Biophys Res Commun ; 648: 36-43, 2023 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-36724558

RESUMO

It is considered that sensory neurons extend into the tumor microenvironment (TME), which could be associated with tumor growth. However, little is known about how sensory signaling could promote tumor progression. In this study, chemogenetic activation of transient receptor potential vanilloid 1 (Trpv1)-positive sensory neurons (C-fibers) by the microinjection of AAV-hSyn-FLEX-hM3Dq-mCherry into the sciatic nerve dramatically increased tumor volume in tumor-bearing Trpv1-Cre mice. This activation in Trpv1::hM3Dq mice that had undergone tumor transplantation significantly reduced the population of tumor-infiltrating CD4+ T cells and increased the mRNA level of the M2-macrophage marker, CX3C motif chemokine receptor 1 (Cx3cr1) in immunosuppressive cells, such as tumor-associated macrophages (TAMs) and tumor-infiltrating monocytic myeloid-derived suppressor cells (M-MDSCs). Under these conditions, we found a significant correlation between the decreased expression of the M1-macrophage marker Tnf and tumor volume. These findings suggest that repeated activation of Trpv1-positive sensory neurons may facilitate tumor growth along with changes in tumor-infiltrating immune cells.


Assuntos
Antineoplásicos , Camundongos , Animais , Antineoplásicos/metabolismo , Macrófagos/metabolismo , Células Receptoras Sensoriais/metabolismo , Linhagem Celular Tumoral , Transplante de Neoplasias , Microambiente Tumoral , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo
3.
Mol Brain ; 16(1): 19, 2023 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-36737827

RESUMO

A growing body of evidence suggests that intractable pain reduces both the quality of life and survival in cancer patients. In the present study, we evaluated whether chronic pain stimuli could directly affect cancer pathology using tumor-bearing mice. For this purpose, we used two different models of chronic pain in mice, neuropathic pain and persistent postsurgical pain, with Lewis lung carcinoma (LLC) as tumor cells. We found that tumor growth was dramatically promoted in these pain models. As well as these pain models, tumor growth of LLC, severe osteosarcoma (AXT) and B16 melanoma cells was significantly promoted by concomitant activation of sensory neurons in AAV6-hM3Dq-injected mice treated with the designer drug clozapine-N-oxide (CNO). Significant increases in mRNA levels of vascular endothelial growth factor-A (Vegfa), tachykinin precursor 1 (Tac1) and calcitonin-related polypeptide alpha (Calca) in the ipsilateral side of dorsal root ganglion of AAV6-hM3Dq-injected mice were observed by concomitant activation of sensory neurons due to CNO administration. Moreover, in a model of bone cancer pain in which mice were implanted with AXT cells into the right femoral bone marrow cavity, the survival period was significantly prolonged by repeated inhibition of sensory neurons of AAV6-hM4Di-injected mice by CNO administration. These findings suggest that persistent pain signals may promote tumor growth by the increased expression of sensory-located peptides and growth factors, and controlling cancer pain may prolong cancer survival.


Assuntos
Neoplasias Ósseas , Dor do Câncer , Dor Crônica , Camundongos , Animais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Dor do Câncer/complicações , Dor Crônica/metabolismo , Qualidade de Vida , Células Receptoras Sensoriais/metabolismo , Neoplasias Ósseas/complicações
4.
Mol Brain ; 16(1): 18, 2023 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-36732798

RESUMO

A growing body of evidence suggests that excess stress could aggravate tumor progression. The paraventricular nucleus (PVN) of the hypothalamus plays an important role in the adaptation to stress because the hypothalamic-pituitary-adrenal (HPA) axis can be activated by inducing the release of corticotropin-releasing hormone (CRH) from the PVN. In this study, we used pharmacogenetic techniques to investigate whether concomitant activation of CRHPVN neurons could directly contribute to tumor progression. Tumor growth was significantly promoted by repeated activation of CRHPVN neurons, which was followed by an increase in the plasma levels of corticosterone. Consistent with these results, chronic administration of glucocorticoids induced tumor progression. Under the concomitant activation of CRHPVN neurons, the number of cytotoxic CD8+ T cells in the tumor microenvironment was dramatically decreased, and the mRNA expression levels of hypoxia inducible factor 1 subunit α (HIF1α), glucocorticoid receptor (GR) and Tsc22d3 were upregulated in inhibitory lymphocytes, tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). Furthermore, the mRNA levels of various kinds of driver molecules related to tumor progression and tumor metastasis were prominently elevated in cancer cells by concomitant activation of CRHPVN neurons. These findings suggest that repeated activation of the PVN-CRHergic system may aggravate tumor growth through a central-peripheral-associated tumor immune system.


