RESUMO
Parkinson's disease (PD) is a debilitating neurodegenerative disorder characterized by progressive motor decline and the aggregation of α-synuclein protein. Growing evidence suggests that α-synuclein aggregates may spread from neurons of the digestive tract to the central nervous system in a prion-like manner, yet the mechanisms of α-synuclein transmission and neurotoxicity remain poorly understood. Animal models that are amenable to high-throughput investigations are needed to facilitate the discovery of disease mechanisms. Here we describe the first Caenorhabditis elegans models in which feeding with α-synuclein preformed fibrils (PFFs) induces dopaminergic neurodegeneration, prion-like seeding of aggregation of human α-synuclein expressed in the host, and an associated motor decline. RNAi-mediated knockdown of the C. elegans syndecan sdn-1, or other enzymes involved in heparan sulfate proteoglycan synthesis, protected against PFF-induced α-synuclein aggregation, motor dysfunction, and dopamine neuron degeneration. This work offers new models by which to investigate gut-derived α-synuclein spreading and propagation of disease.
Assuntos
Doença de Parkinson , Príons , Animais , Caenorhabditis elegans , Dopamina , Neurônios Dopaminérgicos , alfa-Sinucleína/genéticaRESUMO
Fetal hemoglobin (HbF) induction by hydroxyurea (HU) therapy is associated with decreased morbidity and mortality in sickle cell anemia (SCA) patients, but not all patients respond to or tolerate HU. This provides a rationale for developing novel HbF inducers to treat SCA. Thalidomide analogs have the ability to induce HbF production while inhibiting the release of tumor necrosis factor-alpha. Molecular hybridization of HU and thalidomide was used to synthesize 3- (1,3-dioxoisoindolin-2-yl) benzyl nitrate (compound 4C). In this study, we show that compound 4C increases HbF production in a transgenic SCA mouse model and reduces the production of pro-inflammatory cytokines by SCA mouse monocytes cultured ex vivo. Therefore, compound 4C is a novel drug designed to treat SCA with a unique combination of HbF-inducing and anti-inflammatory properties.
Assuntos
Anemia Falciforme/tratamento farmacológico , Citocinas/metabolismo , Hemoglobina Fetal/biossíntese , Hidroxiureia , Talidomida , Anemia Falciforme/genética , Anemia Falciforme/metabolismo , Anemia Falciforme/patologia , Animais , Citocinas/genética , Modelos Animais de Doenças , Hemoglobina Fetal/genética , Hidroxiureia/análogos & derivados , Hidroxiureia/síntese química , Hidroxiureia/química , Hidroxiureia/farmacologia , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Knockout , Talidomida/análogos & derivados , Talidomida/síntese química , Talidomida/química , Talidomida/farmacologiaRESUMO
The molecular mechanisms by which nitric oxide (NO) bioavailability modulates the clinical expression of sickle cell disease (SCD) remain elusive. We investigated the effect of hypoxia and NO bioavailability on sickle red blood cell (sRBC) adhesion using mice deficient for endothelial NO synthase (eNOS) because their NO metabolite levels are similar to those of SCD mice but without hypoxemia. Whereas sRBC adhesion to endothelial cells in eNOS-deficient mice was synergistically upregulated at the onset of hypoxia, leukocyte adhesion was unaffected. Restoring NO metabolite levels to physiological levels markedly reduced sRBC adhesion to levels seen under normoxia. These results indicate that sRBC adherence to endothelial cells increases in response to hypoxia prior to leukocyte adherence, and that low NO bioavailability synergistically upregulates sRBC adhesion under hypoxia. Although multiple adhesion molecules mediate sRBC adhesion, we found a central role for P-selectin in sRBC adhesion. Hypoxia and low NO bioavailability upregulated P-selectin expression in endothelial cells in an additive manner through p38 kinase pathways. These results demonstrate novel cellular and signaling mechanisms that regulate sRBC adhesion under hypoxia and low NO bioavailability. Importantly, these findings point us toward new molecular targets to inhibit cell adhesion in SCD.
Assuntos
Anemia Falciforme/sangue , Eritrócitos Anormais/metabolismo , Hipóxia/sangue , Óxido Nítrico/sangue , Anemia Falciforme/genética , Anemia Falciforme/patologia , Animais , Adesão Celular/fisiologia , Modelos Animais de Doenças , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Eritrócitos Anormais/patologia , Eritrócitos Anormais/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Óxido Nítrico Sintase Tipo III/sangue , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico Sintase Tipo III/genética , Selectina-P/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Although reduction in leukocyte counts following hydroxyurea therapy in sickle cell disease (SCD) predicts fetal hemoglobin (HbF) response, the underlying mechanism remains unknown. We previously reported that leukocyte counts are regulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) in SCD patients. Here we examined the roles of GM-CSF in the regulation of HbF expression in SCD. Upon the analysis of retrospective data in 372 patients, HbF levels were inversely correlated with leukocyte counts and GM-CSF levels in SCD patients without hydroxyurea therapy, while HbF increments after hydroxyurea therapy correlated with a reduction in leukocyte counts, suggesting a negative effect of GM-CSF on HbF expression. Consistently, in vitro studies using primary erythroblasts showed that the addition of GM-CSF to erythroid cells decreased HbF expression. We next examined the intracellular signaling pathway through which GM-CSF reduced HbF expression. Treatment of erythroid cells with GM-CSF resulted in the reduction of intracellular cAMP levels and abrogated phosphorylation of cAMP response-element-binding-protein, suggesting attenuation of the cAMP-dependent pathway, while the phosphorylation levels of mitogen-activated protein kinases were not affected. This is compatible with our studies showing a role for the cAMP-dependent pathway in HbF expression. Together, these results demonstrate that GM-CSF plays a role in regulating both leukocyte count and HbF expression in SCD. Reduction in GM-CSF levels upon hydroxyurea therapy may be critical for efficient HbF induction. The results showing the involvement of GM-CSF in HbF expression may suggest possible mechanisms for hydroxyurea resistance in SCD.
