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1.
Eur Neuropsychopharmacol ; 25(10): 1650-1660, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26049820

RESUMO

Transcription factors play important roles in the control of neuronal function in physiological and pathological conditions. We previously reported reduced levels of transcription factor SP4 protein, but not transcript, in the cerebellum in bipolar disorder and associated with more severe negative symptoms in schizophrenia. We have recently reported phosphorylation of Sp4 at S770, which is regulated by membrane depolarization and NMDA receptor activity. The aim of this study was to investigate SP4 S770 phosphorylation in bipolar disorder and its association with negative symptoms in schizophrenia, and to explore the potential relationship between phosphorylation and protein abundance. Here we report a significant increase in SP4 phosphorylation in the cerebellum, but not the prefrontal cortex, of bipolar disorder subjects (n=10) (80% suicide) compared to matched controls (n=10). We found that SP4 phosphorylation inversely correlated with SP4 levels independently of disease status in both areas of the human brain. Moreover, SP4 phosphorylation in the cerebellum positively correlated with negative symptoms in schizophrenia subjects (n=15). Further, we observed that a phospho-mimetic mutation in truncated Sp4 was sufficient to significantly decrease Sp4 steady-state levels, while a non-phosphorylatable mutant showed increased stability in cultured rat cerebellar granule neurons. Our results indicate that SP4 S770 phosphorylation is increased in the cerebellum in bipolar disorder subjects that committed suicide and in severe schizophrenia subjects, and may be part of a degradation signal that controls Sp4 abundance in cerebellar granule neurons. This opens the possibility that modulation of SP4 phosphorylation may contribute to the molecular pathophysiology of psychotic disorders.


Assuntos
Transtorno Bipolar/metabolismo , Esquizofrenia/metabolismo , Fator de Transcrição Sp4/metabolismo , Adulto , Idoso , Animais , Transtorno Bipolar/genética , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Neurônios/metabolismo , Fosforilação , Córtex Pré-Frontal/metabolismo , Estabilidade Proteica , Ratos , Esquizofrenia/genética , Fator de Transcrição Sp4/genética
2.
PLoS One ; 10(4): e0125115, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25915526

RESUMO

BACKGROUND: Altered expression of transcription factor specificity protein 4 (SP4) has been found in the postmortem brain of patients with psychiatric disorders including schizophrenia and bipolar disorder. Reduced levels of SP4 protein have recently been reported in peripheral blood mononuclear cells in first-episode psychosis. Also, SP4 levels are modulated by lithium treatment in cultured neurons. Phosphorylation of SP4 at S770 is increased in the cerebellum of bipolar disorder subjects and upon inhibition of NMDA receptor signaling in cultured neurons. The aim of this study was to investigate whether SP4 S770 phosphorylation is increased in lymphocytes of first-episode psychosis patients and the effect of lithium treatment on this phosphorylation. METHODS: A cross-sectional study of S770 phosphorylation relative to total SP4 immunoreactivity using specific antibodies in peripheral blood mononuclear cells in first-episode psychosis patients (n = 14, treated with lithium or not) and matched healthy controls (n = 14) by immunoblot was designed. We also determined the effects of the prescribed drugs lithium, olanzapine or valproic acid on SP4 phosphorylation in rat primary cultured cerebellar granule neurons. RESULTS: We found that SP4 S770 phosphorylation was significantly increased in lymphocytes in first-episode psychosis compared to controls and decreased in patients treated with lithium compared to patients who did not receive lithium. Moreover, incubation with lithium but not olanzapine or valproic acid reduced SP4 phosphorylation in rat cultured cerebellar granule neurons. CONCLUSIONS: The findings presented here indicate that SP4 S770 phosphorylation is increased in lymphocytes in first-episode psychosis which may be reduced by lithium treatment in patients. Moreover, our study shows lithium treatment prevents this phosphorylation in vitro in neurons. This pilot study suggests that S770 SP4 phosphorylation could be a peripheral biomarker of psychosis, and may be regulated by lithium treatment in first-episode psychosis.


