RESUMO
Calcium (Ca2+ ) signaling is critical for neuronal functioning and requires the concerted interplay of numerous Ca2+ -binding proteins, including neuronal calcium sensor 1 (NCS1). Although an important role of NCS1 in neuronal processes and in neurodevelopmental and neurodegenerative diseases has been established, the underlying mechanisms remain enigmatic. Here, we systematically investigated the functions of NCS1 in the brain. Using Golgi-Cox staining, we observed a reduction in dendritic complexity and spine density in the prefrontal cortex and the dorsal hippocampus of Ncs1-/- mice, which may underlie concomitantly observed deficits in memory acquisition. Subsequent RNA sequencing of Ncs1-/- and Ncs1+/+ mouse brain tissues revealed that NCS1 modulates gene expression related to neuronal morphology and development. Investigation of developmental databases further supported a molecular role of NCS1 during brain development by identifying temporal gene expression patterns. Collectively, this study provides insights into NCS1-dependent signaling and lays the foundation for a better understanding of NCS1-associated diseases.
Assuntos
Cálcio/metabolismo , Hipocampo/patologia , Doenças do Sistema Nervoso/patologia , Proteínas Sensoras de Cálcio Neuronal/fisiologia , Neurônios/patologia , Neuropeptídeos/fisiologia , Animais , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças do Sistema Nervoso/etiologia , Doenças do Sistema Nervoso/metabolismo , Neurônios/metabolismo , RNA-Seq , Transdução de Sinais , TranscriptomaAssuntos
Autorreceptores/metabolismo , Canais de Cálcio Tipo L/fisiologia , Neurônios Dopaminérgicos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Proteínas Sensoras de Cálcio Neuronal/fisiologia , Neuropeptídeos/fisiologia , Doença de Parkinson/metabolismo , Substância Negra/metabolismo , Área Tegmentar Ventral/metabolismo , Animais , Humanos , MasculinoRESUMO
Dopamine midbrain neurons within the substantia nigra are particularly prone to degeneration in Parkinson's disease. Their selective loss causes the major motor symptoms of Parkinson's disease, but the causes for the high vulnerability of SN DA neurons, compared to neighbouring, more resistant ventral tegmental area dopamine neurons, are still unclear. Consequently, there is still no cure available for Parkinson's disease. Current therapies compensate the progressive loss of dopamine by administering its precursor l-DOPA and/or dopamine D2-receptor agonists. D2-autoreceptors and Cav1.3-containing L-type Ca(2+) channels both contribute to Parkinson's disease pathology. L-type Ca(2+) channel blockers protect SN DA neurons from degeneration in Parkinson's disease and its mouse models, and they are in clinical trials for neuroprotective Parkinson's disease therapy. However, their physiological functions in SN DA neurons remain unclear. D2-autoreceptors tune firing rates and dopamine release of SN DA neurons in a negative feedback loop through activation of G-protein coupled potassium channels (GIRK2, or KCNJ6). Mature SN DA neurons display prominent, non-desensitizing somatodendritic D2-autoreceptor responses that show pronounced desensitization in PARK-gene Parkinson's disease mouse models. We analysed surviving human SN DA neurons from patients with Parkinson's disease and from controls, and detected elevated messenger RNA levels of D2-autoreceptors and GIRK2 in Parkinson's disease. By electrophysiological analysis of postnatal juvenile and adult mouse SN DA neurons in in vitro brain-slices, we observed that D2-autoreceptor desensitization is reduced with postnatal maturation. Furthermore, a transient high-dopamine state in vivo, caused by one injection of either l-DOPA or cocaine, induced adult-like, non-desensitizing D2-autoreceptor responses, selectively in juvenile SN DA neurons, but not ventral tegmental area dopamine neurons. With pharmacological and genetic tools, we identified that the expression of this sensitized D2-autoreceptor phenotype required Cav1.3 L-type Ca(2+) channel activity, internal Ca(2+), and the interaction of the neuronal calcium sensor NCS-1 with D2-autoreceptors. Thus, we identified a first physiological function of Cav1.3 L-type Ca(2+) channels in SN DA neurons for homeostatic modulation of their D2-autoreceptor responses. L-type Ca(2+) channel activity however, was not important for pacemaker activity of mouse SN DA neurons. Furthermore, we detected elevated substantia nigra dopamine messenger RNA levels of NCS-1 (but not Cav1.2 or Cav1.3) after cocaine in mice, as well as in remaining human SN DA neurons in Parkinson's disease. Thus, our findings provide a novel homeostatic functional link in SN DA neurons between Cav1.3- L-type-Ca(2+) channels and D2-autoreceptor activity, controlled by NCS-1, and indicate that this adaptive signalling network (Cav1.3/NCS-1/D2/GIRK2) is also active in human SN DA neurons, and contributes to Parkinson's disease pathology. As it is accessible to pharmacological modulation, it provides a novel promising target for tuning substantia nigra dopamine neuron activity, and their vulnerability to degeneration.
