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1.
J Toxicol Sci ; 45(9): 559-567, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32879255

RESUMO

Lead is a main threat to human health due to its neurotoxicity and the astrocyte is known to be a common deposit site of lead in vivo. However, the detailed mechanisms related to lead exposure in the astrocytes were unclear. In order to deeply investigate this issue, we used Sprague-Dawley (SD) rats and astrocytes isolated from the hippocampus of SD rats to establish the lead-exposed animal and cell models through treating with lead acetate. The expression levels of GFAP, LC3, and p62 in the rat hippocampus were detected by immunofluorescence and Western blot after lead exposure. The effects of autophagy on lead-exposed astrocytes were studied by further autophagy inhibitor 3-methyladenine (3-MA) induction. Transmission electron microscopy was used to observe autophagosomes in astrocytes after lead acetate treatment, followed by assessing related autophagy protein markers. In addition, some inflammatory cytokines and oxidative stress markers were also evaluated after lead exposure and 3-MA administration. We found that lead exposure induced activation of astrocytes, as evidenced by increased GFAP levels and GFAP-positive staining cells in the rat hippocampus. Moreover, lead exposure induced autophagy in astrocytes, as evidenced by increased LC3II and Beclin 1 protein levels and decreased p62 expression in both the rat hippocampus and astrocytes, and it was confirmed that this autophagy was activated through blocking the downstream Akt/target of the rapamycin (mTOR) pathway in astrocytes. Furthermore, it was shown that treatment of lead acetate increased the release of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), and the accumulation of malondialdehyde (MDA) and myeloperoxidase (MPO) in astrocytes, which could be alleviated by further 3-MA induction. Therefore, we conclude that lead exposure can induce the autophagy of astrocytes via blocking the Akt/mTOR pathway, leading to accelerated release of inflammatory factors and oxidative stress indicators in astrocytes.


Assuntos
Astrócitos/metabolismo , Astrócitos/fisiologia , Autofagia/efeitos dos fármacos , Autofagia/genética , Compostos Organometálicos/toxicidade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Células Cultivadas , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/citologia , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Estresse Oxidativo/genética , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
2.
Nat Commun ; 11(1): 4150, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811819

RESUMO

The systemic decline in autophagic activity with age impairs homeostasis in several tissues, leading to age-related diseases. A mechanistic understanding of adipocyte dysfunction with age could help to prevent age-related metabolic disorders, but the role of autophagy in aged adipocytes remains unclear. Here we show that, in contrast to other tissues, aged adipocytes upregulate autophagy due to a decline in the levels of Rubicon, a negative regulator of autophagy. Rubicon knockout in adipocytes causes fat atrophy and hepatic lipid accumulation due to reductions in the expression of adipogenic genes, which can be recovered by activation of PPARγ. SRC-1 and TIF2, coactivators of PPARγ, are degraded by autophagy in a manner that depends on their binding to GABARAP family proteins, and are significantly downregulated in Rubicon-ablated or aged adipocytes. Hence, we propose that age-dependent decline in adipose Rubicon exacerbates metabolic disorders by promoting excess autophagic degradation of SRC-1 and TIF2.


Assuntos
Adipócitos/metabolismo , Envelhecimento/fisiologia , Autofagia/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Doenças Metabólicas/metabolismo , Adipócitos/patologia , Adipogenia/genética , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Adiposidade/genética , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/fisiologia , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Técnicas de Inativação de Genes , Glucose/genética , Glucose/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Coativador 1 de Receptor Nuclear/metabolismo , Coativador 2 de Receptor Nuclear/metabolismo , PPAR gama/metabolismo
3.
Proc Natl Acad Sci U S A ; 117(35): 21391-21402, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817423

RESUMO

Syntaxin17, a key autophagosomal N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, can associate with ATG8 family proteins SNAP29 and VAMP8 to facilitate the membrane fusion process between the double-membraned autophagosome and single-membraned lysosome in mammalian macroautophagy. However, the inherent properties of Syntaxin17 and the mechanistic basis underlying the interactions of Syntaxin17 with its binding proteins remain largely unknown. Here, using biochemical, NMR, and structural approaches, we systemically characterized Syntaxin17 as well as its interactions with ATG8 family proteins, SNAP29 and VAMP8. We discovered that Syntaxin17 alone adopts an autoinhibited conformation mediated by a direct interaction between its Habc domain and the Qa-SNARE motif. In addition, we revealed that the Qa-SNARE region of Syntaxin17 contains one LC3-interacting region (LIR) motif, which preferentially binds to GABARAP subfamily members. Importantly, the GABARAP binding of Syntaxin17 can release its autoinhibited state. The determined crystal structure of the Syntaxin17 LIR-GABARAP complex not only provides mechanistic insights into the interaction between Syntaxin17 and GABARAP but also reveals an unconventional LIR motif with a C-terminally extended 310 helix for selectively binding to ATG8 family proteins. Finally, we also elucidated structural arrangements of the autophagic Syntaxin17-SNAP29-VAMP8 SNARE core complex, and uncovered its conserved biochemical and structural characteristics common to all other SNAREs. In all, our findings reveal three distinct states of Syntaxin17, and provide mechanistic insights into the Syntaxin17-mediated autophagosome-lysosome fusion process.


