Search Results (56)

Tuberous sclerosis complex (TSC) is a systemic disease caused by mutations in either the TSC1 (encoding hamartin) or TSC2 (encoding tuberin) gene, with mutations in the TSC2 gene potentially leading to more severe clinical symptoms. Neurological symptoms are a common clinical manifestation of TSC, and neuroinflammation is thought to play an important role. Glial cells are a major source of neuroinflammation, but whether microglia are involved in the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome and the expression of interleukin-1β (IL-1β) in TSC patients remains unclear. We used a transcriptome sequencing dataset for bioinformatics analysis to explore the differences in the expression of microglial inflammasome-associated hub genes. TSC2 knockdown (TSC2 KD) microglia (HMC3 cell line) were generated by lentivirus, and the expression of inflammasome-associated hub genes, microglial activation, and NLRP3 inflammasome activation were verified. In addition, experiments were performed to explore the regulatory effects of rapamycin. Bioinformatics analysis identified a total of eight inflammasome-associated hub genes. By detecting GFP fluorescence, TSC2 mRNA, TSC2 protein expression, and the phosphorylation of the mammalian target of rapamycin (p-mTOR)/mTOR, we confirmed that the TSC2 KD microglia model was successfully established. Compared with the control group, the TSC2 KD group presented higher mRNA levels and fluorescence intensities of microglia AIF1 and CD68, as well as greater reactive oxygen species (ROS) production. Eight inflammasome-associated hub gene mRNA assays revealed that the expression of the NLRP3 and IL1B genes was increased. Compared with the control group, the TSC2 KD group presented increased levels of NLRP3 and Pro-IL-1β proteins in cells and Cleaved-Caspase 1 and Cleaved-IL-1β proteins in the supernatant, suggesting NLRP3 inflammasome activation. Rapamycin intervention alleviated these changes, demonstrating that the TSC2 gene regulation of microglial activation and NLRP3 inflammasome activation are correlated with mTOR phosphorylation. In conclusion, microglia are activated in TSC patients and participate in the NLRP3 inflammasome-associated neuroinflammatory response, and rapamycin treatment can alleviate these changes. Full article

Neurodegenerative diseases are characterized by a progressive process of degeneration and neuronal death in the nervous system, with neuroinflammation being one of the main factors contributing to the progression of these diseases. Aristotelia chilensis (Maqui) is a native tree of Chile used in the Mapuche folk medicine for wounds and digestive treatment. It produces edible black berries with the highest antioxidant capacity among berries, and the Mapuche people used it for producing an alcoholic beverage. The leaves of Maqui contain indole alkaloids with different pharmacological properties that suggest neuroprotective effects. Here, the isolation and chemical characterization of a new alkaloid, named 9-hydroxyaristoquinolone, and the evaluation of its anti-inflammatory activity in the microglial cell line HMC-3, treated with LPS, are reported. 9-Hydroxyaristoquinolone protects microglia from LPS-induced morphological changes at concentrations as low as 1 µM, with a reduction in IKBα-P levels and inhibition of the NF-κB pathway, which was assessed by THP-1 NF-κB dual cell reporter and Western blot in HMC-3 cells. In silico studies suggest that 9-hydroxyaristoquinolone does not induce hepatotoxicity or genotoxicity and exhibits BBB permeability. Full article

Background: A neuroinflammatory disease such as Alzheimer’s disease, presents a significant challenge in neurotherapeutics, particularly due to the complex etiology and allostatic factors, referred to as CNS stressors, that accelerate the development and progression of the disease. These CNS stressors include cerebral hypo-glucose metabolism, hyperinsulinemia, mitochondrial dysfunction, oxidative stress, impairment of neuronal autophagy, hypoxic insults and neuroinflammation. This study aims to explore the efficacy and safety of DAG-MAG-ΒHB, a novel ketone diester, in mitigating these risk factors by sustaining therapeutic ketosis, independent of conventional metabolic pathways. Methods: We evaluated the intestinal absorption of DAG-MAG-ΒHB and the metabolic impact in human microglial cells. Utilizing the HMC3 human microglia cell line, we examined the compound’s effect on cellular viability, Acetyl-CoA and ATP levels, and key metabolic enzymes under hypoglycemia. Additionally, we assessed the impact of DAG-AG-ΒHB on inflammasome activation, mitochondrial activity, ROS levels, inflammation and phagocytic rates. Results: DAG-MAG-ΒHB showed a high rate of intestinal absorption and no cytotoxic effect. In vitro, DAG-MAG-ΒHB enhanced cell viability, preserved morphological integrity, and maintained elevated Acetyl-CoA and ATP levels under hypoglycemic conditions. DAG-MAG-ΒHB increased the activity of BDH1 and SCOT, indicating ATP production via a ketolytic pathway. DAG-MAG-ΒHB showed remarkable resilience against low glucose condition by inhibiting NLRP3 inflammasome activation. Conclusions: In summary, DAG-MAG-ΒHB emerges as a promising treatment for neuroinflammatory conditions. It enhances cellular health under varying metabolic states and exhibits neuroprotective properties against low glucose conditions. These attributes indicate its potential as an effective component in managing neuroinflammatory diseases, addressing their complex progression. Full article

