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Neural Regeneration Research May 2024Recent studies have revealed that lipid droplets accumulate in neurons after brain injury and evoke lipotoxicity, damaging the neurons. However, how lipids are...
Recent studies have revealed that lipid droplets accumulate in neurons after brain injury and evoke lipotoxicity, damaging the neurons. However, how lipids are metabolized by spinal cord neurons after spinal cord injury remains unclear. Herein, we investigated lipid metabolism by spinal cord neurons after spinal cord injury and identified lipid-lowering compounds to treat spinal cord injury. We found that lipid droplets accumulated in perilesional spinal cord neurons after spinal cord injury in mice. Lipid droplet accumulation could be induced by myelin debris in HT22 cells. Myelin debris degradation by phospholipase led to massive free fatty acid production, which increased lipid droplet synthesis, β-oxidation, and oxidative phosphorylation. Excessive oxidative phosphorylation increased reactive oxygen species generation, which led to increased lipid peroxidation and HT22 cell apoptosis. Bromocriptine was identified as a lipid-lowering compound that inhibited phosphorylation of cytosolic phospholipase A2 by reducing the phosphorylation of extracellular signal-regulated kinases 1/2 in the mitogen-activated protein kinase pathway, thereby inhibiting myelin debris degradation by cytosolic phospholipase A2 and alleviating lipid droplet accumulation in myelin debris-treated HT22 cells. Motor function, lipid droplet accumulation in spinal cord neurons and neuronal survival were all improved in bromocriptine-treated mice after spinal cord injury. The results suggest that bromocriptine can protect neurons from lipotoxic damage after spinal cord injury via the extracellular signal-regulated kinases 1/2-cytosolic phospholipase A2 pathway.
PubMed: 37862220
DOI: 10.4103/1673-5374.385308 -
Environmental Pollution (Barking, Essex... Nov 2023Excessive amounts of iron (Fe), zinc (Zn), and copper (Cu) can be toxic to neuronal cells, even though these are essential trace elements for animals and humans....
Excessive amounts of iron (Fe), zinc (Zn), and copper (Cu) can be toxic to neuronal cells, even though these are essential trace elements for animals and humans. However, the precise mechanisms underlying the neurotoxicity of exposure to mixtures of Fe, Zn, and Cu are still mostly unclear. The research aimed to investigate the influence of co-exposure to iron, zinc and copper and the related mechanisms in HT22 murine hippocampal neuronal cells. Intracellular metal content, markers of oxidative damage, and biomarkers of ferroptosis were respectively detected. Afterward, metabolomic analyses were performed to obtain a comprehensive understanding of the metal mixtures on metabolism, and the functions of key enzymes on metabolic pathways were validated. The results showed that metal co-exposure resulted in cellular iron overload and increased lipid peroxidation, accompanied by significant pathological damage and mitochondrial abnormalities in HT22 cells. Meanwhile, it was found that GSH depletion, decreased GPX4, and increased expression of the lipid metabolism gene ACSL4 play important roles in ferroptosis induced by metal mixture. Further, metabolomic analysis revealed metal co-exposure induced significant alterations in metabolite levels, especially in the glycerophospholipid metabolism pathway and the arachidonic acid metabolism pathway. The levels of cPLA2 and its metabolite, arachidonic acid, were significantly increased after metal co-exposure. Then, inhibition of cPLA2 decreased the level of arachidonic acid and attenuated ferroptosis in neuronal cells. Collectively, our findings unveiled ferroptosis induced by metal co-exposure associated with crucial molecular changes in neuronal cells, providing a novel perspective on the comprehensive toxicity risk assessment of metal mixtures.
Topics: Humans; Mice; Animals; Zinc; Copper; Ferroptosis; Lipid Metabolism; Arachidonic Acid; Iron; Metals; Lipid Metabolism Disorders; Phospholipases A2, Cytosolic
PubMed: 37625769
DOI: 10.1016/j.envpol.2023.122438 -
Cell Reports Sep 2023Most gastrointestinal stromal tumors (GISTs) develop due to gain-of-function mutations in the tyrosine kinase gene, KIT. We recently showed that mutant KIT mislocalizes...
