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BioRxiv : the Preprint Server For... Mar 2024Microglia are highly adaptable innate immune cells that rapidly respond to damage signals in the brain through adoption of a reactive phenotype and production of...
Microglia are highly adaptable innate immune cells that rapidly respond to damage signals in the brain through adoption of a reactive phenotype and production of defensive inflammatory cytokines. Microglia express a distinct transcriptome, encoding receptors that allow them to dynamically respond to pathogens, damage signals, and cellular debris. Expression of one such receptor, the microglia-specific purinergic receptor , is known to be downregulated in reactive microglia. Here, we explore the microglial response to purinergic damage signals in reactive microglia in the TMEV mouse model of viral brain infection and temporal lobe epilepsy. Using two-photon calcium imaging in acute hippocampal brain slices, we found that the ability of microglia to detect damage signals, engage calcium signaling pathways, and chemoattract towards laser-induced tissue damage was dramatically reduced during the peak period of seizures, cytokine production, and infection. Using combined RNAscope hybridization and immunohistochemistry, we found that during this same stage of heightened infection and seizures, microglial expression was reduced, while the pro-inflammatory cytokine expression was upregulated in microglia, suggesting that the depressed ability of microglia to respond to new damage signals via occurs during the time when local elevated cytokine production contributes to seizure generation following infection. Therefore, changes in microglial purinergic receptors during infection likely limit the ability of reactive microglia to respond to new threats in the CNS and locally contain the scale of the innate immune response in the brain.
PubMed: 38558969
DOI: 10.1101/2024.03.06.583768 -
The Journal of Toxicological Sciences 2024Vascular endothelial cells serve as barriers between blood components and subendothelial tissue and regulate the blood coagulation-fibrinolytic system. Ionizing...
Transcriptome analysis of cultured human vascular endothelial cells after γ-ray irradiation and correlation analysis with ATP, ADP, and adenosine as secondary soluble factors.
Vascular endothelial cells serve as barriers between blood components and subendothelial tissue and regulate the blood coagulation-fibrinolytic system. Ionizing radiation is a common physical stimulant that induces a bystander effect whereby irradiated cells influence neighboring cells through signalings, including purinergic receptor signaling, activated by adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine as secondary soluble factors. Human vascular endothelial EA.hy926 cells were cultured and irradiated with γ-rays or treated with ATP, ADP, or adenosine under non-toxic conditions. RNA-seq, gene ontology, and hierarchical clustering analyses were performed. The transcriptome analysis of differentially expressed genes in vascular endothelial cells after γ-ray irradiations suggests that the change of gene expression by γ-irradiation is mediated by ATP and ADP. In addition, the expression and activity of the proteins related to blood coagulation and fibrinolysis systems appear to be secondarily regulated by ATP and ADP in vascular endothelial cells after exposure to γ-irradiation. Although it is unclear whether the changes of the gene expression related to blood coagulation and fibrinolysis systems by γ-irradiation affected the increased hemorrhagic tendency through the exposure to γ-irradiation or the negative feedback to the activated blood coagulation system, the present data indicate that toxicity associated with γ-irradiation involves the dysfunction of vascular endothelial cells related to the blood coagulation-fibrinolytic system, which is mediated by the signalings, including purinergic receptor signaling, activated by ATP and ADP.
Topics: Humans; Adenosine; Endothelial Cells; Adenosine Triphosphate; Receptors, Purinergic; Gene Expression Profiling; Adenosine Diphosphate; Cells, Cultured
PubMed: 38556355
DOI: 10.2131/jts.49.193 -
The Journal of Biological Chemistry May 2024P2X receptors are a family of ligand gated ion channels found in a range of eukaryotic species including humans but are not naturally present in the yeast Saccharomyces...
