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Autophagy Feb 2022Macroautophagy (hereafter referred to as autophagy) is a finely tuned process of programmed degradation and recycling of proteins and cellular components, which is...
Macroautophagy (hereafter referred to as autophagy) is a finely tuned process of programmed degradation and recycling of proteins and cellular components, which is crucial in neuronal function and synaptic integrity. Mounting evidence implicates chromatin remodeling in fine-tuning autophagy pathways. However, this epigenetic regulation is poorly understood in neurons. Here, we investigate the role in autophagy of KANSL1, a member of the nonspecific lethal complex, which acetylates histone H4 on lysine 16 (H4K16ac) to facilitate transcriptional activation. Loss-of-function of KANSL1 is strongly associated with the neurodevelopmental disorder Koolen-de Vries Syndrome (KdVS). Starting from KANSL1-deficient human induced-pluripotent stem cells, both from KdVS patients and genome-edited lines, we identified SOD1 (superoxide dismutase 1), an antioxidant enzyme, to be significantly decreased, leading to a subsequent increase in oxidative stress and autophagosome accumulation. In KANSL1-deficient neurons, autophagosome accumulation at excitatory synapses resulted in reduced synaptic density, reduced GRIA/AMPA receptor-mediated transmission and impaired neuronal network activity. Furthermore, we found that increased oxidative stress-mediated autophagosome accumulation leads to increased MTOR activation and decreased lysosome function, further preventing the clearing of autophagosomes. Finally, by pharmacologically reducing oxidative stress, we could rescue the aberrant autophagosome formation as well as synaptic and neuronal network activity in KANSL1-deficient neurons. Our findings thus point toward an important relation between oxidative stress-induced autophagy and synapse function, and demonstrate the importance of H4K16ac-mediated changes in chromatin structure to balance reactive oxygen species- and MTOR-dependent autophagy.: APO: apocynin; ATG: autophagy related; BAF: bafilomycin A; BSO: buthionine sulfoximine; CV: coefficient of variation; DIV: days in vitro; H4K16ac: histone 4 lysine 16 acetylation; iPSC: induced-pluripotent stem cell; KANSL1: KAT8 regulatory NSL complex subunit 1; KdVS: Koolen-de Vries Syndrome; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEA: micro-electrode array; MTOR: mechanistic target of rapamycin kinase; NSL complex: nonspecific lethal complex; 8-oxo-dG: 8-hydroxydesoxyguanosine; RAP: rapamycin; ROS: reactive oxygen species; sEPSCs: spontaneous excitatory postsynaptic currents; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; SYN: synapsin; WRT: wortmannin.
Topics: Abnormalities, Multiple; Autophagosomes; Autophagy; Chromosome Deletion; Chromosomes, Human, Pair 17; Epigenesis, Genetic; Humans; Intellectual Disability; Lysine; Lysosomes; Reactive Oxygen Species; Sirolimus; Superoxide Dismutase-1; TOR Serine-Threonine Kinases
PubMed: 34286667
DOI: 10.1080/15548627.2021.1936777 -
American Journal of Translational... 2021The aim of this study was to show the effects of autophagy inhibitor Wortmannin and antiangiogenic-proapoptotic Thalidomide on autophagy and apoptosis markers in 4T1...
The aim of this study was to show the effects of autophagy inhibitor Wortmannin and antiangiogenic-proapoptotic Thalidomide on autophagy and apoptosis markers in 4T1 breast cancer cells in vitro and in vivo. The half-maximal inhibitory concentration (IC50) values of 4T1 cells for Wortmannin and Thalidomide were evaluated by Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. After cancer formation in 28 BALB/C female mice, drugs were administered for seven days. Cells and tissue sections were evaluated for anti-phosphoinositide 3-kinase (PI3K), anti- the microtubule-associated protein 1 light chain3 (MAPLC3β), anti-caspase 8, anti-caspase 9, and anti-caspase 3 immunoreactivities by immunohistochemical staining and apoptosis by Terminal Transferase dUTP Nick End Labeling (TUNEL) assay. Both PI3K and MAPLC3β immunoreactivities decreased in all treatments when compared to control group except Thalidomide treatment in primary cancer tissue. The caspase 3, 8, and 9 immunoreactivities were increased in all treatment groups and TUNEL positive cells were the highest in the Wortmannin and Thalidomide group. Our findings suggest that autophagy is an important mechanism for 4T1 cells and both Wortmannin and Thalidomide treatments inhibit autophagy and induce apoptosis. In primary cancer tissues, autophagy was not effective as in vitro. The treatment of Wortmannin and Thalidomide increased the apoptotic cells in vivo independent from autophagy inhibition. Different results may be because of microenvironment. Further studies must be done to elucidate the effect of microenvironment.