Assuntos
Autorreceptores/metabolismo , Canais de Cálcio Tipo L/fisiologia , Neurônios Dopaminérgicos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Proteínas Sensoras de Cálcio Neuronal/fisiologia , Neuropeptídeos/fisiologia , Doença de Parkinson/metabolismo , Substância Negra/metabolismo , Área Tegmentar Ventral/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/patologia , Receptores de Dopamina D2/metabolismo , Substância Negra/citologia , Substância Negra/patologia , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/patologiaRESUMO
Sensing and regulating intracellular levels of calcium are essential for proper cellular function. In neurons, calcium sensing plays important roles in neuronal plasticity, neurotransmitter release, long-term synapse modification and ion channel activity. Neuronal calcium sensor-1 (NCS-1) is a member of the highly conserved neuronal calcium sensor family. Although NCS-1 has been associated with psychiatric conditions including autism, bipolar disorder and schizophrenia, it is unclear which role NCS-1 plays in behavior. To understand the involvement of NCS-1 in psychiatric conditions, we provided a comprehensive behavioral characterization of NCS-1 knockout (KO) mice. These mice grow and develop normally without apparent abnormalities in comparison to wild type littermates. However, open field showed that NCS-1 deficiency impairs novelty-induced exploratory activity in both KO and heterozygote (HT) mice. Moreover, NCS-1-deficiency also resulted in anxiety- and depressive-like behaviors as demonstrated by elevated plus maze, large open field, forced swim and tail suspension tasks. Furthermore, based on spontaneous object recognition test, non-aversive long-term memory was impaired in NCS-1 KO mice. In contrast, neither social behavior nor a kind of aversive memory was affected under NCS-1 deficiency. These data implicate NCS-1 in exploratory activity, memory and mood-related behaviors, suggesting that NCS-1 gene ablation may result in phenotypic abnormalities associated with neuropsychiatric disorders.
Assuntos
Transtornos de Ansiedade/fisiopatologia , Transtorno Depressivo/fisiopatologia , Transtornos da Memória/fisiopatologia , Proteínas Sensoras de Cálcio Neuronal/deficiência , Proteínas Sensoras de Cálcio Neuronal/fisiologia , Neuropeptídeos/deficiência , Neuropeptídeos/fisiologia , Animais , Ansiolíticos/farmacologia , Antidepressivos Tricíclicos/farmacologia , Transtornos de Ansiedade/tratamento farmacológico , Aprendizagem da Esquiva/fisiologia , Transtorno Depressivo/tratamento farmacológico , Diazepam/farmacologia , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Imipramina/farmacologia , Masculino , Memória de Longo Prazo/fisiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Sensoras de Cálcio Neuronal/genética , Neuropeptídeos/genética , Reconhecimento Psicológico/fisiologia , Comportamento SocialRESUMO
The review considers peculiarities of Ca(2+)-signalization in electroexcitable cells of the higher eukaryotes. The light has been thrown upon the problems of Ca(2+)-dependent mechanisms of regulation of muscle contractility and of neuronal synaptic plasticity in the higher vertebrate animals. A particular attention has been paid to analysis of contribution of such poorly studied components of Ca(2+)-signalization as non-selective TRPC-channels, Orai channels, sensory STIMI proteins, Ca(2+)-controlled K(+)-channels of high and low conductivity, and neuronal Ca(2+)-sensors (NCS).