P2X receptors are a family of ligand gated ion channels found in a range of eukaryotic species including humans but are not naturally present in the yeast Saccharomyces cerevisiae. We demonstrate the first recombinant expression and functional gating of the P2X2 receptor in baker's yeast. We leverage the yeast host for facile genetic screens of mutant P2X2 by performing site saturation mutagenesis at residues of interest, including SNPs implicated in deafness and at residues involved in native binding. Deep mutational analysis and rounds of genetic engineering yield mutant P2X2 F303Y A304W, which has altered ligand selectivity toward the ATP analog AMP-PNP. The F303Y A304W variant shows over 100-fold increased intracellular calcium amplitudes with AMP-PNP compared to the WT receptor and has a much lower desensitization rate. Since AMP-PNP does not naturally activate P2X receptors, the F303Y A304W P2X2 may be a starting point for downstream applications in chemogenetic cellular control. Interestingly, the A304W mutation selectively destabilizes the desensitized state, which may provide a mechanistic basis for receptor opening with suboptimal agonists. The yeast system represents an inexpensive, scalable platform for ion channel characterization and engineering by circumventing the more expensive and time-consuming methodologies involving mammalian hosts.
Topics: Humans; Amino Acid Substitution; Ligands; Protein Engineering; Receptors, Purinergic P2X2; Saccharomyces cerevisiae; Models, Molecular; Protein Structure, Tertiary; Protein Structure, Quaternary; Structural Homology, Protein; Mutation
PubMed: 38556082
DOI: 10.1016/j.jbc.2024.107248 -
Brain Sciences Feb 2024In the original publication [...].
Correction: Babiec et al. Alterations of Purinergic Receptors Levels and Their Involvement in the Glial Cell Morphology in a Pre-Clinical Model of Autism Spectrum Disorders. 2023, , 1088.
In the original publication [...].
PubMed: 38539686
DOI: 10.3390/brainsci14030233 -
Cells Mar 2024High blood levels of low-density lipoprotein (LDL)-cholesterol (LDL-C) are associated with atherosclerosis, mainly by promoting foam cell accumulation in vessels. As...
Low Density Lipoprotein Cholesterol Decreases the Expression of Adenosine A Receptor and Lipid Rafts-Protein Flotillin-1: Insights on Cardiovascular Risk of Hypercholesterolemia.
High blood levels of low-density lipoprotein (LDL)-cholesterol (LDL-C) are associated with atherosclerosis, mainly by promoting foam cell accumulation in vessels. As cholesterol is an essential component of cell plasma membranes and a regulator of several signaling pathways, LDL-C excess may have wider cardiovascular toxicity. We examined, in untreated hypercholesterolemia (HC) patients, selected regardless of the cause of LDL-C accumulation, and in healthy participants (HP), the expression of the adenosine A receptor (AR), an anti-inflammatory and vasodilatory protein with cholesterol-dependent modulation, and Flotillin-1, protein marker of cholesterol-enriched plasma membrane domains. Blood cardiovascular risk and inflammatory biomarkers were measured. AR and Flotillin-1 expression in peripheral blood mononuclear cells (PBMC) was lower in patients compared to HP and negatively correlated to LDL-C blood levels. No other differences were observed between the two groups apart from transferrin and ferritin concentrations. AR and Flotillin-1 proteins levels were positively correlated in the whole study population. Incubation of HP PBMCs with LDL-C caused a similar reduction in AR and Flotillin-1 expression. We suggest that LDL-C affects AR expression by impacting cholesterol-enriched membrane microdomains. Our results provide new insights into the molecular mechanisms underlying cholesterol toxicity, and may have important clinical implication for assessment and treatment of cardiovascular risk in HC.
Topics: Humans; Hypercholesterolemia; Cholesterol, LDL; Receptor, Adenosine A2A; Leukocytes, Mononuclear; Adenosine; Cardiovascular Diseases; Risk Factors; Cholesterol; Carrier Proteins; Heart Disease Risk Factors; Membrane Microdomains; Membrane Proteins
PubMed: 38534331
DOI: 10.3390/cells13060488 -
Scientific Reports Mar 2024Type 2 diabetes (T2D) can cause severe cardiac complications at functional, histologic and molecular levels. These pathological complications could be mediated by...