PubMed: 34306363
DOI: No ID Found -
Journal of Veterinary Internal Medicine Jul 2022Outbreaks of liver disease in horses are common but the etiology of most remains unknown. Forage mycotoxins have been suspected to be a cause.
BACKGROUND
Outbreaks of liver disease in horses are common but the etiology of most remains unknown. Forage mycotoxins have been suspected to be a cause.
OBJECTIVES
To examine the association between outbreaks of liver disease and the presence of mycotoxins in forage stored on the same premises.
ANIMALS
Premises were identified where ≥4 horses were contemporaneously affected by liver disease, and a control group was formed from premises where ≥4 horses had been examined and found to have no evidence of liver disease.
METHODS
Forage was collected from 29 case and 12 control premises. The forage was analyzed for mycotoxin content using a liquid chromatography/mass spectrometry method, targeting 54 mycotoxins. The presence and distribution of mycotoxins between case and control samples was compared.
RESULTS
Mycotoxins were found in 23/29 (79%) case samples and 10/12 (83%) control samples (P > .99; relative risk, 0.93; 95% confidence interval [CI], 0.64-1.75). Median (interquartile range [IQR]) total mycotoxin concentration was similar in case and control samples (85.8 μg/kg [1.6-268] vs. 315 μg/kg [6.3-860]; P = .16). Ten mycotoxins were found exclusively in case premises comprising fumonisin B1, 15-acetyldeoxynivalenol, deoxynivalenol, zearalenone, aflatoxins B1 and G1, methylergonovine, nivalenol, verruculogen, and wortmannin. The median (IQR) concentration of fumonisin B1 was significantly higher in case versus control samples (0 μg/kg [0-81.7] vs. 0 μg/kg [0-0]; P = .04).
CONCLUSIONS AND CLINICAL IMPORTANCE
Several mycotoxins with known hepatotoxic potential were found, alone or in combination, exclusively at case premises, consistent with the hypothesis that forage-associated mycotoxicosis may be a cause of outbreaks of liver disease in horses in the United Kingdom.
Topics: Animals; Food Contamination; Horse Diseases; Horses; Liver Diseases; Mycotoxins; United Kingdom; Zearalenone
PubMed: 35792718
DOI: 10.1111/jvim.16486 -
International Journal of Molecular... Jul 2022Hyperglycemia-induced protein glycation and formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of diabetic complications and...
Hyperglycemia-induced protein glycation and formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of diabetic complications and pathological biomineralization. Receptors for AGEs (RAGEs) mediate the generation of reactive oxygen species (ROS) via activation of NADPH-oxidase. It is conceivable that binding of glycated proteins with biomineral particles composed mainly of calcium carbonate and/or phosphate enhances their neutrophil-activating capacity and hence their proinflammatory properties. Our research managed to confirm this hypothesis. Human serum albumin (HSA) was glycated with methylglyoxal (MG), and HSA-MG was adsorbed onto mineral microparticles composed of calcium carbonate nanocrystals (vaterite polymorph, CC) or hydroxyapatite nanowires (CP). As scopoletin fluorescence has shown, HO generation by neutrophils stimulated with HSA-MG was inhibited with diphenyleneiodonium chloride, wortmannin, genistein and EDTA, indicating a key role for NADPH-oxidase, protein tyrosine kinase, phosphatidylinositol 3-kinase and divalent ions (presumably Ca) in HSA-MG-induced neutrophil respiratory burst. Superoxide anion generation assessed by lucigenin-enhanced chemiluminescence (Luc-CL) was significantly enhanced by free HSA-MG and by both CC-HSA-MG and CP-HSA-MG microparticles. Comparing the concentrations of CC-bound and free HSA-MG, one could see that adsorption enhanced the neutrophil-activating capacity of HSA-MG.
