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Biomolecules Jan 2024Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by amyloid-beta (Aβ) plaques and tau neurofibrillary tangles (NFT). Modelling...
Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by amyloid-beta (Aβ) plaques and tau neurofibrillary tangles (NFT). Modelling aspects of AD is challenging due to its complex multifactorial etiology and pathology. The present study aims to establish a cost-effective and rapid method to model the two primary pathologies in organotypic brain slices. Coronal hippocampal brain slices (150 µm) were generated from postnatal (day 8-10) C57BL6 wild-type mice and cultured for 9 weeks. Collagen hydrogels containing either an empty load or a mixture of human Aβ42 and P301S aggregated tau were applied to the slices. The media was further supplemented with various intracellular pathway modulators or heavy metals to augment the appearance of Aβ plaques and tau NFTs, as assessed by immunohistochemistry. Immunoreactivity for Aβ and tau was significantly increased in the ventral areas in slices with a mixture of human Aβ42 and P301S aggregated tau compared to slices with empty hydrogels. Aβ plaque- and tau NFT-like pathologies could be induced independently in slices. Heavy metals (aluminum, lead, cadmium) potently augmented Aβ plaque-like pathology, which developed intracellularly prior to cell death. Intracellular pathway modulators (scopolamine, wortmannin, MHY1485) significantly boosted tau NFT-like pathologies. A combination of nanomolar concentrations of scopolamine, wortmannin, MHY1485, lead, and cadmium in the media strongly increased Aβ plaque- and tau NFT-like immunoreactivity in ventral areas compared to the slices with non-supplemented media. The results highlight that we could harness the potential of the collagen hydrogel-based spreading of human Aβ42 and P301S aggregated tau, along with pharmacological manipulation, to produce pathologies relevant to AD. The results offer a novel ex vivo organotypic slice model to investigate AD pathologies with potential applications for screening drugs or therapies in the future.
Topics: Mice; Animals; Humans; Alzheimer Disease; tau Proteins; Cadmium; Wortmannin; Mice, Transgenic; Amyloid beta-Peptides; Neurofibrillary Tangles; Brain; Plaque, Amyloid; Collagen; Hydrogels; Scopolamine Derivatives
PubMed: 38397402
DOI: 10.3390/biom14020165 -
Frontiers in Cell and Developmental... 2023Optimal mitochondrial functioning is indispensable for acquiring oocyte competence and meiotic maturation, whilst mitochondrial dysfunction may lead to diminished...
Optimal mitochondrial functioning is indispensable for acquiring oocyte competence and meiotic maturation, whilst mitochondrial dysfunction may lead to diminished reproductive potential and impaired fertility. The role of the intra-ovarian IGF system in ovarian follicular dynamics has been implicated earlier. Although several studies have demonstrated the role of the IGF axis in facilitating mitochondrial function over a multitude of cell lines, its role in oocyte energy metabolism remains largely unexplored. Here using zebrafish, the relative importance of IGF1 in modulating oocyte mitochondrial bioenergetics has been investigated. A dramatic increase in ovarian and expression accompanied heightened ATP levels and mitochondrial polarization in full-grown (FG) oocytes resuming meiotic maturation and ovulation . Concomitant with elevated expression and IGF1R phosphorylation, hCG (LH analog) stimulation of FG follicles prompted a sharp increase in NRF-1 and ATP levels, suggesting a positive influence of gonadotropin action on expression vis-à-vis oocyte bioenergetics. While recombinant IGF1 administration enhanced mitochondrial function, IGF1R immunodepletion or priming with PI3K inhibitor wortmannin could abrogate NRF-1 immunoreactivity, expression of respiratory chain subunits, ΔΨ and ATP content. Mechanistically, activation of PI3K/Akt signaling in IGF1-treated follicles corroborated well with the rapid phosphorylation of GSK3β at Ser9 (inactive) followed by PGC-1β accumulation. While selective inhibition of GSK3β promoted PGC-1β, Akt inhibition could abrogate IGF1-induced p-GSK3β (Ser9) and PGC-1β immunoreactive protein indicating Akt-mediated GSK3β inactivation and PGC-1β stabilization. The IGF1-depleted follicles showed elevated superoxide anions, subdued steroidogenic potential, and attenuated G2-M1 transition. In summary, this study highlights the importance of IGF1 signaling in oocyte bioenergetics prior to resumption of meiosis.
PubMed: 37457295
DOI: 10.3389/fcell.2023.1202693 -
Poultry Science Oct 2023Zearalenone (ZEA) is produced mainly by fungi belonging to genus Fusarium in foods and feeds. Heterophil extracellular traps (HETs) are a novel defense mechanism of...
