RESUMO
PURPOSE: The aim of our study is to evaluate the number and the features of admissions to the emergency room (ER) requiring psychiatric consultation, in the period between May 4th and August 31st 2020. METHODS: We carried out a retrospective longitudinal observational study examining the 4 months following the initial lockdown imposed during the COVID-19 outbreak (May 4th and August 31st 2020). More specifically, the ER admissions leading to psychiatric referral were reviewed at all seven public hospitals of AUSL Romagna (Emilia Romagna region, Italy). Socio-demographic variables, history of medical comorbidities or psychiatric disorders, reason for ER admission, psychiatric diagnosis at discharge, and actions taken by the psychiatrist were collected. RESULTS: An 11.3% (p = 0.007) increase in psychiatric assessments was observed when compared with the same period of the previous year (2019). A positive personal history of psychiatric disorders (OR:0.68, CI: 0.53-0.87) and assessments leading to no indication for follow-up (OR: 0.22, CI: 0.13-0.39) were significantly less frequent, while there was a significant increase of cases featuring organic comorbidities (OR: 1.24, CI: 1.00-1.52) and suicidal ideation/self-harm/suicide attempt (OR: 1,71, CI: 1.19-2.45) or psychomotor agitation (OR: 1.46, CI: 1.02-2.07) as reason for admission. CONCLUSIONS: Our results showed an increase in ER psychiatric consultations compared to the previous year, underlying the increased psychological distress caused by the lockdown.
Assuntos
COVID-19 , COVID-19/epidemiologia , Controle de Doenças Transmissíveis , Serviço Hospitalar de Emergência , Humanos , Itália/epidemiologia , Pandemias , Estudos RetrospectivosRESUMO
The G protein-coupled receptor 17 (GPR17) plays crucial roles in myelination. It is highly expressed during transition of oligodendrocyte progenitor cells to immature oligodendrocytes, but, after this stage, it must be down-regulated to allow generation of mature myelinating cells. After endocytosis, GPR17 is sorted into lysosomes for degradation or recycled to the plasma membrane. Balance between degradation and recycling is important for modulation of receptor levels at the cell surface and thus for the silencing/activation of GPR17-signaling pathways that, in turn, affect oligodendrocyte differentiation. The molecular mechanisms at the basis of these processes are still partially unknown and their characterization will allow a better understanding of myelination and provide cues to interpret the consequences of GPR17 dysfunction in diseases. Here, we demonstrate that the endocytic trafficking of GPR17 is mediated by the interaction of a type I PDZ-binding motif located at the C-terminus of the receptor and SNX27, a recently identified protein of the endosome-associated retromer complex and whose functions in oligodendrocytes have never been studied. SNX27 knock-down significantly reduces GPR17 plasma membrane recycling in differentiating oligodendrocytes while accelerating cells' terminal maturation. Interestingly, trisomy-linked down-regulation of SNX27 expression in the brain of Ts65Dn mice, a model of Down syndrome, correlates with a decrease in GPR17(+) cells and an increase in mature oligodendrocytes, which, however, fail in reaching full maturation, eventually leading to hypomyelination. Our data demonstrate that SNX27 modulates GPR17 plasma membrane recycling and stability, and that disruption of the SNX27/GPR17 interaction might contribute to pathological oligodendrocyte differentiation defects. GLIA 2016. GLIA 2016;64:1437-1460.
Assuntos
Transporte Biológico/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Oligodendroglia/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Nexinas de Classificação/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Diferenciação Celular/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/patologia , Modelos Animais de Doenças , Síndrome de Down/metabolismo , Síndrome de Down/patologia , Endocitose/fisiologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oligodendroglia/patologia , Nexinas de Classificação/deficiência , Nexinas de Classificação/genéticaRESUMO
Oligodendrocyte progenitor cells (OPCs) persist in the adult central nervous system and guarantee oligodendrocyte turnover throughout life. It remains obscure how OPCs avoid exhaustion during adulthood. Similar to stem cells, OPCs could self-maintain by undergoing asymmetric divisions generating a mixed progeny either keeping a progenitor phenotype or proceeding to differentiation. To address this issue, we examined the distribution of stage-specific markers in sister OPCs during mitosis and later after cell birth, and assessed its correlation with distinct short-term fates. In both the adult and juvenile cerebral cortex a fraction of dividing OPCs gives rise to sister cells with diverse immunophenotypic profiles and short-term behaviors. Such heterogeneity appears as cells exit cytokinesis, but does not derive from the asymmetric segregation of molecules such as NG2 or PDGFRa expressed in the mother cell. Rather, rapid downregulation of OPC markers and upregulation of molecules associated with lineage progression contributes to generate early sister OPC asymmetry. Analyses during aging and upon exposure to physiological (i.e., increased motor activity) and pathological (i.e., trauma or demyelination) stimuli showed that both intrinsic and environmental factors contribute to determine the fraction of symmetric and asymmetric OPC pairs and the phenotype of the OPC progeny as soon as cells exit mitosis.