Topics: Calcium Carbonate; Glycation End Products, Advanced; Humans; Hydrogen Peroxide; Minerals; NADP; NADPH Oxidases; Neutrophil Activation; Pyruvaldehyde; Serum Albumin; Serum Albumin, Human; Glycated Serum Albumin
PubMed: 35887188
DOI: 10.3390/ijms23147840 -
Cells Mar 2021During infection or certain metabolic disorders, neutrophils can escape from blood vessels, invade and attach to other tissues. The invasion and adhesion of neutrophils...
During infection or certain metabolic disorders, neutrophils can escape from blood vessels, invade and attach to other tissues. The invasion and adhesion of neutrophils is accompanied and maintained by their own secretion. We have previously found that adhesion of neutrophils to fibronectin dramatically and selectively stimulates the release of the free amino acid hydroxylysine. The role of hydroxylysine and lysyl hydroxylase in neutrophil adhesion has not been studied, nor have the processes that control them. Using amino acid analysis, mass spectrometry and electron microscopy, we found that the lysyl hydroxylase inhibitor minoxidil, the matrix metalloproteinase inhibitor doxycycline, the PI3K/Akt pathway inhibitors wortmannin and the Akt1/2 inhibitor and drugs that affect the actin cytoskeleton significantly and selectively block the release of hydroxylysine and partially or completely suppress spreading of neutrophils. The actin cytoskeleton effectors and the Akt 1/2 inhibitor also increase the phenylalanine release. We hypothesize that hydroxylysine release upon adhesion is the result of the activation of lysyl hydroxylase in interaction with matrix metalloproteinase, the PI3K/Akt pathway and intact actin cytoskeleton, which play important roles in the recruitment of neutrophils into tissue through extracellular matrix remodeling.
Topics: Amino Acids; Apoptosis; Humans; Hydroxylysine; Neutrophils
PubMed: 33807594
DOI: 10.3390/cells10030563 -
Journal of Nanobiotechnology Mar 2022Copper oxide nanoparticles (CuONPs) are widely used metal oxide NPs owing to their excellent physical-chemical properties. Circulation translocation of CuONPs after...
Copper oxide nanoparticles (CuONPs) are widely used metal oxide NPs owing to their excellent physical-chemical properties. Circulation translocation of CuONPs after inhalation leads to vascular endothelial injury. Mitochondria, an important regulatory hub for maintaining cell functions, are signaling organelles in responses to NPs-induced injury. However, how mitochondrial dynamics (fission and fusion) and mitophagy (an autophagy process to degrade damaged mitochondria) are elaborately orchestrated to maintain mitochondrial homeostasis in CuONPs-induced vascular endothelial injury is still unclear. In this study, we demonstrated that CuONPs exposure disturbed mitochondrial dynamics through oxidative stress-dependent manner in vascular endothelial cells, as evidenced by the increase of mitochondrial fission and the accumulation of fragmented mitochondria. Inhibition of mitochondrial fission with Mdivi-1 aggravated CuONPs-induced mtROS production and cell death. Furthermore, we found that mitochondrial fission led to the activation of PINK1-mediated mitophagy, and pharmacological inhibition with wortmannin, chloroquine or genetical inhibition with siRNA-mediated knockdown of PINK1 profoundly repressed mitophagy, suggesting that the protective role of mitochondrial fission and PINK1-mediated mitophagy in CuONPs-induced toxicity. Intriguingly, we identified that TAX1BP1 was the primary receptor to link the ubiquitinated mitochondria with autophagosomes, since TAX1BP1 knockdown elevated mtROS production, decreased mitochondrial clearance and aggravated CuONPs-induced cells death. More importantly, we verified that urolithin A, a mitophagy activator, promoted mtROS clearance and the removal of damaged mitochondria induced by CuONPs exposure both in vitro and in vivo. Overall, our findings indicated that modulating mitophagy may be a therapeutic strategy for pathological vascular endothelial injury caused by NPs exposure.