Assuntos
Linfócitos T CD8-Positivos , Núcleo Hipotalâmico Paraventricular , Núcleo Hipotalâmico Paraventricular/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Hipotálamo/metabolismo , Hormônio Liberador da Corticotropina/metabolismo , Corticosterona , Neurônios/metabolismo , RNA Mensageiro/metabolismo
5.
Br J Cancer ; 127(8): 1565-1574, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35945243

RESUMO

BACKGROUND: It has been considered that activation of peripheral µ-opioid receptors (MORs) induces side effects of opioids. In this study, we investigated the possible improvement of the immune system in tumour-bearing mice by systemic administration of the peripheral MOR antagonist naldemedine. METHODS: The inhibitory effect of naldemedine on MOR-mediated signalling was tested by cAMP inhibition and ß-arrestin recruitment assays using cultured cells. We assessed possible changes in tumour progression and the number of splenic lymphocytes in tumour-bearing mice under the repeated oral administration of naldemedine. RESULTS: Treatment with naldemedine produced a dose-dependent inhibition of both the decrease in the cAMP level and the increase in ß-arrestin recruitment induced by the MOR agonists. Repeated treatment with naldemedine at a dose that reversed the morphine-induced inhibition of gastrointestinal transport, but not antinociception, significantly decreased tumour volume and prolonged survival in tumour-transplanted mice. Naldemedine administration significantly decreased the increased expression of immune checkpoint-related genes and recovered the decreased level of toll-like receptor 4 in splenic lymphocytes in tumour-bearing mice. CONCLUSIONS: The blockade of peripheral MOR may induce an anti-tumour effect through the recovery of T-cell exhaustion and promotion of the tumour-killing system.


Assuntos
Neoplasias , Receptores Opioides mu , Analgésicos Opioides/efeitos adversos , Animais , Sistema Imunitário/metabolismo , Camundongos , Derivados da Morfina , Naltrexona/análogos & derivados , Neoplasias/induzido quimicamente , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Receptor 4 Toll-Like/metabolismo , beta-Arrestinas/metabolismo
6.
Mol Brain ; 15(1): 17, 2022 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-35172858

RESUMO

Recent research has suggested that the mesolimbic dopamine network that mainly terminates in the nucleus accumbens may positively control the peripheral immune system. The activation of dopamine receptors in neurons in the nucleus accumbens by the release of endogenous dopamine is thus expected to contribute to efferent immune regulation. As in the stimulation of Gs-coupled dopamine D1-receptors or Gi-coupled D2-receptors by endogenous dopamine, we investigated whether specific stimulation of dopamine D1-receptor-expressing neurons or inhibition of dopamine D2-receptor-expressing neurons in the nucleus accumbens could produce anti-tumor effects and improve the immune system in transgenic mice using pharmacogenetic techniques. Repeated stimulation of D1-receptor-expressing neurons in either the medial shell, lateral shell or core regions of the nucleus accumbens significantly decreased tumor volume under a state of tumor transplantation, whereas repeated suppression of D2-receptor-expressing neurons in these areas had no effect on this event. The number of splenic CD8+ T cells was significantly increased following repeated stimulation of D1-receptor-expressing neurons in the nucleus accumbens of mice with tumor transplantation. Furthermore, this stimulation produced a significant reduction in the population of splenic CD8+ T cells that expressed immune checkpoint-related inhibitory receptors, PD-1, TIM-3 and LAG-3. These findings suggest that repeated stimulation of D1-receptor-expressing neurons (probably D1-receptor-expressing medium spiny neurons) in the nucleus accumbens suppressed tumor progression and improved the immune system by suppressing the exhaustion of splenic CD8+ T cells.


Assuntos
Dopamina , Núcleo Accumbens , Animais , Linfócitos T CD8-Positivos , Camundongos , Camundongos Transgênicos , Neurônios
7.
Mol Brain ; 15(1): 10, 2022 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-34991655

RESUMO

Emerging evidence suggests that the mesolimbic dopaminergic network plays a role in the modulation of pain. As chronic pain conditions are associated with hypodopaminergic tone in the nucleus accumbens (NAc), we evaluated the effects of increasing signaling at dopamine D1/D2-expressing neurons in the NAc neurons in a model of neuropathic pain induced by partial ligation of sciatic nerve. Bilateral microinjection of either the selective D1-receptor (Gs-coupled) agonist Chloro-APB or the selective D2-receptor (Gi-coupled) agonist quinpirole into the NAc partially reversed nerve injury-induced thermal allodynia. Either optical stimulation of D1-receptor-expressing neurons or optical suppression of D2-receptor-expressing neurons in both the inner and outer substructures of the NAc also transiently, but significantly, restored nerve injury-induced allodynia. Under neuropathic pain-like condition, specific facilitation of terminals of D1-receptor-expressing NAc neurons projecting to the VTA revealed a feedforward-like antinociceptive circuit. Additionally, functional suppression of cholinergic interneurons that negatively and positively control the activity of D1- and D2-receptor-expressing neurons, respectively, also transiently elicited anti-allodynic effects in nerve injured animals. These findings suggest that comprehensive activation of D1-receptor-expressing neurons and integrated suppression of D2-receptor-expressing neurons in the NAc may lead to a significant relief of neuropathic pain.