Assuntos
Anemia Falciforme/metabolismo , AMP Cíclico/metabolismo , Regulação para Baixo/efeitos dos fármacos , Hemoglobina Fetal/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/genética , Linhagem Celular , Células Cultivadas , Células Eritroides/efeitos dos fármacos , Hemoglobina Fetal/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Hidroxiureia/uso terapêutico , Contagem de Leucócitos , RNA Mensageiro/análise , Estudos Retrospectivos , Transcrição Gênica/efeitos dos fármacosRESUMO
A novel series of thalidomide derivatives (4a-f) designed by molecular hybridization were synthesized and evaluated in vitro and in vivo for their potential use in the oral treatment of sickle cell disease symptoms. Compounds 4a-f demonstrated analgesic, anti-inflammatory, and NO-donor properties. Compounds 4c and 4d were considered promising candidate drugs and were further evaluated in transgenic sickle cell mice to determine their capacity to reduce the levels of the proinflammatory cytokine tumor necrosis factor α (TNFα). Unlike hydroxyurea, the compounds reduced the concentrations of TNFα to levels similar to those induced with the control dexamethasone (300 µmol/kg). These compounds are novel lead drug candidates with multiple beneficial actions in the treatment of sickle cell disease symptoms and offer an alternative to hydroxyurea treatment.
Assuntos
Anemia Falciforme/tratamento farmacológico , Antidrepanocíticos/síntese química , Antidrepanocíticos/farmacologia , Desenho de Fármacos , Ácido Acético , Analgésicos/síntese química , Analgésicos/química , Analgésicos/farmacologia , Anemia Falciforme/genética , Anemia Falciforme/metabolismo , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Antidrepanocíticos/química , Capsaicina , Cólica/induzido quimicamente , Cólica/metabolismo , Cólica/prevenção & controle , Orelha/patologia , Edema/induzido quimicamente , Edema/metabolismo , Edema/prevenção & controle , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Modelos Químicos , Estrutura Molecular , Peritonite/induzido quimicamente , Peritonite/metabolismo , Peritonite/prevenção & controle , Talidomida/síntese química , Talidomida/química , Talidomida/farmacologia , Tioglicolatos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Pharmacologic induction of fetal hemoglobin (HbF) expression is an effective treatment strategy for sickle cell disease (SCD) and ß-thalassemia. Pomalidomide is a potent structural analog of thalidomide and member of a new class of immunomodulatory drugs. Recent reports demonstrated that pomalidomide reduced or eliminated transfusion requirements in certain hematologic malignancies and induced HbF ex vivo in CD34(+) progenitor cells from healthy and SCD donors. We investigated the effects of pomalidomide on erythropoiesis and hemoglobin synthesis in a transgenic mouse model of SCD. We found that 8 weeks of treatment with pomalidomide induced modest increases of HbF with similar efficacy as hydroxyurea. However, in stark contrast to hydroxyurea's myelosuppressive effects, pomalidomide augmented erythropoiesis and preserved bone marrow function. Surprisingly, combinatory therapy with both drugs failed to mitigate hydroxyurea's myelotoxic effects and caused loss of HbF induction. These findings support further evaluation of pomalidomide as a novel therapy for SCD.
Assuntos
Anemia Falciforme/sangue , Antidrepanocíticos/farmacologia , Medula Óssea/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Hemoglobina Fetal/efeitos dos fármacos , Talidomida/análogos & derivados , Animais , Modelos Animais de Doenças , Hidroxiureia/efeitos adversos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Talidomida/farmacologiaRESUMO
Adhesive interactions between circulating sickle red blood cells (RBCs), leukocytes, and endothelial cells are major pathophysiologic events in sickle cell disease (SCD). To develop new therapeutics that efficiently inhibit adhesive interactions, we generated an anti-P-selectin aptamer and examined its effects on cell adhesion using knockout-transgenic SCD model mice. Aptamers, single-stranded oligonucleotides that bind molecular targets with high affinity and specificity, are emerging as new therapeutics for cardiovascular and hematologic disorders. In vitro studies found that the anti-P-selectin aptamer exhibits high specificity to mouse P-selectin but not other selectins. SCD mice were injected with the anti-P-selectin aptamer, and cell adhesion was observed under hypoxia. The anti-P-selectin aptamer inhibited the adhesion of sickle RBCs and leukocytes to endothelial cells by 90% and 80%, respectively. The anti-P-selectin aptamer also increased microvascular flow velocities and reduced the leukocyte rolling flux. SCD mice treated with the anti-P-selectin aptamer demonstrated a reduced mortality rate associated with the experimental procedures compared with control mice. These results demonstrate that anti-P-selectin aptamer efficiently inhibits the adhesion of both sickle RBCs and leukocytes to endothelial cells in SCD model mice, suggesting a critical role for P-selectin in cell adhesion. Anti-P-selectin aptamer may be useful as a novel therapeutic agent for SCD.