RESUMO
Moyamoya disease (MMD) is closely associated with the Ring Finger Protein 213 (RNF213), a susceptibility gene for MMD. However, its biological function remains unclear. We aimed to elucidate the role of RNF213 in the damage incurred by human endothelial cells under oxygen-glucose deprivation (OGD). We analyzed autophagy in peripheral blood mononuclear cells (PBMCs) derived from patients carrying either RNF213 wildtype (WT) or variant (p.R4810K). Subsequently, human umbilical vein endothelial cells (HUVECs) were transfected with RNF213 WT (HUVECWT) or p.R4810K (HUVECR4810K) and exposed to OGD for 2 h. Immunoblotting was used to analyze autophagy marker proteins, and endothelial function was analyzed by tube formation assay. Autophagic vesicles were observed using transmission electron microscopy. Post-OGD exposure, we administered rapamycin and cilostazol as potential autophagy inducers. The RNF213 variant group during post-OGD exposure (vs. pre-OGD) showed autophagy inhibition, increased protein expression of SQSTM1/p62 (p < 0.0001) and LC3-II (p = 0.0039), and impaired endothelial function (p = 0.0252). HUVECR4810K during post-OGD exposure (versus pre-OGD) showed a remarkable increase in autophagic vesicles. Administration of rapamycin and cilostazol notably restored the function of HUVECR4810K and autophagy. Our findings support the pivotal role of autophagy impaired by the RNF213 variant in MMD-induced endothelial cell dysfunction.
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Tripartite motif (TRIM) proteins are a large family of E3 ubiquitin ligases implicated in antiviral defense systems, tumorigenesis, and protein quality control. TRIM proteins contribute to protein quality control by regulating the ubiquitin-proteasome system, endoplasmic reticulum-associated degradation, and macroautophagy/autophagy. However, the detailed mechanisms through which various TRIM proteins regulate downstream events have not yet been fully elucidated. Herein, we identified a novel function of TRIM22 in the regulation of autophagy. TRIM22 promotes autophagosome-lysosome fusion by mediating the association of GABARAP family proteins with PLEKHM1, thereby inducing the autophagic clearance of protein aggregates, independent of its E3 ubiquitin ligase activity. Furthermore, a TRIM22 variant associated with early-onset familial Alzheimer disease interferes with autophagosome-lysosome fusion and autophagic clearance. These findings suggest TRIM22 as a critical autophagic regulator that orchestrates autophagosome-lysosome fusion by scaffolding autophagy-related proteins, thus representing a potential therapeutic target in neurodegenerative diseases.Abbreviations: AD: Alzheimer disease; ADAOO: AD age of onset; AICD: APP intracellular domain; APP: amyloid beta precursor protein; BSA: bovine serum albumin; cDNAs: complementary DNAs; CQ: chloroquine; CTF: carboxyl-terminal fragment; EBSS: Earle's balanced salt solution; GABARAP: GABA type A receptor-associated protein; GST: glutathione S-transferase; HA: hemagglutinin; HOPS: homotypic fusion and protein sorting; IFN: interferon; IL1A/IL-1α: interleukin 1 alpha; KO: knockout; MTORC1: mechanistic target of rapamycin kinase complex 1; NFKBIA/IκBα: NFKB inhibitor alpha; NFE2L2/NRF2: NFE2 like bZIP transcription factor; PBS: phosphate-buffered saline; PI3K: class I phosphoinositide 3-kinase; PLA: proximity ligation assay; PLEKHM1: pleckstrin homology and RUN domain containing M1; PSEN1: presenilin 1; SEM: standard errors of the means; SNAREs: soluble N-ethylmaleimide-sensitive factor attachment protein receptors; SNCA: synuclein alpha; SNP: single nucleotide polymorphism; TBS: tris-buffered saline; TNF/TNF-α: tumor necrosis factor; TRIM: tripartite motif; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.