Assuntos
Neuralgia , Núcleo Accumbens , Animais , Dopamina , Neurônios Dopaminérgicos/metabolismo , Receptores de Dopamina D2/metabolismo
8.
Mol Brain ; 14(1): 146, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34544461

RESUMO

Chronic postsurgical pain (CPSP) is a serious problem. We developed a mouse model of CPSP induced by electrocautery and examined the mechanism of CPSP. In this mouse model, while both incision and electrocautery each produced acute allodynia, persistent allodynia was only observed after electrocautery. Under these conditions, we found that the mRNA levels of Small proline rich protein 1A (Sprr1a) and Annexin A10 (Anxa10), which are the key modulators of neuropathic pain, in the spinal cord were more potently and persistently increased by electrocautery than by incision. Furthermore, these genes were overexpressed almost exclusively in chronic postsurgical pain-activated neurons. This event was associated with decreased levels of tri-methylated histone H3 at Lys27 and increased levels of acetylated histone H3 at Lys27 at their promoter regions. On the other hand, persistent allodynia and overexpression of Sprr1a and Anxa10 after electrocautery were dramatically suppressed by systemic administration of GSK-J4, which is a selective H3K27 demethylase inhibitor. These results suggest that the effects of electrocautery contribute to CPSP along with synaptic plasticity and epigenetic modification.


Assuntos
Anexinas/biossíntese , Proteínas Ricas em Prolina do Estrato Córneo/biossíntese , Eletrocoagulação/efeitos adversos , Código das Histonas , Hiperalgesia/etiologia , Proteínas do Tecido Nervoso/biossíntese , Neuralgia/genética , Neurônios/fisiologia , Dor Pós-Operatória/genética , Medula Espinal/fisiopatologia , Animais , Anexinas/genética , Benzazepinas/farmacologia , Benzazepinas/uso terapêutico , Proteínas Ricas em Prolina do Estrato Córneo/genética , Modelos Animais de Doenças , Feminino , Traumatismos do Pé/fisiopatologia , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Genes Reporter , Genes fos , Histonas/metabolismo , Hiperalgesia/tratamento farmacológico , Hiperalgesia/fisiopatologia , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Lisina/metabolismo , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Neuralgia/tratamento farmacológico , Neuralgia/fisiopatologia , Neurônios/efeitos dos fármacos , Dor Pós-Operatória/tratamento farmacológico , Dor Pós-Operatória/fisiopatologia , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia
9.
Biochem Biophys Res Commun ; 534: 624-631, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33220930

RESUMO

In the present study, we demonstrated that there is a direct relationship between scratching behaviors induced by itch and functional changes in the brain reward system. Using a conditional place preference test, the rewarding effect was clearly evoked by scratching under both acute and chronic itch stimuli. The induction of ΔFosB, a member of the Fos family of transcription factors, was observed in dopamine transporter (DAT)-positive dopamine neurons in the ventral tegmental area (VTA) of mice suffering from a chronic itch sensation. Based on a cellular analysis of scratching-activated neurons, these neurons highly expressed tyrosine hydroxylase (TH) and DAT genes in the VTA. Furthermore, in an in vivo microdialysis study, the levels of extracellular dopamine in the nucleus accumbens (NAcc) were significantly increased by transient scratching behaviors. To specifically suppress the mesolimbic dopaminergic pathway using pharmacogenetics, we used the TH-cre/hM4Di mice. Pharmacogenetic suppression of mesolimbic dopaminergic neurons significantly decreased scratching behaviors. Under the itch condition with scratching behaviors restricted by an Elizabethan collar, the induction of ΔFosB was found mostly in corticotropin-releasing hormone (CRH)-containing neurons of the hypothalamic paraventricular nucleus (PVN). These findings suggest that repetitive abnormal scratching behaviors under acute and chronic itch stimuli may activate mesolimbic dopamine neurons along with pleasant emotions, while the restriction of such scratching behaviors may initially induce the activation of PVN-CRH neurons associated with stress.


