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Ozone (O3)-induced respiratory toxicity varies considerably within the human population and across inbred mouse strains, indicative of gene-environment interactions (GxE). Though previous studies have identified several quantitative trait loci (QTL) and candidate genes underlying responses to O3 exposure, precise mechanisms of susceptibility remain incompletely described. We sought to update our understanding of the genetic architecture of O3 responsiveness using the Collaborative Cross (CC) recombinant inbred mouse panel. We evaluated hallmark O3-induced inflammation and injury phenotypes in 56 CC strains after exposure to filtered air or 2 ppm O3, and performed focused genetic analysis of variation in lung injury, as reflected by protein in lung lavage fluid. Strain-dependent responses to O3 were clear, and QTL mapping revealed two novel loci on Chr (Chromosomes) 10 (peak, 26.2 Mb; 80% confidence interval [CI], 24.6-43.6 Mb) and 15 (peak, 47.1 Mb; 80% CI, 40.2-54.9 Mb), the latter surpassing the 95% significance threshold. At the Chr 15 locus, C57BL/6J and CAST/EiJ founder haplotypes were associated with higher lung injury responses compared with all other CC founder haplotypes. With further statistical analysis and a weight of evidence approach, we delimited the Chr 15 QTL to an â¼2 Mb region containing 21 genes (10 protein coding) and nominated three candidate genes, namely Oxr1, Rspo2, and Angpt1. Gene and protein expression data further supported Oxr1 and Angpt1 as priority candidate genes. In summary, we have shown that O3-induced lung injury is modulated by genetic variation, identified two high priority candidate genes, and demonstrated the value of the CC for detecting GxE.
Assuntos
Lesão Pulmonar , Ozônio , Animais , Camundongos , Mapeamento Cromossômico , Cromossomos Humanos Par 15 , Camundongos de Cruzamento Colaborativo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Ozônio/toxicidadeRESUMO
Acute ozone (O3) exposure is associated with multiple adverse cardiorespiratory outcomes, the severity of which varies across individuals in human populations and inbred mouse strains. However, molecular determinants of response, including susceptibility biomarkers that distinguish who will develop severe injury and inflammation, are not well characterized. We and others have demonstrated that airway macrophages (AMs) are an important resident immune cell type that are functionally and transcriptionally responsive to O3 inhalation. Here, we sought to explore influences of strain, exposure, and strain-by-O3 exposure interactions on AM gene expression and identify transcriptional correlates of O3-induced inflammation and injury across six mouse strains, including five Collaborative Cross (CC) strains. We exposed adult mice of both sexes to filtered air (FA) or 2 ppm O3 for 3 h and measured inflammatory and injury parameters 21 h later. Mice exposed to O3 developed airway neutrophilia and lung injury with strain-dependent severity. In AMs, we identified a common core O3 transcriptional response signature across all strains, as well as a set of genes exhibiting strain-by-O3 exposure interactions. In particular, a prominent gene expression contrast emerged between a low- (CC017/Unc) and high-responding (CC003/Unc) strain, as reflected by cellular inflammation and injury. Further inspection indicated that differences in their baseline gene expression and chromatin accessibility profiles likely contribute to their divergent post-O3 exposure transcriptional responses. Together, these results suggest that aspects of O3-induced respiratory responses are mediated through altered AM transcriptional signatures and further confirm the importance of gene-environment interactions in mediating differential responsiveness to environmental agents.
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Pulmão/patologia , Macrófagos/metabolismo , Ozônio/efeitos adversos , Animais , Cromatina/metabolismo , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/genética , Inflamação/patologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause autosomal-dominant Parkinson's disease (PD) and contribute to sporadic PD. Common genetic variation in LRRK2 modifies susceptibility to immunological disorders including Crohn's disease and leprosy. Previous studies have reported that LRRK2 is expressed in B lymphocytes and macrophages, suggesting a role for LRRK2 in immunological functions. In this study, we characterized the LRRK2 protein expression and phosphorylation using human lymphoblasts. Lipopolysaccharide (LPS), a proinflammatory agent, induced the increase of LRRK2 expression and kinase activities in human lymphoblasts in a time-dependent manner. Moreover, LPS activated the Toll-like receptor (TLR) signaling pathway, increased TRAF6/LRRK2 interaction, and elevated the phosphorylation levels of MAPK (JNK1/2, p38, and ERK1/2) and IkBα. Treatment with LRRK2 inhibitor 68 reduced LPS-induced TRAF6/LRRK2 interaction and MAPK and IkBα phosphorylation, thereby reducing TNF-α secretion. These results indicate that LRRK2 is actively involved in proinflammatory responses in human lymphoblasts, and inhibition of GTP binding by 68 results in an anti-inflammation effect against proinflammatory stimuli. These findings not only provide novel insights into the mechanisms of LRRK2-linked immune and inflammatory responses in B-cell-like lymphoblasts, but also suggest that 68 may also have potential therapeutic value for LRRK2-linked immunological disorders.