Zearalenone (ZEA) is produced mainly by fungi belonging to genus Fusarium in foods and feeds. Heterophil extracellular traps (HETs) are a novel defense mechanism of chicken innate immunity involving activated heterophils. However, the conditions and requirements for ZEA-triggered HET release remain unknown. In this study, immunostaining analysis demonstrated that ZEA-triggered extracellular fibers were composed of histone and elastase assembled on DNA skeleton, showing that ZEA can induce the formation of HETs. Further experiments indicated that ZEA-induced HET release was concentration-dependent (ranging from 20 to 80 μM ZEA) and time-dependent (ranging from 30 to 180 min). Moreover, in 80 μM ZEA-exposed chicken heterophils, reactive oxygen species (ROS) level, catalase (CAT), superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and glutathione (GSH) content were increased. Simultaneously, ZEA at 80 μM activated ERK and p38 MAPK signaling pathways by increasing the phosphorylation level of ERK and p38 proteins. Pharmacological inhibition assays revealed that blocking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, ERK, and p38 mitogen-activated protein kinase (MAPK) reduced ZEA-induced ROS levels but had no impact on HET formation. Furthermore, immunostaining analysis indicated that the heterophil underwent the formation of autophagosome based on being stained with LC3B. The pharmacological inhibition assays demonstrated that rapamycin-, wortmannin-, and 3-methyladenine (3-MA)-treatments modulated ZEA-triggered HET formation, indicating that heterophil autophagy played a key role in ZEA-induced HET formation. Further studies on energy metabolism showed that inhibition of lactate/glucose transport, hexokinase-2 (HK-2), fructose-2,6-biphosphatase 3 (PFKFB3) in glycolysis abated ZEA-induced HETs, implying that glycolysis was one of the factors influencing the ZEA-induced HET formation. Besides, inhibition of the peptidylarginine deiminase (PAD) enzyme and P2X significantly reduced the ZEA-induced HET formation. In conclusion, we demonstrated that ZEA-triggered HET formation, which was associated with glycolysis, autophagy, PAD enzyme, and P2X receptor activation, providing valuable insight into the negative effect of ZEA on chicken innate immunity.
Topics: Animals; Extracellular Traps; Reactive Oxygen Species; Chickens; Zearalenone; Protein-Arginine Deiminases; Autophagy; Glycolysis
PubMed: 37542939
DOI: 10.1016/j.psj.2023.102946 -
International Journal of Molecular... Nov 2023Ouabain, a substance originally obtained from plants, is now classified as a hormone because it is produced endogenously in certain animals, including humans. However,...
Ouabain, a substance originally obtained from plants, is now classified as a hormone because it is produced endogenously in certain animals, including humans. However, its precise effects on the body remain largely unknown. Previous studies have shown that ouabain can influence the phenotype of epithelial cells by affecting the expression of cell-cell molecular components and voltage-gated potassium channels. In this study, we conducted whole-cell clamp assays to determine whether ouabain affects the activity and/or expression of TRPV4 channels. Our findings indicate that ouabain has a statistically significant effect on the density of TRPV4 currents (dI), with an EC50 of 1.89 nM. Regarding treatment duration, dI reaches its peak at around 1 h, followed by a subsequent decline and then a resurgence after 6 h, suggesting a short-term modulatory effect related to on TRPV4 channel activity and a long-term effect related to the promotion of synthesis of new TRPV4 channel units. The enhancement of dI induced by ouabain was significantly lower in cells seeded at low density than in cells in a confluent monolayer, indicating that the action of ouabain depends on intercellular contacts. Furthermore, the fact that U73122 and neomycin suppress the effect caused by ouabain in the short term suggests that the short-term induced enhancement of dI is due to the depletion of PIP2 stores. In contrast, the fact that the long-term effect is inhibited by PP2, wortmannin, PD, FR18, and IKK16 suggests that cSrc, PI3K, Erk1/2, and NF-kB are among the components included in the signaling pathways.
Topics: Humans; Animals; Ouabain; TRPV Cation Channels; Signal Transduction; Epithelial Cells; Sodium-Potassium-Exchanging ATPase
PubMed: 38069012
DOI: 10.3390/ijms242316687 -
Cell Death & Disease May 2024The targeted elimination of radio- or chemotherapy-induced senescent cells by so-called senolytic substances represents a promising approach to reduce tumor relapse as...