Topics: Copper; Endothelial Cells; Mitophagy; Nanoparticles; Oxides; Protein Kinases
PubMed: 35305662
DOI: 10.1186/s12951-022-01338-4 -
Frontiers in Pharmacology 2020Ischemic/reperfusion (I/R) injury is the primary cause of acute kidney injury (AKI). Gastrin, a gastrointestinal hormone, is involved in the regulation of kidney... (Review)
Review
Ischemic/reperfusion (I/R) injury is the primary cause of acute kidney injury (AKI). Gastrin, a gastrointestinal hormone, is involved in the regulation of kidney function of sodium excretion. However, whether gastrin has an effect on kidney I/R injury is unknown. Here we show that cholecystokinin B receptor (CCKBR), the gastrin receptor, was significantly up-regulated in I/R-injured mouse kidneys. While pre-administration of gastrin ameliorated I/R-induced renal pathological damage, as reflected by the levels of serum creatinine and blood urea nitrogen, hematoxylin and eosin staining and periodic acid-Schiff staining. The protective effect could be ascribed to the reduced apoptosis for gastrin reduced tubular cell apoptosis both and . studies also showed gastrin preserved the viability of hypoxia/reoxygenation (H/R)-treated human kidney 2 (HK-2) cells and reduced the lactate dehydrogenase release, which were blocked by CI-988, a specific CCKBR antagonist. Mechanistically, the PI3K/Akt/Bad pathway participates in the pathological process, because gastrin treatment increased phosphorylation of PI3K, Akt and Bad. While in the presence of wortmannin (1 μM), a PI3K inhibitor, the gastrin-induced phosphorylation of Akt after H/R treatment was blocked. Additionally, wortmannin and Akt inhibitor VIII blocked the protective effect of gastrin on viability of HK-2 cells subjected to H/R treatment. These studies reveals that gastrin attenuates kidney I/R injury via a PI3K/Akt/Bad-mediated anti-apoptosis signaling. Thus, gastrin can be considered as a promising drug candidate to prevent AKI.
PubMed: 33343341
DOI: 10.3389/fphar.2020.540479 -
Journal of Pharmacological Sciences Jun 2023Microglia hyperactivation is an important cause of neuroinflammation in Alzheimer's disease (AD). Paeoniflorin (PF), ferulic acid (FA), and atractylenolide III (ATL) are...
Microglia hyperactivation is an important cause of neuroinflammation in Alzheimer's disease (AD). Paeoniflorin (PF), ferulic acid (FA), and atractylenolide III (ATL) are potent in anti-inflammation and neuroprotection. Multiple components can act on different targets simultaneously to exert synergistic therapeutic effects and exploring the synergistic potential between compounds is an important area of research. We investigated the effects of PF, FA, and ATL, alone or in combination, on LPS-induced neuroinflammation and autophagy in BV2 microglia cells. We found that PF, FA, and ATL, alone or in combination, significantly reduced the production of inflammatory factors such as IL-6, IL-1β, and TNF-α, especially in the PF + FA + ATL group, which performed the best. In addition, the combination of PF, FA, and ATL significantly increased the expression of autophagy-related proteins p-AMPK, p-ULK1, Beclin1, LC3, and TFEB and decreased the expression of p62. Moreover, the restoration of autophagic flux by the combination of PF, FA, and ATL was abrogated by the addition of the autophagy inhibitor Wortmannin. In conclusion, PF, FA, and ATL have a synergistic effect in reducing LPS-induced inflammatory factor release from BV2 microglia cells, and its protective effect may be through activation of the AMPK/ULK1/TFEB autophagic signaling pathway.
Topics: Humans; Microglia; Lipopolysaccharides; AMP-Activated Protein Kinases; Neuroinflammatory Diseases; Autophagy
PubMed: 37169480
DOI: 10.1016/j.jphs.2023.04.007 -
Frontiers in Genetics 2022Craniopharyngioma is a benign tumor, and the predominant treatment methods are surgical resection and radiotherapy. However, both treatments may lead to complex...