Assuntos
Envelhecimento , Mitose/fisiologia , Oligodendroglia/fisiologia , Células-Tronco/fisiologia , Análise de Variância , Animais , Antígenos/genética , Antígenos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bromodesoxiuridina , Ciclo Celular/fisiologia , Diferenciação Celular , Células Cultivadas , Sistema Nervoso Central/citologia , Regulação da Expressão Gênica/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteoglicanas/genética , Proteoglicanas/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
GPR17 is a G(i) -coupled dual receptor activated by uracil-nucleotides and cysteinyl-leukotrienes. These mediators are massively released into hypoxic tissues. In the normal heart, GPR17 expression has been reported. By contrast, its role in myocardial ischaemia has not yet been assessed. In the present report, the expression of GPR17 was investigated in mice before and at early stages after myocardial infarction by using immunofluorescence, flow cytometry and RT-PCR. Before induction of ischaemia, results indicated the presence of the receptor in a population of stromal cells expressing the stem-cell antigen-1 (Sca-1). At early stages after ligation of the coronary artery, the receptor was expressed in Sca-1(+) cells, and cells stained with Isolectin-B4 and anti-CD45 antibody. GPR17(+) cells also expressed mesenchymal marker CD44. GPR17 function was investigated in vitro in a Sca-1(+)/CD31(-) cell line derived from normal hearts. These experiments showed a migratory function of the receptor by treatment with UDP-glucose and leukotriene LTD4, two GPR17 pharmacological agonists. The GPR17 function was finally assessed in vivo by treating infarcted mice with Cangrelor, a pharmacological receptor antagonist, which, at least in part, inhibited early recruitment of GPR17(+) and CD45(+) cells. These findings suggest a regulation of heart-resident mesenchymal cells and blood-borne cellular species recruitment following myocardial infarction, orchestrated by GPR17.
Assuntos
Células-Tronco Mesenquimais/fisiologia , Infarto do Miocárdio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Animais , Antígenos Ly/metabolismo , Movimento Celular , Receptores de Hialuronatos , Antígenos Comuns de Leucócito/metabolismo , Leucotrieno D4/farmacologia , Leucotrieno D4/fisiologia , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/patologia , Proteínas do Tecido Nervoso/agonistas , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Uridina Difosfato Glucose/farmacologia , Uridina Difosfato Glucose/fisiologiaRESUMO
GPR17 is a G-protein-coupled receptor that is activated by two classes of molecules: uracil-nucleotides and cysteinyl-leukotrienes. GPR17 is required for initiating the differentiation of oligodendrocyte precursors but has to be down-regulated to allow cells to undergo terminal maturation. Although a great deal has been learned about GPR17 expression and signaling, no information is currently available about the trafficking of native receptors after the exposure of differentiating oligodendrocytes to endogenous agonists. Here, we demonstrate that neuron-conditioned medium induces the transcriptionally mediated, time-regulated expression of GPR17 in Oli-neu, an oligodendrocyte precursor cell line, making these cells suitable for studying the endocytic traffic of the native receptor. Agonist-induced internalization, intracellular trafficking, and membrane recycling of GPR17 were analyzed by biochemical and immunofluorescence assays using an ad hoc-developed antibody against the extracellular N-terminal of GPR17. Both UDP-glucose and LTD(4) increased GPR17 internalization, although with different efficiency. At early time points, internalized GPR17 co-localized with transferrin receptor, whereas at later times it partially co-localized with the lysosomal marker Lamp1, suggesting that a portion of GPR17 is targeted to lysosomes upon ligand binding. An analysis of receptor recycling and degradation demonstrated that a significant aliquot of GPR17 is recycled to the cell surface. Furthermore, internalized GPR17 displayed a co-localization with the marker of the "short loop" recycling endosomes, Rab4, while showing very minor co-localization with the "long loop" recycling marker, Rab11. Our results provide the first data on the agonist-induced trafficking of native GPR17 in oligodendroglial cells and may have implications for both physiological and pathological myelination.