Type 2 diabetes (T2D) can cause severe cardiac complications at functional, histologic and molecular levels. These pathological complications could be mediated by ATP-releasing channels such as Panx1 and ATP receptors, in particular P2X7. The aim of our study was to investigate the effect of high-intensity interval training (HIIT) on T2D-induced cardiac complications at the functional, histopathological and molecular levels, with a particular focus on ATP-releasing channels. 48 male Wistar rats at the age of 8 weeks were randomly allocated into four groups: control (Con), Diabetes (T2D), Training (TR), and Diabetes + Training (T2D + TR). T2D was induced by a high-fat diet plus a low dose (35 mg/kg) of STZ administration. Rats in the TR and T2D + TR groups underwent an 8-weeks training program involving intervals ranging from 80 to 100% of their maximum running speed (Vmax), with 4-10 intervals per session. Protein expression of Interleukin 1β (IL1β), Interleukin 10 (IL-10), Pannexin 1 (Panx1), P2X7R (purinergic P2X receptor 7), NLRP1 (NLR Family Pyrin Domain Containing 1), BAX, and Bcl2 were measured in the heart tissue. Additionally, we assessed heart function, histopathological changes, as well as insulin resistance using the homeostasis model assessment of insulin resistance (HOMA-IR). In contrast to the T2D group, HIIT led to increased protein expression of Bcl2 and IL-10 in the heart. It also resulted in improvements in systolic and diastolic blood pressures, heart rate, ± dp/dt (maximum and minimum changes in left ventricular pressure), while reducing protein expression of IL-1β, Panx1, P2X7R, NLRP1, and BAX levels in the heart. Furthermore, left ventricular diastolic pressure (LVDP) was reduced (P ≤ 0.05). Moreover, heart lesion scores increased with T2D but decreased with HIIT, along with a reduction in fibrosis percentage (P ≤ 0.05). The results of this study suggest that the cardioprotective effects of HIIT on the diabetic heart may be mediated by the modulation of ATP-releasing channels. This modulation may lead to a reduction in inflammation and apoptosis, improve cardiac function, and attenuate cardiac injury and fibrosis.
Topics: Male; Rats; Animals; Diabetes Mellitus, Type 2; Insulin Resistance; High-Intensity Interval Training; Interleukin-10; bcl-2-Associated X Protein; Rats, Wistar; Fibrosis; Adenosine Triphosphate
PubMed: 38532054
DOI: 10.1038/s41598-024-57818-0 -
Journal of Neuroinflammation Mar 2024Guillain-Barré syndrome (GBS), a post-infectious, immune-mediated, acute demyelinating disease of the peripheral nerves and nerve roots, represents the most prevalent...
BACKGROUND
Guillain-Barré syndrome (GBS), a post-infectious, immune-mediated, acute demyelinating disease of the peripheral nerves and nerve roots, represents the most prevalent and severe acute paralyzing neuropathy. Purinergic P2X7 receptors (P2X7R) play a crucial role in central nervous system inflammation. However, little is known about their role in the immune-inflammatory response within the peripheral nervous system.
METHODS
Initially, we assessed the expression of purinergic P2X7R in the peripheral blood of patients with GBS using flow cytometry and qRT-PCR. Next, we explored the expression of P2 X7R in CD4 T cells, CD8 T cells, and macrophages within the sciatic nerves and spleens of rats using immunofluorescence labeling and flow cytometry. The P2X7R antagonist brilliant blue G (BBG) was employed to examine its therapeutic impact on rats with experimental autoimmune neuritis (EAN) induced by immunization with the P0 peptide. We analyzed CD4 T cell differentiation in splenic mononuclear cells using flow cytometry, assessed Th17 cell differentiation in the sciatic nerve through immunofluorescence staining, and examined the expression of pro-inflammatory cytokine mRNA using RT-PCR. Additionally, we performed protein blotting to assess the expression of P2X7R and NLRP3-related inflammatory proteins within the sciatic nerve. Lastly, we utilized flow cytometry and immunofluorescence labeling to examine the expression of NLRP3 on CD4 T cells in rats with EAN.