Assuntos
Autofagossomos/fisiologia , Lisossomos/fisiologia , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Motivos de Aminoácidos , Proteínas Reguladoras de Apoptose/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Escherichia coli , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo
4.
Gene ; 762: 144974, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32707305

RESUMO

BACKGROUND: There exists considerable evidence conforming that autophagy may play an important role in the biological process of breast cancer. This study aimed to construct and evaluate a novel autophagy-related gene signature as a potential prognostic factor and therapeutic target in breast cancer patients based on high-throughput sequencing datasets. MATERIALS & METHODS: Autophagy-related genes obtained from the Human Autophagy Database and high-sequencing data obtained from The Cancer Genome Atlas (TCGA) were analyzed to identify differential expressed genes (DEGs) between tumor and normal tissues. Then GO and KEGG analysis were performed to explore potential biological and pathological functions of DEGs. Autophagy-related prognostic genes were identified by univariate COX regression analysis. Subsequently stepwise model selection using the Alkaike information criterion (AIC) and multivariate COX regression model was performed to construct autophagy-related gene signature. Then patients were divided into high- and low-risk groups based on the risk score identified by the autophagy-related gene signature. Multivariate COX regression model and stratification analysis were used to specify the prognostic value of this gene signature in whole cohort and various subgroups. T-test and ANOVA analysis were used to compare the expression differences of continuous variables (5 prognostic genes and risk score) in binary and multiple category groups respectively. Kaplan-Meier analysis, log-rank tests and the area under receiver operating characteristic (ROC) curve (AUC) were conducted to validate the accuracy and precise of the autophagy-related gene signature based on GSE20685 and GSE21653 datasets. RESULTS: We profiled autophagy-related DEGs in normal and breast tumor tissues. GO and KEGG analysis indicated that autophagy-related DEGs might participate in breast cancer occurrence, development and drug resistance. Then we identified five autophagy-related genes (EIF4EBP1, ATG4A, BAG1, MAP1LC3A and SERPINA1) that had significantly prognostic values for breast cancer. Autophagy-related gene signature was constructed and patients were divided into high- and low- risk groups based on their risk score. Patients in the high-risk group tended to have shorter overall survival (OS) and relapse-free survival (RFS) times than those in the low-risk group (OS: HR = 1.620, 95%CIs: 1.345-1.950; P < 0.001; RFS: HR = 1.487, 95%CIs: 1.248-1.771, P < 0.001). Autophagy-related gene signature had significant prognostic value in stratified subgroups especially in advanced breast cancer subgroups (T3-4; N2-3; stage III-IV). Its prognostic value was further confirmed in two GEO validation datasets (GSE20685: P = 6.795e-03; GSE21653: P = 1.383e-03). Finally, association analysis between clinicopathological factors and gene signature showed the risk score was higher in patients with ER/PR negative, higher clinical stage or T stage (P < 0.01). CONCLUSION: We established and confirmed a novel autophagy-related gene signature for patients with breast cancer that had independent survival prognostic value especially in advanced breast cancer subgroups. Our research might promote the molecular mechanism study of autophagy-related genes in breast cancer.


Assuntos
Autofagia , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Transcriptoma , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo
5.
Mol Med ; 26(1): 69, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641037