Craniopharyngioma is a benign tumor, and the predominant treatment methods are surgical resection and radiotherapy. However, both treatments may lead to complex complications, seriously affecting patients' survival rate and quality of life. Adamantinomatous craniopharyngioma (ACP), as one of the histological subtypes of craniopharyngioma, is associated with a high incidence and poor prognosis, and there is a gap in the targeted therapy of immune-related genes for ACP. In this study, two gene expression profiles of ACP, namely GSE68015 and GSE94349, were downloaded from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were identified by the Limma package, and 271 differentially expressed immune-related genes (DEIRGs) were obtained from the Immport database. The gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were performed for annotation, visualization, and integrated discovery. Five hub genes, including CXCL6, CXCL10, CXCL11, CXCL13, and SAA1, were screened out through protein-protein interaction (PPI) network interaction construction. Two diagnostic markers, namely S100A2 and SDC1 (both of which have the Area Under Curve value of 1), were screened by the machine learning algorithm. CIBERSORT analysis showed that M2 macrophages, activated NK cells, and gamma delta T cells had higher abundance in ACP infiltration, while CD8+ T cells, regulatory T cells, and Neutrophils had less abundance in ACP infiltration. The expression of gamma delta T cells was positively correlated with CXCL6, S100A2, SDC1, and SAA1, while CD8+ T cells expression was negatively correlated with CXCL6, S100A2, SDC1, and CXCL10. ACP with high CXCL6 showed remarkable drug sensitivity to Pentostatin and Wortmannin via CellMiner database analysis. Our results deepened the understanding of the molecular immune mechanism in ACP and provided potential biomarkers for the precisely targeted therapy for ACP.
PubMed: 35432485
DOI: 10.3389/fgene.2022.830793 -
Frontiers in Immunology 2022Polyphosphates are linear polymers of inorganic phosphates that exist in all living cells and serve pleiotropic functions. Bacteria produce long-chain polyphosphates,...
Polyphosphates are linear polymers of inorganic phosphates that exist in all living cells and serve pleiotropic functions. Bacteria produce long-chain polyphosphates, which can interfere with host defense to infection. In contrast, short-chain polyphosphates are released from platelet dense granules and bind to the chemokine CXCL4. Here, we report that long-chain polyphosphates induced the release of CXCL4 from mouse bone marrow-derived macrophages and peritoneal macrophages in a dose-/time-dependent fashion resulting from an induction of CXCL4 mRNA. This polyphosphate effect was lost after pre-incubation with recombinant exopolyphosphatase (PPX) Fc fusion protein, demonstrating the potency of long chains over monophosphates and ambient cations. In detail, polyphosphate chains >70 inorganic phosphate residues were required to reliably induce CXCL4. Polyphosphates acted independently of the purinergic P2Y1 receptor and the MyD88/TRIF adaptors of Toll-like receptors. On the other hand, polyphosphates augmented LPS/MyD88-induced CXCL4 release, which was explained by intracellular signaling convergence on PI3K/Akt. Polyphosphates induced Akt phosphorylation at threonine-308. Pharmacologic blockade of PI3K (wortmannin, LY294002) antagonized polyphosphate-induced CXCL4 release from macrophages. Intratracheal polyphosphate administration to C57BL/6J mice caused histologic signs of lung injury, disruption of the endothelial-epithelial barrier, influx of Ly6G polymorphonuclear neutrophils, depletion of CD11cSiglecF alveolar macrophages, and release of CXCL4. Long-chain polyphosphates synergized with the complement anaphylatoxin, C5a, which was partly explained by upregulation of C5aR1 on myeloid cells. C5aR1 mice were protected from polyphosphate-induced lung injury. C5a generation occurred in the lungs and bronchoalveolar lavage fluid (BALF) of polyphosphate-treated C57BL/6J mice. In conclusion, we demonstrate that polyphosphates govern immunomodulation in macrophages and promote acute lung injury.
Topics: Mice; Animals; Complement C5a; Anaphylatoxins; Platelet Factor 4; Polyphosphates; Proto-Oncogene Proteins c-akt; Phosphatidylinositol 3-Kinases; Myeloid Differentiation Factor 88; Mice, Inbred C57BL; Immunologic Factors; Acute Lung Injury; Bacteria
PubMed: 36405694
DOI: 10.3389/fimmu.2022.980733