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BACKGROUND: Isolated rapid-eye-movement (REM) sleep behavior disorder (iRBD) is considered as a prodromal stage of either multiple system atrophy (MSA) or Lewy body disease (LBD; Parkinson's disease and dementia with Lewy bodies). However, current knowledge is limited in predicting and differentiating the type of future phenoconversion in iRBD patients. We investigated the role of plasma neurofilament light chain (NfL) and cardiac metaiodobenzylguanidine (MIBG) uptake as predictors for phenoconversion. METHODS: Forty patients with iRBD were enrolled between April 2018 and October 2019 and prospectively followed every 3 months to determine phenoconversion to either MSA or LBD. Plasma NfL levels were measured at enrollment. Cardiac MIBG uptake and striatal dopamine transporter uptake were assessed at baseline. RESULTS: Patients were followed for a median of 2.92 years. Four patients converted to MSA and 7 to LBD. Plasma NfL level at baseline was significantly higher in future MSA-converters (median 23.2 pg/mL) when compared with the rest of the samples (median 14.1 pg/mL, p = 0.003). NfL level above 21.3 pg/mL predicted phenoconversion to MSA with the sensitivity of 100% and specificity of 94.3%. Baseline MIBG heart-to-mediastinum ratio of LBD-converters (median 1.10) was significantly lower when compared with the rest (median 2.00, p < 0.001). Heart-to-mediastinum ratio below 1.545 predicted phenoconversion to LBD with the sensitivity of 100% and specificity of 92.9%. CONCLUSIONS: Plasma NfL and cardiac MIBG uptake may be useful biomarkers in predicting phenoconversion of iRBD. Elevated plasma NfL levels may suggest imminent phenoconversion to MSA, whereas low cardiac MIBG uptake suggests phenoconversion to LBD.
Assuntos
Doença por Corpos de Lewy , Atrofia de Múltiplos Sistemas , Doença de Parkinson , Transtorno do Comportamento do Sono REM , Humanos , 3-Iodobenzilguanidina , Transtorno do Comportamento do Sono REM/diagnóstico por imagem , Filamentos Intermediários , Doença por Corpos de Lewy/diagnóstico por imagem , Atrofia de Múltiplos Sistemas/diagnóstico por imagemRESUMO
OBJECTIVE: We aimed to present the study design and baseline cross-sectional participant characteristics of biobank innovations for chronic cerebrovascular disease with Alzheimer's disease study (BICWALZS) participants. METHODS: A total of 1,013 participants were enrolled in BICWALZS from October 2016 to December 2020. All participants underwent clinical assessments, basic blood tests, and standardized neuropsychological tests (n=1,013). We performed brain magnetic resonance imaging (MRI, n=817), brain amyloid positron emission tomography (PET, n=713), single nucleotide polymorphism microarray chip (K-Chip, n=949), locomotor activity assessment (actigraphy, n=200), and patient-derived dermal fibroblast sampling (n=175) on a subset of participants. RESULTS: The mean age was 72.8 years, and 658 (65.0%) were females. Based on clinical assessments, total of 168, 534, 211, 80, and 20 had subjective cognitive decline, mild cognitive impairment (MCI), Alzheimer's dementia, vascular dementia, and other types of dementia or not otherwise specified, respectively. Based on neuroimaging biomarkers and cognition, 199, 159, 78, and 204 were cognitively normal (CN), Alzheimer's disease (AD)-related cognitive impairment, vascular cognitive impairment, and not otherwise specified due to mixed pathology (NOS). Each group exhibited many differences in various clinical, neuropsychological, and neuroimaging results at baseline. Baseline characteristics of BICWALZS participants in the MCI, AD, and vascular dementia groups were generally acceptable and consistent with 26 worldwide dementia cohorts and another independent AD cohort in Korea. CONCLUSION: The BICWALZS is a prospective and longitudinal study assessing various clinical and biomarker characteristics in older adults with cognitive complaints. Details of the recruitment process, methodology, and baseline assessment results are described in this paper.
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Cardiac 123I-metaiodobenzylguanidine (MIBG) uptake correlates with the extent of cardiac sympathetic denervation found in disease with Lewy pathology, such as Parkinson's disease (PD). Protein α-synuclein, the main component of Lewy body, is a candidate biomarker of PD, but its relationship with cardiac MIBG uptake has never been explored. Plasma α-synuclein levels were measured in 37 patients with early PD. Cardiac 123I-MIBG scintigraphy and 18F-FP-CIT brain PET were performed, and striatal dopamine transporter (DAT) uptake was quantified using automated segmentation. The relationships of plasma α-synuclein levels with cardiac MIBG and striatal DAT uptake were investigated. The plasma α-synuclein level correlated with early (R = 0.38, P = 0.033) and delayed (R = 0.49, P = 0.0055) MIBG heart-to-mediastinum (H/M) ratios, and its correlation with delayed H/M ratio remained significant after adjustment with age, disease duration, motor severity, and striatal DAT uptake (P = 0.016). The regional SUVRs of any subregions of caudate and putamen did not correlate with plasma α-synuclein level. In the patients with early PD, the plasma α-synuclein level correlated with cardiac sympathetic denervation, but not with nigrostriatal degeneration. This may suggest that plasma α-synuclein levels more readily reflect the peripheral deposition of Lewy bodies than their central deposition.
