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1.
Neurochem Int ; 126: 74-85, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30633953

RESUMO

Antinociception caused by cannabinoids may have a partial peripheral origin in addition to its central site of action. In fact, we have observed that anandamide selectively and reversibly inhibits GABAA receptors of putative nociceptive neurons of mouse trigeminal sensory ganglia via CB1 receptor activation to inhibit adenylyl cyclase and decrease cAMP with downstream posttranslational alterations. Since cannabinoids are often used chronically, we studied changes in cAMP levels and GABA-mediated currents of trigeminal neurons following 24 h application of anandamide (0.5 µM) or the synthetic cannabinoid WIN 55,212-2 (5 µM). With this protocol GABA responses were similar to control despite persistent fall in cAMP levels. Inhibition by WIN 55,212-2 of GABA effects recovered after 30 min washout and was not associated with changes in CB1 receptor expression, indicating lack of CB1 receptor inactivation and transient loss of negative coupling between CB1 receptors and GABAA receptors. The phosphodiesterase inhibitor rolipram (100 µM; 24 h) enhanced cAMP levels and GABA-mediated currents, suggesting GABAA receptors were sensitive to persistent upregulation via cAMP. While the adenylyl cyclase activator forskolin (1-20 µM) facilitated cAMP levels and GABA currents following 30 min application, this action was lost after 24 h in line with the drug limited lifespan. The PKA inhibitor PKI 14-22 (10 µM) increased cAMP without changing GABA currents. These data indicate that modulation of GABAA receptors by intracellular cAMP could be lost following persistent application of cannabinoids. Thus, these observations provide an insight into the waning antinociceptive effects of these compounds.


Assuntos
Canabinoides/administração & dosagem , AMP Cíclico/metabolismo , Receptores de GABA-A/metabolismo , Células Receptoras Sensoriais/metabolismo , Gânglio Trigeminal/metabolismo , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Esquema de Medicação , Camundongos , Camundongos Endogâmicos C57BL , Células Receptoras Sensoriais/efeitos dos fármacos , Fatores de Tempo , Gânglio Trigeminal/efeitos dos fármacos
2.
Nat Commun ; 9(1): 3351, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-30120221

RESUMO

The originally published version of this article contained an error in the name of the author Flóra Gölöncsér, which was incorrectly given as Flóra Göröncsér. This has now been corrected in both the PDF and HTML versions of the article.

3.
Nat Commun ; 9(1): 1354, 2018 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-29636447

RESUMO

Two subclasses of acid-sensing ion channels (ASIC3) and of ATP-sensitive P2X receptors (P2X3Rs) show a partially overlapping expression in sensory neurons. Here we report that both recombinant and native receptors interact with each other in multiple ways. Current measurements with the patch-clamp technique prove that ASIC3 stimulation strongly inhibits the P2X3R current partly by a Ca2+-dependent mechanism. The proton-binding site is critical for this effect and the two receptor channels appear to switch their ionic permeabilities during activation. Co-immunoprecipation proves the close association of the two protein structures. BN-PAGE and SDS-PAGE analysis is also best reconciled with the view that ASIC3 and P2X3Rs form a multiprotein structure. Finally, in vivo measurements in rats reveal the summation of pH and purinergically induced pain. In conclusion, the receptor subunits do not appear to form a heteromeric channel, but tightly associate with each other to form a protein complex, mediating unidirectional inhibition.


Assuntos
Canais Iônicos Sensíveis a Ácido/genética , Cálcio/metabolismo , Gânglios Espinais/metabolismo , Hiperalgesia/genética , Dor/genética , Prótons , Receptores Purinérgicos P2X3/genética , Canais Iônicos Sensíveis a Ácido/metabolismo , Animais , Animais Recém-Nascidos , Células CHO , Cricetulus , Gânglios Espinais/citologia , Concentração de Íons de Hidrogênio , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Ativação do Canal Iônico , Masculino , Oócitos/citologia , Oócitos/metabolismo , Dor/metabolismo , Dor/patologia , Técnicas de Patch-Clamp , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Ratos Wistar , Receptores Purinérgicos P2X3/metabolismo , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologia , Xenopus laevis
4.
Neuroscience ; 351: 47-64, 2017 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-28363781

