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Scientific Reports Jun 2024Neurological and cardiac injuries are significant contributors to morbidity and mortality following pediatric in-hospital cardiac arrest (IHCA). Preservation of...
Neurological and cardiac injuries are significant contributors to morbidity and mortality following pediatric in-hospital cardiac arrest (IHCA). Preservation of mitochondrial function may be critical for reducing these injuries. Dimethyl fumarate (DMF) has shown potential to enhance mitochondrial content and reduce oxidative damage. To investigate the efficacy of DMF in mitigating mitochondrial injury in a pediatric porcine model of IHCA, toddler-aged piglets were subjected to asphyxia-induced CA, followed by ventricular fibrillation, high-quality cardiopulmonary resuscitation, and random assignment to receive either DMF (30 mg/kg) or placebo for four days. Sham animals underwent similar anesthesia protocols without CA. After four days, tissues were analyzed for mitochondrial markers. In the brain, untreated CA animals exhibited a reduced expression of proteins of the oxidative phosphorylation system (CI, CIV, CV) and decreased mitochondrial respiration (p < 0.001). Despite alterations in mitochondrial content and morphology in the myocardium, as assessed per transmission electron microscopy, mitochondrial function was unchanged. DMF treatment counteracted 25% of the proteomic changes induced by CA in the brain, and preserved mitochondrial structure in the myocardium. DMF demonstrates a potential therapeutic benefit in preserving mitochondrial integrity following asphyxia-induced IHCA. Further investigation is warranted to fully elucidate DMF's protective mechanisms and optimize its therapeutic application in post-arrest care.
Topics: Animals; Heart Arrest; Asphyxia; Swine; Disease Models, Animal; Dimethyl Fumarate; Mitochondria; Brain; Humans; Myocardium; Oxidative Phosphorylation
PubMed: 38879681
DOI: 10.1038/s41598-024-64317-9 -
Acta Neuropathologica Communications Jun 2024Wasteosomes (or corpora amylacea) are polyglucosan bodies that appear in the human brain with aging and in some neurodegenerative diseases, and have been suggested to...
Wasteosomes (or corpora amylacea) are polyglucosan bodies that appear in the human brain with aging and in some neurodegenerative diseases, and have been suggested to have a potential role in a nervous system cleaning mechanism. Despite previous studies in several neurodegenerative disorders, their status in frontotemporal lobar degeneration (FTLD) remains unexplored. Our study aims to characterize wasteosomes in the three primary FTLD proteinopathies, assessing frequency, distribution, protein detection, and association with aging or disease duration. Wasteosome scores were obtained in various brain regions from 124 post-mortem diagnosed sporadic FTLD patients, including 75 participants with tau (FTLD-tau), 42 with TAR DNA-binding protein 43 (FTLD-TDP), and 7 with Fused in Sarcoma (FTLD-FUS) proteinopathies, along with 29 control subjects. The wasteosome amount in each brain region for the different FLTD patients was assessed with a permutation test with age at death and sex as covariables, and multiple regressions explored associations with age at death and disease duration. Double immunofluorescence studies examined altered proteins linked to FTLD in wasteosomes. FTLD patients showed a higher accumulation of wasteosomes than control subjects, especially those with FTLD-FUS. Unlike FTLD-TDP and control subjects, wasteosome accumulation did not increase with age in FTLD-tau and FTLD-FUS. Cases with shorter disease duration in FTLD-tau and FTLD-FUS seemed to exhibit higher wasteosome quantities, whereas FTLD-TDP appeared to show an increase with disease progression. Immunofluorescence studies revealed the presence of tau and phosphorylated-TDP-43 in the periphery of isolated wasteosomes in some patients with FTLD-tau and FTLD-TDP, respectively. Central inclusions of FUS were observed in a higher number of wasteosomes in FTLD-FUS patients. These findings suggest a role of wasteosomes in FTLD, especially in the more aggressive forms of FLTD-FUS. Detecting these proteins, particularly FUS, in wasteosomes from cerebrospinal fluid could be a potential biomarker for FTLD.
Topics: Humans; Frontotemporal Lobar Degeneration; Female; Male; RNA-Binding Protein FUS; Aged; tau Proteins; Middle Aged; Aged, 80 and over; DNA-Binding Proteins; Brain
PubMed: 38879502
DOI: 10.1186/s40478-024-01812-0 -
Molecular Medicine (Cambridge, Mass.) Jun 2024Myocardial infarction (MI) leads to enhanced activity of cardiac fibroblasts (CFs) and abnormal deposition of extracellular matrix proteins, resulting in cardiac...