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3-Iodobenzilguanidina/farmacologia , Encéfalo/diagnóstico por imagem , Coração/diagnóstico por imagem , Doença de Parkinson/diagnóstico por imagem , Compostos Radiofarmacêuticos/farmacologia , alfa-Sinucleína/sangue , Idoso , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/sangue , Tomografia por Emissão de Pósitrons , Cintilografia , Tropanos/farmacocinéticaRESUMO
To delineate the impact of non-motor markers (REM sleep behavior disorder (RBD), orthostatic hypotension (OH), cardiac sympathetic denervation, hyposmia) on neuronal injury in early-stage Parkinson's disease (PD), we measured the plasma neurofilament light chain (NFL) level of PD patients and evaluated its relationship with these markers. The study population comprised a cohort of 77 patients with PD and 54 controls. OH was assessed using 5-min head-up tilt-table test. Other clinical parameters such as RBD, Unified Parkinson's Disease Rating Scale (UPDRS), cognition, Cross-Cultural Smell Identification Test (CCSIT), white matter hyperintensity (WMH), cardiac metaiodobenzylguanidine (MIBG) and striatal dopamine transporter (DAT) uptake were assessed. Plasma NFL levels were measured using Simoa platform. During mean 24.8 months of follow-up, 70 patients remained PD, 5 patients converted to Parkinson-plus syndrome (P + converter), and 2 were lost to follow-up. NFL level did not differ between PD and control groups (age-adjusted means 10.40 pg/mL vs 9.51 pg/mL, p = 0.151), but PD patients with OH (median 15.31 pg/mL) had higher levels compared with those without OH (median 9.2 pg/mL, p = 0.008), as well as the control group (median 9.7 pg/mL, p = 0.002). P + converter group had the highest plasma NFL level (38.17 pg/mL, p < 0.001). In a multiple regression analysis, OH, age, and disease duration independently correlated with plasma NFL level. This finding adds biomarker-based evidence for poor clinical outcomes associated with OH in patients with PD.
Assuntos
Hipotensão Ortostática , Doença de Parkinson , Transtorno do Comportamento do Sono REM , 3-Iodobenzilguanidina , Humanos , Hipotensão Ortostática/etiologia , Filamentos Intermediários , Doença de Parkinson/complicaçõesRESUMO
Although Alzheimer's disease (AD) is the most common neurodegenerative disease, there are still no drugs available to treat or prevent AD effectively. Here, we examined changes in levels of selected proteins implicated in the pathogenesis of AD using plasma samples of control subjects and patients with cognition impairment. To precisely categorize the disease, fifty-six participants were examined with clinical cognitive tests, amyloid positron emission tomography (PET) scan, and white matter hyperintensities scored by magnetic resonance imaging. Plasma cathepsin D levels of the subjects were examined by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Correlation of plasma cathepsin D levels with AD-related factors and clinical characteristics were examined by statistical analysis. By analyzing quantitative immunoblot and ELISA, we found that the plasma level of cathepsin D, a major lysosomal protease, was decreased in the group with amyloid plaque deposition at the brain compared to the control group. The level of plasma cathepsin D was negatively correlated with clinical dementia rating scale sum of boxes (CDR-SB) scores. In addition, our integrated multivariable logistic regression model suggests the high performance of plasma cathepsin D level for discriminating AD from non-AD. These results suggest that the plasma cathepsin D level could be developed as a diagnostic biomarker candidate for AD.
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Doença de Alzheimer/sangue , Catepsina D/sangue , Fatores Etários , Idoso , Apolipoproteína E4/genética , Biomarcadores/sangue , Escolaridade , Feminino , Humanos , Masculino , Testes de Estado Mental e Demência , Análise Multivariada , Curva ROC , Reprodutibilidade dos TestesRESUMO
The current diagnosis of Parkinson's disease (PD) mostly relies on clinical rating scales related to motor dysfunction. Given that clinical symptoms of PD appear after significant neuronal cell death in the brain, it is required to identify accessible, objective, and quantifiable biomarkers for early diagnosis of PD. In this study, a total of 20 patients with idiopathic PD and 20 age-matched patients with essential tremor according to the UK Brain Bank Criteria were consecutively enrolled to identify peripheral blood biomarkers for PD. Clinical data were obtained by clinical survey and assessment. Using albumin-depleted and immunoglobulin G-depleted plasma samples, we performed immunoblot analysis of seven autophagy-related proteins and compared the levels of proteins to those of the control group. We also analyzed the correlation between the levels of candidate proteins and clinical characteristics. Finally, we validated our biomarker models using receiver operating characteristic curve analysis. We found that the levels of BCL2-associated athanogene 2 (BAG2) and cathepsin D were significantly decreased in plasma of patients with PD (P = 0.009 and P = 0.0077, respectively). The level of BAG2 in patients with PD was significantly correlated with Cross-Culture Smell Identification Test score, which indicates olfactory dysfunction. We found that our biomarker model distinguishes PD with 87.5% diagnostic accuracy (area under the curve (AUC) = 0.875, P < 0.0001). Our result suggests BAG2 and cathepsin D as candidates for early-diagnosis plasma biomarkers for PD. We provide the possibility of plasma biomarkers related to the autophagy pathway, by which decreased levels of BAG2 and cathepsin D might lead to dysfunction of autophagy.