RESUMO

Transgenic knock-in (KI) mice that express CaV2.1 channels containing an R192Q gain-of-function mutation in the α1A subunit known to cause familial hemiplegic migraine type-1 in patients, exhibit key disease characteristics and provide a useful tool to investigate pathophysiological mechanisms of pain transduction. Previously, KI trigeminal sensory neurons were shown to exhibit constitutive hyperexcitability due to up-regulation of ATP-gated P2X3 receptors that trigger spike activity at a more negative threshold. This implies that intrinsic neuronal conductances may shape action potential generation in response to ATP, which could act as a mediator of migraine headache. Here we investigated whether the hyperpolarization-activated conductance Ih, mediated by hyperpolarization activated cyclic nucleotide-gated channels (HCN), contributes to sub-threshold behavior and firing in wild-type (WT) and KI trigeminal ganglia (TG) neurons. Whereas most WT and KI trigeminal neurons expressed Ih current, blocked by the specific inhibitor ZD7288, it was smaller in KI neurons despite similar activation and deactivation kinetics. HCN1 and HCN2 were the most abundantly expressed subunits in TG, both in situ and in culture. In KI TG neurons, HCN2 subunits were predominantly present in the cytoplasm, not at the plasma membrane, likely accounting for the smaller Ih of such cells. ZD7288 hyperpolarized the membrane potential, thereby raising the firing threshold, and prolonging the spike trajectory to generate fewer spikes due to P2X3 receptor activation. The low amplitude of Ih in KI TG neurons suggests that down-regulation of Ih current in sub-threshold behavior acts as a compensatory mechanism to limit sensory hyperexcitability, manifested under certain stressful stimuli.


Assuntos
Ataxia Cerebelar/fisiopatologia , Transtornos de Enxaqueca/fisiopatologia , Células Receptoras Sensoriais/efeitos dos fármacos , Gânglio Trigeminal/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Ataxia Cerebelar/induzido quimicamente , Ataxia Cerebelar/genética , Ataxia Cerebelar/metabolismo , Modelos Animais de Doenças , Técnicas de Introdução de Genes/métodos , Potenciais da Membrana/efeitos dos fármacos , Camundongos Transgênicos , Transtornos de Enxaqueca/induzido quimicamente , Transtornos de Enxaqueca/genética , Transtornos de Enxaqueca/metabolismo , Pirimidinas/farmacologia , Receptores Purinérgicos P2X3/genética , Receptores Purinérgicos P2X3/metabolismo , Células Receptoras Sensoriais/metabolismo , Gânglio Trigeminal/metabolismo
5.
Mol Pain ; 122016.
Artigo em Inglês | MEDLINE | ID: mdl-27175010

RESUMO

BACKGROUND: On trigeminal ganglion neurons, pain-sensing P2X3 receptors are constitutively inhibited by brain natriuretic peptide via its natriuretic peptide receptor-A. This inhibition is associated with increased P2X3 serine phosphorylation and receptor redistribution to non-lipid raft membrane compartments. The natriuretic peptide receptor-A antagonist anantin reverses these effects. We studied whether P2X3 inhibition is dysfunctional in a genetic familial hemiplegic migraine type-1 model produced by introduction of the human pathogenic R192Q missense mutation into the mouse CACNA1A gene (knock-in phenotype). This model faithfully replicates several properties of familial hemiplegic migraine type-1, with gain-of-function of CaV2.1 Ca(2+) channels, raised levels of the algogenic peptide calcitonin gene-related peptide, and enhanced activity of P2X3 receptors in trigeminal ganglia. RESULTS: In knock-in neurons, anantin did not affect P2X3 receptor activity, membrane distribution, or serine phosphorylation level, implying ineffective inhibition by the constitutive brain natriuretic peptide/natriuretic peptide receptor-A pathway. However, expression and functional properties of this pathway remained intact together with its ability to downregulate TRPV1 channels. Reversing the familial hemiplegic migraine type-1 phenotype with the CaV2.1-specific antagonist, ω-agatoxin IVA restored P2X3 activity to wild-type level and enabled the potentiating effects of anantin again. After blocking calcitonin gene-related peptide receptors, P2X3 receptors exhibited wild-type properties and were again potentiated by anantin. CONCLUSIONS: P2X3 receptors on mouse trigeminal ganglion neurons are subjected to contrasting modulation by inhibitory brain natriuretic peptide and facilitatory calcitonin gene-related peptide that both operate via complex intracellular signaling. In the familial hemiplegic migraine type-1 migraine model, the action of calcitonin gene-related peptide appears to prevail over brain natriuretic peptide, thus suggesting that peripheral inhibition of P2X3 receptors becomes insufficient and contributes to trigeminal pain sensitization.