Most gastrointestinal stromal tumors (GISTs) develop due to gain-of-function mutations in the tyrosine kinase gene, KIT. We recently showed that mutant KIT mislocalizes to the Golgi area and initiates uncontrolled signaling. However, the molecular mechanisms underlying its Golgi retention remain unknown. Here, we show that protein kinase D2 (PKD2) is activated by the mutant, which causes Golgi retention of KIT. In PKD2-inhibited cells, KIT migrates from the Golgi region to lysosomes and subsequently undergoes degradation. Importantly, delocalized KIT cannot trigger downstream activation. In the Golgi/trans-Golgi network (TGN), KIT activates the PKD2-phosphatidylinositol 4-kinase IIIβ (PKD2-PI4KIIIβ) pathway through phospholipase Cγ2 (PLCγ2) to generate a PI4P-rich membrane domain, where the AP1-GGA1 complex is aberrantly recruited. Disruption of any factors in this cascade results in the release of KIT from the Golgi/TGN. Our findings show the molecular mechanisms underlying KIT mislocalization and provide evidence for a strategy for inhibition of oncogenic signaling.
Topics: Humans; Gastrointestinal Stromal Tumors; Protein Kinase D2; Phospholipase C gamma; Golgi Apparatus; trans-Golgi Network; Proto-Oncogene Proteins c-kit
PubMed: 37616163
DOI: 10.1016/j.celrep.2023.113035 -
Kidney International Nov 2023Much akin to the explosion in number of known target antigens in membranous nephropathy, there has been a rapid expansion in the availability of animal models involving...
Much akin to the explosion in number of known target antigens in membranous nephropathy, there has been a rapid expansion in the availability of animal models involving the first 2 antigens discovered in adult disease, phospholipase A2 receptor and thrombospondin type 1 domain-containing 7A. In this issue, Tomas et al. describe a novel mouse model of phospholipase A2 receptor-associated membranous nephropathy that shows great promise for investigating molecular mechanisms of disease and as an experimental system for testing existing and emerging therapies.
Topics: Animals; Mice; Glomerulonephritis, Membranous; Receptors, Phospholipase A2; Disease Models, Animal; Autoantibodies; Thrombospondins
PubMed: 37863634
DOI: 10.1016/j.kint.2023.09.003 -
Proceedings of the National Academy of... Feb 2024A missense variant in patatin-like phospholipase domain-containing protein 3 [PNPLA3(I148M)] is the most impactful genetic risk factor for fatty liver disease (FLD). We...
A missense variant in patatin-like phospholipase domain-containing protein 3 [PNPLA3(I148M)] is the most impactful genetic risk factor for fatty liver disease (FLD). We previously showed that PNPLA3 is ubiquitylated and subsequently degraded by proteasomes and autophagosomes and that the PNPLA3(148M) variant interferes with this process. To define the machinery responsible for PNPLA3 turnover, we used small interfering (si)RNAs to inactivate components of the ubiquitin proteasome system. Inactivation of bifunctional apoptosis regulator (BFAR), a membrane-bound E3 ubiquitin ligase, reproducibly increased PNPLA3 levels in two lines of cultured hepatocytes. Conversely, overexpression of BFAR decreased levels of endogenous PNPLA3 in HuH7 cells. BFAR and PNPLA3 co-immunoprecipitated when co-expressed in cells. BFAR promoted ubiquitylation of PNPLA3 in vitro in a reconstitution assay using purified, epitope-tagged recombinant proteins. To confirm that BFAR targets PNPLA3, we inactivated in mice. Levels of PNPLA3 protein were increased twofold in hepatic lipid droplets of mice with no associated increase in PNPLA3 mRNA levels. Taken together these data are consistent with a model in which BFAR plays a role in the post-translational degradation of PNPLA3. The identification of BFAR provides a potential target to enhance PNPLA3 turnover and prevent FLD.
Topics: Animals; Mice; Acyltransferases; Hepatocytes; Non-alcoholic Fatty Liver Disease; Phospholipases A2, Calcium-Independent; Ubiquitin; Ubiquitin-Protein Ligases; Membrane Proteins; Adaptor Proteins, Signal Transducing; Apoptosis Regulatory Proteins; Humans; Cell Line, Tumor
PubMed: 38294943
DOI: 10.1073/pnas.2312291121 -
Phytotherapy Research : PTR Dec 2023Acute kidney injury (AKI) is a common clinical condition associated with increased incidence and mortality rates. Hederasaponin C (HSC) is one of the main active...
Acute kidney injury (AKI) is a common clinical condition associated with increased incidence and mortality rates. Hederasaponin C (HSC) is one of the main active components of Pulsatilla chinensis (Bunge) Regel. HSC possesses various pharmacological activities, including anti-inflammatory activity. However, the protective effect of HSC against lipopolysaccharide (LPS)-induced AKI in mice remains unclear. Therefore, we investigated the protective effect of HSC against LPS-induced renal inflammation and the underlying molecular mechanisms. Herein, using MTT and LDH assays to assess both cell viability and LDH activity; using dual staining techniques to identify different cell death patterns; conducting immunoblotting, QRT-PCR, and immunofluorescence analyses to evaluate levels of protein and mRNA expression; employing immunoblotting, molecular docking, SPR experiments, and CETSA to investigate the interaction between HSC and TLR4; and studying the anti-inflammatory effects of HSC in the LPS-induced AKI. The results indicate that HSC inhibits the expression of TLR4 and the activation of NF-κB and PIP2 signaling pathways, while simultaneously suppressing the activation of the NLRP3 inflammasome. In animal models, HSC ameliorated LPS-induced AKI and diminished inflammatory response and the level of renal injury markers. These findings suggest that HSC has potential as a therapeutic agent to mitigate sepsis-related AKI.
