RESUMO
The physical and functional interaction between transient receptor potential channel ankyrin 1 (TRPA1) and neuronal calcium sensor 1 (NCS-1) was assessed. NCS-1 is a calcium (Ca2+) sensor found in many tissues, primarily neurons, and TRPA1 is a Ca2+ channel involved not only in thermal and pain sensation but also in conditions such as cancer and chemotherapy-induced peripheral neuropathy, in which NCS-1 is also a regulatory component.We explored the interactions between these two proteins by employing western blot, qRT-PCR, co-immunoprecipitation, Ca2+ transient monitoring with Fura-2 spectrophotometry, and electrophysiology assays in breast cancer cells (MDA-MB-231) with different levels of NCS-1 expression and neuroblastoma cells (SH-SY5Y).Our findings showed that the expression of TRPA1 was directly correlated with NCS-1 levels at both the protein and mRNA levels. Additionally, we found a physical and functional association between these two proteins. Physically, the NCS-1 and TRPA1 co-immunoprecipitate. Functionally, NCS-1 enhanced TRPA1-dependent Ca2+ influx, current density, open probability, and conductance, where the functional effects depended on PI3K. Conclusion: NCS-1 appears to act not only as a Ca2+ sensor but also modulates TRPA1 protein expression and channel function in a direct fashion through the PI3K pathway. These results contribute to understanding how Ca2+ homeostasis is regulated and provides a mechanism underlying conditions where Ca2+ dynamics are compromised, including breast cancer. With a cellular pathway identified, targeted treatments can be developed for breast cancer and neuropathy, among other related diseases.
Assuntos
Neoplasias da Mama , Proteínas Sensoras de Cálcio Neuronal , Neuropeptídeos , Canal de Cátion TRPA1 , Feminino , Humanos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular Tumoral , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Proteínas Sensoras de Cálcio Neuronal/genética , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Neuropeptídeos/metabolismo , Neuropeptídeos/genética , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Canal de Cátion TRPA1/metabolismo , Canal de Cátion TRPA1/genéticaRESUMO
Peripheral biomarker and post-mortem brains studies have shown alterations of neuronal calcium sensor 1 (Ncs-1) expression in people with bipolar disorder or schizophrenia. However, its engagement by psychiatric medications and potential contribution to behavioral regulation remains elusive. We investigated the effect on Ncs-1 expression of valproic acid (VPA), a mood stabilizer used for the management of bipolar disorder. Treatment with VPA induced Ncs-1 gene expression in cell line while chronic administration of this drug to mice increased both Ncs-1 protein and mRNA levels in the mouse frontal cortex. Inhibition of histone deacetylases (HDACs), a known biochemical effect of VPA, did not alter the expression of Ncs-1. In contrast, pharmacological inhibition or genetic downregulation of glycogen synthase kinase 3ß (Gsk3ß) increased Ncs-1 expression, whereas overexpression of a constitutively active Gsk3ß had the opposite effect. Moreover, adeno-associated virus-mediated Ncs-1 overexpression in mouse frontal cortex caused responses similar to those elicited by VPA or lithium in tests evaluating social and mood-related behaviors. These findings indicate that VPA increases frontal cortex Ncs-1 gene expression as a result of Gsk3 inhibition. Furthermore, behavioral changes induced by Ncs-1 overexpression support a contribution of this mechanism in the regulation of behavior by VPA and potentially other psychoactive medications inhibiting Gsk3 activity.