Assuntos
Prurido/fisiopatologia , Prurido/psicologia , Recompensa , Área Tegmentar Ventral/fisiopatologia , Doença Aguda , Animais , Comportamento Animal/fisiologia , Doença Crônica , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Expressão Gênica , Histamina/administração & dosagem , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Núcleo Accumbens/fisiopatologia , Testes Farmacogenômicos , Cloreto de Picrila/administração & dosagem , Prurido/genética , Tirosina 3-Mono-Oxigenase/genética
10.
Biochem Biophys Res Commun ; 534: 988-994, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33139013

RESUMO

TRV130 (oliceridine), a G protein-biased ligand for µ-opioid receptor, has recently been synthesized. It is considered to have strong antinociceptive effects and only minor adverse effects. However, whether or not oliceridine actually exhibits an ideal pharmacological profile as an analgesic has not yet been fully clarified in animal studies. This study examined the pharmacological profile of oliceridine in cells and animals. Oliceridine (10 µM) did not produce any µ-opioid receptor internalization in cells even though it increased impedance, which reflects the activation of Gi protein using the CellKey™ system, and inhibited the formation of cAMP. In mice, oliceridine (0.3-10 mg/kg) produced a dose-dependent antinociceptive effect with a rapid-onset and short-duration action in the hot-plate test, as well as antihyperalgesia after sciatic nerve ligation without the development of antinociceptive tolerance using the thermal hyperalgesia test. On the other hand, oliceridine inhibited gastrointestinal transit. Furthermore, oliceridine produced rapid-onset hyperlocomotion at antinociceptive doses; sensitization developed in mice and an emetic effect was observed in ferrets. These results indicate that, although oliceridine may produce dopamine-related behaviors even through selective stimulation of the G-protein-biased µ-opioid receptor pathway, it still offers advantages for breakthrough pain without antinociceptive tolerance with adequate doses.


Assuntos
Analgésicos/uso terapêutico , Proteínas de Ligação ao GTP/metabolismo , Neuralgia/tratamento farmacológico , Receptores Opioides mu/metabolismo , Compostos de Espiro/uso terapêutico , Tiofenos/uso terapêutico , Analgésicos/farmacologia , Animais , Linhagem Celular , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Neuralgia/metabolismo , Receptores Opioides mu/agonistas , Transdução de Sinais/efeitos dos fármacos , Compostos de Espiro/farmacologia , Tiofenos/farmacologia , Fatores de Tempo
11.
Brain ; 142(6): 1675-1689, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31135049

RESUMO

The mechanism by which dopaminergic neurons are selectively affected in Parkinson's disease is not fully understood. In this study, we found a dramatic increase in the expression of catechol-O-methyltransferase (COMT), along with a lower level of DNA methylation, in induced pluripotent stem cell-derived dopaminergic neurons from patients with parkin (PARK2) gene mutations compared to those from healthy controls. In addition, a significant increase in the expression of COMT was found in dopaminergic neurons of isogenic PARK2 induced pluripotent stem cell lines that mimicked loss of function of PARK2 by CRISPR Cas9 technology. In dopamine transporter (DAT)-Cre mice, overexpression of COMT, specifically in dopaminergic neurons of the substantia nigra, produced cataleptic behaviours associated with impaired motor coordination. These findings suggest that upregulation of COMT, likely resulting from DNA hypomethylation, in dopaminergic neurons may contribute to the initial stage of neuronal dysfunction in Parkinson's disease.


Assuntos
Catecol O-Metiltransferase/genética , Neurônios Dopaminérgicos/metabolismo , Doença de Parkinson/metabolismo , Ubiquitina-Proteína Ligases/genética , Animais , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos Transgênicos , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , Substância Negra/metabolismo
12.
J Nat Med ; 73(3): 468-479, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30739283

RESUMO

The Kampo medicine yokukansan (YKS) has a wide variety of properties such as anxiolytic, anti-inflammatory and analgesic effects, and is also thought to regulate tumor suppression. In this study, we investigated the anti-tumor effect of YKS. We used Lewis lung carcinoma (LLC)-bearing mice that were fed food pellets containing YKS and then performed a fecal microbiota analysis, a microarray analysis for microRNAs (miRNAs) and an in vitro anti-tumor assay. The fecal microbiota analysis revealed that treatment with YKS partly reversed changes in the microbiota composition due to LLC implantation. Furthermore, a miRNA array analysis using blood serum showed that treatment with YKS restored the levels of miR-133a-3p/133b-3p, miR-1a-3p and miR-342-3p following LLC implantation to normal levels. A TargetScan analysis revealed that the epidermal growth factor receptor 1 signaling pathway is one of the major target pathways for these miRNAs. Furthermore, treatment with YKS restored the levels of miR-200b-3p and miR-200c-3p, a recognized mediator of cancer progression and controller of emotion, in the hypothalamus of mice bearing LLC. An in vitro assay revealed that a mixture of pachymic acid, saikosaponins a and d and isoliquiritigenin, which are all contained in YKS, exerted direct and additive anti-tumor effects. The present findings constitute novel evidence that YKS may exert an anti-tumor effect by reversing changes in the fecal microbiota and miRNAs circulating in the blood serum and hypothalamus, and the compounds found in YKS could have direct and additive anti-tumor effects.