Microglia play a crucial role as immune cells responsible for the brain’s defense mechanisms. Similar to the actions of macrophages in the body, microglial cells elicit an inflammatory immune response in the brain. Recent papers highlight activated microglial cells as pivotal contributors to inflammatory responses in the brain, leading to damage to nerve tissue and the onset of Alzheimer’s disease (AD). In the brains of AD patients, elevated levels of inflammatory cytokines such as interleukin-6 (IL-6) and oxidized cholesterol metabolites (oxysterols) are observed. These factors are closely associated with inflammatory diseases in the brain. 4′-O-Methylalpinum isoflavone (mAI), derived from Cudrania tricuspidata fruit, possesses antioxidant, neuroprotective, and anti-inflammatory properties. Consequently, this study examined the effect of mAI on the expression of IL-6, a major inflammatory cytokine. The HMC3 microglial cell line was treated with oxysterols to assess the effectiveness of mAI in mitigating this inflammatory response. The results indicated that mAI inhibited the gene expression and protein secretion of IL-6 induced by 25-hydroxycholesterol (25OHChol) and 27-hydroxycholesterol (27OHChol). Furthermore, the expression of MHC class II, a marker for microglial activation, was reduced to baseline levels. These findings suggest that mAI may serve as a viable option for suppressing and treating brain inflammatory diseases induced by cholesterol oxidation products. This is achieved by curtailing the expression of the inflammatory cytokine resulting from the activation of microglial cells by immuno-oxysterol. Full article

Parkinson’s disease (PD) is a widespread age-related neurodegenerative disorder characterized by the presence of an aggregated protein, α-synuclein (α-syn), which is encoded by the SNCA gene and localized to presynaptic terminals in a normal human brain. The α-syn aggregation is induced by the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mitochondrial neurotoxin and is therefore used to mimic PD-like pathology in various in vitro and in vivo models. However, in vitro PD-like pathology using α-syn and MPTP in human microglial cells has not yet been reported. Malvidin-3-O-glucoside (M3G) is a major anthocyanin primarily responsible for pigmentation in various fruits and beverages and has been reported to possess various bioactivities. However, the neuroprotective effects of M3G in humanized in vitro PD-like pathologies have not been reported. Therefore, individual and co-treatments of α-syn and MPTP in a human microglial (HMC3) cell line were used to establish a humanized PD-like pathology model in vitro. The individual treatments were significantly less cytotoxic when compared to the α-syn and MPTP co-treatment. This study examined the neuroprotective effects of M3G by treating HMC3 cells with α-syn (8 μg/mL) and MPTP (2 mM) individually or in a co-treatment in the presence or absence of M3G (50 μM). M3G demonstrated anti-apoptotic, anti-inflammatory, and antioxidative properties against the α-syn- and MPTP-generated humanized in vitro PD-like pathology. This study determined that the cytoprotective effects of M3G are mediated by nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase (HO)-1 signaling. Full article

Microglia, essential immune cells in the brain, play crucial roles in neuroinflammation by performing various functions such as neurogenesis, synaptic pruning, and pathogen defense. These cells are activated by inflammatory factors like β-amyloid (Aβ) and oxysterols, leading to morphological and functional changes, including the secretion of inflammatory cytokines and the upregulation of MHC class II molecules. This study focused on identifying specific markers for microglial activation, with a particular emphasis on the roles of oxysterols in this process. We used the HMC3 human microglial cell line to investigate the induction of heat shock protein 60 (HSP60), a chaperonin protein by oxysterols, specifically in the presence of 25-hydroxycholesterol (25OHChol) and 27-hydroxycholesterol (27OHChol). Our findings obtained by the proteomics approach revealed that these oxysterols significantly increased HSP60 expression on microglial cells. This induction was further confirmed using Western blot analysis and immunofluorescence microscopy. Additionally, Aβ_1–42 also promoted HSP60 expression, indicating its role as a microglial activator. HSP60 involved in protein folding and immune modulation was identified as a potential marker for microglial activation. This study underscores the importance of HSP60 in the inflammatory response of microglia, suggesting its utility as a target for new therapeutic approaches in neuroinflammatory diseases such as Alzheimer’s disease (AD). Full article