Topics: Animals; Mice; Acute Kidney Injury; Anti-Inflammatory Agents; Lipopolysaccharides; Molecular Docking Simulation; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Signal Transduction; Toll-Like Receptor 4; Saponins; Phosphoinositide Phospholipase C
PubMed: 37778741
DOI: 10.1002/ptr.8014 -
American Journal of Kidney Diseases :... Mar 2024Rituximab is the first-choice therapy for patients with primary membranous nephropathy (MN) and nephrotic syndrome. However, approximately 30% of patients are...
RATIONALE & OBJECTIVE
Rituximab is the first-choice therapy for patients with primary membranous nephropathy (MN) and nephrotic syndrome. However, approximately 30% of patients are treatment-resistant or become treatment-intolerant with hypersensitivity reactions upon repeated drug exposures. We aimed to assess whether ofatumumab, a fully human second-generation anti-CD20 antibody, could be a valuable alternative to rituximab in this population.
STUDY DESIGN
Case series.
SETTING & PARTICIPANTS
7 rituximab-intolerant and 10 rituximab-resistant patients with MN who consented to receive ofatumumab (50-300mg, single intravenous infusion) and were followed at the nephrology unit of Azienda Socio-Sanitaria Territoriale Papa Giovanni XXIII (Bergamo, Italy) between September 2015 and January 2019.
FINDINGS
Over a median (IQR) follow-up of 5.0 (3.0-9.8) months, all 7 rituximab-intolerant and 3 of the 10 rituximab-resistant patients exhibited complete (proteinuria<0.3g/d) or partial (proteinuria<3.5g/d with≥50% reduction vs baseline) remission of nephrotic syndrome. Circulating B cells were similarly depleted in all patients by 1 week, and serum anti-phospholipase A receptor antibody concentrations decreased to<2.7 relative units/mL in 3 of 4 rituximab-intolerant and 4 of 8 rituximab-resistant patients with phospholipase A receptor-related disease. Ofatumumab significantly reduced 24-hour urinary protein and immunoglobulin G excretion and increased serum albumin and immunoglobulin G levels. These effects were greater in rituximab-intolerant than in rituximab-resistant patients. Measured glomerular filtration rate significantly increased by an average of 13.4% at 24 months compared with baseline (P=0.036) among all patients in the series. There were 14 nonserious infusion-related adverse events in 9 patients that recovered with temporary infusion interruption.
LIMITATIONS
Retrospective design, limited number of patients.
CONCLUSIONS
Ofatumumab may represent an effective and safe treatment for rituximab-intolerant cases of MN. Larger prospective studies will be needed to validate these preliminary findings and explore the effectiveness of other second-generation anti-CD20 antibodies in this clinical setting.
PLAIN-LANGUAGE SUMMARY
Primary membranous nephropathy (MN) is one of the most frequent causes of nephrotic syndrome (NS) in adults. In this case series, we explored the efficacy of ofatumumab, a fully human second-generation anti-CD20 antibody, in 17 patients with MN and NS who were intolerant or unresponsive to rituximab. All 7 rituximab-intolerant patients exhibited complete or partial clinical remission, compared with only 3 of the 10 rituximab-resistant patients. Autoantibody levels decreased in all patients with phospholipase A receptor-related disease. Ofatumumab achieved a significant reduction in urinary protein and immunoglobulin G excretion while increasing serum albumin and immunoglobulin G levels. Ofatumumab may be a promising option for patients with MN who are rituximab-intolerant. Further investigations are warranted to validate these preliminary findings.
Topics: Adult; Humans; Rituximab; Nephrotic Syndrome; Glomerulonephritis, Membranous; Retrospective Studies; Prospective Studies; Antibodies, Monoclonal, Humanized; Immunoglobulin G; Proteinuria; Serum Albumin; Phospholipases; Immunosuppressive Agents; Receptors, Phospholipase A2
PubMed: 37777061
DOI: 10.1053/j.ajkd.2023.08.010 -
Proceedings of the National Academy of... Apr 2024Nonalcoholic fatty liver disease, recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with...