The targeted elimination of radio- or chemotherapy-induced senescent cells by so-called senolytic substances represents a promising approach to reduce tumor relapse as well as therapeutic side effects such as fibrosis. We screened an in-house library of 178 substances derived from marine sponges, endophytic fungi, and higher plants, and determined their senolytic activities towards DNA damage-induced senescent HCT116 colon carcinoma cells. The Pan-PI3K-inhibitor wortmannin and its clinical derivative, PX-866, were identified to act as senolytics. PX-866 potently induced apoptotic cell death in senescent HCT116, MCF-7 mammary carcinoma, and A549 lung carcinoma cells, independently of whether senescence was induced by ionizing radiation or by chemotherapeutics, but not in proliferating cells. Other Pan-PI3K inhibitors, such as the FDA-approved drug BAY80-6946 (Copanlisib, Aliqopa®), also efficiently and specifically eliminated senescent cells. Interestingly, only the simultaneous inhibition of both PI3K class I alpha (with BYL-719 (Alpelisib, Piqray®)) and delta (with CAL-101 (Idelalisib, Zydelig®)) isoforms was sufficient to induce senolysis, whereas single application of these inhibitors had no effect. On the molecular level, inhibition of PI3Ks resulted in an increased proteasomal degradation of the CDK inhibitor p21 in all tumor cell lines analyzed. This led to a timely induction of apoptosis in senescent tumor cells. Taken together, the senolytic properties of PI3K-inhibitors reveal a novel dimension of these promising compounds, which holds particular potential when employed alongside DNA damaging agents in combination tumor therapies.
Topics: Humans; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p21; HCT116 Cells; Proteasome Endopeptidase Complex; Apoptosis; Phosphoinositide-3 Kinase Inhibitors; MCF-7 Cells; Proteolysis; A549 Cells; Wortmannin; Senotherapeutics; Class I Phosphatidylinositol 3-Kinases; DNA Damage; Pyrimidines; Quinazolines
PubMed: 38811535
DOI: 10.1038/s41419-024-06755-x -
Scientific Reports Mar 2024The destruction of the microvascular structure and function can seriously affect the survival and prognosis of patients with acute myocardial infarction (AMI)....
The destruction of the microvascular structure and function can seriously affect the survival and prognosis of patients with acute myocardial infarction (AMI). Nuciferine has a potentially beneficial effect in the treatment of cardiovascular disease, albeit its role in microvascular structure and function during AMI remains unclear. This study aimed to investigate the protective effect and the related mechanisms of nuciferine in microvascular injury during AMI. Cardiac functions and pathological examination were conducted in vivo to investigate the effect of nuciferine on AMI. The effect of nuciferine on permeability and adherens junctions in endothelial cells was evaluated in vitro, and the phosphorylation level of the PI3K/AKT pathway (in the presence or absence of PI3K inhibitors) was also analyzed. In vivo results indicated that nuciferine inhibited ischemia-induced cardiomyocyte damage and vascular leakage and improved cardiac function. In addition, the in vitro results revealed that nuciferine could effectively inhibit oxygen-glucose deprivation (OGD) stimulated breakdown of the structure and function of human coronary microvascular endothelial cells (HCMECs). Moreover, nuciferine could significantly increase the phosphorylation level of the PI3K/AKT pathway. Finally, the inhibitor wortmannin could reverse the protective effect of nuciferine on HCMECs. Nuciferine inhibited AMI-induced microvascular injury by regulating the PI3K/AKT pathway and protecting the endothelial barrier function in mice.
Topics: Animals; Humans; Mice; Apoptosis; Aporphines; Endothelial Cells; Myocardial Infarction; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction
PubMed: 38528077
DOI: 10.1038/s41598-024-57595-w -
International Journal of Molecular... Apr 2024Estrogen plays an important role in osteoporosis prevention. We herein report the possible novel signaling pathway of 17β-estradiol (E2) in the matrix mineralization of...
Estrogen plays an important role in osteoporosis prevention. We herein report the possible novel signaling pathway of 17β-estradiol (E2) in the matrix mineralization of MC3T3-E1, an osteoblast-like cell line. In the culture media-containing stripped serum, in which small lipophilic molecules such as steroid hormones including E2 were depleted, matrix mineralization was significantly reduced. However, the E2 treatment induced this. The E2 effects were suppressed by ICI182,780, the estrogen receptor (ER)α, and the ERβ antagonist, as well as their mRNA knockdown, whereas Raloxifene, an inhibitor of estrogen-induced transcription, and G15, a G-protein-coupled estrogen receptor (GPER) 1 inhibitor, had little or no effect. Furthermore, the E2-activated matrix mineralization was disrupted by PMA, a PKC activator, and SB202190, a p38 MAPK inhibitor, but not by wortmannin, a PI3K inhibitor. Matrix mineralization was also induced by the culture media from the E2-stimulated cell culture. This effect was hindered by PMA or heat treatment, but not by SB202190. These results indicate that E2 activates the p38 MAPK pathway via ERs independently from actions in the nucleus. Such activation may cause the secretion of certain signaling molecule(s), which inhibit the PKC pathway. Our study provides a novel pathway of E2 action that could be a therapeutic target to activate matrix mineralization under various diseases, including osteoporosis.
Topics: Animals; Mice; Estradiol; Osteoblasts; Signal Transduction; Calcification, Physiologic; Cell Line; p38 Mitogen-Activated Protein Kinases; Receptors, Estrogen; Estrogens; Estrogen Receptor alpha
PubMed: 38731947
DOI: 10.3390/ijms25094727