Glucagon-like peptide-1 (GLP-1)-based drugs have been approved by the United States Food and Drug Administration (FDA) and are widely used to treat type 2 diabetes mellitus (T2DM) and obesity. More recent developments of unimolecular peptides targeting multiple incretin-related receptors (“multi-agonists”), including the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) and the glucagon (Gcg) receptor (GcgR), have emerged with the aim of enhancing drug benefits. In this study, we utilized human and mouse microglial cell lines, HMC3 and IMG, respectively, together with the human neuroblastoma SH-SY5Y cell line as cellular models of neurodegeneration. Using these cell lines, we studied the neuroprotective and anti-inflammatory capacity of several multi-agonists in comparison with a single GLP-1 receptor (GLP-1R) agonist, exendin-4. Our data demonstrate that the two selected GLP-1R/GIPR dual agonists and a GLP-1R/GIPR/GcgR triple agonist not only have neurotrophic and neuroprotective effects but also have anti-neuroinflammatory properties, as indicated by the decreased microglial cyclooxygenase 2 (COX2) expression, nitrite production, and pro-inflammatory cytokine release. In addition, our results indicate that these multi-agonists have the potential to outperform commercially available single GLP-1R agonists in neurodegenerative disease treatment. Full article

Microglia-mediated inflammatory response is one key cause of many central nervous system diseases, like Alzheimer’s disease. We hypothesized that a novel C15orf39 (MAPK1 substrate) plays a critical role in the microglial inflammatory response. To confirm this hypothesis, we used lipopolysaccharide (LPS)-and interferon-gamma (IFN-γ)-induced human microglia HMC3 cells as a representative indicator of the microglial in vitro inflammatory response. We found that C15orf39 was down-regulated when interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα) expression increased in LPS/IFN-γ-stimulated HMC3 cells. Once C15orf39 was overexpressed, IL-6 and TNFα expression were reduced in LPS/IFN-γ-stimulated HMC3 cells. In contrast, C15orf39 knockdown promoted IL-6 and TNFα expression in LPS/IFN-γ-stimulated HMC3 cells. These results suggest that C15orf39 is a suppressive factor in the microglial inflammatory response. Mechanistically, C15orf39 interacts with the cytoplasmic protein arginine methyltransferase 2 (PRMT2). Thus, we termed C15orf39 a PRMT2 interaction protein (PRMT2 IP). Furthermore, the interaction of C15orf39 and PRMT2 suppressed the activation of NF-κB signaling via the PRMT2-IκBα signaling axis, which then led to a reduction in transcription of the inflammatory factors IL6 and TNF-α. Under inflammatory conditions, NF-κBp65 was found to be activated and to suppress C15orf39 promoter activation, after which it canceled the suppressive effect of the C15orf39-PRMT2-IκBα signaling axis on IL-6 and TNFα transcriptional expression. In conclusion, our findings demonstrate that in a steady condition, the interaction of C15orf39 and PRMT2 stabilizes IκBα to inhibit IL-6 and TNFα expression by suppressing NF-κB signaling, which reversely suppresses C15orf39 transcription to enhance IL-6 and TNFα expression in the microglial inflammatory condition. Our study provides a clue as to the role of C15orf39 in microglia-mediated inflammation, suggesting the potential therapeutic efficacy of C15orf39 in some central nervous system diseases. Full article