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Catepsina D/sangue , Tremor Essencial/diagnóstico , Chaperonas Moleculares/sangue , Doença de Parkinson/diagnóstico , Biomarcadores/sangue , Diagnóstico Diferencial , Tremor Essencial/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/sangue , Estudos Prospectivos , Curva ROCRESUMO
Mutations in WASHC5 (also known as KIAA0196) cause autosomal dominant hereditary spastic paraplegia (HSP) type SPG8. WASHC5, commonly called strumpellin, is a core component of the Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex that activates actin nucleation at endosomes. Although various other cellular roles for strumpellin have also been described, none account for how SPG8-associated mutations lead to HSP. Here, we identified protein interactors of the WASH complex by immunoprecipitation and mass spectrometry and assessed the functions of strumpellin in cultured cells using both overexpression and RNA interference along with cell-spreading assays to investigate cell adhesion. We uncovered a decrease in CAV1 protein abundance as well as endosomal fission defects resulting from pathogenic SPG8 mutations. CAV1, a key component of caveolae, interacted with strumpellin in cells, and strumpellin inhibited the lysosomal degradation of CAV1. SPG8-associated missense mutations in strumpellin did not rescue endosomal tubulation defects, reduction in CAV1 protein abundance, or integrin-mediated cell adhesion in strumpellin-deficient cells. Mechanistically, we demonstrated that the WASH complex maintained CAV1 and integrin protein amounts by inhibiting their lysosomal degradation through its endosomal actin nucleation activity. In addition, the interaction of strumpellin with CAV1 stimulated integrin recycling, thereby promoting cell adhesion. These findings provide a molecular link between WASHC5 mutations and impairment of CAV1- and integrin-mediated cell adhesion, providing insights into the cellular pathogenesis of SPG8.
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Caveolina 1/metabolismo , Integrinas/metabolismo , Paraplegia/metabolismo , Proteínas/metabolismo , Paraplegia Espástica Hereditária/metabolismo , Animais , Caveolina 1/genética , Adesão Celular/genética , Células HEK293 , Humanos , Integrinas/genética , Lisossomos/genética , Lisossomos/metabolismo , Lisossomos/patologia , Mutação , Paraplegia/genética , Paraplegia/patologia , Proteínas/genética , Proteólise , Ratos , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/patologiaRESUMO
BACKGROUND AND PURPOSE: Cerebrospinal fluid (CSF) biomarkers of Alzheimer's disease (AD) could be misleading in idiopathic normal-pressure hydrocephalus (iNPH). We therefore investigated the CSF biomarkers in 18F-florbetaben amyloid-negative positron-emission tomography (PET) [amyloid PET(-)] iNPH, amyloid-positive PET [amyloid PET(+)] AD, and cognitively normal (CN) subjects. METHODS: Ten amyloid PET(+) AD patients (56.7±5.6 years old, mean±standard deviation), 10 amyloid PET(-) iNPH patients (72.8±4.5 years old), and 8 CN subjects (61.2±6.5 years old) were included. We measured the levels of ß-amyloid (Aß)40, Aß42, total tau (t-tau) protein, and phosphorylated tau (p-tau) protein in the CSF using enzyme-linked immunosorbent assays. RESULTS: The level of Aß42 and the Aß42/Aß40 ratio in the CSF were significantly lower in AD than in iNPH or CN subjects. The Aß40 level did not differ significantly between AD and iNPH (p=1.000), but it did between AD and CN subjects (p=0.032). The levels of both t-tau and p-tau were higher in AD than in iNPH or CN subjects. The levels of Aß42, Aß40, t-tau, and p-tau were lower in iNPH than in CN subjects, but there was no significant difference after controlling for age. CONCLUSIONS: Our results suggest that the mechanism underlying low CSF Aß levels differs between amyloid PET(-) iNPH and amyloid PET(+) AD subjects. The lower levels of all CSF biomarkers in iNPH patients might be due to reduced clearances from extracellular fluid and decreased brain metabolism of the periventricular zone in iNPH resulting from glymphatic dysfunction.