Assuntos
Enxaqueca com Aura/genética , Enxaqueca com Aura/metabolismo , Peptídeo Natriurético Encefálico/metabolismo , Receptores Purinérgicos P2X3/metabolismo , Células Receptoras Sensoriais/patologia , Gânglio Trigeminal/patologia , Animais , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Camundongos , Enxaqueca com Aura/patologia , Modelos Biológicos , Peptídeos Cíclicos/farmacologia , Fenótipo , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores do Fator Natriurético Atrial/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Canais de Cátion TRPV/metabolismo , Gânglio Trigeminal/efeitos dos fármacos , Gânglio Trigeminal/metabolismo , ômega-Agatoxina IVA/farmacologia
6.
Neurosci Lett ; 620: 104-10, 2016 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-27021026

RESUMO

Migraine is a neurovascular brain disorder suggested to be due to dysfunction of the trigeminovascular system with sensitization of trigeminal ganglion (TG) nociceptors. Since the neuropeptide calcitonin gene-related peptide (CGRP) has been established as a key player in the pathogenesis of migraine, CGRP receptor antagonists have been considered useful compounds to block headache originating from hyperactivation of such TG neurons. Whereas there is some information on the expression of CGRP receptors in postmortem human tissue, data are lacking for migraineurs suffering from common or genetic migraine. To help to clarify these issues it is very useful to study a transgenic knock-in (KI) mouse model of hemiplegic migraine expressing a R192Q missense mutation in the α1 subunit of CaV2.1 calcium channels previously found in patients with familial hemiplegic migraine type-1 (FHM-1). The aim of the present study, therefore, was to compare CGRP receptor expression and function in wildtype (WT) versus KI mouse TG. The principal components of the CGRP receptor, namely the CLR and RAMP-1 proteins, were similarly expressed in WT and KI TG neurons (in situ or in culture) and responded to exogenous CGRP with a strong rise in cAMP concentration. Hence, the previously reported phenotype of sensitization of KI TG neurons is not due to up-regulation of CGRP receptors but is likely caused by a constitutively larger release of CGRP. This observation implies that, in FHM-1 TG, normal TG sensory neuron signaling can be restored once the extracellular concentration of CGRP returns to control level with targeted treatment.


Assuntos
Canais de Cálcio Tipo N/genética , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Gânglio Trigeminal/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Células Cultivadas , Ataxia Cerebelar/genética , Técnicas de Introdução de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transtornos de Enxaqueca/genética , Mutação de Sentido Incorreto , Neurônios/metabolismo , Cultura Primária de Células , Proteína 1 Modificadora da Atividade de Receptores/metabolismo
7.
Mol Pain ; 11: 71, 2015 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-26576636