Assuntos
Membrana Celular/metabolismo , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Clonagem Molecular , Endocitose , Humanos , Lisossomos/metabolismo , Camundongos , Modelos Biológicos , Oligodendroglia/citologia , Interferência de RNA , RatosRESUMO
Mutations in the CACNA1A gene, which encodes the pore-forming α1A subunit of the CaV2.1 voltage-gated calcium channel, cause a number of human neurologic diseases including familial hemiplegic migraine. We have analyzed the functional impact of the E1015K amino acid substitution located in the "synprint" domain of the α1A subunit. This variant was identified in two families with hemiplegic migraine and in one patient with migraine with aura. The wild type (WT) and the E1015K forms of the GFP-tagged α1A subunit were expressed in cultured hippocampal neurons and HEK cells to understand the role of the variant in the transport activity and physiology of CaV2.1. The E1015K variant does not alter CaV2.1 protein expression, and its transport to the cell surface and synaptic terminals is similar to that observed for WT channels. Electrophysiological data demonstrated that E1015K channels have increased current density and significantly altered inactivation properties compared with WT. Furthermore, the SNARE proteins syntaxin 1A and SNAP-25 were unable to modulate voltage-dependent inactivation of E1015K channels. Overall, our findings describe a genetic variant in the synprint site of the CaV2.1 channel which is characterized by a gain-of-function and associated with both hemiplegic migraine and migraine with aura in patients.
Assuntos
Canais de Cálcio Tipo N , Hipocampo , Enxaqueca com Aura , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso , Terminações Pré-Sinápticas , Adolescente , Adulto , Substituição de Aminoácidos , Animais , Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/metabolismo , Criança , Feminino , Células HEK293 , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Transporte de Íons/genética , Masculino , Pessoa de Meia-Idade , Enxaqueca com Aura/genética , Enxaqueca com Aura/metabolismo , Enxaqueca com Aura/patologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/patologia , Coelhos , Ratos , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/genética , Sintaxina 1/metabolismoRESUMO
The rat adenine receptor (rAdeR) was the first member of a family of G protein-coupled receptors (GPCRs) activated by adenine and designated as P0-purine receptors. The present study aimed at gaining insights into structural aspects of ligand binding and function of the rAdeR. We exchanged amino acid residues predicted to be involved in ligand binding (Phe110(3.24), Asn115(3.29), Asn173(4.60), Phe179(45.39), Asn194(5.40), Phe195(5.41), Leu201(5.47), His252(6.54), and Tyr268(7.32)) for alanine and expressed them in Spodoptera frugiperda (Sf9) insect cells. Membrane preparations subjected to [(3)H]adenine binding studies revealed only minor effects indicating that none of the exchanged amino acids is part of the ligand binding pocket, at least in the inactive state of the receptor. Furthermore, we coexpressed the rAdeR and its mutants with mammalian Gi proteins in Sf9 insect cells to probe receptor activation. Two amino acid residues, Asn194(5.40) and Leu201(5.47), were found to be crucial for activation since their alanine mutants did not respond to adenine. Moreover we showed that-in contrast to most other rhodopsin-like GPCRs-the rAdeR does not contain essential disulfide bonds since preincubation with dithiothreitol neither altered adenine binding in Sf9 cell membranes, nor adenine-induced inhibition of adenylate cyclase in 1321N1 astrocytoma cells transfected with the rAdeR. To detect rAdeRs by Western blot analysis, we developed a specific antibody. Finally, we were able to show that the extended N-terminal sequence of the rAdeR constitutes a putative signal peptide of unknown function that is cleaved off in the mature receptor. Our results provide important insights into this new, poorly investigated family of purinergic receptors.
Assuntos
Receptores Purinérgicos/química , Receptores Purinérgicos/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Western Blotting , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Quaternária de Proteína , RatosRESUMO
Unveiling the mechanisms participating in the damage and repair of traumatic brain injury (TBI) is fundamental to develop new therapies. The P2Y-like GPR17 receptor has recently emerged as a sensor of damage and a key actor in lesion remodeling/repair in the rodent brain, but its role in humans is totally unknown. Here, we characterized GPR17 expression in brain specimens from seven intensive care unit TBI patients undergoing neurosurgery for contusion removal and from 28 autoptic TBI cases (and 10 control subjects of matched age and gender) of two university hospitals. In both neurosurgery and autoptic samples, GPR17 expression was strong inside the contused core and progressively declined distally according to a spatio-temporal gradient. Inside and around the core, GPR17 labeled dying neurons, reactive astrocytes, and activated microglia/macrophages. In peri-contused parenchyma, GPR17 decorated oligodendrocyte precursor cells (OPCs) some of which had proliferated, indicating re-myelination attempts. In autoptic cases, GPR17 expression positively correlated with death for intracranial complications and negatively correlated with patients' post-traumatic survival. Data indicate lesion-specific sequential involvement of GPR17 in the (a) death of irreversibly damaged neurons, (b) activation of microglia/macrophages remodeling the lesion, and (c) activation/proliferation of multipotent parenchymal progenitors (both reactive astrocytes and OPCs) starting repair processes. Data validate GPR17 as a target for neurorepair and are particularly relevant to setting up new therapies for TBI patients.