BACKGROUND
Myocardial infarction (MI) leads to enhanced activity of cardiac fibroblasts (CFs) and abnormal deposition of extracellular matrix proteins, resulting in cardiac fibrosis. Tartrate-resistant acid phosphatase 5 (ACP5) has been shown to promote cell proliferation and phenotypic transition. However, it remains unclear whether ACP5 is involved in the development of cardiac fibrosis after MI. The present study aimed to investigate the role of ACP5 in post-MI fibrosis and its potential underlying mechanisms.
METHODS
Clinical blood samples were collected to detect ACP5 concentration. Myocardial fibrosis was induced by ligation of the left anterior descending coronary artery. The ACP5 inhibitor, AubipyOMe, was administered by intraperitoneal injection. Cardiac function and morphological changes were observed on Day 28 after injury. Cardiac CFs from neonatal mice were extracted to elucidate the underlying mechanism in vitro. The expression of ACP5 was silenced by small interfering RNA (siRNA) and overexpressed by adeno-associated viruses to evaluate its effect on CF activation.
RESULTS
The expression of ACP5 was increased in patients with MI, mice with MI, and mice with Ang II-induced fibrosis in vitro. AubipyOMe inhibited cardiac fibrosis and improved cardiac function in mice after MI. ACP5 inhibition reduced cell proliferation, migration, and phenotypic changes in CFs in vitro, while adenovirus-mediated ACP5 overexpression had the opposite effect. Mechanistically, the classical profibrotic pathway of glycogen synthase kinase-3β (GSK3β)/β-catenin was changed with ACP5 modulation, which indicated that ACP5 had a positive regulatory effect. Furthermore, the inhibitory effect of ACP5 deficiency on the GSK3β/β-catenin pathway was counteracted by an ERK activator, which indicated that ACP5 regulated GSK3β activity through ERK-mediated phosphorylation, thereby affecting β-catenin degradation.
CONCLUSION
ACP5 may influence the proliferation, migration, and phenotypic transition of CFs, leading to the development of myocardial fibrosis after MI through modulating the ERK/GSK3β/β-catenin signaling pathway.
Topics: Animals; Fibrosis; Myocardial Infarction; Mice; Humans; Tartrate-Resistant Acid Phosphatase; Male; Cell Proliferation; Disease Models, Animal; Fibroblasts; Myocardium; Glycogen Synthase Kinase 3 beta; Mice, Inbred C57BL; Signal Transduction; Cell Movement
PubMed: 38879488
DOI: 10.1186/s10020-024-00856-1 -
The Journal of Biological Chemistry Jun 2024Resistance to inhibitors of cholinesterases (ric-8 proteins) are involved in modulating G-protein function but little is known of their potential physiological...
Resistance to inhibitors of cholinesterases (ric-8 proteins) are involved in modulating G-protein function but little is known of their potential physiological importance in the heart. In the present study, we assessed the role of resistance to inhibitors of cholinesterase 8b (Ric-8b) in determining cardiac contractile function. We developed a murine model in which it was possible to conditionally delete ric-8b in cardiac tissue in the adult animal after the addition of tamoxifen. Deletion of ric-8b led to severely reduced contractility as measured using echocardiography days after administration of tamoxifen. Histological analysis of the ventricular tissue showed highly variable myocyte size, prominent fibrosis and an increase in cellular apoptosis. RNA sequencing revealed transcriptional remodelling in response to cardiac ric-8b deletion involving the extracellular matrix and inflammation. Phosphoproteomic analysis revealed substantial downregulation of phosphopeptides related to myosin light chain 2. At the cellular level, the deletion of ric-8b led to loss of activation of the L-type calcium channel through the β-adrenergic pathways. Using fluorescence resonance energy transfer-based assays we showed ric-8b protein selectively interacts with the stimulatory G-protein, Gαs. We explored if deletion of Gnas (the gene encoding Gα) in cardiac tissue using a similar approach in the mouse led to an equivalent phenotype. The conditional deletion of the Gα gene in the ventricle led to comparable effects on contractile function and cardiac histology. We conclude that ric-8b is essential to preserve cardiac contractile function likely through an interaction with the stimulatory G-protein and downstream phosphorylation of myosin light chain 2.
PubMed: 38879012
DOI: 10.1016/j.jbc.2024.107470 -
Journal of Experimental & Clinical... Jun 2024Pancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer with an aggressive metastatic phenotype and very poor clinical prognosis. Interestingly, a lower...
BACKGROUND
Pancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer with an aggressive metastatic phenotype and very poor clinical prognosis. Interestingly, a lower occurrence of PDAC has been described in individuals with severe and long-standing asthma. Here we explored the potential link between PDAC and the glucocorticoid (GC) budesonide, a first-line therapy to treat asthma.