RESUMO

BACKGROUND: ATP-gated P2X3 receptors are important transducers of nociceptive stimuli and are almost exclusively expressed by sensory ganglion neurons. In mouse trigeminal ganglion (TG), P2X3 receptor function is unexpectedly enhanced by pharmacological block of natriuretic peptide receptor-A (NPR-A), outlining a potential inhibitory role of endogenous natriuretic peptides in nociception mediated by P2X3 receptors. Lack of change in P2X3 protein expression indicates a complex modulation whose mechanisms for downregulating P2X3 receptor function remain unclear. RESULTS: To clarify this process in mouse TG cultures, we suppressed NPR-A signaling with either siRNA of the endogenous agonist BNP, or the NPR-A blocker anantin. Thus, we investigated changes in P2X3 receptor distribution in the lipid raft membrane compartment, their phosphorylation state, as well as their function with patch clamping. Delayed onset of P2X3 desensitization was one mechanism for the anantin-induced enhancement of P2X3 activity. Anantin application caused preferential P2X3 receptor redistribution to the lipid raft compartment and decreased P2X3 serine phosphorylation, two phenomena that were not interdependent. An inhibitor of cGMP-dependent protein kinase and siRNA-mediated knockdown of BNP mimicked the effect of anantin. CONCLUSIONS: We demonstrated that in mouse trigeminal neurons endogenous BNP acts on NPR-A receptors to determine constitutive depression of P2X3 receptor function. Tonic inhibition of P2X3 receptor activity by BNP/NPR-A/PKG pathways occurs via two distinct mechanisms: P2X3 serine phosphorylation and receptor redistribution to non-raft membrane compartments. This novel mechanism of receptor control might be a target for future studies aiming at decreasing dysregulated P2X3 receptor activity in chronic pain.


Assuntos
Peptídeo Natriurético Encefálico/fisiologia , Nociceptividade/fisiologia , Receptores Purinérgicos P2X3/metabolismo , Animais , Dor Crônica/fisiopatologia , Regulação para Baixo , Gânglios Sensitivos , Camundongos , Fosforilação , Receptores do Fator Natriurético Atrial/metabolismo , Transdução de Sinais , Gânglio Trigeminal
8.
PLoS One ; 8(11): e81138, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24312267

RESUMO

Important pain transducers of noxious stimuli are small- and medium-diameter sensory neurons that express transient receptor vanilloid-1 (TRPV1) channels and/or adenosine triphosphate (ATP)-gated P2X3 receptors whose activity is upregulated by endogenous neuropeptides in acute and chronic pain models. Little is known about the role of endogenous modulators in restraining the expression and function of TRPV1 and P2X3 receptors. In dorsal root ganglia, evidence supports the involvement of the natriuretic peptide system in the modulation of nociceptive transmission especially via the B-type natriuretic peptide (BNP) that activates the natriuretic peptide receptor-A (NPR-A) to downregulate sensory neuron excitability. Since the role of BNP in trigeminal ganglia (TG) is unclear, we investigated the expression of BNP in mouse TG in situ or in primary cultures and its effect on P2X3 and TRPV1 receptors of patch-clamped cultured neurons. Against scant expression of BNP, almost all neurons expressed NPR-A at membrane level. While BNP rapidly increased cGMP production and Akt kinase phosphorylation, there was no early change in passive neuronal properties or responses to capsaicin, α,ß-meATP or GABA. Nonetheless, 24 h application of BNP depressed TRPV1 mediated currents (an effect blocked by the NPR-A antagonist anantin) without changing responses to α,ß-meATP or GABA. Anantin alone decreased basal cGMP production and enhanced control α,ß-meATP-evoked responses, implying constitutive regulation of P2X3 receptors by ambient BNP. These data suggest a slow modulatory action by BNP on TRPV1 and P2X3 receptors outlining the role of this peptide as a negative regulator of trigeminal sensory neuron excitability to nociceptive stimuli.


Assuntos
Peptídeo Natriurético Encefálico/metabolismo , Receptores Purinérgicos P2X3/metabolismo , Células Receptoras Sensoriais/metabolismo , Canais de Cátion TRPV/metabolismo , Gânglio Trigeminal/citologia , Animais , Regulação da Expressão Gênica , Camundongos , Peptídeo Natriurético Encefálico/genética , Nociceptividade , Receptores do Fator Natriurético Atrial/genética , Receptores do Fator Natriurético Atrial/metabolismo , Gânglio Trigeminal/fisiologia
9.
PLoS One ; 8(1): e52394, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23326332