Assuntos
Lesões Encefálicas/metabolismo , Regeneração Nervosa/fisiologia , Neuroglia/metabolismo , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adulto , Idoso , Lesões Encefálicas/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Neuroglia/patologia , Neurônios/patologia , Adulto JovemRESUMO
The developing and mature central nervous system contains neural precursor cells expressing the proteoglycan NG2. Some of these cells continuously differentiate to myelin-forming oligodendrocytes; knowledge of the destiny of NG2(+) precursors would benefit from the characterization of new key functional players. In this respect, the G protein-coupled membrane receptor GPR17 has recently emerged as a new timer of oligodendrogliogenesis. Here, we used purified oligodendrocyte precursor cells (OPCs) to fully define the immunophenotype of the GPR17-expressing cells during OPC differentiation, unveil its native signaling pathway, and assess the functional consequences of GPR17 activation by its putative endogenous ligands, uracil nucleotides and cysteinyl leukotrienes (cysLTs). GPR17 presence was restricted to very early differentiation stages and completely segregated from that of mature myelin. Specifically, GPR17 decorated two subsets of slowly proliferating NG2(+) OPCs: (i) morphologically immature cells expressing other early proteins like Olig2 and PDGF receptor-α, and (ii) ramified preoligodendrocytes already expressing more mature factors, like O4 and O1. Thus, GPR17 is a new marker of these transition stages. In OPCs, GPR17 activation by either uracil nucleotides or cysLTs resulted in potent inhibition of intracellular cAMP formation. This effect was counteracted by GPR17 antagonists and receptor silencing with siRNAs. Finally, uracil nucleotides promoted and GPR17 inhibition, by either antagonists or siRNAs, impaired the normal program of OPC differentiation. These data have implications for the in vivo behavior of NG2(+) OPCs and point to uracil nucleotides and cysLTs as main extrinsic local regulators of these cells under physiological conditions and during myelin repair.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células , Regulação da Expressão Gênica/fisiologia , Oligodendroglia/metabolismo , Receptores Acoplados a Proteínas G/biossíntese , Células-Tronco/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Bainha de Mielina/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Fator de Transcrição 2 de Oligodendrócitos , Oligodendroglia/citologia , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores do Fator de Crescimento Derivado de Plaquetas/biossíntese , Células-Tronco/citologia , Nucleotídeos de Uracila/metabolismo , Nucleotídeos de Uracila/farmacologiaRESUMO
Cholesterol and sphingolipids are abundant in neuronal membranes, where they help the organisation of the membrane microdomains involved in major roles such as axonal and dendritic growth, and synapse and spine stability. The aim of this study was to analyse their roles in presynaptic physiology. We first confirmed the presence of proteins of the exocytic machinery (SNARES and Ca(v)2.1 channels) in the lipid microdomains of cultured neurons, and then incubated the neurons with fumonisin B (an inhibitor of sphingolipid synthesis), or with mevastatin or zaragozic acid (two compounds that affect the synthesis of cholesterol by inhibiting HMG-CoA reductase or squalene synthase). The results demonstrate that fumonisin B and zaragozic acid efficiently decrease sphingolipid and cholesterol levels without greatly affecting the viability of neurons or the expression of synaptic proteins. Electron microscopy showed that the morphology and number of synaptic vesicles in the presynaptic boutons of cholesterol-depleted neurons were similar to those observed in control neurons. Zaragozic acid (but not fumonisin B) treatment impaired synaptic vesicle uptake of the lipophilic dye FM1-43 and an antibody directed against the luminal epitope of synaptotagmin-1, effects that depended on the reduction in cholesterol because they were reversed by cholesterol reloading. The time-lapse confocal imaging of neurons transfected with ecliptic SynaptopHluorin showed that cholesterol depletion affects the post-depolarisation increase in fluorescence intensity. Taken together, these findings show that reduced cholesterol levels impair synaptic vesicle exocytosis in cultured neurons.