Neurodegenerative diseases (NDDs) are progressive multifactorial disorders of the nervous system sharing common pathogenic features, including intracellular misfolded protein aggregation, mitochondrial deficit, and inflammation. Taking into consideration the multifaceted nature of NDDs, development of multitarget-directed ligands (MTDLs) has evolved as an attractive therapeutic strategy. Compounds that target the cannabinoid receptor type II (CB2R) are rapidly emerging as novel effective MTDLs against common NDDs, such as Alzheimer’s disease (AD). We recently developed the first CB2R bitopic/dualsteric ligand, namely FD22a, which revealed the ability to induce neuroprotection with fewer side effects. To explore the potential of FD22a as a multitarget drug for the treatment of NDDs, we investigated here its ability to prevent the toxic effect of β-amyloid (Aβ_25–35 peptide) on human cellular models of neurodegeneration, such as microglia (HMC3) and glioblastoma (U87-MG) cell lines. Our results displayed that FD22a efficiently prevented Aβ_25–35 cytotoxic and proinflammatory effects in both cell lines and counteracted β-amyloid-induced depression of autophagy in U87-MG cells. Notably, a quantitative proteomic analysis of U87-MG cells revealed that FD22a was able to potently stimulate the autophagy–lysosomal pathway (ALP) by activating its master transcriptional regulator TFEB, ultimately increasing the potential of this novel CB2R bitopic/dualsteric ligand as a multitarget drug for the treatment of NDDs. Full article

The unfolded protein response is an intricate system of sensor proteins in the endoplasmic reticulum (ER) that recognizes misfolded proteins and transmits information via transcription factors to either regain proteostasis or, depending on the severity, to induce apoptosis. The main transmembrane sensor is IRE1α, which contains cytoplasmic kinase and RNase domains relevant for its activation and the mRNA splicing of the transcription factor XBP1. Mast cell leukemia (MCL) is a severe form of systemic mastocytosis. The inhibition of IRE1α in the MCL cell line HMC-1.2 has anti-proliferative and pro-apoptotic effects, motivating us to elucidate the IRE1α interactors/regulators in HMC-1.2 cells. Therefore, the TurboID proximity labeling technique combined with MS analysis was applied. Gene Ontology and pathway enrichment analyses revealed that the majority of the enriched proteins are involved in vesicle-mediated transport, protein stabilization, and ubiquitin-dependent ER-associated protein degradation pathways. In particular, the AAA ATPase VCP and the oncoprotein MTDH as IRE1α-interacting proteins caught our interest for further analyses. The pharmacological inhibition of VCP activity resulted in the increased stability of IRE1α and MTDH as well as the activation of IRE1α. The interaction of VCP with both IRE1α and MTDH was dependent on ubiquitination. Moreover, MTDH stability was reduced in IRE1α-knockout cells. Hence, pharmacological manipulation of IRE1α–MTDH–VCP complex(es) might enable the treatment of MCL. Full article

Hyperchloremia and hypernatremia are associated with higher mortality in ischemic stroke, but it remains unclear whether their influence directly contributes to ischemic injury. We investigated the impact of 0.9% sodium chloride (154 mM NaCl), 0.9% sodium acetate (167 mM CH₃COONa), and their different combinations (3:1, 2:1, and 1:1) on microglial (HMC-3) and neuronal (differentiated SH-SY5Y) survival during oxygen–glucose deprivation/reperfusion (OGD/R). Further, we assessed the effect of hyperchloremia and hypernatremia-treated and OGD/R-induced HMC-3-conditioned media on differentiated SH-SY5Y cells under OGD/R conditions. We performed cell viability, cell toxicity, and nitric oxide (NO) release assays and studied the alteration in expression of caspase-1 and caspase-3 in different cell lines when exposed to hyperchloremia and hypernatremia. Cell survival was decreased in 0.9% NaCl, 0.9% CH₃COONa, combinations of HMC-3 and differentiated SH-SY5Y, and differentiated SH-SY5Y cells challenged with HMC-3-conditioned media under normal and OGD/R conditions. Under OGD/R conditions, differentiated SH-SY5Y cells were less likely to survive exposure to 0.9% NaCl. Expression of caspase-1 and caspase-3 in HMC-3 and differentiated SH-SY5Y cells was altered when exposed to 0.9% NaCl, 0.9% CH₃COONa, and their combinations. A total of 0.9% NaCl and 0.9% CH₃COONa and their combinations decreased the NO production in HMC-3 cells under normal and OGD/R conditions. Both hypernatremia and hyperchloremia reduced the survival of HMC-3 and differentiated SH-SY5Y cells under OGD/R conditions. Based on the OGD/R in vitro model that mimics human ischemic stroke conditions, it possibly provides a link for the increased death associated with hyperchloremia or hypernatremia in stroke patients. Full article