Assuntos
Antipsicóticos/administração & dosagem , Lítio/administração & dosagem , Neurônios/efeitos dos fármacos , Transtornos Psicóticos/tratamento farmacológico , Transtornos Psicóticos/metabolismo , Fator de Transcrição Sp4/sangue , Adolescente , Adulto , Animais , Antipsicóticos/farmacologia , Benzodiazepinas/farmacologia , Células Cultivadas , Estudos Transversais , Feminino , Humanos , Lítio/farmacologia , Masculino , Modelos Biológicos , Neurônios/citologia , Olanzapina , Fosforilação/efeitos dos fármacos , Projetos Piloto , Transtornos Psicóticos/sangue , Ratos , Serina/metabolismo , Ácido Valproico/farmacologia , Adulto Jovem
3.
Dev Neurobiol ; 75(1): 93-108, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25045015

RESUMO

Glutamatergic signaling through N-methyl-d-aspartate receptors (NMDARs) is important for neuronal development and plasticity and is often dysregulated in psychiatric disorders. Mice mutant for the transcription factor Sp4 have reduced levels of NMDAR subunit 1 (NR1) protein, but not mRNA, and exhibit behavioral and memory deficits (Zhou et al., [2010] Human Molecular Genetics 19: 3797-3805). In developing cerebellar granule neurons (CGNs), Sp4 controls dendrite patterning (Ramos et al., [2007] Proc Natl Acad Sci USA 104: 9882-9887). Sp4 target genes that regulate dendrite pruning or NR1 levels are not known. Here we report that Sp4 activates transcription of Nervous Wreck 2 (Nwk2; also known as Fchsd1) and, further, that Nwk2, an F-BAR domain-containing protein, mediates Sp4-dependent regulation of dendrite patterning and cell surface expression of NR1. Knockdown of Nwk2 in CGNs increased primary dendrite number, phenocopying Sp4 knockdown, and exogenous expression of Nwk2 in Sp4-depleted neurons rescued dendrite number. We observed that acute Sp4 depletion reduced levels of surface, but not total, NR1, and this was rescued by Nwk2 expression. Furthermore, expression of Nr1 suppressed the increase in dendrite number in Sp4- or Nwk2- depleted neurons. We previously reported that Sp4 protein levels were reduced in cerebellum of subjects with bipolar disorder (BD) (Pinacho et al., [2011] Bipolar Disorders 13: 474-485). Here we report that Nwk2 mRNA and NR1 protein levels were also reduced in postmortem cerebellum of BD subjects. Our data suggest a role for Sp4-regulated Nwk2 in NMDAR trafficking and identify a Sp4-Nwk2-NMDAR1 pathway that regulates neuronal morphogenesis during development and may be disrupted in bipolar disorder.


Assuntos
Transtorno Bipolar/metabolismo , Proteínas de Transporte/metabolismo , Cerebelo/metabolismo , Dendritos/fisiologia , Proteínas de Membrana/metabolismo , Morfogênese/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais/fisiologia , Fator de Transcrição Sp4/fisiologia , Adulto , Idoso , Animais , Cerebelo/citologia , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade
4.
Sci Signal ; 7(328): ra51, 2014 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-24894994

RESUMO

Calcium (Ca(2+)) signaling activated in response to membrane depolarization regulates neuronal maturation, connectivity, and plasticity. Store-operated Ca(2+) entry (SOCE) occurs in response to depletion of Ca(2+) from endoplasmic reticulum (ER), mediates refilling of this Ca(2+) store, and supports Ca(2+) signaling in nonexcitable cells. We report that maximal activation of SOCE occurred in cerebellar granule neurons cultured under resting conditions and that this Ca(2+) influx promoted the degradation of transcription factor Sp4, a regulator of neuronal morphogenesis and function. Lowering the concentration of extracellular potassium, a condition that reduces neuronal excitability, stimulated depletion of intracellular Ca(2+) stores, resulted in the relocalization of the ER Ca(2+) sensor STIM1 into punctate clusters consistent with multimerization and accumulation at junctions between the ER and plasma membrane, and induced a Ca(2+) influx with characteristics of SOCE. Compounds that block SOCE prevented the ubiquitylation and degradation of Sp4 in neurons exposed to a low concentration of extracellular potassium. Knockdown of STIM1 blocked degradation of Sp4, whereas expression of constitutively active STIM1 decreased Sp4 abundance under depolarizing conditions. Our findings indicated that, in neurons, SOCE is induced by hyperpolarization, and suggested that this Ca(2+) influx pathway is a distinct mechanism for regulating neuronal gene expression.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Cálcio/metabolismo , Regulação da Expressão Gênica/fisiologia , Glicoproteínas de Membrana/metabolismo , Neurônios/metabolismo , Proteólise/efeitos dos fármacos , Fator de Transcrição Sp4/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Western Blotting , Células Cultivadas , Cerebelo/citologia , Primers do DNA/genética , Imunofluorescência , Regulação da Expressão Gênica/genética , Imidazóis/farmacologia , Imuno-Histoquímica , Imunoprecipitação , Proteínas Luminescentes/metabolismo , Plasmídeos/genética , Potássio/metabolismo , Ratos , Ratos Long-Evans , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Molécula 1 de Interação Estromal , Ubiquitinação
5.
J Neurochem ; 129(4): 743-52, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24475768