Assuntos
Colesterol/metabolismo , Exocitose/fisiologia , Vesículas Sinápticas/fisiologia , Animais , Anticolesterolemiantes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Canais de Cálcio Tipo N/metabolismo , Células Cultivadas , Exocitose/efeitos dos fármacos , Fumonisinas/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Imunoglobulina G/metabolismo , Lovastatina/análogos & derivados , Lovastatina/farmacologia , Microdomínios da Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Modelos Neurológicos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurônios/ultraestrutura , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Proteínas SNARE/metabolismo , Esfingolipídeos/metabolismo , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/ultraestrutura , Sinaptotagmina I/antagonistas & inibidores , Sinaptotagmina I/imunologia , Sinaptotagmina I/metabolismo , Ácidos Tricarboxílicos/farmacologiaRESUMO
Acid sphingomyelinase (A-SMase) is an important enzyme in sphingolipid metabolism and plays key roles in apoptosis, immunity, development, and cancer. In addition, it mediates cytotoxicity of cisplatin and some other chemotherapeutic drugs. The mechanism of A-SMase activation is still undefined. We now demonstrate that, upon CD95 stimulation, A-SMase is activated through translocation from intracellular compartments to the plasma membrane in an exocytic pathway requiring the t-SNARE protein syntaxin 4. Indeed, down-regulation of syntaxin 4 inhibits A-SMase translocation and activation induced by CD95 stimulation. This leads to inhibition of the CD95-triggered signaling events, including caspase 3 and 9 activation and apoptosis, activation of the survival pathway involving the protein kinase Akt, and important changes in cell cycle and proliferation. The molecular interaction between A-SMase and syntaxin 4 was not known and clarifies the mechanism of A-SMase activation. The novel actions of syntaxin 4 in sphingolipid metabolism and exocytosis we describe here define signaling mechanisms of broad relevance in cell pathophysiology.
Assuntos
Apoptose/fisiologia , Membrana Celular/enzimologia , Exocitose/fisiologia , Proteínas Qa-SNARE/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Receptor fas/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Ativação Enzimática/fisiologia , Humanos , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esfingomielinas/metabolismo , Células U937RESUMO
NG2-expressing cells comprise a population of cycling precursors that can exit the cell cycle and differentiate into mature oligodendrocytes. As a whole, they display heterogeneous properties and behaviors that remain unresolved at the molecular level, although partly interpretable as distinct maturation stages. To address this issue, we analyzed the expression of the GPR17 receptor, recently shown to decorate NG2-expressing cells and to operate as an early sensor of brain damage, in immature and adult oligodendrocyte progenitors in the intact brain and after injury. In both the early postnatal and adult cerebral cortex, distinct GPR17 protein localizations and expression levels define different stages of oligodendroglial maturation, ranging from the precursor phase to the premyelinating phenotype. As soon as cells exit mitosis, a fraction of NG2-expressing cells displays accumulation of GPR17 protein in the Golgi apparatus. GPR17 expression is subsequently upregulated and distributed to processes of cells that stop dividing, progressively lose NG2 positivity and assume premyelinating features. Absence of colabeling with mature markers or myelin proteins indicates that GPR17 is downregulated when cells complete their final maturation. BrdU-based fate-mapping demonstrated that a significant fraction of newly generated oligodendrocyte progenitors transiently upregulates GPR17 during maturation. Importantly, we also found that GPR17 does not participate to the early reaction of NG2-expressing cells to damage, while it is induced at postacute stages after injury. These findings identify GPR17 as a marker for progenitor progression within the oligodendroglial lineage and highlight its participation to postacute reactivity of NG2 cells in different injury paradigms.
Assuntos
Antígenos/biossíntese , Dano Encefálico Crônico/metabolismo , Lesões Encefálicas/metabolismo , Diferenciação Celular/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Oligodendroglia/metabolismo , Proteoglicanas/biossíntese , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/metabolismo , Doença Aguda , Animais , Antígenos/genética , Biomarcadores/metabolismo , Dano Encefálico Crônico/patologia , Lesões Encefálicas/patologia , Modelos Animais de Doenças , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Camundongos , Camundongos Endogâmicos C57BL , Regeneração Nervosa/fisiologia , Oligodendroglia/patologia , Cultura Primária de Células , Proteoglicanas/genética , Células-Tronco/patologiaRESUMO
The P2Y-like receptor GPR17 is expressed by adult neural progenitor cells, suggesting a role in lineage determination. Here, we characterized GPR17 expression and function in mouse cortical primary astrocytes/precursor cell cultures. GPR17 is expressed by a subpopulation of oligodendrocyte precursor cells (OPCs), but not by astrocytes. This expression pattern was also confirmed in vivo. In vitro, GPR17 expression was markedly influenced by culturing conditions. In the presence of growth factors (GFs), no significant GPR17 expression was found. When cultures were shifted to a differentiating medium, a dramatic, time-dependent increase in the number of highly branched GPR17-positive cells was observed. Under these conditions, GPR17 was induced in the totality of O4-positive immature oligodendrocytes. Instead, in cultures originally grown in the absence of GFs, GPR17 was already expressed in morphologically more mature OPCs. Shifting of these cultures to differentiating conditions induced GPR17 only in a subpopulation of O4-positive cells. Under both culture protocols, appearance of more mature CNPase- and MBP-positive cells was associated to a progressive loss of GPR17. GPR17 expression also sensitized cells to adenine nucleotide-induced cytotoxicity, whereas activation with uracil nucleotides promoted differentiation towards a more mature phenotype. We suggest that GFs may keep OPCs in a less differentiated stage by restraining GPR17 expression, and that, under permissive conditions, GPR17 contributes to OPCs differentiation. However, upon high extracellular adenine nucleotide concentrations, as during trauma and ischemia, GPR17 sensitizes cells to cytotoxicity. This double-edged sword role may be exploited to unveil new therapeutic approaches to acute and chronic brain disorders.