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Considering that cigarette smoke condensate (CSC) is primarily absorbed through the alveoli in the lungs, herein, we used a mouse alveolar macrophage cell line (MH-S cells). After 24â¯h exposure, CSC decreased dose-dependently cell viability accompanying an increase in intracellular ROS and NO level. CSC structurally or functionally damaged organelles including mitochondria, ER and lysosome and enhanced the expression of proteins related to apoptosis, ER stress and DNA damage accompanying an elevated proportion of annexin V-bound cells. Meanwhile, the expression of certain proteins related to mitochondrial dynamics (OPA1 and DRP1) and autophagy (ATG5) did not overall show significant dose-dependent change in cells exposed to CSC. More importantly, conversion of LC3-I to LC3B-II, a representative marker for autophagy, was also unclear. Considering that intracellular organelles work together in harmony to perform defense mechanism against foreign bodies, we investigated changes in immune response following CSC exposure. The level of IFN-γ and MIP-1α was elevated in CSC-exposed cells, whereas the MCP-1α level decreased. The expression of chemokine receptors (CD195 and CXCR2) and an adhesion molecule (CD54) increased by CSC treatment, the expression of certain antigen presentation-related proteins (MHC class II, CD40, and CD80) were also enhanced. Meanwhile, the expression of CD86, a co-stimulatory molecule for antigen presentation, dose-dependently decreased. In conclusion, we suggest that CSC may induce apoptotic cell death and disturbance in host defense mechanisms by impairing function of cellular components.
Assuntos
Macrófagos Alveolares/efeitos dos fármacos , Organelas/efeitos dos fármacos , Fumaça/efeitos adversos , Produtos do Tabaco , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular , Citocinas/metabolismo , Dano ao DNA , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/ultraestrutura , Camundongos , Microscopia Eletrônica de Transmissão , Óxido Nítrico/metabolismo , Organelas/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo , Receptores CCR5/metabolismo , Receptores de Interleucina-8B/metabolismoRESUMO
Mechanisms by which long corticospinal axons degenerate in hereditary spastic paraplegia (HSP) are largely unknown. Here, we have generated induced pluripotent stem cells (iPSCs) from patients with two autosomal recessive forms of HSP, SPG15 and SPG48, which are caused by mutations in the ZFYVE26 and AP5Z1 genes encoding proteins in the same complex, the spastizin and AP5Z1 proteins, respectively. In patient iPSC-derived telencephalic glutamatergic and midbrain dopaminergic neurons, neurite number, length and branching are significantly reduced, recapitulating disease-specific phenotypes. We analyzed mitochondrial morphology and noted a significant reduction in both mitochondrial length and their densities within axons of these HSP neurons. Mitochondrial membrane potential was also decreased, confirming functional mitochondrial defects. Notably, mdivi-1, an inhibitor of the mitochondrial fission GTPase DRP1, rescues mitochondrial morphology defects and suppresses the impairment in neurite outgrowth and late-onset apoptosis in HSP neurons. Furthermore, knockdown of these HSP genes causes similar axonal defects, also mitigated by treatment with mdivi-1. Finally, neurite outgrowth defects in SPG15 and SPG48 cortical neurons can be rescued by knocking down DRP1 directly. Thus, abnormal mitochondrial morphology caused by an imbalance of mitochondrial fission and fusion underlies specific axonal defects and serves as a potential therapeutic target for SPG15 and SPG48.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas de Transporte/genética , GTP Fosfo-Hidrolases/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Mitocondriais/genética , Paraplegia Espástica Hereditária/genética , Axônios/efeitos dos fármacos , Axônios/patologia , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Dinaminas , Humanos , Células-Tronco Pluripotentes Induzidas , Potencial da Membrana Mitocondrial/genética , Mesencéfalo/metabolismo , Mesencéfalo/patologia , Mitocôndrias/genética , Mitocôndrias/patologia , Dinâmica Mitocondrial/genética , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Mutação , Crescimento Neuronal/efeitos dos fármacos , Crescimento Neuronal/genética , Quinazolinonas/farmacologia , Paraplegia Espástica Hereditária/tratamento farmacológico , Paraplegia Espástica Hereditária/fisiopatologiaRESUMO