Assuntos
Medicina Kampo/métodos , Neoplasias/tratamento farmacológico , Animais , Ansiolíticos/farmacologia , Humanos , Masculino , Camundongos
13.
Mol Brain ; 11(1): 6, 2018 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-29458391

RESUMO

Ghrelin exerts a wide range of physiological actions throughout the body and appears to be a promising target for disease therapy. Endogenous ghrelin receptors (GHSRs) are present in extrahypothalamic sites including the substantia nigra pars compacta (SNc), which is related to phenotypic dysregulation or frank degeneration in Parkinson's disease (PD). Here we found a dramatic decrease in the expression of GHSR in PD-specific induced pluripotent stem cell (iPSC)-derived dopaminergic (DAnergic) neurons generated from patients carrying parkin gene (PARK2) mutations compared to those from healthy controls. Consistently, a significant decrease in the expression of GHSR was found in DAnergic neurons of isogenic PARK2-iPSC lines that mimicked loss of function of the PARK2 gene through CRISPR Cas9 technology. Furthermore, either intracerebroventricular injection or microinjection into the SNc of the selective GHSR1a antagonist [D-Lys3]-GHRP6 in normal mice produced cataleptic behaviors related to dysfunction of motor coordination. These findings suggest that the down-regulation of GHSRs in SNc-DA neurons induced the initial dysfunction of DA neurons, leading to extrapyramidal disorder under PD.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Regulação para Baixo , Atividade Motora , Doença de Parkinson/patologia , Doença de Parkinson/fisiopatologia , Receptores de Grelina/genética , Substância Negra/metabolismo , Substância Negra/patologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Neurônios Dopaminérgicos/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Injeções Intraventriculares , Masculino , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Oligopeptídeos/farmacologia , Doença de Parkinson/metabolismo , Receptores de Grelina/antagonistas & inibidores , Receptores de Grelina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
14.
Biochem Biophys Res Commun ; 496(4): 1102-1108, 2018 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-29378186

RESUMO

Ghrelin plays roles in a wide range of central functions by activating the growth hormone secretagogue receptor (GHSR). This receptor has recently been found in the substantia nigra (SN) to control dopamine (DA)-related physiological functions. The dysregulation of DA neurons in the SN pars compacta (SNc) and the consequent depletion of striatal DA are known to underlie the motor deficits observed in Parkinson's disease (PD). In the present study, we further investigated the role of the SN-ghrelin system in motor function under the stereotaxic injection of AAV-CMV-FLEX-diphtheria toxin A (DTA) into the SN of dopamine transporter (DAT)-Cre (DATSN::DTA) mice to expunge DA neurons of the SNc. First, we confirmed the dominant expression of GHSR1a, which is a functional GHSR, in tyrosine hydroxylase (TH)-positive DA neurons in the SNc of control mice. In DATSN::DTA mice, we clearly observed motor dysfunction using several behavioral tests. An immunohistochemical study revealed a dramatic loss of TH-positive DA neurons in the SNc and DAT-labeled axon terminals in the striatum, and an absence of mRNAs for TH and DAT in the SN of DATSN::DTA mice. The mRNA level of GHSR1a was drastically decreased in the SN of these mice. In normal mice, we also found the mRNA expression of GHSR1a within GABAergic neurons in the SN pars reticulata (SNr). Under these conditions, a single injection of ghrelin into the SN failed to improve the motor deficits caused by ablation of the nigrostriatal DA network using DATSN::DTA mice, whereas intra-SN injection of ghrelin suppressed the motor dysfunction caused by the administration of haloperidol, which is associated with the transient inhibition of DA transmission. These findings suggest that phasic activation of the SNc-ghrelin system could improve the dysregulation of nigrostriatal DA transmission related to the initial stage of PD, but not the motor deficits under the depletion of nigrostriatal DA. Although GHSRs are found in non-DA cells of the SNr, GHSRs on DA neurons in the SNc may play a crucial role in motor function.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Grelina/administração & dosagem , Transtornos dos Movimentos/tratamento farmacológico , Transtornos dos Movimentos/fisiopatologia , Parte Compacta da Substância Negra/fisiopatologia , Receptores Dopaminérgicos/metabolismo , Técnicas de Ablação , Animais , Antagonistas de Dopamina/administração & dosagem , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Relação Dose-Resposta a Droga , Masculino , Camundongos , Camundongos Transgênicos , Procedimentos Neurocirúrgicos , Parte Compacta da Substância Negra/efeitos dos fármacos , Recuperação de Função Fisiológica/efeitos dos fármacos , Resultado do Tratamento
15.
Mol Pain ; 14: 1744806918754934, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29310499