Assuntos
Trifosfato de Adenosina/toxicidade , Diferenciação Celular/genética , Proteínas do Tecido Nervoso/genética , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Purinérgicos P2Y1/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Morte Celular/genética , Células Cultivadas , Técnicas de Cocultura , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/fisiologia , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/fisiologia , Receptores Purinérgicos P2Y1/biossínteseRESUMO
Upon central nervous system injury, the extracellular concentrations of nucleotides and cysteinyl-leukotrienes, two unrelated families of endogenous signalling molecules, are markedly increased at the site of damage, suggesting that they may act as 'danger signals' to alert responses to tissue damage and start repair. Here we show that, in non-injured spinal cord parenchyma, GPR17, a P2Y-like receptor responding to both uracil nucleotides (e.g. UDP-glucose) and cysteinyl-leukotrienes (e.g. LTD4 and LTC4), is present on a subset of neurons and of oligodendrocytes at different stages of maturation, whereas it is not expressed by astrocytes. GPR17 immunoreactivity was also found on ependymal cells lining the central canal that still retain some of the characteristics of stem/progenitor cells during adulthood. Induction of spinal cord injury (SCI) by acute compression resulted in marked cell death of GPR17+ neurons and oligodendrocytes inside the lesion followed by the appearance of proliferating GPR17+ microglia/macrophages migrating to and infiltrating into the lesioned area. Moreover, 72 h after SCI, GPR17+ ependymal cells started to proliferate and to express GFAP, suggesting their activation and 'de-differentiation' to pluripotent progenitor cells. The in vivo knock down of GPR17 by an antisense oligonucleotide strategy during SCI induction markedly reduced tissue damage and related histological and motor deficits, thus confirming the crucial role played by this receptor in the early phases of tissue damage development. Taken together, our findings suggest a dual and spatiotemporal-dependent role for GPR17 in SCI. At very early times after injury, GPR17 mediates neuronal and oligodendrocyte death inside the lesioned area. At later times, GPR17+ microglia/macrophages are recruited from distal parenchymal areas and move toward the lesioned zone, to suggest a role in orchestrating local remodelling responses. At the same time, the induction of the stem cell marker GFAP in GPR17+ ependymal cells suggests initiation of repair mechanisms. Thus, GPR17 may act as a 'sensor' of damage that is activated by nucleotides and cysteinyl-leukotrienes released in the lesioned area, and could also participate in post-injury responses. Moreover, its presence on spinal cord pre-oligodendrocytes and precursor-like cells suggests GPR17 as a novel target for therapeutic manipulation to foster remyelination and functional repair in SCI.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Traumatismos da Medula Espinal/metabolismo , Animais , Biomarcadores/metabolismo , Movimento Celular/fisiologia , Proliferação de Células , Epêndima/metabolismo , Técnicas de Silenciamento de Genes , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Microglia/metabolismo , Microglia/patologia , Transtornos dos Movimentos/metabolismo , Transtornos dos Movimentos/patologia , Transtornos dos Movimentos/prevenção & controle , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/fisiologia , Neurônios/metabolismo , Oligodendroglia/metabolismo , Oligodesoxirribonucleotídeos Antissenso , Células-Tronco Pluripotentes/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/fisiologia , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/prevenção & controleRESUMO
The heterogeneity of cognitive profiles among psychiatric patients has been reported to carry significant clinical information. However, how to best characterize such cognitive heterogeneity is still a matter of debate. Despite being well suited for clinical data, cluster analysis techniques, like the Two-Step and the Latent Class, received little to no attention in the literature. The present study aimed to test the validity of the cluster solutions obtained with Two-Step and Latent Class cluster analysis on the cognitive profile of a cross-diagnostic sample of 387 psychiatric inpatients. Two-Step and Latent Class cluster analysis produced similar and reliable solutions. The overall results reported that it is possible to group all psychiatric inpatients into Low and High Cognitive Profiles, with a higher degree of cognitive heterogeneity in schizophrenia and bipolar disorder patients than in depressive disorders and personality disorder patients.