Nonalcoholic fatty liver disease, recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene , encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD. Despite its discovery 20 y ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3-I148M and characterize changes induced by endogenous expression of the disease-causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3-I148M are not endoplasmic reticulum-resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3-I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3-I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild-type variant, PNPLA3-I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet-Golgi contact sites, which were also observed in I148M-expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3-I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3-I148M biology and identify the Golgi apparatus as a central hub of PNPLA3-I148M-driven cellular change.
Topics: Humans; Acyltransferases; Golgi Apparatus; Lipase; Lipid Droplets; Membrane Proteins; Non-alcoholic Fatty Liver Disease; Phospholipases A2, Calcium-Independent
PubMed: 38657050
DOI: 10.1073/pnas.2318619121 -
International Journal of Molecular... Aug 2023Phospholipase C (PLC) enzymes represent crucial participants in the plasma membrane of mammalian cells, including the cardiac sarcolemmal (SL) membrane of... (Review)
Review
Phospholipase C (PLC) enzymes represent crucial participants in the plasma membrane of mammalian cells, including the cardiac sarcolemmal (SL) membrane of cardiomyocytes. They are responsible for the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P) into 1,2-diacylglycerol (DAG) and inositol (1,4,5) trisphosphate (Ins(1,4,5)P), both essential lipid mediators. These second messengers regulate the intracellular calcium (Ca) concentration, which activates signal transduction cascades involved in the regulation of cardiomyocyte activity. Of note, emerging evidence suggests that changes in cardiomyocytes' phospholipid profiles are associated with an increased occurrence of cardiovascular diseases, but the underlying mechanisms are still poorly understood. This review aims to provide a comprehensive overview of the significant impact of PLC on the cardiovascular system, encompassing both physiological and pathological conditions. Specifically, it focuses on the relevance of PLCβ isoforms as potential cardiac biomarkers, due to their implications for pathological disorders, such as cardiac hypertrophy, diabetic cardiomyopathy, and myocardial ischemia/reperfusion injury. Gaining a deeper understanding of the mechanisms underlying PLCβ activation and regulation is crucial for unraveling the complex signaling networks involved in healthy and diseased myocardium. Ultimately, this knowledge holds significant promise for advancing the development of potential therapeutic strategies that can effectively target and address cardiac disorders by focusing on the PLCβ subfamily.
Topics: Animals; Humans; Isoenzymes; Phospholipase C beta; Heart Diseases; Myocytes, Cardiac; Biomarkers; Mammals
PubMed: 37685903
DOI: 10.3390/ijms241713096 -
Renal Failure Dec 2023Hepatitis B virus-associated glomerulonephritis (HBV-GN) is one of the main types of secondary glomerular diseases, and podocyte injury is an important pathogenic...
INTRODUCTION
Hepatitis B virus-associated glomerulonephritis (HBV-GN) is one of the main types of secondary glomerular diseases, and podocyte injury is an important pathogenic mechanism of HBV-GN, participating in the occurrence and development of HBV-GN. However, the specific mechanism of podocyte injury remains to be studied.
METHODS
Human renal podocytes cultured were divided into six groups. The podocyte morphology was observed under a transmission electron microscope, and the expression of M-type phospholipase A receptor (M-PLAR) on the podocyte membrane was observed by indirect immunofluorescence staining under a fluorescence microscope. The pyroptosis rate and reactive oxygen species (ROS) of podocytes were assessed by FLICA/PI double staining and flow cytometry. Western blot (WB) and quantitative real-time PCR (qPCR) were used to determine the expression of PLAR, nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing card (ASC), caspase-1, IL-1β, and IL-18.
RESULTS
Hepatitis B virus X (HBx) transfected into human renal podocytes induced the overexpression of PLAR. Moreover, the overexpressed PLAR combined with secretory phospholipase A group IB (sPLA-IB) aggravated podocyte injury and increased the pyroptosis rate. In addition, the expression of ROS, the NLRP3 inflammasome and downstream inflammatory factors was increased. In contrast, after inhibiting the expression of PLAR and ROS, podocyte damage was alleviated, and the pyroptosis rate and the expression of genes related to the ROS-NLRP3 signaling pathway were decreased.
CONCLUSION
HBx-induced PLAR overexpression on the podocyte membrane can significantly upregulate the ROS-NLRP3 signaling pathway, thereby mediating podocyte pyroptosis.
Topics: Humans; Podocytes; NLR Family, Pyrin Domain-Containing 3 Protein; Pyroptosis; Reactive Oxygen Species; Signal Transduction; Phospholipases; Polyesters
PubMed: 36698326
DOI: 10.1080/0886022X.2023.2170808