Polyhexamethylene guanidine (PHMG) has been widely used in the industry owing to its excellent biocidal, anti-corrosive, and anti-biofouling properties. In Korea, consumers exposed to PHMG-phosphate (PHMG-P)-containing humidifier disinfectant have begun to suffer from fibrotic lung injury-related symptoms for unknown reasons. However, no appropriate treatment has yet been found because the detail toxic mechanism has not been identified. Herein, we first studied the toxic mechanism of PHMG-P-containing solution using human normal bronchial epithelial cells (BEAS-2B cells). When exposed for 24â¯h, PHMG-P-containing solution rapidly decreased cell viability from around 6â¯h after exposure and significantly increased of the phosphatidylserine exposure and the LDH release. At 6â¯h of exposure, the material contained in the solution was found to be bound to the cell membrane and the inner wall of vacuoles, and damaged the cell membrane and organelles. In addition, a significant increase of IFN-γ was observed among cytokines, the expression of apoptosis-, autophagy-, and membrane and DNA damage-related proteins was also enhanced. Meanwhile, the level of intracellular ROS and the secretion of IL-8 and CXCL-1, which are chemokines for professional phagocytes, decreased. Thus, we treated dead BEAS-2B cells to lung fibroblasts (HFL-1), non-professional phagocytes, and then we observed that the dead cells rapidly attached to HFL-1 cells and were taken up. Additionally, increased secretion of IL-8 and CXCL-1 was observed in the cells. Based on these results, we suggest that pulmonary exposure to PHMG-P induces apoptosis of bronchial epithelial cells and lung fibroblasts might play an important role in the clearance of the apoptotic debris.
Assuntos
Apoptose/efeitos dos fármacos , Brônquios/efeitos dos fármacos , Citofagocitose , Desinfetantes/toxicidade , Células Epiteliais/efeitos dos fármacos , Fibroblastos/metabolismo , Guanidinas/toxicidade , Brônquios/metabolismo , Brônquios/ultraestrutura , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CXCL1/metabolismo , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Fibroblastos/ultraestrutura , Humanos , Interferon gama/metabolismo , Interleucina-8/metabolismo , L-Lactato Desidrogenase/metabolismo , Fosfatidilserinas/metabolismo , Fatores de TempoRESUMO
Nanoparticles (NPs) have recently emerged as an inhalable pollutant, owing to their applications, aluminum-based NPs (Al-NPs) have been prioritized for toxicity testing. In the current study, we compared the pulmonary biopersistence and subsequent toxicity of four different types of Al-NPs (two rod-type aluminum oxide NPs [AlONPs] with different aspect ratios [short (S)- and long (L)-AlONPs], spherical aluminum cerium oxide NPs [AlCeO3 , AlCeONPs] and spherical γ-aluminum oxide hydroxide nanoparticles [AlOOHNPs]) 13weeks after a single intratracheal instillation, considering the importance of their properties in their toxicity. We found that the pulmonary biopersistence of Al-NPs was strengthened by a high aspect ratio in the rod-type AlONPs and by the presence of hydroxyl groups in the spherical-type Al-NPs. The highest toxicity was observed in the mice treated with AlOOHNPs, which showed low biostability. More importantly, we identified that the commercially available AlCeONPs were Al2 O3 -coated CeO2 NPs, but not AlCeO3 NPs, although they have been sold under the trade name of AlCeONPs. In conclusion, the aspect ratio and biostability may be important factors in the determination of the biopersistence of NPs and the subsequent biological response. In addition, the physicochemical properties of NPs should be examined in detail before their release into the market to prevent unexpected adverse health effects.
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Alumínio/toxicidade , Nanopartículas Metálicas/toxicidade , Alumínio/administração & dosagem , Animais , Basófilos/efeitos dos fármacos , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Ensaio de Imunoadsorção Enzimática , Eosinófilos/efeitos dos fármacos , L-Lactato Desidrogenase/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Contagem de Leucócitos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Contagem de Linfócitos , Linfócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Masculino , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/química , Camundongos , Camundongos Endogâmicos ICR , Neutrófilos/efeitos dos fármacosRESUMO
With the rapid development of synthetic alternatives to mineral fibers, their possible effects on the environment and human health have become recognized as important issues worldwide. This study investigated effects of four fibrous materials, i.e. nanofibrillar/nanocrystalline celluloses (NCF and CNC), single-walled carbon nanotubes (CNTs), and crocidolite asbestos (ASB), on pulmonary inflammation and immune responses found in the lungs, as well as the effects on spleen and peripheral blood immune cell subsets. BALB/c mice were given NCF, CNC, CNT, and ASB on Day 1 by oropharyngeal aspiration. At 14 days post-exposure, the animals were evaluated. Total cell number, mononuclear phagocytes, polymorphonuclear leukocytes, lymphocytes, and LDH levels were significantly increased in ASB and CNT-exposed mice. Expression of cytokines and chemokines in bronchoalveolar lavage (BAL) was quite different in mice exposed to four particle types, as well as expression of antigen presentation-related surface proteins on BAL cells. The results revealed that pulmonary exposure to fibrous materials led to discrete local immune cell polarization patterns with a TH2-like response caused by ASB and TH1-like immune reaction to NCF, while CNT and CNC caused non-classical or non-uniform responses. These alterations in immune response following pulmonary exposure should be taken into account when testing the applicability of new nanosized materials with fibrous morphology.