RESUMO

Latent changes in trigeminal ganglion structure and function resembling inflammatory conditions may predispose to acute attacks of migraine pain. Here, we investigated whether, in trigeminal sensory ganglia, cytokines such as TNFα might contribute to a local inflammatory phenotype of a transgenic knock-in (KI) mouse model of familial hemiplegic migraine type-1 (FHM-1). To this end, macrophage occurrence and cytokine expression in trigeminal ganglia were compared between wild type (WT) and R192Q mutant Ca(V)2.1 Ca(2+) channel (R192Q KI) mice, a genetic model of FHM-1. Cellular and molecular characterization was performed using a combination of confocal immunohistochemistry and cytokine assays. With respect to WT, R192Q KI trigeminal ganglia were enriched in activated macrophages as suggested by their morphology and immunoreactivity to the markers Iba1, CD11b, and ED1. R192Q KI trigeminal ganglia constitutively expressed higher mRNA levels of IL1ß, IL6, IL10 and TNFα cytokines and the MCP-1 chemokine. Consistent with the report that TNFα is a major factor to sensitize trigeminal ganglia, we observed that, following an inflammatory reaction evoked by LPS injection, TNFα expression and macrophage occurrence were significantly higher in R192Q KI ganglia with respect to WT ganglia. Our data suggest that, in KI trigeminal ganglia, the complex cellular and molecular environment could support a new tissue phenotype compatible with a neuroinflammatory profile. We propose that, in FHM patients, this condition might contribute to trigeminal pain pathophysiology through release of soluble mediators, including TNFα, that may modulate the crosstalk between sensory neurons and resident glia, underlying the process of neuronal sensitisation.


Assuntos
Macrófagos/metabolismo , Enxaqueca com Aura/metabolismo , Gânglio Trigeminal/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Western Blotting , Antígeno CD11b/metabolismo , Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Microscopia de Fluorescência , Enxaqueca com Aura/genética , Enxaqueca com Aura/patologia , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Gânglio Trigeminal/efeitos dos fármacos , Gânglio Trigeminal/patologia , Fator de Necrose Tumoral alfa/genética
10.
J Neurochem ; 122(3): 557-67, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22639984

RESUMO

ATP-activated P2X3 receptors of sensory ganglion neurons contribute to pain transduction and are involved in chronic pain signaling. Although highly homologous (97%) in rat and human species, it is unclear whether P2X3 receptors have identical function. Studying human and rat P2X3 receptors expressed in patch-clamped human embryonic kidney (HEK) cells, we investigated the role of non-conserved tyrosine residues in the C-terminal domain (rat tyrosine-393 and human tyrosine-376) as key determinants of receptor function. In comparison with rat P2X3 receptors, human P2X3 receptors were more expressed and produced larger responses with slower desensitization and faster recovery. In general, desensitization was closely related to peak current amplitude for rat and human receptors. Downsizing human receptor expression to the same level of the rat one still yielded larger responses retaining slower desensitization and faster recovery. Mutating phenylalanine-376 into tyrosine in the rat receptor did not change current amplitude; yet, it retarded desensitization onset, demonstrating how this residue was important to functionally link these two receptor states. Conversely, removing tyrosine from position 376 strongly down-regulated human receptor function. The different topology of tyrosine residues in the C-terminal domain has contrasting functional consequences and is sufficient to account for species-specific properties of this pain-transducing channel.


Assuntos
Regulação da Expressão Gênica/genética , Ativação do Canal Iônico/fisiologia , Receptores Purinérgicos P2X3/química , Receptores Purinérgicos P2X3/fisiologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Fenômenos Biofísicos/efeitos dos fármacos , Fenômenos Biofísicos/genética , Biotinilação , Proteína Tirosina Quinase CSK , Estimulação Elétrica , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Imunoprecipitação , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Mutagênese/fisiologia , Mutação/genética , Técnicas de Patch-Clamp , Fenilalanina/genética , Proteínas Tirosina Quinases/metabolismo , Agonistas do Receptor Purinérgico P2X/farmacologia , Interferência de RNA/fisiologia , RNA Interferente Pequeno/farmacologia , Ratos , Receptores Purinérgicos P2X3/genética , Especificidade da Espécie , Transfecção , Tirosina/genética , Quinases da Família src
11.
PLoS One ; 7(4): e35051, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22532838