RESUMO

Various small molecules act as neurotransmitters and orchestrate neural communication. Growing evidence suggests that not only classical neurotransmitters but also several small molecules, including amino acid derivatives, modulate synaptic transmission. As conditions of acute and chronic pain alter neuronal excitability in the nucleus accumbens, we hypothesized that small molecules released in the nucleus accumbens might play important roles in modulating the pain sensation. However, it is not easy to identify possible pain modulators owing to the absence of a method for comprehensively measuring extracellular small molecules in the brain. In this study, through the use of an emerging metabolomics technique, namely ion chromatography coupled with high-resolution mass spectrometry, we simultaneously analyzed the dynamics of more than 60 small molecules in brain fluids collected by microdialysis, under both the application of pain stimuli and the administration of analgesics. We identified N-acetylaspartylglutamate as a potential pain modulator that is endogenously released in the nucleus accumbens. Infusion of N-acetylaspartylglutamate into the nucleus accumbens significantly attenuated the pain induced by the activation of sensory nerves through optical stimulation. These findings suggest that N-acetylaspartylglutamate released in the nucleus accumbens could modulate pain sensation.


Assuntos
Dipeptídeos/metabolismo , Espaço Extracelular/metabolismo , Espectrometria de Massas , Microdiálise , Núcleo Accumbens/metabolismo , Dor/metabolismo , Sensação , Analgesia , Animais , Comportamento Animal , Líquidos Corporais/metabolismo , Channelrhodopsins/metabolismo , Dopamina/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Optogenética , Dor/patologia , Limiar da Dor , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologia , Bibliotecas de Moléculas Pequenas/metabolismo
16.
Mol Pain ; 14: 1744806918756406, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29357732

RESUMO

Chronic pain induced by nerve damage due to trauma or invasion of cancer to the bone elicits severe ongoing pain as well as hyperalgesia and allodynia likely reflecting adaptive changes within central circuits that amplify nociceptive signals. The present study explored the possible contribution of the mesolimbic dopaminergic circuit in promoting allodynia related to neuropathic and cancer pain. Mice with ligation of the sciatic nerve or treated with intrafemoral osteosarcoma cells showed allodynia to a thermal stimulus applied to the paw on the injured side. Patch clamp electrophysiology revealed that the intrinsic neuronal excitability of ventral tegmental area (VTA) dopamine neurons projecting to the nucleus accumbens (N.Acc.) was significantly reduced in those mice. We used tyrosine hydroxylase (TH)-cre mice that were microinjected with adeno-associated virus (AAV) to express channelrhodopsin-2 (ChR2) to allow optogenetic stimulation of VTA dopaminergic neurons in the VTA or in their N.Acc. terminals. Optogenetic activation of these cells produced a significant but transient anti-allodynic effect in nerve injured or tumor-bearing mice without increasing response thresholds to thermal stimulation in sham-operated animals. Suppressed activity of mesolimbic dopaminergic neurons is likely to contribute to decreased inhibition of N.Acc. output neurons and to neuropathic or cancer pain-induced allodynia suggesting strategies for modulation of pathological pain states.


Assuntos
Neoplasias Ósseas/complicações , Neurônios Dopaminérgicos/patologia , Hiperalgesia/etiologia , Hiperalgesia/patologia , Nervo Isquiático/lesões , Área Tegmentar Ventral/patologia , Animais , Neoplasias Ósseas/fisiopatologia , Dor do Câncer/etiologia , Dor do Câncer/patologia , Dor do Câncer/fisiopatologia , Linhagem Celular Tumoral , Neurônios Dopaminérgicos/metabolismo , Hiperalgesia/fisiopatologia , Ligadura , Masculino , Camundongos Endogâmicos C57BL , Neuralgia/patologia , Núcleo Accumbens/patologia , Núcleo Accumbens/fisiopatologia , Nervo Isquiático/patologia , Nervo Isquiático/fisiopatologia , Área Tegmentar Ventral/fisiopatologia
17.
Mol Pain ; 13: 1744806917740030, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29056067