RESUMO
The interaction of Prep1 and Pbx homeodomain transcription factors regulates their activity, nuclear localization, and likely, function in development. To understand the in vivo role of Prep1, we have analyzed an embryonic lethal hypomorphic mutant mouse (Prep1(i/i)). Prep1(i/i) embryos die at embryonic day 17.5 (E17.5) to birth with an overall organ hypoplasia, severe anemia, impaired angiogenesis, and eye anomalies, particularly in the lens and retina. The anemia correlates with delayed differentiation of erythroid progenitors and may be, at least in part, responsible for intrauterine death. At E14.5, Prep1 is present in fetal liver (FL) cMyb-positive cells, whose deficiency causes a marked hematopoietic phenotype. Prep1 is also localized to FL endothelial progenitors, consistent with the observed angiogenic phenotype. Likewise, at the same gestational day, Prep1 is present in the eye cells that bear Pax6, implicated in eye development. The levels of cMyb and Pax6 in FL and in the retina, respectively, are significantly decreased in Prep1(i/i) embryos, consistent with the hematopoietic and eye phenotypes. Concomitantly, Prep1 deficiency results in the overall decrease of protein levels of its related family member Meis1 and its partners Pbx1 and Pbx2. As both Prep1 and Meis interact with Pbx, the overall Prep1/Meis-Pbx DNA-binding activity is strongly reduced in whole Prep1(i/i) embryos and their organs. Our data indicate that Prep1 is an essential gene that acts upstream of and within a Pbx-Meis network that regulates multiple aspects of embryonic development.
Assuntos
Embrião de Mamíferos , Proteínas de Homeodomínio/metabolismo , Mutação , Proteínas de Neoplasias/metabolismo , Fenótipo , Fatores de Transcrição/metabolismo , Alantoide/citologia , Anemia/genética , Animais , Células Cultivadas , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Olho/anatomia & histologia , Olho/embriologia , Olho/patologia , Feminino , Marcação de Genes , Idade Gestacional , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteína Meis1 , Proteínas de Neoplasias/genética , Neovascularização Fisiológica , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B , Gravidez , RNA Mensageiro/metabolismo , Fatores de Transcrição/genéticaRESUMO
Most of the hypomorphic Prep1(i/i) embryos (expressing 3-10% of the Prep1 protein), die between E17.5 and P0, with profound anemia, eye malformations and angiogenic anomalies [Ferretti, E., Villaescusa, J.C., Di Rosa, P., Fernandez-Diaz, L.-C., Longobardi, E., Mazzieri, R., Miccio, A., Micali, N., Selleri, L., Ferrari G., Blasi, F. (2006). Hypomorphic mutation of the TALE gene Prep1 (pKnox1) causes a major reduction of Pbx and Meis proteins and a pleiotropic embryonic phenotype. Mol. Cell. Biol. 26, 5650-5662]. We now report on the hematopoietic phenotype of these embryos. Prep1(i/i) fetal livers (FL) are hypoplastic, produce less common myeloid progenitors colonies (CFU-GEMM) in cytokine-supplemented methylcellulose and have an increased number of B-cells precursors that differentiate poorly. Prep1(i/i) FL is able to protect lethally irradiated mice only at high cell doses but the few protected mice show major anomalies in all hematopoietic lineages in both bone marrow (BM) and peripheral organs. Prep1(i/i) FL cells compete inefficiently with wild type bone marrow in competitive repopulation experiments, suggesting that the major defect lies in long-term repopulating hematopoietic stem cells (LTR-HSC). Indeed, wt embryonic expression of Prep1 in the aorta-gonad-mesonephros (AGM) region, fetal liver (FL), cKit(+)Sca1(+)Lin(-)AA4.1(+) (KSLA) cells and B-lymphocytes precursors agrees with the observed phenotype. We therefore conclude that Prep1 is required for a correct and complete hematopoiesis.
Assuntos
Hematopoese/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/fisiologia , Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Diferenciação Celular , Linhagem da Célula , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco/citologia , Fatores de Transcrição/genéticaRESUMO
Although extensive studies provided molecular and pharmacological characterization of metabotropic P2Y receptors for extracellular nucleotides, little is still known about their quaternary structure. By the use of transfected cellular systems and SDS-PAGE, in our previous work we established the propensity of P2Y(4) receptor to form dimeric interactions. Here we focused on endogenously expressed P2Y(4) and P2Y(6) subtypes, comparing their oligomeric complexes under Blue Native (BN) gel electrophoresis. We provided evidence that P2Y(4) and P2Y(6) receptors form high order complexes in native neuronal phenotypes and that the oligomers can be disaggregated down to the dimeric P2Y(4) or to the dimeric and monomeric P2Y(6) receptor. Moreover, dimeric P2Y(4) and monomeric P2Y(6) proteins display selective microdomain partitioning in lipid rafts from specialized subcellular compartments such as synaptosomes. Ligand activation by UTP shifted the oligomerization of P2Y(6) but not of P2Y(4) receptor, as analysed by BN electrophoresis. Finally, whereas transfected P2Y(4) and P2Y(6) proteins homo-interact and posses the appropriate domains to associate with all P2Y(1,2,4,6,11) subtypes, in naive PC12 cells the endogenous P2Y(4) forms hetero-oligomers only with the P2Y(6) subunit. In conclusion, our results indicate that quaternary structure distinguishing P2Y(4) from P2Y(6) receptors might be crucial for specific ligand activation, membrane partitioning and consequent functional regulation.