RESUMO

BACKGROUND: We previously showed that the autophagy inhibitor chloroquine (CQ) increases inflammatory cleaved caspase-1 activity in myocytes, and that caspase-1/11 is protective in sterile liver injury. However, the role of caspase-1/11 in the recovery of muscle from ischemia caused by peripheral arterial disease is unknown. We hypothesized that caspase-1/11 mediates recovery in muscle via effects on autophagy and this is modulated by CQ. METHODS: C57Bl/6 J (WT) and caspase-1/11 double-knockout (KO) mice underwent femoral artery ligation (a model of hind-limb ischemia) with or without CQ (50 mg/kg IP every 2nd day). CQ effects on autophagosome formation, microtubule associated protein 1A/1B-light chain 3 (LC3), and caspase-1 expression was measured using electron microscopy and immunofluorescence. Laser Doppler perfusion imaging documented perfusion every 7 days. After 21 days, in situ physiologic testing in tibialis anterior muscle assessed peak force contraction, and myocyte size and fibrosis was also measured. Muscle satellite cell (MuSC) oxygen consumption rate (OCR) and extracellular acidification rate was measured. Caspase-1 and glycolytic enzyme expression was detected by Western blot. RESULTS: CQ increased autophagosomes, LC3 consolidation, total caspase-1 expression and cleaved caspase-1 in muscle. Perfusion, fibrosis, myofiber regeneration, muscle contraction, MuSC fusion, OCR, ECAR and glycolytic enzyme expression was variably affected by CQ depending on presence of caspase-1/11. CQ decreased perfusion recovery, fibrosis and myofiber size in WT but not caspase-1/11KO mice. CQ diminished peak force in whole muscle, and myocyte fusion in MuSC and these effects were exacerbated in caspase-1/11KO mice. CQ reductions in maximal respiration and ATP production were reduced in caspase-1/11KO mice. Caspase-1/11KO MuSC had significant increases in protein kinase isoforms and aldolase with decreased ECAR. CONCLUSION: Caspase-1/11 signaling affects the response to ischemia in muscle and effects are variably modulated by CQ. This may be critically important for disease treated with CQ and its derivatives, including novel viral diseases (e.g. COVID-19) that are expected to affect patients with comorbidities like cardiovascular disease.


Assuntos
Caspase 1/metabolismo , Caspases Iniciadoras/metabolismo , Cloroquina/farmacologia , Infecções por Coronavirus/patologia , Isquemia/patologia , Músculo Esquelético/patologia , Pneumonia Viral/patologia , Animais , Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Betacoronavirus , Infecções por Coronavirus/tratamento farmacológico , Glicólise/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Células Musculares/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Neovascularização Fisiológica , Fosforilação Oxidativa , Pandemias , Doença Arterial Periférica/patologia , Pneumonia Viral/tratamento farmacológico , Regeneração , Transdução de Sinais
6.
Nat Commun ; 11(1): 3284, 2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32601292

RESUMO

The inner nuclear membrane (INM) selectively accumulates proteins that are essential for nuclear functions; however, overaccumulation of INM proteins results in a range of rare genetic disorders. So far, little is known about how defective, mislocalized, or abnormally accumulated membrane proteins are actively removed from the INM, especially in plants and animals. Here, via analysis of a proximity-labeling proteomic profile of INM-associated proteins in Arabidopsis, we identify critical components for an INM protein degradation pathway. We show that this pathway relies on the CDC48 complex for INM protein extraction and 26S proteasome for subsequent protein degradation. Moreover, we show that CDC48 at the INM may be regulated by a subgroup of PUX proteins, which determine the substrate specificity or affect the ATPase activity of CDC48. These PUX proteins specifically associate with the nucleoskeleton underneath the INM and physically interact with CDC48 proteins to negatively regulate INM protein degradation in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Membrana Nuclear/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Plantas Geneticamente Modificadas , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , Proteoma/genética , Coloração e Rotulagem/métodos , Espectrometria de Massas em Tandem/métodos , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
7.
Nat Commun ; 11(1): 3521, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665556

RESUMO

Microtubules (MTs) mediate mitosis, directional signaling, and are therapeutic targets in cancer. Yet in vivo analysis of cancer cell MT behavior within the tumor microenvironment remains challenging. Here we developed an imaging pipeline using plus-end tip tracking and intravital microscopy to quantify MT dynamics in live xenograft tumor models. Among analyzed features, cancer cells in vivo displayed higher coherent orientation of MT dynamics along their cell major axes compared with 2D in vitro cultures, and distinct from 3D collagen gel cultures. This in vivo MT phenotype was reproduced in vitro when cells were co-cultured with IL4-polarized MΦ. MΦ depletion, MT disruption, targeted kinase inhibition, and altered MΦ polarization via IL10R blockade all reduced MT coherence and/or tumor cell elongation. We show that MT coherence is a defining feature for in vivo tumor cell dynamics and migration, modulated by local signaling from pro-tumor macrophages.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animais , Ciclo Celular/genética , Ciclo Celular/fisiologia , Movimento Celular/genética , Movimento Celular/fisiologia , Feminino , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Mitose/genética , Mitose/fisiologia , Análise de Componente Principal , Células RAW 264.7
8.
Nat Commun ; 11(1): 3172, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576838