RESUMO

Background: µ-Opioid receptor internalization is considered to be critically linked to antinociceptive tolerance. Although µ-opioid receptor agonists have been administered simultaneously with other drugs to control pain, little information is available regarding opioid­opioid interactions. Therefore, the present study was designed to further investigate the utility of a new G protein-biased ligand for µ-opioid receptors, TRV130, which has an antinociceptive effect without ß-arrestin-dependent µ-opioid receptor internalization, and its combination with fentanyl using µ-opioid receptor-expressing cells and mice. Results: In the present study, we confirmed that fentanyl produced a profound increase in ß-arrestin-2 recruitment accompanied by µ-opioid receptor internalization, whereas TRV130 did not induce either the recruitment of ß-arrestin-2 or µ-opioid receptor internalization in µ-opioid receptor-expressing cells. Under these conditions, ß-arrestin-2 recruitment accompanied by µ-opioid receptor internalization induced by fentanyl was abolished by TRV130, whereas TRV130 did not alter the reduction of cyclic adenosine monophosphate formation by fentanyl in µ-opioid receptor-expressing cells. In a behavioral assay, TRV130 exerted an antinociceptive effect in a hot-plate test in mice. In a combination test, the antinociceptive effect of TRV130 was synergistically increased by fentanyl. Fentanyl induced antihyperalgesia and development of its tolerance under a neuropathic pain-like state following sciatic nerve ligation. However, treatment of mice with an antinociceptive dose of TRV130 did not induce the rapid development of tolerance to its antihyperalgesic effect under a neuropathic pain-like state. Furthermore, the rapid development of tolerance to the antihyperalgesic effect induced by fentanyl plus TRV130 in mice with sciatic nerve ligation was not observed, unlike in the case of fentanyl alone. Conclusions: These findings provide evidence that activation of the G protein-biased pathway through µ-opioid receptors can alter signaling in the ß-arrestin-2 pathway linked to the stimulation of µ-opioid receptors. Furthermore, the combination of G protein-biased and ß-arrestin-biased ligands of µ-opioid receptors exerts an ideal antinociceptive effect without the rapid development of antinociceptive tolerance.


Assuntos
Tolerância a Medicamentos/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Receptores Opioides mu/metabolismo , beta-Arrestinas/metabolismo , Analgésicos Opioides/farmacologia , Animais , Fentanila/farmacologia , Ligantes , Masculino , Camundongos , Morfina/farmacologia , Neuralgia/tratamento farmacológico , Receptores Opioides/metabolismo , Receptores Opioides mu/efeitos dos fármacos
18.
PLoS One ; 12(2): e0172115, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28225782

RESUMO

BACKGROUND: The EGFR tyrosine kinase inhibitor gefitinib is used in therapy for non-small-cell lung cancer (NSCLC). However, its application is limited by resistance-accelerated disease progression, which is accompanied by the epithelial-to-mesenchymal transition (EMT). In the present study, we performed multiple expression analyses of microRNAs (miRNAs) and quantified the expression of several related EMT players in gefitinib-resistant NSCLC cells. METHODS AND RESULTS: To establish gefitinib-resistant NSCLC cells, gefitinib-sensitive HCC827 cells, which exhibit an in-frame deletion [E746-A750] in EGFR exon 19, were exposed to gefitinib for at least 1.5 months. Next, to profile "gefitinib-resistant HCC827 (HCC827GR)" cells, which have a secondary T790M mutation in EGFR exon 20, a miRNA array analysis was performed in HCC827 and HCC827GR cells. The greatest differences were seen in the levels of miR-155 and miR-200c, which essentially disappeared in HCC827GR cells. In addition to these reductions, the levels of smad2 and zeb1, which are both key players in EMT and targets for miR-155 and miR-200c, respectively, were dramatically increased in HCC827GR cells. In HCC827GR cells, the expression of epithelial-cadherin (E-cadherin) was greatly reduced with repressive histone modifications, whereas vimentin, which is expressed in mesenchymal cells, was dramatically increased with active histone modifications. In another gefitinib-resistant NSCLC cell line (H1975 cells), similar to the findings in HCC827GR cells, both miR-155 and miR-200c were absent, and the EMT was induced along with epigenetic modifications. Interestingly, the inhibition of both miR-155 and miR-200c in HCC827 cells without gefitinib induced significant increases in smad2 and zeb1 along with a dramatic decrease in E-cadherin and a slight increase in vimentin. Furthermore, although the inhibition of these miRNAs in HCC827 cells decreased gefitinib sensitivity, this dual-inhibition in HCC827 cells without gefitinib did not produce a secondary T790M mutation in EGFR exon 20. CONCLUSION AND IMPLICATIONS: These results suggest that chronic treatment of NSCLC cells with gefitinib changes the expression of miRNAs, including dramatic reductions in miR-155 and miR-200c along with an EGFR mutation. Furthermore, this depletion of miR-155 and miR-200c may be associated with the EMT along with histone modifications, and may contribute to the decrease in the sensitivity to gefitinib independent of a secondary EGFR mutation.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Epigênese Genética/efeitos dos fármacos , Neoplasias Pulmonares/genética , MicroRNAs/genética , Quinazolinas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Gefitinibe , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , MicroRNAs/metabolismo
19.
Breast Cancer Res Treat ; 161(2): 269-278, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27888420