Assuntos
Guanosina Trifosfato/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Lipopolissacarídeos/farmacologia , Linfócitos/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Linfócitos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Biológicos , Inibidor de NF-kappaB alfa/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Fator 6 Associado a Receptor de TNF/metabolismoRESUMO
Airway neutrophilia is correlated with disease severity in a number of chronic and acute pulmonary diseases, and dysregulation of neutrophil chemotaxis can lead to host tissue damage. The gene Zfp30 was previously identified as a candidate regulator of neutrophil recruitment to the lungs and secretion of CXCL1, a potent neutrophil chemokine, in a genome-wide mapping study using the Collaborative Cross. ZFP30 is a putative transcriptional repressor with a KRAB domain capable of inducing heterochromatin formation. Using a CRISPR-mediated knockout mouse model, we investigated the role that Zfp30 plays in recruitment of neutrophils to the lung using models of allergic airway disease and acute lung injury. We found that the Zfp30 null allele did not affect CXCL1 secretion or neutrophil recruitment to the lungs in response to various innate immune stimuli. Intriguingly, despite the lack of neutrophil phenotype, we found there was a significant reduction in the proportion of live Zfp30 homozygous female mutant mice produced from heterozygous matings. This deviation from the expected Mendelian ratios implicates Zfp30 in fertility or embryonic development. Overall, our results indicate that Zfp30 is an essential gene but does not influence neutrophilic inflammation in this particular knockout model.
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Proteínas de Ligação a DNA/deficiência , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/genética , Imunomodulação/genética , Fatores de Transcrição/deficiência , Alelos , Animais , Biomarcadores , Sistemas CRISPR-Cas , Células Cultivadas , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Edição de Genes , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Masculino , Camundongos , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fenótipo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Parkinson's disease (PD) is one of the most common movement disorders with loss of dopaminergic neurons and the presence of Lewy bodies in certain brain areas. However, it is not clear how Lewy body (inclusion with protein aggregation) formation occurs. Mutations in leucine-rich repeat kinase 2 (LRRK2) can cause a genetic form of PD and contribute to sporadic PD with the typical Lewy body pathology. Here, we used our recently identified LRRK2 GTP-binding inhibitors as pharmacological probes to study the LRRK2-linked ubiquitination and protein aggregation. Pharmacological inhibition of GTP-binding by GTP-binding inhibitors (68 and Fx2149) increased LRRK2-linked ubiquitination predominantly via K27 linkage. Compound 68- or Fx2149 increased G2019S-LRRK2-linked ubiquitinated aggregates, which occurred through the atypical linkage types K27 and K63. Coexpression of K27R and K63R, which prevented ubiquitination via K27 and K63 linkages, reversed the effects of 68 and Fx2149. Moreover, 68 and Fx2149 also promoted G2019S-LRRK2-linked aggresome (Lewy body-like inclusion) formation via K27 and K63 linkages. These findings demonstrate that LRRK2 GTP-binding activity is critical in LRRK2-linked ubiquitination and aggregation formation. These studies provide novel insight into the LRRK2-linked Lewy body-like inclusion formation underlying PD pathogenesis.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Corpos de Lewy/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Corpos de Lewy/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , UbiquitinaçãoRESUMO
Ambient ozone (O3) exposure has serious consequences on respiratory health, including airway inflammation and injury. Decades of research have yielded thorough descriptions of these outcomes; however, less is known about the molecular processes that drive them. The aim of this study was to further describe the cellular and molecular responses to O3 exposure in murine airways, with a particular focus on transcriptional responses in 2 critical pulmonary tissue compartments: conducting airways (CA) and airway macrophages (AM). After exposing adult, female C57BL/6J mice to filtered air, 1 or 2 ppm O3, we assessed hallmark responses including airway inflammation (cell counts and cytokine secretion) and injury (epithelial permeability), followed by gene expression profiling of CA and AM by RNA-seq. As expected, we observed concentration-dependent increases in airway inflammation and injury. Conducting airways and AM both exhibited changes in gene expression to both 1 and 2 ppm O3 that were largely compartment-specific. In CA, genes associated with epithelial barrier function, detoxification processes, and cellular proliferation were altered, while O3 affected genes involved in innate immune signaling, cytokine production, and extracellular matrix remodeling in AM. Further, CA and AM also exhibited notable differences in concentration-response expression patterns for large numbers of genes. Overall, our study has described transcriptional responses to acute O3 exposure, revealing both shared and unique gene expression patterns across multiple concentrations of O3 and in 2 important O3-responsive tissues. These profiles provide broad mechanistic insight into pulmonary O3 toxicity, and reveal a variety of targets for focused follow-up studies.
Assuntos
Poluentes Atmosféricos/toxicidade , Ozônio/toxicidade , Animais , Líquido da Lavagem Broncoalveolar , Feminino , Inflamação/fisiopatologia , Pulmão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Testes de Toxicidade AgudaRESUMO
BACKGROUND: Induction chemotherapy by isolated limb perfusion (ILP) with melphalan and tumour necrosis factor-α is an effective strategy to facilitate limb-conserving surgery in locally advanced extremity sarcoma. In a comparison of cohorts matched for grade, size and surgical resectability, we compared the outcome of patients undergoing induction ILP prior to limb-conserving surgery and selective post-operative radiotherapy with patients undergoing limb-conserving surgery and routine post-operative radiotherapy. METHODS: Patients with primary, grade 2/3 sarcomas of the lower limbs over 10 cm in size were identified from prospectively maintained databases at 3 centres. Patients treated at a UK centre underwent limb-conserving surgery and post-operative radiotherapy (Standard cohort). Patients at two German centres underwent induction ILP, limb-conserving surgery and selective post-operative radiotherapy (ILP cohort). RESULTS: The Standard cohort comprised 80 patients and the ILP cohort 44 patients. Both cohorts were closely matched in terms of tumour size, grade, histological subtype and surgical resectability. The median age was greater in the Standard vs the ILP cohort (60.5 years vs 56 years, p = 0.033). The median size was 13 cm in both cohorts. 5-year local-recurrence (ILP 12.2%, Standard 20.1%, p = 0.375) and distant metastases-free survival rates (ILP 49.6%, Standard 46.0% p = 0.821) did not differ significantly between cohorts. Fewer patients received post-operative radiotherapy in the ILP cohort compared with the Standard cohort (27% vs 82%, p < 0.001). CONCLUSION: In comparative cohorts, the outcomes of patients undergoing induction ILP prior to surgery did not differ from those undergoing standard management, although induction ILP was associated with a reduced need for adjuvant radiation.