Assuntos
Proteínas do Tecido Nervoso/genética , Receptores Purinérgicos P2/genética , Animais , Western Blotting , Dimerização , Eletroforese em Gel de Poliacrilamida , Imunoprecipitação , Células PC12 , Estrutura Quaternária de Proteína , RatosRESUMO
Prep1 is a homeodomain transcription factor that acts by dimerizing with Pbx. Since Prep1 null embryos die at gastrulation, we studied Prep1(i/i) hypomorphic mice to study the physiological role of Prep1. A low percentage of homozygous Prep1(i/i) mice survived at birth, and their postnatal functions could be investigated. Reduced Prep1 expression caused an abnormal thymic T-cell development: increased CD4(-) CD8(-) double-negative thymocytes, decrease in alphabetaTCR(high) cells (cells with high levels of the alphabetaTau-cell receptor [alphabetaTCR]) and CD4(+) and CD8(+) single-positive (SP) thymocytes, and increase in gammadeltaTCR cells. Peripheral lymphoid organs of Prep1(i/i) mice contained fewer alphabetaTCR mature T cells and more gammadeltaTCR T cells than wild-type littermates. Moreover, Prep1(i/i) CD4(+) CD8(+) double-positive thymocytes underwent more apoptosis, and SP thymocytes proliferated less than control littermates. Mice that were lethally irradiated and then had Prep1(i/i) fetal liver cells transplanted showed the same defects as the Prep1(i/i) mice did. Among PBC family members, Pbx2 and very low levels of Pbx3 were observed in the thymi of wild-type mice. In Prep1(i/i) mice, the level of Pbx2 protein was profoundly decreased, while for Pbx3 no definitive conclusion could be reached. Therefore, the deficient postnatal T-lymphocytic potential of the Prep1 hematopoietic progenitors depends on the combined, not compensated, absence of Prep1 and at least Pbx2.
Assuntos
Células-Tronco Hematopoéticas/citologia , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/citologia , Timo/crescimento & desenvolvimento , Animais , Antígenos de Diferenciação de Linfócitos T/análise , Linfócitos T CD4-Positivos/química , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/química , Linfócitos T CD8-Positivos/citologia , Proliferação de Células , Células-Tronco Hematopoéticas/química , Proteínas de Homeodomínio/genética , Linfopoese/genética , Camundongos , Camundongos Mutantes , Mutação , Proteínas Proto-Oncogênicas/genética , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Transdução de Sinais , Linfócitos T/química , Timo/citologiaRESUMO
Tumor microenvironment is fundamental for cancer progression and chemoresistance. Among stromal cells tumor-associated macrophages (TAMs) represent the largest population of infiltrating inflammatory cells in malignant tumors, promoting their growth, invasion, and immune evasion. M2-polarized TAMs are endowed with the nitric oxide (NO)-generating enzyme inducible nitric oxide synthase (iNOS). NO has divergent effects on tumors, since it can either stimulate tumor cells growth or promote their death depending on the source of it; likewise the role of iNOS in cancer differs depending on the cell type. The role of NO generated by TAMs has not been investigated. Using different tumor models in vitro and in vivo we found that NO generated by iNOS of M2-polarized TAMs is able to protect tumor cells from apoptosis induced by the chemotherapeutic agent cisplatin (CDDP). Here, we demonstrate that the protective effect of NO depends on the inhibition of acid sphingomyelinase (A-SMase), which is activated by CDDP in a pathway involving the death receptor CD95. Mechanistic insights indicate that NO actions occur via generation of cyclic GMP and activation of protein kinase G (PKG), inducing phosphorylation of syntaxin 4 (synt4), a SNARE protein responsible for A-SMase trafficking and activation. Noteworthy, phosphorylation of synt4 at serine 78 by PKG is responsible for the proteasome-dependent degradation of synt4, which limits the CDDP-induced exposure of A-SMase to the plasma membrane of tumor cells. This inhibits the cytotoxic mechanism of CDDP reducing A-SMase-triggered apoptosis. This is the first demonstration that endogenous NO system is a key mechanism through which TAMs protect tumor cells from chemotherapeutic drug-induced apoptosis. The identification of the pathway responsible for A-SMase activity downregulation in tumors leading to chemoresistance warrants further investigations as a means to identify new anti-cancer molecules capable of specifically inhibiting synt4 degradation.