RESUMO

Mutations in PARK7/DJ-1 gene are associated to autosomal recessive early onset forms of Parkinson's disease (PD). Although large gene deletions have been linked to a loss-of-function phenotype, the pathogenic mechanism of missense mutations is less clear. The L166P mutation causes misfolding of DJ-1 protein and its degradation. L166P protein may also accumulate into insoluble cytoplasmic aggregates with a mechanism facilitated by the E3 ligase TNF receptor associated factor 6 (TRAF6). Upon proteasome impairment L166P activates the JNK/p38 MAPK apoptotic pathway by its interaction with TRAF and TNF Receptor Associated Protein (TTRAP). When proteasome activity is blocked in the presence of wild-type DJ-1, TTRAP forms aggregates that are localized to the cytoplasm or associated to nucleolar cavities, where it is required for a correct rRNA biogenesis. In this study we show that in post-mortem brains of sporadic PD patients TTRAP is associated to the nucleolus and to Lewy Bodies, cytoplasmic aggregates considered the hallmark of the disease. In SH-SY5Y neuroblastoma cells, misfolded mutant DJ-1 L166P alters rRNA biogenesis inhibiting TTRAP localization to the nucleolus and enhancing its recruitment into cytoplasmic aggregates with a mechanism that depends in part on TRAF6 activity. This work suggests that TTRAP plays a role in the molecular mechanisms of both sporadic and familial PD. Furthermore, it unveils the existence of an interplay between cytoplasmic and nucleolar aggregates that impacts rRNA biogenesis and involves TRAF6.


Assuntos
Nucléolo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Doença de Parkinson/metabolismo , RNA Ribossômico/metabolismo , Substância Negra/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Fatores de Transcrição/metabolismo , Fracionamento Celular , Linhagem Celular Tumoral , Nucléolo Celular/genética , Proliferação de Células , Proteínas de Ligação a DNA , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Corpos de Lewy/genética , Corpos de Lewy/metabolismo , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Doença de Parkinson/genética , Diester Fosfórico Hidrolases , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Desglicase DJ-1 , RNA Ribossômico/genética , Fator 6 Associado a Receptor de TNF/genética , Fatores de Transcrição/genética
12.
J Biol Chem ; 286(28): 25108-17, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21454471

RESUMO

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of polyglutamines in the first exon of huntingtin (HTT), which confers aggregation-promoting properties to amino-terminal fragments of the protein (N-HTT). Mutant N-HTT aggregates are enriched for ubiquitin and contain ubiquitin E3 ligases, thus suggesting a role for ubiquitination in aggregate formation. Here, we report that tumor necrosis factor receptor-associated factor 6 (TRAF6) binds to WT and polyQ-expanded N-HTT in vitro as well as to endogenous full-length proteins in mouse and human brain in vivo. Endogenous TRAF6 is recruited to cellular inclusions formed by mutant N-HTT. Transient overexpression of TRAF6 promotes WT and mutant N-HTT atypical ubiquitination with Lys(6), Lys(27), and Lys(29) linkage formation. Both interaction and ubiquitination seem to be independent from polyQ length. In cultured cells, TRAF6 enhances mutant N-HTT aggregate formation, whereas it has no effect on WT N-HTT protein localization. Mutant N-HTT inclusions are enriched for ubiquitin staining only when TRAF6 and Lys(6), Lys(27), and Lys(29) ubiquitin mutants are expressed. Finally, we show that TRAF6 is up-regulated in post-mortem brains from HD patients where it is found in the insoluble fraction. These results suggest that TRAF6 atypical ubiquitination warrants investigation in HD pathogenesis.


Assuntos
Encéfalo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Peptídeos/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitinação , Animais , Encéfalo/patologia , Células HEK293 , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Camundongos , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Peptídeos/genética , Ligação Proteica , Transporte Proteico/genética , Fator 6 Associado a Receptor de TNF/genética
13.
Hum Mol Genet ; 19(19): 3759-70, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20634198