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Neutrophil chemotaxis to the airways is a key aspect of host response to microbes and a feature of multiple pulmonary diseases including asthma. Tight regulation of this recruitment is critical to prevent unwanted host tissue damage and inflammation. Using a mouse (Mus musculus) model of asthma applied to the Collaborative Cross population, we previously identified a lung gene expression quantitative trait locus (eQTL) for Zinc finger protein 30 (Zfp30) that was also a QTL for neutrophil recruitment and the hallmark neutrophil chemokine CXCL1. The Zfp30 eQTL is defined by three functionally distinct haplotypes. In this study, we searched for causal genetic variants that underlie the Zfp30 eQTL to gain a better understanding of this candidate repressor's regulation. First, we identified a putative regulatory region spanning 500 bp upstream of Zfp30, which contains 10 SNPs that form five haplotypes. In reporter gene assays in vitro, these haplotypes recapitulated the three previously identified in vivo expression patterns. Second, using site-directed mutagenesis followed by reporter gene assays, we identified a single variant, rs51434084, which explained the majority of variation in expression between two out of three haplotype groups. Finally, using a combination of in silico predictions and electrophoretic mobility shift assays, we identified ZFP148 as a transcription factor that differentially binds to the Zfp30 promoter region harboring rs51434084. In conclusion, we provide evidence in support of rs51434084 being a causal variant for the Zfp30 eQTL, and have identified a mechanism by which this variant alters Zfp30 expression, namely differential binding of ZFP148.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Polimorfismo de Nucleotídeo Único , Mucosa Respiratória/metabolismo , Fatores de Transcrição/genética , Animais , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Haplótipos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Locos de Características Quantitativas , Fatores de Transcrição/metabolismoRESUMO
INTRODUCTION: With modern techniques facilitating limb conservation, amputation for extremity soft-tissue sarcoma (ESTS) is now rare. We sought to determine the indications and outcomes following major amputation for ESTS and whether amputation is prognostic of oncological outcomes in primary disease. PATIENTS AND METHODS: Patients undergoing major amputations for ESTS from 2004 to 2014 were identified from electronic patient records. RESULTS: The amputation rate in primary localized disease was 4.1%. Overall, 69 patients were identified, including 23 (33.3%) amputations for primary localized disease, 36 (52.2%) amputations for recurrent disease, and 10 (14.5%) amputations for metastatic disease. The local recurrence rate for localized disease at 3 years was 10.4%. Three-year overall survival (OS) was 50.3% following curative amputation, with a median survival of 41 months, and median OS following palliative amputation was 6 months. In the context of primary, localized disease, patients undergoing amputation had a greater proportion of high-grade tumors (69.6% vs. 41.1%; p = 0.009) of greater size (median 16.0 vs. 9.0 cm; p = 0.003) when compared with patients undergoing limb-conserving surgery. The rates of systemic relapse and disease-specific survival were poorer following amputation compared with limb-conserving surgery, however mode of surgery (amputation vs. limb conservation) was only prognostic for OS. CONCLUSIONS: Amputation maintains an important role in ESTS and achieves durable local control in those unsuitable for limb-conserving surgery. Survival following amputation in the presence of metastatic disease is poor and should be reserved for patients with significant symptoms.
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Amputação Cirúrgica/mortalidade , Extremidades/cirurgia , Recidiva Local de Neoplasia/mortalidade , Complicações Pós-Operatórias/mortalidade , Sarcoma/cirurgia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Extremidades/patologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/cirurgia , Prognóstico , Sarcoma/patologia , Taxa de Sobrevida , Adulto JovemRESUMO
Although the pathogenesis of Parkinson's disease (PD) remains unclear, mutations in leucine-rich repeat kinase 2 (Lrrk2) are among the major causes of familial PD. Most of these mutations disrupt Lrrk2 kinase and (or) GTPase domain function, resulting in neuronal degeneration. However, the signal pathways underlying Lrrk2-induced neuronal degeneration are not fully understood. There is an expanding body of evidence that suggests a link between Lrrk2 function and MAP kinase (MAPK) cascades. To further investigate this link in vivo, genetic RNAi screens of the MAPK pathways were performed in a Drosophila model to identify genetic modifier(s) that can suppress G2019S-Lrrk2-induced PD-like phenotypes. The results revealed that the knockdown of hemipterous (hep, or JNKK) increased fly survival time, improved locomotor function, and reduced loss of dopaminergic neurons in G2019S-Lrrk2 transgenic flies. Expression of the dominant-negative allele of JNK (JNK-DN), a kinase that is downstream of hep in G2019S-Lrrk2 transgenic flies, elicited a similar effect. Moreover, treatment with the JNK inhibitor SP600125 partially reversed the G2019S-Lrrk2-induced loss of dopaminergic neurons. These results indicate that the hep pathway plays an important role in Lrrk2-linked Parkinsonism in flies. These studies provide new insights into the molecular mechanisms underlying Lrrk2-linked PD pathogenesis and aid in identifying potential therapeutic targets.