RESULTS
P2X7R expression was elevated not only in the peripheral blood of patients with GBS but also in rats with EAN. In rats with EAN, inhibiting P2X7R with BBG alleviated neurological symptoms, reduced demyelination, decreased inflammatory cell infiltration of the peripheral nerves, and improved nerve conduction. BBG also limited the production of pro-inflammatory molecules, down-regulated the expression of P2X7R and NLRP3, and suppressed the differentiation of Th1 and Th17 cells, thus protecting against EAN. These effects collectively contribute to modifying the inflammatory environment and enhancing outcomes in EAN rats.
CONCLUSIONS
Suppression of P2X7R relieved EAN manifestation by regulating CD4 T cell differentiation and NLRP3 inflammasome activation. This finding underscores the potential significance of P2X7R as a target for anti-inflammatory treatments, advancing research and management of GBS.
Topics: Animals; Humans; Rats; CD8-Positive T-Lymphocytes; Cell Differentiation; Guillain-Barre Syndrome; Inflammasomes; Neuritis, Autoimmune, Experimental; NLR Family, Pyrin Domain-Containing 3 Protein; Purinergic P2X Receptor Antagonists; Sciatic Nerve; Th17 Cells; Th1 Cells
PubMed: 38528529
DOI: 10.1186/s12974-024-03057-z -
Pharmacological Research May 2024Cardiovascular diseases (CVDs) are the leading cause of death worldwide, and morbidity and mortality rates continue to rise. Atherosclerosis constitutes the principal...
Cardiovascular diseases (CVDs) are the leading cause of death worldwide, and morbidity and mortality rates continue to rise. Atherosclerosis constitutes the principal etiology of CVDs. Endothelial injury, inflammation, and dysfunction are the initiating factors of atherosclerosis. Recently, we reported that endothelial adenosine receptor 2 A (ADORA2A), a G protein-coupled receptor (GPCR), plays critical roles in neovascularization disease and cerebrovascular disease. However, the precise role of endothelial ADORA2A in atherosclerosis is still not fully understood. Here, we showed that ADORA2A expression was markedly increased in the aortic endothelium of humans with atherosclerosis or Apoe mice fed a high-cholesterol diet. In vivo studies unraveled that endothelial-specific Adora2a deficiency alleviated endothelial-to-mesenchymal transition (EndMT) and prevented the formation and instability of atherosclerotic plaque in Apoe mice. Moreover, pharmacologic inhibition of ADORA2A with KW6002 recapitulated the anti-atherogenic phenotypes observed in genetically Adora2a-deficient mice. In cultured human aortic endothelial cells (HAECs), siRNA knockdown of ADORA2A or KW6002 inhibition of ADORA2A decreased EndMT, whereas adenoviral overexpression of ADORA2A induced EndMT. Mechanistically, ADORA2A upregulated ALK5 expression via a cAMP/PKA/CREB axis, leading to TGFβ-Smad2/3 signaling activation, thereby promoting EndMT. In conclusion, these findings, for the first time, demonstrate that blockade of ADORA2A attenuated atherosclerosis via inhibition of EndMT induced by the CREB1-ALK5 axis. This study discloses a new link between endothelial ADORA2A and EndMT and indicates that inhibiting endothelial ADORA2A could be an effective novel strategy for the prevention and treatment of atherosclerotic CVDs.