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio , Proteínas Sensoras de Cálcio Neuronal , Plasticidade Neuronal , Canais de Cátion TRPC , Animais , Evolução Biológica , Cálcio/metabolismo , Cálcio/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/fisiologia , Humanos , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Proteínas Sensoras de Cálcio Neuronal/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Canais de Cátion TRPC/metabolismo , Canais de Cátion TRPC/fisiologia , Vertebrados/fisiologiaRESUMO
Calcium (Ca(2+)) is an important intracellular messenger, regulating myocyte contraction via excitation-contraction (EC) coupling and gene transcription underlying hypertrophy in the heart. Although the mechanisms of EC coupling in the immature heart are believed to be different from those in the adult heart because of the structural immaturity of the sarcoplasmic reticulum in the young heart, the details of these mechanisms are not completely understood. Neuronal Ca(2+) sensor-1 (NCS-1) is an EF-hand Ca(2+)-binding protein that is highly expressed in young hearts; however, little is known about its cardiac functions. In this review, we summarize our recent findings indicating that NCS-1 acts as a novel regulator enhancing Ca(2+) signals in the heart and hence promoting contraction in the immature heart and hypertrophy in the adult heart. Possible signal transduction pathways are also discussed.
Assuntos
Sistema Cardiovascular , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Direita/patologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos , Proteínas Sensoras de Cálcio Neuronal/genética , Neuropeptídeos/genética , Humanos , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Direita/genética , Proteínas Sensoras de Cálcio Neuronal/fisiologia , Neuropeptídeos/fisiologia , Retículo Sarcoplasmático/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
PURPOSE: CERKL encodes for a ceramide kinase (CERK)-like protein. CERKL mutations are associated with severe retinal degeneration. Several studies have been conducted to prove a biochemical similarity between CERK and CERKL enzymatic activities. However, so far there has been no evidence that CERKL phosphorylates ceramide or any other lipid substrate in vitro or in vivo. The purpose of this work was to characterize CERKL's function by identification of CERKL-interacting proteins in the mammalian retina. METHODS: CERKL-interacting proteins were identified implementing the Ras-recruitment system (RRS) on a bovine retina cDNA library. Co-immunoprecipitation (co-IP) in transfected cells and in photoreceptor outer segments was used to verify the identified interactions. Serial deletion constructs were used to map the interacting sites. CERKL's kinase activity was tested by a CERK activity assay. RESULTS: We identified an interaction between CERKL and several neuronal calcium sensor (NCS) proteins, including guanylate cyclase activating protein 1 (GCAP1), GCAP2, and recoverin. These interactions were confirmed by co-IP experiments in transfected mammalian cells. Moreover, the interaction between endogenous CERKL and GCAP2 was confirmed by co-IP in photoreceptor outer segments. We found that CERKL-GCAP interaction is cation dependent and is mediated by CERKL's N-terminal region and by GCAPs cation-binding domains (EF-hands 2-4). CONCLUSIONS: This study, which is the first to describe the interactions of CERKL with other retinal proteins, links CERKL to proteins involved in the photoresponse and Ca(2+) signaling, providing important clues for future research required in this direction.