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Neurônios Dopaminérgicos/metabolismo , Proteínas de Drosophila/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Doenças Neurodegenerativas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Drosophila melanogaster , Mutação/genética , Transdução de Sinais/fisiologiaRESUMO
Mucus hyper-secretion is a hallmark feature of asthma and other muco-obstructive airway diseases. The mucin proteins MUC5AC and MUC5B are the major glycoprotein components of mucus and have critical roles in airway defense. Despite the biomedical importance of these two proteins, the loci that regulate them in the context of natural genetic variation have not been studied. To identify genes that underlie variation in airway mucin levels, we performed genetic analyses in founder strains and incipient lines of the Collaborative Cross (CC) in a house dust mite mouse model of asthma. CC founder strains exhibited significant differences in MUC5AC and MUC5B, providing evidence of heritability. Analysis of gene and protein expression of Muc5ac and Muc5b in incipient CC lines (n = 154) suggested that post-transcriptional events were important regulators of mucin protein content in the airways. Quantitative trait locus (QTL) mapping identified distinct, trans protein QTL for MUC5AC (chromosome 13) and MUC5B (chromosome 2). These two QTL explained 18 and 20% of phenotypic variance, respectively. Examination of the MUC5B QTL allele effects and subsequent phylogenetic analysis allowed us to narrow the MUC5B QTL and identify Bpifb1 as a candidate gene. Bpifb1 mRNA and protein expression were upregulated in parallel to MUC5B after allergen challenge, and Bpifb1 knockout mice exhibited higher MUC5B expression. Thus, BPIFB1 is a novel regulator of MUC5B.
Assuntos
Proteínas de Transporte/metabolismo , Mucina-5AC/metabolismo , Mucina-5B/metabolismo , Locos de Características Quantitativas , Mucosa Respiratória/metabolismo , Animais , Proteínas de Transporte/genética , Linhagem Celular , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucina-5AC/genética , Mucina-5B/genéticaRESUMO
AIM: Aggressive angiomyxomas (AA) are rare tumors, most commonly presenting in the pelvis of women of childbearing age. This study presents the results of selective marginal resection of this disease in patients managed at a single institution. METHODS: Patients diagnosed with AA from July 2001 to July 2015 were identified from a prospectively maintained histopathology database. RESULTS: Seventeen patients were diagnosed with AA in the study period. The median age at diagnosis was 48 years. Females were more commonly affected with a M:F of 1:8.5. The most common differential diagnoses were an ischiorectal abscess or Bartholin's cyst. Fifteen cases occurred in the pelvis, with two cases at other sites. Median maximum tumor diameter was 10 cm. Of the pelvic cases, 12 were managed operatively via perineal, abdominal, or abdominoperineal approaches. Excision was performed in a marginal fashion with minimal morbidity. Local recurrence developed in 58.3% with a median local recurrence free survival of 25 months. No patients developed metastatic disease or died from disease. CONCLUSION: AA are rare tumors with a propensity for local recurrence. Atypical presentations of other perineal pathologies should prompt further investigation. Surgery should be reserved for symptomatic patients and is associated with low rates of morbidity. J. Surg. Oncol. 2016;114:828-832. © 2016 2016 Wiley Periodicals, Inc.
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Margens de Excisão , Mixoma/cirurgia , Neoplasias Pélvicas/cirurgia , Adolescente , Adulto , Idoso , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Mixoma/diagnóstico , Mixoma/patologia , Recidiva Local de Neoplasia/epidemiologia , Neoplasias Pélvicas/diagnóstico , Neoplasias Pélvicas/patologia , Estudos Retrospectivos , Resultado do Tratamento , Adulto JovemRESUMO
Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal-dominant Parkinsonism with pleomorphic pathology including deposits of aggregated protein and neuronal degeneration. The pathogenesis of LRRK2-linked Parkinson's disease (PD) is not fully understood. Here, using co-immunoprecipitation, we found that LRRK2 interacted with synphilin-1 (SP1), a cytoplasmic protein that interacts with α-synuclein and has implications in PD pathogenesis. LRRK2 interacted with the N-terminus of SP1 whereas SP1 predominantly interacted with the C-terminus of LRRK2, including kinase domain. Co-expression of SP1 with LRRK2 increased LRRK2-induced cytoplasmic aggregation in cultured cells. Moreover, SP1 also attenuated mutant LRRK2-induced toxicity and reduced LRRK2 kinase activity in cultured cells. Knockdown of SP1 by siRNA enhanced LRRK2 neuronal toxicity. In vivo Drosophila studies, co-expression of SP1 and mutant G2019S-LRRK2 in double transgenic Drosophila increased survival and improved locomotor activity. Expression of SP1 protects against G2019S-LRRK2-induced dopamine neuron loss and reduced LRRK2 phosphorylation in double transgenic fly brains. Our findings demonstrate that SP1 attenuates mutant LRRK2-induced PD-like phenotypes and plays a neural protective role.