RESUMO

Bone marrow engraftment of the hematopoietic stem and progenitor cells (HSPCs) involves homing to the vasculatures and lodgment to their niches. How HSPCs transmigrate from the vasculature to the niches is unclear. Here, we show that loss of diaphanous-related formin mDia2 leads to impaired engraftment of long-term hematopoietic stem cells and loss of competitive HSPC repopulation. These defects are likely due to the compromised trans-endothelial migration of HSPCs since their homing to the bone marrow vasculatures remained intact. Mechanistically, loss of mDia2 disrupts HSPC polarization and induced cytoplasmic accumulation of MAL, which deregulates the activity of serum response factor (SRF). We further reveal that beta2 integrins are transcriptional targets of SRF. Knockout of beta2 integrins in HSPCs phenocopies mDia2 deficient mice. Overexpression of SRF or beta2 integrins rescues HSPC engraftment defects associated with mDia2 deficiency. Our findings show that mDia2-SRF-beta2 integrin signaling is critical for HSPC lodgment to the niches.


Assuntos
Antígenos CD18/metabolismo , Forminas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , NADPH Desidrogenase/metabolismo , Animais , Medula Óssea/metabolismo , Modelos Animais de Doenças , Forminas/genética , Transplante de Células-Tronco Hematopoéticas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , NADPH Desidrogenase/genética , Transdução de Sinais
9.
J Vis Exp ; (159)2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32478717

RESUMO

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology.  To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.


Assuntos
Dendritos/metabolismo , Drosophila melanogaster/metabolismo , Imageamento Tridimensional , Microtúbulos/metabolismo , Junção Neuromuscular/metabolismo , Imagem Individual de Molécula , Sinapses/metabolismo , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Larva/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Interferência de RNA , Software
10.
Proc Natl Acad Sci U S A ; 117(26): 15230-15241, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32513711

RESUMO

Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerations. However, the mechanism by which the UBQLN2 mutations cause disease remains unclear. Alterations in proteins involved in autophagy are prominent in neuronal tissue of human ALS UBQLN2 patients and in a transgenic P497S UBQLN2 mouse model of ALS/FTD, suggesting a pathogenic link. Here, we show UBQLN2 functions in autophagy and that ALS/FTD mutant proteins compromise this function. Inactivation of UBQLN2 expression in HeLa cells reduced autophagic flux and autophagosome acidification. The defect in acidification was rescued by reexpression of wild type (WT) UBQLN2 but not by any of the five different UBQLN2 ALS/FTD mutants tested. Proteomic analysis and immunoblot studies revealed P497S mutant mice and UBQLN2 knockout HeLa and NSC34 cells have reduced expression of ATP6v1g1, a critical subunit of the vacuolar ATPase (V-ATPase) pump. Knockout of UBQLN2 expression in HeLa cells decreased turnover of ATP6v1g1, while overexpression of WT UBQLN2 increased biogenesis of ATP6v1g1 compared with P497S mutant UBQLN2 protein. In vitro interaction studies showed that ATP6v1g1 binds more strongly to WT UBQLN2 than to ALS/FTD mutant UBQLN2 proteins. Intriguingly, overexpression of ATP6v1g1 in UBQLN2 knockout HeLa cells increased autophagosome acidification, suggesting a therapeutic approach to overcome the acidification defect. Taken together, our findings suggest that UBQLN2 mutations drive pathogenesis through a dominant-negative loss-of-function mechanism in autophagy and that UBQLN2 functions as an important regulator of the expression and stability of ATP6v1g1. These findings may have important implications for devising therapies to treat UBQLN2-linked ALS/FTD.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Amiotrófica Lateral/genética , Autofagossomos/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/genética , Demência/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Proteínas Relacionadas à Autofagia/genética , Biomarcadores/metabolismo , Linhagem Celular , Demência/metabolismo , Demência/patologia , Predisposição Genética para Doença , Humanos , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Ligação Proteica , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Regulação para Cima , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
11.
Nat Commun ; 11(1): 2725, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483152

RESUMO

The functional study of lncRNAs in skeletal muscle satellite cells (SCs) remains at the infancy stage. Here we identify SAM (Sugt1 asssociated muscle) lncRNA that is enriched in the proliferating myoblasts. Global deletion of SAM has no overt effect on mice but impairs adult muscle regeneration following acute damage; it also exacerbates the chronic injury-induced dystrophic phenotype in mdx mice. Consistently, inducible deletion of SAM in SCs leads to deficiency in muscle regeneration. Further examination reveals that SAM loss results in a cell-autonomous defect in the proliferative expansion of myoblasts. Mechanistically, we find SAM interacts and stabilizes Sugt1, a co-chaperon protein key to kinetochore assembly during cell division. Loss of SAM or Sugt1 both disrupts kinetochore assembly in mitotic cells due to the mislocalization of two components: Dsn1 and Hec1. Altogether, our findings identify SAM as a regulator of SC proliferation through facilitating Sugt1 mediated kinetochore assembly during cell division.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Proliferação de Células/genética , Cinetocoros/metabolismo , Mioblastos/metabolismo , RNA Longo não Codificante/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Células Cultivadas , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mioblastos/citologia , Estabilidade Proteica , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo
12.
Life Sci ; 256: 117959, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32531375