RESUMO

Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the Substantia Nigra and the formation of ubiquitin- and alpha-synuclein (aSYN)-positive cytoplasmic inclusions called Lewy bodies (LBs). Although most PD cases are sporadic, families with genetic mutations have been found. Mutations in PARK7/DJ-1 have been associated with autosomal recessive early-onset PD, while missense mutations or duplications of aSYN (PARK1, PARK4) have been linked to dominant forms of the disease. In this study, we identify the E3 ubiquitin ligase tumor necrosis factor-receptor associated factor 6 (TRAF6) as a common player in genetic and sporadic cases. TRAF6 binds misfolded mutant DJ-1 and aSYN. Both proteins are substrates of TRAF6 ligase activity in vivo. Interestingly, rather than conventional K63 assembly, TRAF6 promotes atypical ubiquitin linkage formation to both PD targets that share K6-, K27- and K29- mediated ubiquitination. Importantly, TRAF6 stimulates the accumulation of insoluble and polyubiquitinated mutant DJ-1 into cytoplasmic aggregates. In human post-mortem brains of PD patients, TRAF6 protein colocalizes with aSYN in LBs. These results reveal a novel role for TRAF6 and for atypical ubiquitination in PD pathogenesis.


Assuntos
Encéfalo/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Corpos de Lewy/metabolismo , Proteínas Mutantes/metabolismo , Proteínas Oncogênicas/metabolismo , Doença de Parkinson/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , alfa-Sinucleína/metabolismo , Encéfalo/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Corpos de Lewy/patologia , Proteínas Mutantes/química , Proteínas Oncogênicas/química , Doença de Parkinson/patologia , Ligação Proteica , Proteína Desglicase DJ-1 , Dobramento de Proteína , Estrutura Quaternária de Proteína , Transporte Proteico , Especificidade por Substrato , Ubiquitinação
14.
J Biol Chem ; 285(24): 18565-74, 2010 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-20395301

RESUMO

Mutations in PARK7/DJ-1 are associated with autosomal recessive, early onset Parkinson disease (PD). DJ-1 is an atypical peroxiredoxin-like peroxidase that may act as a redox-dependent chaperone and a regulator of transcription. Here we show that DJ-1 plays an essential role in the expression of rearranged during transfection (RET), a receptor for the glial cell line-derived neurotrophic factor, a neuroprotective molecule for dopaminergic neurons, the main target of degeneration in PD. The inducible loss of DJ-1 triggers the establishment of hypoxia and the production of reactive oxygen species that stabilize the hypoxia-inducible factor-1alpha (HIF-1a). HIF-1a expression is required for RET down-regulation. This study establishes for the first time a molecular link between the lack of functional DJ-1 and the glial cell line-derived neurotrophic factor signaling pathway that may explain the adult-onset loss of dopaminergic neurons. Furthermore, it suggests that hypoxia may play an important role in PD.


Assuntos
Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Mutação , Neuroblastoma/metabolismo , Proteínas Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-ret/metabolismo , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neuroglia/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Oncogênicas/metabolismo , Oxirredução , Proteína Desglicase DJ-1 , Espécies Reativas de Oxigênio , Transdução de Sinais
15.
Proteomics ; 10(8): 1645-57, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20186750

RESUMO

Unconjugated bilirubin (UCB) is a powerful antioxidant and a modulator of cell growth through the interaction with several signal transduction pathways. Although newborns develop a physiological jaundice, in case of severe hyperbilirubinemia UCB may become neurotoxic causing severe long-term neuronal damages, also known as bilirubin encephalopathy. To investigate the mechanisms of UCB-induced neuronal toxicity, we used the human neuroblastoma cell line SH-SY5Y as an in vitro model system. We verified that UCB caused cell death, in part due to oxidative stress, which leads to DNA damage and cell growth reduction. The mechanisms of cytotoxicity and cell adaptation to UCB were studied through a proteomic approach that identified differentially expressed proteins involved in cell proliferation, intracellular trafficking, protein degradation and oxidative stress response. In particular, the results indicated that cells exposed to UCB undertake an adaptive response that involves DJ-1, a multifunctional neuroprotective protein, crucial for cellular oxidative stress homeostasis. This study sheds light on the mechanisms of bilirubin-induced neurotoxicity and might help to design a strategy to prevent or ameliorate the neuronal damages leading to bilirubin encephalopathy.


Assuntos
Bilirrubina/toxicidade , Citoproteção , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neuroblastoma/química , Proteínas Oncogênicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Estresse Oxidativo/efeitos dos fármacos , Proteína Desglicase DJ-1 , Proteômica
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