Assuntos
Proteínas Sensoras de Cálcio Neuronal/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Retina/enzimologia , Animais , Cátions , Bovinos , Células Cultivadas , Imunofluorescência , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Humanos , Imunoprecipitação , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
The calcium-binding protein frequenin (Frq), discovered in the fruit fly Drosophila, and its mammalian homologue neuronal calcium sensor 1 (NCS-1) have been reported to affect several aspects of synaptic transmission, including basal levels of neurotransmission and short- and long-term synaptic plasticities. However, discrepant reports leave doubts about the functional roles of these conserved proteins. In this review, we attempt to resolve some of these seemingly contradictory reports. We discuss how stimulation protocols, sources of calcium (voltage-gated channels versus internal stores), and expression patterns (presynaptic versus postsynaptic) of Frq may result in the activation of various protein targets, leading to different synaptic effects. In addition, the potential interactions of Frq's C-terminal and N-terminal domains with other proteins are discussed. Frq also has a role in regulating neurite outgrowth, axonal regeneration, and synaptic development. We examine whether the effects of Frq on neurotransmitter release and neurite outgrowth are distinct or interrelated through homeostatic mechanisms. Learning and memory are affected by manipulations of Frq probably through changes in synaptic transmission and neurite outgrowth, raising the possibility that Frq may be implicated in human pathological conditions, including schizophrenia, bipolar disorder, and X-linked mental retardation.
Assuntos
Diferenciação Celular/fisiologia , Proteínas Sensoras de Cálcio Neuronal/fisiologia , Neuropeptídeos/fisiologia , Terminações Pré-Sinápticas/fisiologia , Transmissão Sináptica/fisiologia , Animais , Sinalização do Cálcio/fisiologia , Humanos , Transtornos Neurocognitivos/metabolismo , Transtornos Neurocognitivos/patologia , Transtornos Neurocognitivos/fisiopatologiaRESUMO
RATIONALE: Neuronal calcium sensor-1 (NCS-1) regulates various neuronal functions. Although it is expressed in the heart, very little is known about its cardiac functions. OBJECTIVE: This study aimed to identify the physiological and pathological roles of NCS-1 in the heart. METHODS AND RESULTS: We characterized the cardiac functions of knockout mice (Ncs1(-/-)) and identified NCS-1 as a novel regulator of cardiac Ca(2+) signaling, specifically in immature and hypertrophic hearts. NCS-1 was highly expressed in young hearts, and its deletion decreased survival and contractile function in young mice. Intracellular Ca(2+) levels and sarcoplasmic reticulum Ca(2+) content were significantly lower in Ncs1(-/-) myocytes than in wild-type cells. This was due to reduced Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity in Ncs1(-/-) myocytes, which led to reduced sarcoplasmic reticulum Ca(2+) uptake and release. NCS-1 physically and functionally interacted with inositol 1,4,5-trisphosphate receptors (IP(3)Rs) in the heart. In addition, IP(3)R stimulation resulted in phosphorylation of CaMKII-δ, which was enhanced by NCS-1 overexpression. These results suggest that a functional link exists between NCS-1, IP(3)R function, and CaMKII activation that may affect global Ca(2+) signals in the immature heart. Furthermore, NCS-1 was upregulated in hypertrophic hearts, and hormone-induced hypertrophy was largely prevented in Ncs1(-/-) hearts. Inhibitors of IP(3)Rs, CaMKII, and calcineurin all prevented NCS-1-induced hypertrophy, which suggests the involvement of these pathways. CONCLUSIONS: NCS-1 is an important regulator of immature heart function and hypertrophy, and it functions in part by promoting IP(3)R function, followed by CaMKII-dependent signal activation.