Assuntos
Ansiedade/induzido quimicamente , Lobo Frontal/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas Sensoras de Cálcio Neuronal/genética , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Ácido Valproico/efeitos adversos , Animais , Ansiedade/genética , Ansiedade/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Regulação para Baixo , Glicogênio Sintase Quinase 3 beta/genética , Células HEK293 , Humanos , Masculino , Camundongos , Células PC12 , Ratos , Comportamento Social , Regulação para Cima , Ácido Valproico/administração & dosagemRESUMO
Sepsis is defined as the host's reaction to infection and it is characterized by a systemic inflammatory response with important clinical implications. Central nervous system dysfunction secondary to sepsis is associated with local generation of pro- and anti-inflammatory cytokines, impaired cerebral microcirculation, disturbance of neurotransmitters, apoptosis, and cognitive impairment. It is known that during the process of learning and memory formation several pathways are involved such as dopaminergic and cholinergic systems. Thus, the objective of this study is to evaluate the neuronal calcium sensor (NCS-1) and dopamine-cAMP regulated phosphoprotein of 32,000 kDa (DARPP-32) expression as well as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) levels in prefrontal cortex and hippocampus of rats 12, 24, and 48 h after sepsis induction. To this aim, we used sham-operated Wistar rats or submitted to the cecal ligation and perforation procedure. After 12 and 24 h, there was an increase of NGF levels in hippocampus; and up to 48 h, a decrease of NCS-1 expression in prefrontal cortex, a decrease of BDNF levels in hippocampus and an increase of NGF levels in hippocampus. In conclusion, we believe that the low expression of NCS-1 in prefrontal cortex and low levels of BDNF in hippocampus may be associated with the pathophysiology of cognitive impairment during sepsis and a putative role of the dopaminergic system.
Assuntos
Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Hipocampo/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Neuropeptídeos/metabolismo , Córtex Pré-Frontal/metabolismo , Sepse/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Immunoblotting , Masculino , Fator de Crescimento Neural/metabolismo , Ratos Wistar , Fatores de TempoRESUMO
It is well known that dopamine imbalances are associated with many psychiatric disorders and that the dopaminergic receptor D2 is the main target of antipsychotics. Recently it was shown that levels of two proteins implicated in dopaminergic signaling, Neuronal calcium sensor-1 (NCS-1) and DARPP-32, are altered in the prefrontal cortex (PFC) of both schizophrenic and bipolar disorder patients. NCS-1, which inhibits D2 internalization, is upregulated in the PFC of both patients. DARPP-32, which is a downstream effector of dopamine signaling, integrates the pathways of several neurotransmitters and is downregulated in the PFC of both patients. Here, we used PC12 cells stably overexpressing NCS-1 (PC12-NCS-1 cells) to address the function of this protein in DARPP-32 signaling pathway in vitro. PC12-NCS-1 cells displayed downregulation of the cAMP/PKA pathway, with decreased levels of cAMP and phosphorylation of CREB at Ser133. We also observed decreased levels of total and phosphorylated DARPP-32 at Thr34. However, these cells did not show alterations in the levels of D2 and phosphorylation of DARPP-32 at Thr75. These results indicate that NCS-1 modulates PKA/cAMP signaling pathway. Identification of the cellular mechanisms linking NCS-1 and DARPP-32 may help in the understanding the signaling machinery with potential to be turned into targets for the treatment of schizophrenia and other debilitating psychiatric disorders.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteínas Sensoras de Cálcio Neuronal/genética , Neuropeptídeos/genética , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Regulação para Baixo/genética , Regulação para Baixo/fisiologia , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Proteínas Sensoras de Cálcio Neuronal/fisiologia , Neuropeptídeos/metabolismo , Neuropeptídeos/fisiologia , Células PC12 , Fosforilação , Ratos , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D2/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Transfecção , Regulação para Cima/genéticaRESUMO
BACKGROUND: Schizophrenia is the major psychiatry disorder, which the exact cause remains unknown. However, it is well known that dopamine-mediated neurotransmission imbalance is associated with this pathology and the main target of antipsychotics is the dopamine receptor D2. Recently, it was described alteration in levels of two dopamine signaling related proteins in schizophrenic prefrontal cortex (PFC): Neuronal Calcium Sensor-1 (NCS-1) and DARPP-32. NCS-1, which is upregulated in PFC of schizophrenics, inhibits D2 internalization. DARPP-32, which is decreased in PFC of schizophrenics, is a key downstream effector in transducing dopamine signaling. We previously demonstrated that antipsychotics do not change levels of both proteins in rat's brain. However, since NCS-1 and DARPP-32 levels are not altered in wild type rats, we treated wild type PC12 cells (PC12 WT) and PC12 cells stably overexpressing NCS-1 (PC12 Clone) with antipsychotics to investigate if NCS-1 upregulation modulates DARPP-32 expression in response to antipsychotics treatment. RESULTS: We chronically treated both PC12 WT and PC12 Clone cells with typical (Haloperidol) or atypical (Clozapine and Risperidone) antipsychotics for 14 days. Using western blot technique we observed that there is no change in NCS-1 and DARPP-32 protein levels in both PC12 WT and PC12 Clone cells after typical and atypical antipsychotic treatments. CONCLUSIONS: Because we observed no alteration in NCS-1 and DARPP-32 levels in both PC12 WT and Clone cells treated with typical or atypical antipsychotics, we suggest that the alteration in levels of both proteins in schizophrenic's PFC is related to psychopathology but not with antipsychotic treatment.