Topics: Animals; Humans; Male; Mice; Adenosine A2 Receptor Antagonists; Atherosclerosis; Cells, Cultured; Cyclic AMP Response Element-Binding Protein; Endothelial Cells; Epithelial-Mesenchymal Transition; Mice, Inbred C57BL; Mice, Knockout; Receptor, Adenosine A2A; Receptor, Transforming Growth Factor-beta Type I; Signal Transduction
PubMed: 38522762
DOI: 10.1016/j.phrs.2024.107156 -
Journal of Orthopaedic Surgery and... Mar 2024The lack of effective understanding of the pain mechanism of McCune-Albright syndrome (MAS) has made the treatment of pain in this disease a difficult clinical... (Review)
Review
BACKGROUND
The lack of effective understanding of the pain mechanism of McCune-Albright syndrome (MAS) has made the treatment of pain in this disease a difficult clinical challenge, and new therapeutic targets are urgently needed to address this dilemma.
OBJECTIVE
This paper summarizes the novel mechanisms, targets, and treatments that may produce pain in MAS and fibrous dysplasia (polyfibrous dysplasia, or FD).
METHODS
We conducted a systematic search in the PubMed database, Web of Science, China Knowledge Network (CNKI) with the following keywords: "McCune-Albright syndrome (MAS); polyfibrous dysplasia (FD); bone pain; bone remodeling; G protein coupled receptors; GDNF family receptors; purinergic receptors and glycogen synthase kinase", as well as other keywords were systematically searched. Papers published between January 2018 and May 2023 were selected for finding. Initial screening was performed by reading the titles and abstracts, and available literature was screened against the inclusion and exclusion criteria.
RESULTS
In this review, we systematically analyzed the cutting-edge advances in this disease, synthesized the findings, and discussed the differences. With regard to the complete mechanistic understanding of the pain condition in FD/MAS, in particular, we collated new findings on new pathways, neurotrophic factor receptors, purinergic receptors, interferon-stimulating factors, potassium channels, protein kinases, and corresponding hormonal modulation and their respective strengths and weaknesses.
CONCLUSION
This paper focuses on basic research to explore FD/MAS pain mechanisms. New nonneuronal and molecular mechanisms, mechanically loaded responsive neurons, and new targets for potential clinical interventions are future research directions, and a large number of animal experiments, tissue engineering techniques, and clinical trials are still needed to verify the effectiveness of the targets in the future.
Topics: Animals; Fibrous Dysplasia, Polyostotic; Fibrous Dysplasia of Bone; Pain; Bone Remodeling; China
PubMed: 38515135
DOI: 10.1186/s13018-024-04687-y -
Cell Reports. Medicine Mar 2024Metabolic (dysfunction)-associated steatohepatitis (MASH) is the advanced stage of metabolic (dysfunction)-associated fatty liver disease (MAFLD) lacking approved...
Metabolic (dysfunction)-associated steatohepatitis (MASH) is the advanced stage of metabolic (dysfunction)-associated fatty liver disease (MAFLD) lacking approved clinical drugs. Adenosine A1 receptor (AR), belonging to the G-protein-coupled receptors (GPCRs) superfamily, is mainly distributed in the central nervous system and major peripheral organs with wide-ranging physiological functions; however, the exact role of hepatic AR in MAFLD remains unclear. Here, we report that liver-specific depletion of AR aggravates while overexpression attenuates diet-induced metabolic-associated fatty liver (MAFL)/MASH in mice. Mechanistically, activation of hepatic AR promotes the competitive binding of sterol-regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) to sequestosome 1 (SQSTM1), rather than protein kinase A (PKA) leading to SCAP degradation in lysosomes. Reduced SCAP hinders SREBP1c/2 maturation and thus suppresses de novo lipogenesis and inflammation. Higher hepatic AR expression is observed in patients with MAFL/MASH and high-fat diet (HFD)-fed mice, which is supposed to be a physiologically adaptive response because AR agonists attenuate MAFL/MASH in an AR-dependent manner. These results highlight that hepatic AR is a potential target for MAFL/MASH therapy.
Topics: Humans; Mice; Animals; Receptor, Adenosine A1; Fatty Liver; Lipogenesis; Diet, High-Fat
PubMed: 38508143
DOI: 10.1016/j.xcrm.2024.101477