Assuntos
Sinalização do Cálcio/fisiologia , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Coração/crescimento & desenvolvimento , Miócitos Cardíacos/metabolismo , Proteínas Sensoras de Cálcio Neuronal/fisiologia , Neuropeptídeos/fisiologia , Animais , Animais Recém-Nascidos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomegalia/prevenção & controle , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Miócitos Cardíacos/enzimologia , Proteínas Sensoras de Cálcio Neuronal/biossíntese , Neuropeptídeos/biossíntese , Regulação para Cima/genéticaRESUMO
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
It has been suggested that overexpression of neuronal Ca2+ sensor-1 (NCS-1) protein is implicated in the pathophysiology of neurodisorders such as schizophrenia, bipolar disturbance and X-linked mental retardation. The mechanism by which NCS-1 would be involved in the causes and/or consequences of these neurodisorders is still far from elucidation. Independent evidence has pointed NCS-1 as a key regulator of synaptic efficacy by altering the expression and activity of voltage-gated channels, inhibiting internalization of dopaminergic receptors, and altering phosphoinositide metabolism. In this study, we examined the possible participation of NCS-1 protein in signal transmission dependent on muscarinic receptor activation, using PC12 cells stably expressing NCS-1 (PC12-NCS-1). Carbachol (CCH; 300 microM) was able to evoke glutamate release more efficiently from PC12-NCS-1 (15.3+/-1.0nmol/mg of protein) than wild type cells (PC12-wt; 8.3+/-0.9nmol/mg of protein). This increase of glutamate release induced by CCH was independent on extracellular Ca2+ influx. Additionally, a larger increase of cytoplasmic levels of InsP3 (663.0+/-63.0 and 310.0+/-39.0% of fluorescence in A.U.) and [Ca2+]i (766.4+/-40.0 and 687.8+/-37.1nmol/L) was observed after CCH stimulus of PC12-NCS-1 compared with PC12-wt. Clearly distinction between intracellular Ca2+ dynamics was also observed in PC12-NCS-1 and PC12-wt. A larger increase followed by fast decay of [Ca2+]i was observed in PC12-NCS-1. A plateau with a delayed decay of [Ca2+]i was characteristic of PC12-wt [Ca2+]i response. Both enhancement of InsP3 production and glutamate release observed in PC12-NCS-1 were blocked by atropine (10 microM). Together, our data show that overexpression of NCS-1 in PC12 cells induces an enhancement of intracellular second messenger and transmitter release dependent on CCH response, suggesting that muscarinic signaling is "up-regulated" in this cell model.
Assuntos
Ácido Glutâmico/metabolismo , Proteínas Sensoras de Cálcio Neuronal/fisiologia , Neuropeptídeos/fisiologia , Receptores Muscarínicos/metabolismo , Transdução de Sinais/fisiologia , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Carbacol/farmacologia , Quelantes/farmacologia , Agonistas Colinérgicos/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência , Proteínas Sensoras de Cálcio Neuronal/genética , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Células PC12 , Transporte Proteico/efeitos dos fármacos , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
OBJECTIVE: To evaluate whether the thermotaxis tracking model is suitable for assessing long-term memory (LTM) in the nematode Caenorhabditis elegans. METHODS: Animals were trained at 20 degrees C overnight in presence of food. The percentage of animals performing isothermal tracking (IT) behavior was measured at different time intervals after the training. RESULTS: The percentage of animals performing IT behavior, the numbers of body bends inside and outside the training temperature, and the expression patterns of AFD and AIY neurons were similar to those in control animals at 36 and 48 h after training; whereas when extending to 60, 72, and 84 h, locomotory behavior defects were observed in the assayed animals, suggesting that this thermal tracking model is feasible for analyzing LTM at 36 and 48 h after training. Moreover, the percentage of animals performing IT behavior was reduced at 18, 36, and 48 h after training in neuronal calcium sensor-1 gene (nsc-1) mutant animals compared with that in wild-type N2 animals. In addition, exposure to plumbum (Pb) significantly repressed the LTM at 18, 36, and 48 h after training in both wild-type N2 and ncs-1 mutant animals. CONCLUSION: The thermotaxis tracking model is suitable for evaluating the LTM regulated by NCS-1, and can be employed for elucidating regulatory functions of specific genes or effects of stimuli on memory in C. elegans.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Memória de Longo Prazo/fisiologia , Atividade Motora , Proteínas Sensoras de Cálcio Neuronal/fisiologia , Sensação Térmica/fisiologia , Animais , Caenorhabditis elegans , Modelos NeurológicosRESUMO
In neurons, intracellular calcium signals have crucial roles in activating neurotransmitter release and in triggering alterations in neuronal function. Calmodulin has been widely studied as a Ca(2+) sensor that has several defined roles in neuronal Ca(2+) signalling, but members of the neuronal calcium sensor protein family have also begun to emerge as key components in a number of regulatory pathways and have increased the diversity of neuronal Ca(2+) signalling pathways. The differing properties of these proteins allow them to have discrete, non-redundant functions.