Assuntos
Antipsicóticos/farmacologia , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Neuropeptídeos/metabolismo , Animais , Western Blotting , Células Clonais , Células PC12 , RatosRESUMO
Step-down inhibitory avoidance is usually acquired in one single trial, which makes it ideal for studying processes initiated by training, uncontaminated by prior or further trials, rehearsals, or retrievals. Neuronal calcium sensor 1 (NCS-1) is a dopamine receptor interacting protein that has been associated with associative learning and memory. We evaluated whether inhibitory avoidance can alter NCS-1 levels in rat brain. We focused our analysis on the striatum, entorhinal cortex, hippocampus and prefrontal cortex. Protein levels were measured using immunoblotting normalized by actin levels. Our results indicate that NCS-1 levels are not altered after step-down inhibitory avoidance in rat striatum, entorhinal cortex, hippocampus and prefrontal cortex. The link between protein interactions and the varied physiological roles of NCS-1 still remains to be fully established. Furthermore, other experiments are needed to shed more light on the role of NCS-1 and other mechanisms linked to signaling pathways related to inhibitory avoidance task.
Assuntos
Aprendizagem da Esquiva/fisiologia , Encéfalo/metabolismo , Regulação da Expressão Gênica/fisiologia , Inibição Psicológica , Memória/fisiologia , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Neuropeptídeos/metabolismo , Animais , Comportamento Animal , Encéfalo/anatomia & histologia , Eletrochoque/efeitos adversos , Masculino , Ratos , Ratos Wistar , Estatísticas não Paramétricas , Fatores de TempoRESUMO
Schizophrenia (SCZ) and bipolar disorder (BPD) are severe illnesses representing an enormous social, familiar and individual burden that affect 1% of the population world-wide. Several evidences indicate abnormalities of the dopamine system in both SCZ and BPD. Neuronal calcium sensor-1 (NCS-1) is a protein that has many functions in neurotransmission such as inhibition of dopamine D(2) receptor desensitization, regulation of ionic channels and enhancement of exocytosis of neurotransmitters. In addition, NCS-1 protein expression and mRNA levels were found increased in pre-frontal cortex (PFC) of SCZ and BPD patients. NCS-1 expression in neural and neuroendocrine cells is well documented and, recently, it was shown that NCS-1 is also expressed in mast cells and neutrophils. NCS-1 has important functions in mast cells since it stimulates Fc epsilon RI-triggered exocytosis and the release of arachidonic acid metabolites. Then, due to the known close relation between the nervous and immune systems, we sought to investigate the NCS-1 expression in lymphocytes and monocytes (CD4+ T lymphocytes, CD56+ NK cells, CD19+ B lymphocytes and CD14+ monocytes) of SCZ and BPD patients. Using flow cytometry, our results have shown that NCS-1 expression was diminished in CD4+T lymphocytes, CD19+ B lymphocytes and CD14+ monocytes of BPD patients and also decreased in CD4+ T lymphocytes and CD56+ NK cells of SCZ patients. Results suggest that immune cells might be a cellular model for studies with SCZ and BPD patients considering NCS-1 functions. Efforts need to be done to investigate the motive of the decreased percentage of immune cells expressing NCS-1 in patients with SCZ and BPD.