RESUMO

Resveratrol has the ability to promote functional recovery after sciatic nerve crush injury (SNCI), though the mechanism through which this occurs in not fully understood. Resveratrol can promote autophagy, a key process in Wallerian degeneration; thus, we hypothesized that resveratrol could promote recovery from SNCI by promoting Schwann cell autophagy and acceleration of Wallerian degeneration. Motor function recovery was assessed by calculating Sciatic Function Indexes (SFIs) at days 7, 14, 21, 28 post SNCI. Autophagy and myelin clearance were assessed by microtubule-associated protein light chain 3B (LC3B) and myelin protein zero (MPZ) immunofluorescence and Western blot analysis on the fourth day after SNCI. The autophagy of Schwann cells following resveratrol administration was quantified by immunofluorescence in RSC96 cells. Immunofluorescence and Transmission electron microscopy (TEM) were also used in Resveratrol treated sciatic nerve four days post-SNCI to find LC3B positive areas and typical double membrane structures represent for autophagy. The SNCI+resveratrol (crush+Res) groups recovered faster than the SNCI+vehicles (crush+V) group. On day four, almost all of the myelin had regenerated in the crush+Res rats, while the crush+V group's myelin remained intact and the expression levels of LC3-II/I was the highest. On day 28 post-injury, both the control and crush+Res groups' myelin neurofibers reached peak numbers as did the thickness of the myelin sheath. Both in vitro and in vivo immunofluorescence showed that LC3B was colocalized with Schwann cells. This is the first study to observe that resveratrol can promote recovery from SCNI by accelerating the myelin clearance process by promoting autophagy of Schwann cells.


Assuntos
Autofagia/efeitos dos fármacos , Lesões por Esmagamento/fisiopatologia , Compressão Nervosa , Recuperação de Função Fisiológica/efeitos dos fármacos , Resveratrol/farmacologia , Células de Schwann/patologia , Nervo Isquiático/patologia , Nervo Isquiático/fisiopatologia , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Lesões por Esmagamento/patologia , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Atividade Motora/efeitos dos fármacos , Proteína P0 da Mielina/metabolismo , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/metabolismo , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/patologia , Regeneração Nervosa/efeitos dos fármacos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Nervo Isquiático/efeitos dos fármacos
13.
Proc Natl Acad Sci U S A ; 117(25): 14376-14385, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513718

RESUMO

Temporally harmonized elimination of damaged or unnecessary organelles and cells is a prerequisite of health. Under Type 2 inflammatory conditions, human airway epithelial cells (HAECs) generate proferroptotic hydroperoxy-arachidonoyl-phosphatidylethanolamines (HpETE-PEs) as proximate death signals. Production of 15-HpETE-PE depends on activation of 15-lipoxygenase-1 (15LO1) in complex with PE-binding protein-1 (PEBP1). We hypothesized that cellular membrane damage induced by these proferroptotic phospholipids triggers compensatory prosurvival pathways, and in particular autophagic pathways, to prevent cell elimination through programmed death. We discovered that PEBP1 is pivotal to driving dynamic interactions with both proferroptotic 15LO1 and the autophagic protein microtubule-associated light chain-3 (LC3). Further, the 15LO1-PEBP1-generated ferroptotic phospholipid, 15-HpETE-PE, promoted LC3-I lipidation to stimulate autophagy. This concurrent activation of autophagy protects cells from ferroptotic death and release of mitochondrial DNA. Similar findings are observed in Type 2 Hi asthma, where high levels of both 15LO1-PEBP1 and LC3-II are seen in HAECs, in association with low bronchoalveolar lavage fluid mitochondrial DNA and more severe disease. The concomitant activation of ferroptosis and autophagy by 15LO1-PEBP1 complexes and their hydroperoxy-phospholipids reveals a pathobiologic pathway relevant to asthma and amenable to therapeutic targeting.


Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , Asma/imunologia , Autofagia/imunologia , Células Epiteliais/patologia , Ferroptose/imunologia , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Adulto , Animais , Asma/diagnóstico , Asma/patologia , Líquido da Lavagem Broncoalveolar/citologia , Linhagem Celular , Sobrevivência Celular/imunologia , Células Epiteliais/imunologia , Feminino , Técnicas de Inativação de Genes , Humanos , Ácidos Hidroxieicosatetraenoicos/imunologia , Ácidos Hidroxieicosatetraenoicos/metabolismo , Interleucina-13/imunologia , Interleucina-13/metabolismo , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Simulação de Dinâmica Molecular , Proteína de Ligação a Fosfatidiletanolamina/genética , Fosfatidiletanolaminas/imunologia , Fosfatidiletanolaminas/metabolismo , Cultura Primária de Células , Ligação Proteica/imunologia , Índice de Gravidade de Doença
14.
PLoS One ; 15(6): e0234180, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32511278

RESUMO

The autophagy-endolysosomal pathway is an evolutionally conserved degradation system that is tightly linked to a wide variety of physiological processes. Dysfunction of this system is associated with many pathological conditions such as cancer, inflammation and neurodegenerative diseases. Therefore, monitoring the cellular autophagy-endolysosomal activity is crucial for studies on the pathogenesis as well as therapeutics of such disorders. To this end, we here sought to create a novel means exploiting Keima, an acid-stable fluorescent protein possessing pH-dependent fluorescence excitation spectra, for precisely monitoring the autophagy-endolysosomal system. First, we generated three lines of transgenic (tg) mouse expressing monomeric Keima-fused MAP1LC3B (mKeima-LC3B). Then, these tg mice were subjected to starvation by food-restriction, and also challenged to neurodegeneration by genetically crossing with a mouse model of amyotrophic lateral sclerosis; i.e., SOD1H46R transgenic mouse. Unexpectedly, despite that a lipidated-form of endogenous LC3 (LC3-II) was significantly increased, those of mKeima-LC3B (mKeima-LC3B-II) were not changed under both stressed conditions. It was also noted that mKeima-LC3B-positive aggregates were progressively accumulated in the spinal cord of SOD1H46R;mKeima-LC3B double-tg mice, suggestive of acid-resistance and aggregate-prone natures of long-term overexpressed mKeima-LC3B in vivo. Next, we characterized mouse embryonic fibroblasts (MEFs) derived from mKeima-LC3B-tg mice. In contrast with in vivo, levels of mKeima-LC3B-I were decreased under starved conditions. Furthermore, when starved MEFs were treated with chloroquine (CQ), the abundance of mKeima-LC3B-II was significantly increased. Remarkably, when cultured medium was repeatedly changed between DMEM (nutrient-rich) and EBSS (starvation), acidic/neutral signal ratios of mKeima-LC3B-positive compartments were rapidly and reversibly shifted, which were suppressed by the CQ treatment, indicating that intraluminal pH of mKeima-LC3B-positive vesicles was changeable upon nutritional conditions of culture media. Taken together, although mKeima-LC3B-tg mice may not be an appropriate tool to monitor the autophagy-endolysosomal system in vivo, mKeima-LC3B must be one of the most sensitive reporter molecules for monitoring this system under in vitro cultured conditions.


Assuntos
Autofagia/fisiologia , Endossomos/metabolismo , Proteínas Luminescentes/genética , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Animais , Células Cultivadas , Meios de Cultura/farmacologia , Endossomos/genética , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Proteínas Luminescentes/metabolismo , Lisossomos/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Inanição , Superóxido Dismutase-1/genética , Imagem com Lapso de Tempo
15.
Nat Commun ; 11(1): 2993, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32532970

RESUMO

The accumulation of protein aggregates is involved in the onset of many neurodegenerative diseases. Aggrephagy is a selective type of autophagy that counteracts neurodegeneration by degrading such aggregates. In this study, we found that LC3C cooperates with lysosomal TECPR1 to promote the degradation of disease-related protein aggregates in neural stem cells. The N-terminal WD-repeat domain of TECPR1 selectively binds LC3C which decorates matured autophagosomes. The interaction of LC3C and TECPR1 promotes the recruitment of autophagosomes to lysosomes for degradation. Augmented expression of TECPR1 in neural stem cells reduces the number of protein aggregates by promoting their autophagic clearance, whereas knockdown of LC3C inhibits aggrephagy. The PH domain of TECPR1 selectively interacts with PtdIns(4)P to target TECPR1 to PtdIns(4)P containing lysosomes. Exchanging the PH against a tandem-FYVE domain targets TECPR1 ectopically to endosomes. This leads to an accumulation of LC3C autophagosomes at endosomes and prevents their delivery to lysosomes.