RESUMO

The regulation of transcription factor function in response to neuronal activity is important for development and function of the nervous system. The transcription factor Sp4 regulates the developmental patterning of dendrites, contributes to complex processes including learning and memory, and has been linked to psychiatric disorders such as schizophrenia and bipolar disorder. Despite its many roles in the nervous system, the molecular mechanisms regulating Sp4 activity are poorly understood. Here, we report a site of phosphorylation on Sp4 at serine 770 that is decreased in response to membrane depolarization. Inhibition of the voltage-dependent NMDA receptor increased Sp4 phosphorylation. Conversely, stimulation with NMDA reduced the levels of Sp4 phosphorylation, and this was dependent on the protein phosphatase 1/2A. A phosphomimetic substitution at S770 impaired the Sp4-dependent maturation of cerebellar granule neuron primary dendrites, whereas a non-phosphorylatable Sp4 mutant behaved like wild type. These data reveal that transcription factor Sp4 is regulated by NMDA receptor-dependent activation of a protein phosphatase 1/2A signaling pathway. Our findings also suggest that the regulated control of Sp4 activity is an important mechanism governing the developmental patterning of dendrites.


Assuntos
N-Metilaspartato/farmacologia , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Fator de Transcrição Sp4/metabolismo , Animais , Calcineurina/fisiologia , Inibidores de Calcineurina , Canais de Cálcio/fisiologia , Linhagem Celular , Cerebelo/citologia , Dendritos/ultraestrutura , Maleato de Dizocilpina/farmacologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Neurogênese , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Ácido Okadáico/farmacologia , Mutação Puntual , Cloreto de Potássio/farmacologia , Proteína Fosfatase 1/antagonistas & inibidores , Proteína Fosfatase 1/fisiologia , Proteína Fosfatase 2/fisiologia , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno/farmacologia , Ratos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Fator de Transcrição Sp4/química , Transfecção
6.
Prostate ; 70(8): 807-16, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20054820

RESUMO

BACKGROUND: p21-activated kinase 6 (PAK6) is a serine/threonine kinase belonging to the p21-activated kinase (PAK) family. We investigated the role of PAK6 in radiation-induced cell death in human prostate cancer cells. METHODS: We used a short hairpin RNA (shRNA) strategy to stably knock down PAK6 in PC3 and DU145 cells. Radiation sensitivities were compared in PAK6 stably knockdown cells versus the scrambled shRNA-expressing control cells. RESULTS: PAK6 mRNA and protein levels in PC3 and DU145 cells were upregulated upon exposure to 6 Gy of radiation. After irradiation, an increased percentage of apoptotic cells and cleaved caspase-3 levels were demonstrated in combination with a decrease in cell viability and a reduction in clonogenic survival in PAK6-knockdown cells. In addition, transfection with PAK6 shRNA blocked cells in a more radiosensitive G2-M phase and increased levels of DNA double-strand breaks. We further explored the potential mechanisms by which PAK6 mediates resistance to radiation-induced apoptosis. Inhibition of PAK6 caused a decrease in Ser(112) phosphorylation of BAD, a proapoptotic member of the Bcl-2 family, which led to enhanced binding of BAD to Bcl-2 and Bcl-X(L) and release of cytochrome c culminating into caspase activation and cell apoptosis. CONCLUSIONS: The combination of PAK6 inhibition and irradiation resulted in significantly decreased survival of prostate cancer cells. The underlying mechanisms by which targeting PAK6 may improve radiation response seem to be multifaceted, and involve alterations in cell cycle distribution and impaired DNA double-strand break repair as well as relieved BAD phosphorylation.


Assuntos
Próstata/metabolismo , Próstata/efeitos da radiação , Quinases Ativadas por p21/metabolismo , Apoptose/genética , Apoptose/efeitos da radiação , Western Blotting , Ciclo Celular/genética , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Humanos , Masculino , Próstata/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/efeitos da radiação , Tolerância a Radiação/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Regulação para Cima/genética , Regulação para Cima/efeitos da radiação , Quinases Ativadas por p21/genética
7.
Discov Med ; 7(38): 75-81, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18093469

RESUMO

Glioblastoma multiforme is the most aggressive fatal brain tumor with a median survival of about a year after diagnosis. The current available treatment options only marginally improve patient outcome. The complexity and heterogeneity of the disease pose an extensive challenge to the development of novel therapeutic agents. Through molecular and genetic profiling, it has been possible to both identify mechanisms of disease progression and its therapeutic resistance. This paper highlights the significance of these advances.


Assuntos
Perfilação da Expressão Gênica , Glioma/metabolismo , Apoptose/genética , Apoptose/fisiologia , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioma/genética , Glioma/patologia , Humanos , Modelos Biológicos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
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