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Materiais Biomiméticos/química , Celulose/química , Imunidade Celular , Pulmão/imunologia , Nanoestruturas/química , Nanotubos de Carbono/química , Pneumonia/imunologia , Animais , Apresentação de Antígeno , Asbesto Crocidolita/efeitos adversos , Materiais Biomiméticos/efeitos adversos , Líquido da Lavagem Broncoalveolar/imunologia , Celulose/efeitos adversos , Citocinas/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Fibras Minerais/efeitos adversos , Nanoestruturas/efeitos adversos , Nanotubos de Carbono/efeitos adversos , Pneumonia/etiologia , Aspiração Respiratória , Equilíbrio Th1-Th2RESUMO
Methylisothiazolinone (MIT) is a powerful biocide and preservative, which is widely used alone or in a 1:3 ratio with methylchloroisothiazolinone (MCIT) under the trade name of Kathons in the manufacture of numerous personal and household products. Considering that Kathons injected intravenously is distributed in the blood and then in the liver, we explored the toxic mechanism of MIT on human liver epithelium cells. At 24 h after exposure, MIT bound to the plasma membrane and the inner wall of vacuoles in the cells, and rupture of the cell membrane and nuclear envelop, autophagosome-like vacuoles formation and mitochondrial damage were observed. Cell viability dose-dependently decreased accompanying an increase of apoptotic cells, and the level of LDH, NO, IFN-gamma, IL-10 and IL-8, but not IL-1ß, significantly increased in the culture media of cells exposed to MIT. Additionally, expression of autophagy-, membrane damage- and apoptosis-related proteins was notably enhanced, and the produced ATP level dose-dependently decreased with the reduced mitochondrial activity. Furthermore, the increased DNA damage and the decreased transcription activity were observed in MIT-treated cells. Meanwhile, the intracellular ROS level did not show dose-dependent change at the same time-point. Then we explored the role of autophagy in MIT-induced cytotoxicity by inhibiting or inducing the autophagic signal. Intriguingly, no additional cell death induced by autophagic modulation occurred when MIT was treated. Taken together, we suggest that MIT may induce multiple pathways of cell death and inflammatory response through DNA damage caused by rupture of the nuclear envelope.
Assuntos
Apoptose/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Tiazóis/toxicidade , Trifosfato de Adenosina/metabolismo , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Interferon gama/análise , Interleucina-10/análise , Interleucina-8/análise , Fígado/citologia , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Ambient fine particulate matter (AFP) is a main risk factor for the cornea as ultraviolet light. However, the mechanism of corneal damage following exposure to AFP has been poorly understood. In this study, we first confirmed that AFP can penetrate the cornea of mice, considering that two-dimensional cell culture systems are limited in reflecting the situation in vivo. Then, we investigated the toxic mechanism using human corneal epithelial (HCET) cells. At 24h after exposure, AFP located within the autophagosome-like vacuoles, and cell proliferation was clearly inhibited in all the tested concentration. Production of ROS and NO and secretion of pro-inflammatory cytokines were elevated in a dose-dependent manner. Additionally, conversion of LC3B from I-type to II-type and activation of caspase cascade which show autophagic- and apoptotic cell death, respectively, were observed in cells exposed to AFP. Furthermore, AFP decreased mitochondrial volume, inhibited ATP production, and altered the expression of metabolism-related genes. Taken together, we suggest that AFP induces cell death and inflammatory response by influencing mitochondrial function in HCET cells. In addition, we recommend that stringent air quality regulations are needed for eye health.