RESUMO

PURPOSE: Triple-negative breast cancer (TNBC) has aggressive characteristics and fewer treatment options than other subtypes. The purpose of this study was to explore prognostic biomarkers for TNBC that can be easily detected from the blood samples. METHODS: MDA-MB-231 and MDA-MB-231BR, a brain metastatic variant of the human TNBC cell line MDA-MB-231, were used as less and more aggressive models of TNBC, respectively. The extent to which the candidate gene/protein identified by RNA sequencing correlated well with aggressiveness of TNBC and how much protein was detected from the blood of tumor-bearing mice were evaluated. RESULTS: Both the in vitro proliferation and in vivo tumor growth of MDA-MB-231BR were more rapid than those of MDA-MB-231. RNA sequencing identified ESM1 as a gene that was expressed significantly more in MDA-MB-231BR than in MDA-MB-231, and qRT-PCR confirmed a significantly higher expression of ESM1 in MDA-MB-231BR xenograft in vivo. Furthermore, Kaplan-Meier analysis of relapse-free survival demonstrated that TNBC patients with high ESM1 expression had clearly worse relapse-free survival than those with low ESM1 expression, which was consistent with our preclinical findings. Endocan, a protein of ESM1 gene product, was successfully detected in both conditioned medium from MDA-MB-231BR and plasma samples from mice bearing MDA-MB-231BR xenograft, which showed a significantly distinct pattern from less aggressive MDA-MB-231. Moreover, bisulfite sequence analysis revealed that overexpression of ESM1 in MDA-MB-231BR might be attributed to DNA demethylation in an upstream region of the ESM1 gene. CONCLUSION: This study indicates that endocan could be used as a blood-based prognostic biomarker in TNBC patients.


Assuntos
Biomarcadores Tumorais , Proteínas de Neoplasias/metabolismo , Proteoglicanas/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/mortalidade , Animais , Linhagem Celular Tumoral , Ilhas de CpG , Metilação de DNA , Modelos Animais de Doenças , Espaço Extracelular/metabolismo , Feminino , Expressão Gênica , Xenoenxertos , Humanos , Camundongos , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/genética , Prognóstico , Proteoglicanas/sangue , Proteoglicanas/genética , Neoplasias de Mama Triplo Negativas/genética
20.
Mol Pain ; 122016.
Artigo em Inglês | MEDLINE | ID: mdl-27909152

RESUMO

BACKGROUND: Exercise alleviates pain and it is a central component of treatment strategy for chronic pain in clinical setting. However, little is known about mechanism of this exercise-induced hypoalgesia. The mesolimbic dopaminergic network plays a role in positive emotions to rewards including motivation and pleasure. Pain negatively modulates these emotions, but appropriate exercise is considered to activate the dopaminergic network. We investigated possible involvement of this network as a mechanism of exercise-induced hypoalgesia. METHODS: In the present study, we developed a protocol of treadmill exercise, which was able to recover pain threshold under partial sciatic nerve ligation in mice, and investigated involvement of the dopaminergic reward network in exercise-induced hypoalgesia. To temporally suppress a neural activation during exercise, a genetically modified inhibitory G-protein-coupled receptor, hM4Di, was specifically expressed on dopaminergic pathway from the ventral tegmental area to the nucleus accumbens. RESULTS: The chemogenetic-specific neural suppression by Gi-DREADD system dramatically offset the effect of exercise-induced hypoalgesia in transgenic mice with hM4Di expressed on the ventral tegmental area dopamine neurons. Additionally, anti-exercise-induced hypoalgesia effect was significantly observed under the suppression of neurons projecting out of the ventral tegmental area to the nucleus accumbens as well. CONCLUSION: Our findings suggest that the dopaminergic pathway from the ventral tegmental area to the nucleus accumbens is involved in the anti-nociception under low-intensity exercise under a neuropathic pain-like state.


Assuntos
Dopamina/metabolismo , Terapia por Exercício/métodos , Neuralgia/patologia , Neuralgia/reabilitação , Núcleo Accumbens/metabolismo , Área Tegmentar Ventral/fisiopatologia , Animais , Clozapina/análogos & derivados , Clozapina/farmacologia , Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Teste de Esforço , Hiperalgesia/etiologia , Hiperalgesia/reabilitação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Núcleo Accumbens/efeitos dos fármacos , Medição da Dor , Limiar da Dor/fisiologia , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Antagonistas da Serotonina/farmacologia , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo
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