Assuntos
Autofagossomos/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Neurais/metabolismo , Autofagossomos/ultraestrutura , Autofagia/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular , Endossomos/metabolismo , Células HeLa , Humanos , Lisossomos/ultraestrutura , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microscopia Confocal , Microscopia Imunoeletrônica , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Células-Tronco Neurais/citologia , Doenças Neurodegenerativas/metabolismo , Agregados Proteicos , Agregação Patológica de Proteínas , Ligação Proteica , Transporte Proteico , Proteólise , Interferência de RNA
16.
Proc Natl Acad Sci U S A ; 117(27): 15989-15999, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32581130

RESUMO

Huntington disease (HD) is caused by an expansion mutation of the N-terminal polyglutamine of huntingtin (mHTT). mHTT is ubiquitously present, but it induces noticeable damage to the brain's striatum, thereby affecting motor, psychiatric, and cognitive functions. The striatal damage and progression of HD are associated with the inflammatory response; however, the underlying molecular mechanisms remain unclear. Here, we report that cGMP-AMP synthase (cGAS), a DNA sensor, is a critical regulator of inflammatory and autophagy responses in HD. Ribosome profiling revealed that the cGAS mRNA has high ribosome occupancy at exon 1 and codon-specific pauses at positions 171 (CCG) and 172 (CGT) in HD striatal cells. Moreover, the protein levels and activity of cGAS (based on the phosphorylated STING and phosphorylated TBK1 levels), and the expression and ribosome occupancy of cGAS-dependent inflammatory genes (Ccl5 and Cxcl10) are increased in HD striatum. Depletion of cGAS diminishes cGAS activity and decreases the expression of inflammatory genes while suppressing the up-regulation of autophagy in HD cells. In contrast, reinstating cGAS in cGAS-depleted HD cells activates cGAS activity and promotes inflammatory and autophagy responses. Ribosome profiling also revealed that LC3A and LC3B, the two major autophagy initiators, show altered ribosome occupancy in HD cells. We also detected the presence of numerous micronuclei, which are known to induce cGAS, in the cytoplasm of neurons derived from human HD embryonic stem cells. Collectively, our results indicate that cGAS is up-regulated in HD and mediates inflammatory and autophagy responses. Thus, targeting the cGAS pathway may offer therapeutic benefits in HD.


Assuntos
Autofagia/fisiologia , Doença de Huntington/genética , Doença de Huntington/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Animais , Quimiocina CCL5/metabolismo , Quimiocina CXCL10/metabolismo , Corpo Estriado/metabolismo , Células-Tronco Embrionárias , Humanos , Proteína Huntingtina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Neostriado/metabolismo , Neurônios/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Transcriptoma , Regulação para Cima
17.
Proc Natl Acad Sci U S A ; 117(24): 13571-13579, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32482850

RESUMO

Synchronized beating of cilia on multiciliated cells (MCCs) generates a directional flow of mucus across epithelia. This motility requires a "9 + 2" microtubule (MT) configuration in axonemes and the unidirectional array of basal bodies of cilia on the MCCs. However, it is not fully understood what components are needed for central MT-pair assembly as they are not continuous with basal bodies in contrast to the nine outer MT doublets. In this study, we discovered that a homozygous knockdown mouse model for MT minus-end regulator calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), Camsap3 tm1a/tm1a , exhibited multiple phenotypes, some of which are typical of primary ciliary dyskinesia (PCD), a condition caused by motile cilia defects. Anatomical examination of Camsap3 tm1a/tm1a mice revealed severe nasal airway blockage and abnormal ciliary morphologies in nasal MCCs. MCCs from different tissues exhibited defective synchronized beating and ineffective generation of directional flow likely underlying the PCD-like phenotypes. In normal mice, CAMSAP3 localized to the base of axonemes and at the basal bodies in MCCs. However, in Camsap3 tm1a/tm1a , MCCs lacked CAMSAP3 at the ciliary base. Importantly, the central MT pairs were missing in the majority of cilia, and the polarity of the basal bodies was disorganized. These phenotypes were further confirmed in MCCs of Xenopus embryos when CAMSAP3 expression was knocked down by morpholino injection. Taken together, we identified CAMSAP3 as being important for the formation of central MT pairs, proper orientation of basal bodies, and synchronized beating of motile cilia.


Assuntos
Corpos Basais/metabolismo , Cílios/metabolismo , Transtornos da Motilidade Ciliar/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animais , Axonema/metabolismo , Polaridade Celular , Transtornos da Motilidade Ciliar/genética , Células Epiteliais/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Xenopus
18.
Am J Chin Med ; 48(4): 945-966, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32476431