Assuntos
Apoptose/efeitos dos fármacos , Córnea/efeitos dos fármacos , Material Particulado/toxicidade , Animais , Células Cultivadas , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Epitélio Corneano/efeitos dos fármacos , Camundongos , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
The tissue distribution and toxicity of nanoparticles (NPs) depend on their physical and chemical properties both in the manufactured condition and within the biological system. We characterized three types of commercially available aluminum-based NPs (Al-NPs), two rod-type aluminum oxide NPs (Al2 O3 , AlONPs), with different aspect ratios (short [S]- and long [L]-AlONPs), and spherical aluminum cerium oxide NPs (AlCeO3 , AlCeONPs). The surface area was in order of the S-AlONPs > L-AlONPs > AlCeONPs. Very importantly, we found that AlCeONPs is Al2 O3 -coated CeO2 NPs, but not AlCeO3 NPs, and that the Al level in AlCeONPs is approximately 20% of those in S- and L-AlONPs. All three types of Al-NPs were slightly ionized in gastric fluid and rapidly particlized in the intestinal fluid. There were no significant differences in the body weight gain following 28 days of repeated oral administration of the three different types of Al-NPs. All Al-NPs elevated Al level in the heart, spleen, kidney and blood at 24 hours after the final dose, accompanied by the altered tissue level of redox reaction-related trace elements. Subsequently, in four types of cells derived from the organs which Al-NPs are accumulated, H9C2 (heart), HEK-293 (kidney), splenocytes and RAW264.7 (blood), S-AlONPs showed a very low uptake level and did not exert significant cytotoxicity. Meanwhile, cytotoxicity and uptake level were the most remarkable in cells treated with AlCeONPs. In conclusion, we suggest that the physicochemical properties of NPs should be examined in detail before the release into the market to prevent unexpected adverse health effects.
Assuntos
Compostos de Alumínio , Cério/química , Nanopartículas Metálicas , Administração Oral , Compostos de Alumínio/química , Compostos de Alumínio/farmacocinética , Compostos de Alumínio/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Células HEK293 , Humanos , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Masculino , Nanopartículas Metálicas/química , Nanopartículas Metálicas/toxicidade , Camundongos Endogâmicos ICR , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Especificidade de Órgãos , Oxirredução , Tamanho da Partícula , Ratos , Propriedades de Superfície , Distribuição TecidualRESUMO
Centrioles are assembled during S phase and segregated into 2 daughter cells at the end of mitosis. The initiation of centriole assembly is regulated by polo-like kinase 4 (PLK4), the major serine/threonine kinase in centrioles. Despite its importance in centriole duplication, only a few substrates have been identified, and the detailed mechanism of PLK4 has not been fully elucidated. CP110 is a coiled-coil protein that plays roles in centriolar length control and ciliogenesis in mammals. Here, we revealed that PLK4 specifically phosphorylates CP110 at the S98 position. The phospho-resistant CP110 mutant inhibited centriole assembly, whereas the phospho-mimetic CP110 mutant induced centriole assembly, even in PLK4-limited conditions. This finding implies that PLK4 phosphorylation of CP110 is an essential step for centriole assembly. The phospho-mimetic form of CP110 augmented the centrosomal SAS6 level. Based on these results, we propose that the phosphorylated CP110 may be involved in the stabilization of cartwheel SAS6 during centriole assembly.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfoproteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Mutação de Sentido Incorreto , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilação , Proteínas Serina-Treonina Quinases/química , Transporte ProteicoRESUMO
Iron is closely associated with an ambient particulate matters-induced inflammatory response, and the cornea that covers the front of the eye, is among tissues exposed directly to ambient particulate matters. Prior to this study, we confirmed that nano-sized iron particles (FeNPs) can penetrate the cornea. Thus, we identified the toxic mechanism of FeNPs using human corneal epithelial cells. At 24h after exposure, FeNPs located inside autophagosome-like vacuoles or freely within human corneal epithelial cells. Level of inflammatory mediators including nitric oxide, cytokines, and a chemokine was notably elevated accompanied by the increased generation of reactive oxygen species. Additionally, cell proliferation dose-dependently decreased, and level of multiple pathways of cell death-related indicators was clearly altered following exposure to FeNPs. Furthermore, expression of gene encoding DNA binding protein inhibitor (1, 2, and 3), which are correlated to inhibition of the binding of mistranscripted RNA, was significantly down-regulated. More importantly, expression of p-Akt and caspase-3 and conversion to LC3B-II from LC3B-I was enhanced by pretreatment with a caspase-1 inhibitor. Taken together, we suggest that FeNPs may induce multiple pathways of cell death via generation of mistranscripted RNA, and these cell death pathways may influence by cross-talk. Furthermore, we propose the need of further study for the possibility of tumorigenesis following exposure to FeNPs.