In this study, we investigated the beneficial effects of grapefruit IntegroPectin, derived from industrial waste grapefruit peels via hydrodynamic cavitation, on microglia cells exposed to oxidative stress conditions. Grapefruit IntegroPectin fully counteracted cell death and the apoptotic process induced by cell exposure to tert-butyl hydroperoxide (TBH), a powerful hydroperoxide. The protective effects of the grapefruit IntegroPectin were accompanied with a decrease in the amount of ROS, and were strictly dependent on the activation of the phosphoinositide 3-kinase (PI3K)/Akt cascade. Finally, IntegroPectin treatment inhibited the neuroinflammatory response and the basal microglia activation by down-regulating the PI3K- nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB)- inducible nitric oxide synthase (iNOS) cascade. These data strongly support further investigations aimed at exploring IntegroPectin’s therapeutic role in in vivo models of neurodegenerative disorders, characterized by a combination of chronic neurodegeneration, oxidative stress and neuroinflammation. Full article

A major problem in SARS-CoV-2-infected patients is the massive tissue inflammation in certain target organs, including the lungs. Mast cells (MC), basophils (BA), and eosinophils (EO) are key effector cells in inflammatory processes. These cells have recently been implicated in the pathogenesis of SARS-CoV-2 infections. We explored coronavirus receptor (CoV-R) expression profiles in primary human MC, BA, and EO, and in related cell lines (HMC-1, ROSA, MCPV-1, KU812, and EOL-1). As determined using flow cytometry, primary MC, BA, and EO, and their corresponding cell lines, displayed the CoV-R CD13 and CD147. Primary skin MC and BA, as well as EOL-1 cells, also displayed CD26, whereas primary EO and the MC and BA cell lines failed to express CD26. As assessed using qPCR, most cell lines expressed transcripts for CD13, CD147, and ABL2, whereas ACE2 mRNA was not detectable, and CD26 mRNA was only identified in EOL-1 cells. We also screened for drug effects on CoV-R expression. However, dexamethasone, vitamin D, and hydroxychloroquine did not exert substantial effects on the expression of CD13, CD26, or CD147 in the cells. Together, MC, BA, and EO express distinct CoV-R profiles. Whether these receptors mediate virus–cell interactions and thereby virus-induced inflammation remains unknown at present. Full article

Epigenetic dysregulation, particularly alterations in DNA methylation and hydroxymethylation, plays a pivotal role in cancer initiation and progression. Ten-eleven translocation (TET) proteins catalyze the successive oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidized methylcytosines in DNA, thereby serving as central modulators of DNA methylation–demethylation dynamics. TET loss of function is causally related to neoplastic transformation across various cell types while its genetic or pharmacological activation exhibits anti-cancer effects, making TET proteins promising targets for epigenetic cancer therapy. Here, we developed a robust cell-based screening system to identify novel TET activators and evaluated their potential as anti-cancer agents. Using a carefully curated library of 4533 compounds provided by the National Cancer Institute, Bethesda, MD, USA, we identified mitoxantrone as a potent TET agonist. Through rigorous validation employing various assays, including immunohistochemistry and dot blot studies, we demonstrated that mitoxantrone significantly elevated 5hmC levels. Notably, this elevation manifested only in wild-type (WT) but not TET-deficient mouse embryonic fibroblasts, primary bone marrow-derived macrophages, and leukemia cell lines. Furthermore, mitoxantrone-induced cell death in leukemia cell lines occurred in a TET-dependent manner, indicating the critical role of TET proteins in mediating its anti-cancer effects. Our findings highlight mitoxantrone’s potential to induce tumor cell death via a novel mechanism involving the restoration of TET activity, paving the way for targeted epigenetic therapies in cancer treatment. Full article

The behavior and migration of human mesenchymal stromal cells (hMSCs) are focal points of research in the biomedical field. One of the major aspects is potential therapy using hMCS, but at present, the safety of their use is still controversial owing to limited data on changes that occur with hMSCs in the long term. Fluorescent photoconvertible proteins are intensively used today as “gold standard” to mark the individual cells and study single-cell interactions, migration processes, and the formation of pure lines. A crucial disadvantage of this method is the need for genetic modification of the primary culture, which casts doubt on the possibility of exploring the resulting clones in personalized medicine. Here we present a new approach for labeling and tracking hMSCs without genetic modification based on the application of cell-internalizable photoconvertible polyelectrolyte microcapsules (size: 2.6 ± 0.5 μm). These capsules were loaded with rhodamine B, and after thermal treatment, exhibited fluorescent photoconversion properties. Photoconvertible capsules demonstrated low cytotoxicity, did not affect the immunophenotype of the hMSCs, and maintained a high level of fluorescent signal for at least seven days. The developed approach was tested for cell tracking for four days and made it possible to trace the destiny of daughter cells without the need for additional labeling. Full article