Assuntos
Proteínas de Transporte/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Doença de Parkinson/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Dopamina/metabolismo , Drosophila , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Camundongos , Mutação , Degeneração Neural/genética , Proteínas do Tecido Nervoso/genética , Neurônios/enzimologia , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Fosforilação , Domínios e Motivos de Interação entre Proteínas , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Leucine-rich repeat kinase 2 is a large protein with implications in genetic and sporadic causes of Parkinson's disease. The physiological functions of LRRK2 are largely unknown. In this report, we investigated whether LRRK2 alters neural transport using live-cell imaging techniques and human neuroblastoma SH-SY5Y cells. Our results demonstrated that expression of the PD-linked mutant, LRRK2-R1441C, induced mitochondrial, and lysosomal transport defects in neurites of SH-SY5Y cells. Most importantly, recently identified GTP-binding inhibitors, 68 and FX2149, can reduce LRRK2 GTP-binding activity and attenuates R1441C-induced mitochondrial and lysosomal transport impairments. These results provide direct evidence and an early mechanism for neurite injury underlying LRRK2-induced neurodegeneration. This is the first report to show that LRRK2 GTP-binding activity plays a critical role during neurite transport, suggesting inhibition of LRRK2 GTP-binding could be a potential novel strategy for PD intervention.
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Leucine-rich repeat kinase-2 (LRRK2), a cytoplasmic protein containing both GTP binding and kinase activities, has emerged as a highly promising drug target for Parkinson's disease (PD). The majority of PD-linked mutations in LRRK2 dysregulate its GTP binding and kinase activities, which may contribute to neurodegeneration. While most known LRRK2 inhibitors are developed to target the kinase domain, we have recently identified the first LRRK2 GTP binding inhibitor, 68, which not only inhibits LRRK2 GTP binding and kinase activities with high potency in vitro, but also reduces neurodegeneration. However, the in vivo effects of 68 are low due to its limited brain penetration. To address this problem, we reported herein the design and synthesis of a novel analog of 68, FX2149, aimed at increasing the in vivo efficacy. Pharmacological characterization of FX2149 exhibited inhibition of LRRK2 GTP binding activity by ~90% at a concentration of 10 nM using in vitro assays. Furthermore, FX2149 protected against mutant LRRK2-induced neurodegeneration in SH-SY5Y cells at 50-200 nM concentrations. Importantly, FX2149 at 10 mg/kg (i.p.) showed significant brain inhibition efficacy equivalent to that of 68 at 20 mg/kg (i.p.), determined by mouse brain LRRK2 GTP binding and phosphorylation assays. Furthermore, FX2149 at 10 mg/kg (i.p.) attenuated lipopolysaccharide (LPS)-induced microglia activation and LRRK2 upregulation in a mouse neuroinflammation model comparable to 68 at 20 mg/kg (i.p.). Our results highlight a novel GTP binding inhibitor with better brain efficacy, which represents a new lead compound for further understanding PD pathogenesis and therapeutic studies.