METHODS
We tested the effect of budesonide and the classical GCs on the morphology, proliferation, migration and invasiveness of patient-derived PDAC cells and pancreatic cancer cell lines, using 2D and 3D cultures in vitro. Furthermore, a xenograft model was used to investigate the effect of budesonide on PDAC tumor growth in vivo. Finally, we combined genome-wide transcriptome analysis with genetic and pharmacological approaches to explore the mechanisms underlying budesonide activities in the different environmental conditions.
RESULTS
We found that in 2D culture settings, high micromolar concentrations of budesonide reduced the mesenchymal invasive/migrating features of PDAC cells, without affecting proliferation or survival. This activity was specific and independent of the Glucocorticoid Receptor (GR). Conversely, in a more physiological 3D environment, low nanomolar concentrations of budesonide strongly reduced PDAC cell proliferation in a GR-dependent manner. Accordingly, we found that budesonide reduced PDAC tumor growth in vivo. Mechanistically, we demonstrated that the 3D environment drives the cells towards a general metabolic reprogramming involving protein, lipid, and energy metabolism (e.g., increased glycolysis dependency). This metabolic change sensitizes PDAC cells to the anti-proliferative effect of budesonide, which instead induces opposite changes (e.g., increased mitochondrial oxidative phosphorylation). Finally, we provide evidence that budesonide inhibits PDAC growth, at least in part, through the tumor suppressor CDKN1C/p57Kip2.
CONCLUSIONS
Collectively, our study reveals that the microenvironment influences the susceptibility of PDAC cells to GCs and provides unprecedented evidence for the anti-proliferative activity of budesonide on PDAC cells in 3D conditions, in vitro and in vivo. Our findings may explain, at least in part, the reason for the lower occurrence of pancreatic cancer in asthmatic patients and suggest a potential suitability of budesonide for clinical trials as a therapeutic approach to fight pancreatic cancer.
Topics: Humans; Budesonide; Mice; Pancreatic Neoplasms; Energy Metabolism; Cell Proliferation; Animals; Cell Line, Tumor; Carcinoma, Pancreatic Ductal; Xenograft Model Antitumor Assays; Cell Movement
PubMed: 38877560
DOI: 10.1186/s13046-024-03072-1 -
Scientific Reports Jun 2024Glucagon-like peptide 1 receptor (GLP-1R) agonist is an emerging anti-diabetic medication whose effects on the risk and progression of cholangiocarcinoma (CCA) are...
Glucagon-like peptide 1 receptor (GLP-1R) agonist is an emerging anti-diabetic medication whose effects on the risk and progression of cholangiocarcinoma (CCA) are controversial. This study aimed to elucidate the roles of GLP-1R and its agonists on intrahepatic CCA (iCCA) progression. Expressions of GLP-1R in iCCA tissues investigated by immunohistochemistry showed that GLP-1R expressions were significantly associated with poor histological grading (P = 0.027). iCCA cell lines, KKU-055 and KKU-213A, were treated with exendin-4 and liraglutide, GLP-1R agonists, and their effects on proliferation and migration were assessed. Exendin-4 and liraglutide did not affect CCA cell proliferation in vitro, but liraglutide significantly suppressed the migration of CCA cells, partly by inhibiting epithelial-mesenchymal transition. In contrast, liraglutide significantly reduced CCA tumor volumes and weights in xenografted mice (P = 0.046). GLP-1R appeared downregulated when CCA cells were treated with liraglutide in vitro and in vivo. In addition, liraglutide treatment significantly suppressed Akt and STAT3 signaling in CCA cells, by reducing their phosphorylation levels. These results suggested that liraglutide potentially slows down CCA progression, and further clinical investigation would benefit the treatment of CCA with diabetes mellitus.
Topics: Liraglutide; Cholangiocarcinoma; Humans; Animals; Cell Line, Tumor; Glucagon-Like Peptide-1 Receptor; Mice; Male; Bile Duct Neoplasms; Cell Proliferation; Epithelial-Mesenchymal Transition; Cell Movement; Female; Xenograft Model Antitumor Assays; Disease Progression; Middle Aged; Signal Transduction; Aged; Antineoplastic Agents; STAT3 Transcription Factor; Exenatide; Mice, Nude; Proto-Oncogene Proteins c-akt
PubMed: 38877189
DOI: 10.1038/s41598-024-64774-2 -
Nature Communications Jun 2024Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a determinant of cardiac myofilament function. Although cMyBP-C phosphorylation by various protein...
Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a determinant of cardiac myofilament function. Although cMyBP-C phosphorylation by various protein kinases has been extensively studied, the influence of protein phosphatases on cMyBP-C's multiple phosphorylation sites has remained largely obscure. Here we provide a detailed biochemical characterization of cMyBP-C dephosphorylation by protein phosphatases 1 and 2 A (PP1 and PP2A), and develop an integrated kinetic model for cMyBP-C phosphorylation using data for both PP1, PP2A and various protein kinases known to phosphorylate cMyBP-C. We find strong site-specificity and a hierarchical mechanism for both phosphatases, proceeding in the opposite direction of sequential phosphorylation by potein kinase A. The model is consistent with published data from human patients and predicts complex non-linear cMyBP-C phosphorylation patterns that are validated experimentally. Our results suggest non-redundant roles for PP1 and PP2A under both physiological and heart failure conditions, and emphasize the importance of phosphatases for cMyBP-C regulation.
Topics: Phosphorylation; Humans; Protein Phosphatase 1; Carrier Proteins; Animals; Protein Phosphatase 2; Myocardium; Protein Kinases; Kinetics
PubMed: 38877002
DOI: 10.1038/s41467-024-49408-5 -
Nature Communications Jun 2024Inositol hexaphosphate (InsP) is the major storage form of phosphorus in seeds. Reducing seed InsP content is a breeding objective in agriculture, as InsP negatively...
Inositol hexaphosphate (InsP) is the major storage form of phosphorus in seeds. Reducing seed InsP content is a breeding objective in agriculture, as InsP negatively impacts animal nutrition and the environment. Nevertheless, how InsP accumulation is regulated remains largely unknown. Here, we identify a clade of receptor-like cytoplasmic kinases (RLCKs), named Inositol Polyphosphate-related Cytoplasmic Kinases 1-6 (IPCK1-IPCK6), deeply involved in InsP accumulation. The InsP concentration is dramatically reduced in seeds of ipck quadruple (T-4m/C-4m) and quintuple (C-5m) mutants, accompanied with the obviously increase of phosphate (Pi) concentration. The plasma membrane-localized IPCKs recruit IPK1 involved in InsP synthesis, and facilitate its binding and activity via phosphorylation of GRF 14-3-3 proteins. IPCKs also recruit IPK2s and PI-PLCs required for InsP/InsP and InsP biosynthesis respectively, to form a potential IPCK-GRF-PLC-IPK2-IPK1 complex. Our findings therefore uncover a regulatory mechanism of InsP accumulation governed by IPCKs, shedding light on the mechanisms of InsP biosynthesis in eukaryotes.
Topics: 14-3-3 Proteins; Phytic Acid; Arabidopsis; Arabidopsis Proteins; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Mutation; Cell Membrane; Gene Expression Regulation, Plant; Inositol Phosphates
PubMed: 38877001
DOI: 10.1038/s41467-024-49102-6 -
Nature Communications Jun 2024Stomatal movement is vital for plants to exchange gases and adaption to terrestrial habitats, which is regulated by environmental and phytohormonal signals. Here, we...
Stomatal movement is vital for plants to exchange gases and adaption to terrestrial habitats, which is regulated by environmental and phytohormonal signals. Here, we demonstrate that hydrogen peroxide (HO) is required for light-induced stomatal opening. HO accumulates specifically in guard cells even when plants are under unstressed conditions. Reducing HO content through chemical treatments or genetic manipulations results in impaired stomatal opening in response to light. This phenomenon is observed across different plant species, including lycopodium, fern, and monocotyledonous wheat. Additionally, we show that HO induces the nuclear localization of KIN10 protein, the catalytic subunit of plant energy sensor SnRK1. The nuclear-localized KIN10 interacts with and phosphorylates the bZIP transcription factor bZIP30, leading to the formation of a heterodimer between bZIP30 and BRASSINAZOLE-RESISTANT1 (BZR1), the master regulator of brassinosteroid signaling. This heterodimer complex activates the expression of amylase, which enables guard cell starch degradation and promotes stomatal opening. Overall, these findings suggest that HO plays a critical role in light-induced stomatal opening across different plant species.
Topics: Plant Stomata; Hydrogen Peroxide; Light; Gene Expression Regulation, Plant; Plant Proteins; Arabidopsis; Triticum; Arabidopsis Proteins; Signal Transduction; Phosphorylation; Ferns; Basic-Leucine Zipper Transcription Factors
PubMed: 38876991
DOI: 10.1038/s41467-024-49377-9 -
The Journal of Biological Chemistry Jun 2024Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains,...
Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinson's disease (PD) has led to intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.
PubMed: 38876305
DOI: 10.1016/j.jbc.2024.107469