RESUMO

Tetramethylpyrazine has shown neuroprotective and axonal outgrowth-promoting effects and can improve cognitive deficit in a rat model of chronic hypoperfusion. However, the role of tetramethylpyrazine in sevoflurane-induced neurotoxicity is still vague. Therefore, this study was designed to investigate the effects and mechanisms of tetramethylpyrazine on sevoflurane-induced autophagy, apoptosis, and the expression of BACE1 and A[Formula: see text] in SH-SY5Y cells. We measured the expression levels of the apoptosis protein markers Bax and Bcl-2, autophagy protein markers Atg5 and LC3-II, BACE1, and A[Formula: see text] in SH-SY5Y cells after sevoflurane treatment and determined the effects of tetramethylpyrazine on sevoflurane-induced expression of these proteins after silencing GPR50 or Atg5 with siRNA in vitro. We found that exposure to 3.4% sevoflurane for 6 h decreased the expression of autophagy protein markers and increased the expression of the apoptosis protein markers, BACE1, and A[Formula: see text] in SH-SY5Y cells. The number of red puncta (autolysosomes) and yellow puncta (autophagosomes) in each SH-SY5Y cell decreased after transient transfection with the mRFP-GFP-LC3 expression plasmid. Silencing of GPR50 decreased the expression of pCREB, Atg5, and LC3-II, while silencing of Atg5 increased the expression of BACE1 and A[Formula: see text] in SH-SY5Y cells. Our results demonstrate that tetramethylpyrazine attenuated sevoflurane-induced neurotoxicity by enhancing autophagy through the GPR50/CREB pathway in SH-SY5Y cells.


Assuntos
Autofagia/efeitos dos fármacos , Autofagia/genética , Proteína de Ligação a CREB/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroprostanos , Pirazinas/farmacologia , Pirazinas/uso terapêutico , Receptores Acoplados a Proteínas-G/metabolismo , Sevoflurano/toxicidade , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Células Tumorais Cultivadas
19.
J Vis Exp ; (160)2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32597870

RESUMO

Neurospheres are primary cell aggregates that comprise neural stem cells and progenitor cells. These 3D structures are an excellent tool to determine the differentiation and proliferation potential of neural stem cells, as well as to generate cell lines than can be assayed over time. Also, neurospheres can create a niche (in vitro) that allows the modeling of the dynamic changing environment, such as varying growth factors, hormones, neurotransmitters, among others. Microtus ochrogaster (prairie vole) is a unique model for understanding the neurobiological basis of socio-sexual behaviors and social cognition. However, the cellular mechanisms involved in these behaviors are not well known. The protocol aims to obtain neural progenitor cells from the neurogenic niches of the adult prairie vole, which are cultured under non-adherent conditions, to generate neurospheres. The size and number of neurospheres depend on the region (subventricular zone or dentate gyrus) and sex of the prairie vole. This method is a remarkable tool to study sex-dependent differences in neurogenic niches in vitro and the neuroplasticity changes associated with social behaviors such as pair bonding and biparental care. Also, cognitive conditions that entail deficits in social interactions (autism spectrum disorders and schizophrenia) could be examined.


Assuntos
Arvicolinae/fisiologia , Pradaria , Neurogênese , Neurônios/citologia , Animais , Adesão Celular , Células Cultivadas , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Imageamento Tridimensional , Antígeno Ki-67/metabolismo , Masculino , Microdissecção , Proteínas Associadas aos Microtúbulos/metabolismo , Nestina/metabolismo , Neuropeptídeos/metabolismo , Esferoides Celulares/citologia
20.
Anticancer Res ; 40(5): 2537-2548, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32366398

RESUMO

BACKGROUND/AIM: Radiotherapy-induced autophagy affects radiation-sensitivity and radiotherapy efficacy. Histone modifications also occur during radiotherapy. This study assessed radiotherapy effects on histone modification and autophagy in non-small cell lung cancer (NSCLC) cells. MATERIALS AND METHODS: NSCLC cells were subjected to γ-irradiation. Autophagy was detected using western blotting and acridine orange staining. Radiation effect on cell growth was evaluated by clonogenic assay. Histone modifications were assessed by western blotting. Next generation sequencings (NGSs) were conducted to identify histone modification target genes. RESULTS: Radio-protective autophagy and histone H4 lysine 20 trimethylation (H4K20me3) were up-regulated after irradiation. By NGSs, genes that are differentially expressed upon irradiation were identified, including the candidate H4K20me3 target gene GABARAPL1. Furthermore, we showed that GABARAPL1 is essential for the radiation-induced autophagy. CONCLUSION: Our findings revealed the regulatory axis of radiation-induced H4K20me3-GABARAPL1 in radio-protective autophagy. Modulation of this axis may be a new strategy to enhance radiotherapy efficacy in NSCLC.


Assuntos
Autofagia/efeitos da radiação , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Histonas/metabolismo , Neoplasias Pulmonares/metabolismo , Lisina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Linhagem Celular Tumoral , Epigênese Genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Metilação , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , RNA Interferente Pequeno/genética
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