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Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/síntese química , Doença de Parkinson/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Encéfalo/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos , Microglia/efeitos dos fármacos , Doença de Parkinson/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Regulação para CimaRESUMO
Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause autosomal-dominant Parkinson's disease (PD) and contribute to sporadic PD. LRRK2 contains Guanosine-5'-triphosphate (GTP) binding, GTPase and kinase activities that have been implicated in the neuronal degeneration of PD pathogenesis, making LRRK2, a potential drug target. To date, there is no disease-modifying drug to slow the neuronal degeneration of PD and no published LRRK2 GTP domain inhibitor. Here, the biological functions of two novel GTP-binding inhibitors of LRRK2 were examined in PD cell and mouse models. Through a combination of computer-aided drug design (CADD) and LRRK2 bio-functional screens, two novel compounds, 68: and 70: , were shown to reduce LRRK2 GTP binding and to inhibit LRRK2 kinase activity in vitro and in cultured cell assays. Moreover, these two compounds attenuated neuronal degeneration in human SH-SY5Y neuroblastoma cells and mouse primary neurons expressing mutant LRRK2 variants. Although both compounds inhibited LRRK2 kinase activity and reduced neuronal degeneration, solubility problems with 70: prevented further testing in mice. Thus, only 68: was tested in a LRRK2-based lipopolysaccharide (LPS)-induced pre-inflammatory mouse model. 68: reduced LRRK2 GTP-binding activity and kinase activity in brains of LRRK2 transgenic mice after intraperitoneal injection. Moreover, LPS induced LRRK2 upregulation and microglia activation in mouse brains. These findings suggest that disruption of GTP binding to LRRK2 represents a potential novel therapeutic approach for PD intervention and that these novel GTP-binding inhibitors provide both tools and lead compounds for future drug development.
Assuntos
Guanosina Trifosfato/metabolismo , Neurônios/efeitos dos fármacos , Doença de Parkinson/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Sulfonas/farmacologia , Tiazóis/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Humanos , Inflamação/metabolismo , Inflamação/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Mutação , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/enzimologia , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Sulfonas/uso terapêutico , Tiazóis/uso terapêuticoRESUMO
Airway remodelling is a feature of asthma that contributes to loss of lung function. One of the central components of airway remodelling is subepithelial fibrosis. Interleukin (IL)-13 is a key T-helper 2 cytokine and is believed to be the central mediator of allergic asthma including remodelling, but the mechanism driving the latter has not been elucidated in human asthma. We hypothesised that IL-13 stimulates collagen type-1 production by the airway fibroblast in a matrix metalloproteinase (MMP)- and transforming growth factor (TGF)-ß1-dependent manner in human asthma as compared to healthy controls. Fibroblasts were cultured from endobronchial biopsies in 14 subjects with mild asthma and 13 normal controls that underwent bronchoscopy. Airway fibroblasts were treated with various mediators including IL-13 and specific MMP-inhibitors. IL-13 significantly stimulated collagen type-1 production in asthma compared to normal controls. Inhibitors of MMP-2 significantly attenuated collagen production in asthma but had no effect in normal controls. IL-13 significantly increased total and active forms of TGF-ß1, and this activation was blocked using an MMP-2 inhibitor. IL-13 activated endogenous MMP-2 in asthma patients as compared to normal controls. In an ex vivo model, IL-13 potentiates airway remodelling through a mechanism involving TGF-ß1 and MMP-2. These effects provide insights into the mechanism involved in IL-13-directed airway remodelling in asthma.
Assuntos
Asma/metabolismo , Colágeno Tipo I/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Interleucina-13/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Actinas/metabolismo , Adulto , Biópsia , Brônquios/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Testes de Função RespiratóriaRESUMO
TCR signaling plays a critical role in regulatory T cell (Treg) development. However, the mechanism for tissue-specific induction of Tregs in the periphery remains unclear. We observed that surfactant protein A (SP-A)-deficient mice have impaired expression of Foxp3 and fewer CD25(+)Foxp3(+) Tregs after ex vivo stimulation and after stimulation with LPS in vivo. The addition of exogenous SP-A completely reversed this phenotype. Although SP-A is known to inhibit T cell proliferation under certain activation conditions, both IL-2 levels as well as active TGF-ß levels increase on extended culture with exogenous SP-A, providing a key mechanism for the maintenance and induction of Tregs. In addition, kinetic suppression assays demonstrate that SP-A enhances the frequency of functional Foxp3(+) Tregs in responder T cell populations in a TGF-ß-dependent manner. In mice treated with LPS in vivo, Tregs increased â¼160% in wild-type mice compared with only a 50% increase in LPS-treated SP-A(-/-) mice 8 d after exposure. Taken together, these findings support the hypothesis that SP-A affects T cell immune function by the induction of Tregs during activation.
Assuntos
Ativação Linfocitária/fisiologia , Proteína A Associada a Surfactante Pulmonar/imunologia , Linfócitos T Reguladores/imunologia , Animais , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Interleucina-2/metabolismo , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Knockout , Proteína A Associada a Surfactante Pulmonar/biossíntese , Proteína A Associada a Surfactante Pulmonar/genética , Linfócitos T Reguladores/metabolismoRESUMO
Graft-versus-host disease (GVHD) is a severe and frequent complication of allogeneic bone marrow transplantation (BMT) that involves the gastrointestinal (GI) tract and lungs. The pathobiology of GVHD is complex and involves immune cell recognition of host Ags as foreign. We hypothesize a central role for the collectin surfactant protein A (SP-A) in regulating the development of GVHD after allogeneic BMT. C57BL/6 (H2b; WT) and SP-A-deficient mice on a C57BL/6 background (H2b; SP-A(-/-)) mice underwent allogeneic or syngeneic BMT with cells from either C3HeB/FeJ (H2k; SP-A-deficient recipient mice that have undergone an allogeneic BMT [SP-A(-/-)alloBMT] or SP-A-sufficient recipient mice that have undergone an allogeneic BMT) or C57BL/6 (H2b; SP-A-deficient recipient mice that have undergone a syngeneic BMT or SP-A-sufficient recipient mice that have undergone a syngeneic BMT) mice. Five weeks post-BMT, mice were necropsied, and lung and GI tissue were analyzed. SP-A(-/-) alloBMT or SP-A-sufficient recipient mice that have undergone an allogeneic BMT had no significant differences in lung pathology; however, SP-A(-/-)alloBMT mice developed marked features of GI GVHD, including decreased body weight, increased tissue inflammation, and lymphocytic infiltration. SP-A(-/-)alloBMT mice also had increased colon expression of IL-1ß, IL-6, TNF-α, and IFN-γ and as well as increased Th17 cells and diminished regulatory T cells. Our results demonstrate the first evidence, to our knowledge, of a critical role for SP-A in modulating GI GVHD. In these studies, we demonstrate that mice deficient in SP-A that have undergone an allogeneic BMT have a greater incidence of GI GVHD that is associated with increased Th17 cells and decreased regulatory T cells. The results of these studies demonstrate that SP-A protects against the development of GI GVHD and establishes a role for SP-A in regulating the immune response in the GI tract.
Assuntos
Gastroenteropatias/imunologia , Gastroenteropatias/metabolismo , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/metabolismo , Proteína A Associada a Surfactante Pulmonar/fisiologia , Animais , Transplante de Medula Óssea/imunologia , Transplante de Medula Óssea/patologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Gastroenteropatias/genética , Doença Enxerto-Hospedeiro/genética , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína A Associada a Surfactante Pulmonar/deficiência , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Células Th17/imunologia , Células Th17/patologiaRESUMO
During pulmonary infections, a careful balance between activation of protective host defense mechanisms and potentially injurious inflammatory processes must be maintained. Surfactant protein A (SP-A) is an immune modulator that increases pathogen uptake and clearance by phagocytes while minimizing lung inflammation by limiting dendritic cell (DC) and T cell activation. Recent publications have shown that SP-A binds to and is bacteriostatic for Mycoplasma pneumoniae in vitro. In vivo, SP-A aids in maintenance of airway homeostasis during M. pneumoniae pulmonary infection by preventing an overzealous proinflammatory response mediated by TNF-α. Although SP-A was shown to inhibit maturation of DCs in vitro, the consequence of DC/SP-A interactions in vivo has not been elucidated. In this article, we show that the absence of SP-A during M. pneumoniae infection leads to increased numbers of mature DCs in the lung and draining lymph nodes during the acute phase of infection and, consequently, increased numbers of activated T and B cells during the course of infection. The findings that glycyrrhizin, a specific inhibitor of extracellular high-mobility group box-1 (HMGB-1) abrogated this effect and that SP-A inhibits HMGB-1 release from immune cells suggest that SP-A inhibits M. pneumoniae-induced DC maturation by regulating HMGB-1 cytokine activity.