Plasma Membrane Rafts Research Articles

Inhibiting the cholesterol storage enzyme ACAT1/SOAT1 in aging Apolipoprotein E4 mice alter their brains inflammatory profiles.

Aging and Apolipoprotein E4 (APOE4) are the two most significant risk factors for late-onset Alzheimer's disease (LOAD). Compared to APOE3, APOE4 disrupts cholesterol homeostasis, increases cholesteryl esters (CEs), and exacerbates neuroinflammation in brain cells including microglia. Targeting CEs and neuroinflammation could be a novel strategy to ameliorate APOE4 dependent phenotypes. Toll-like receptor 4 (TLR4) is a key player in inflammation, its regulation is associated with cholesterol content of lipid rafts in cell membranes. We previously demonstrated that in normal microglia expressing APOE3, inhibiting the cholesterol storage enzyme acylCoA:cholesterol acyltransferase 1 (ACAT1/SOAT1) reduces CEs, dampened neuroinflammation via modulating the fate of TLR4. We also showed that treating myelin debris-loaded normal microglia with ACAT inhibitor F12511 reduced cellular CEs and activated ABC transporter 1 (ABCA1) for cholesterol efflux. In this study, we found that treating primary microglia expressing APOE4 with F12511 also reduces CEs, activated ABCA1, and dampened LPS dependent NFkB activation. In vivo, a two-week injections of nanoparticle F12511, which consists of DSPE-PEG 2000 , phosphatidylcholine, and F12511, to aged female APOE4 mice reduced TLR4 protein content and decreased proinflammatory cytokines including IL-1β in APOE4 mice brains. Overall, our work suggests nanoparticle F12511 is a novel agent to ameliorate LOAD.

The causal relationship between gut microbiota and diabetic neuropathy: a bi-directional two-sample Mendelian randomization study.

Many studies suggest a strong correlation between gut microbiota (GM) and diabetic neuropathy (DN). However, the precise causal relationship between GM and DN has yet to be fully elucidated. Hence, a bi-directional Mendelian randomization (MR) analysis was used to examine the association between GM and DN. Widely known genome-wide association study (GWAS) of GM was collected from the MiBio Gen project. Summary-level datasets for DN were taken from the FinnGen project. Inverse variance weighted approach was used for evaluating the causal relationship between GM and DN. Subsequently, pleiotropy and heterogeneity tests were performed to verify the reliability of the data. Furthermore, a bidirectional two-sample MR analysis was done to investigate the directionality of the causal relationships. Gene Ontology analysis was conducted to identify the associations that could indicate biological functions. We identified potential causal associations between GM and DN (p< 0.05 in all three MR methods). Among them, we found increased levels of Christensenellaceae R-7 (Odds ratio, OR= 1.52; 95% confidence interval, CI = 1.03-2.23; p = 0.03), Ruminococcaceae UCG013 (OR =1.35; 95% CI = 1.00-1.85; p = 0.04), and Eggerthella groups (OR = 1.27; 95% CI = 1.05-1.55; p = 0.01), which may be associated with a higher risk of DN, while increased levels of Peptococcaceae (OR = 0.69; 95% CI = 0.54-0.90; p< 0.01) and Eubacterium coprostanoligenes groups (OR = 0.68; 95% CI = 0.49-0.93; p = 0.01) could be associated with a lower risk. Gene Ontology pathway analysis revealed enrichment of genes regulated by the associated single-nucleotide polymorphisms (SNPs) in the apical plasma membrane, glycosyltransferase activity, hexosyltransferase activity and membrane raft. Reverse MR analyses indicated that DN was associated with five microbial taxa in all three MR methods. The results of our study validate the possible causative relationship between GM and DN. This discovery gives new perspectives into the mechanism on how GM influences DN, and establishes a theoretical foundation for future investigations into targeted preventive measures.

Network Pharmacological Study of the Active Ingredient of Panax Notoginseng Saponins for the Treatment of Lung Cancer by Inhibiting AKR1C3

Background: Lung cancer is the malignancy with the second highest diagnostic rate and the leading cause of cancer-related death. Objective: This study aims to investigate the potential mechanism and molecular targets of Panax notoginseng saponins (PNS) in inhibiting lung cancer through network pharmacology. Methods: Pharmacodynamic targets of each compound of PNS were searched from TargetNet, SwissTargetPrediction, and BatMan-TCM databases. Next, the differential expression genes (DEGs) in lung cancer were obtained from the Gene Expression Omnibus (GEO) database and screened by R package. Later, the STRING 11.0 database was utilized to analyze the protein-protein interaction (PPI) network of common targets of PNS-lung cancer, clusterProfiler to perform gene ontology (GO) annotation, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for the common targets, and Cytoscape 3.8.0 to construct and analyze the "ingredient-target" network for the common targets of PNS-lung cancer. Results: A total of 154 potential pharmacodynamic targets of PNS, 2399 DEGs of lung cancer-related diseases, and 21 common targets of PNS-lung cancer were obtained by database search and screening. The 21 common targets were mainly involved in biological processes (such as small molecule metabolism and cytokine production) and were major components of cellular structures (such as neuronal cell bodies and membrane rafts). Besides, these targets could function as carboxylic ester hydrolases, G protein-coupled amine receptors, and oxidoreductase. They were mainly enriched in 14 signaling pathways, like neuroactive ligand-receptor interaction, regulation of lipolysis in adipocytes, and calcium signaling pathway. Furthermore, the molecular docking results revealed that aldo-keto reductase family 1 member C3 (AKR1C3) and melanin metabolic enzyme (MME) may be direct targets of ginsenoside Rg1 and notoginsenoside R2. Conclusion: Our study showed that ginsenosides inhibit the progression of lung cancer through multiple targets and pathways. More importantly, PNS may treat lung cancer by directly inhibiting AKR1C3.

Flotillins affect LPS-induced TLR4 signaling by modulating the trafficking and abundance of CD14

Lipopolysaccharide (LPS) induces a strong pro-inflammatory reaction of macrophages upon activation of Toll-like receptor 4 (TLR4) with the assistance of CD14 protein. Considering a key role of plasma membrane rafts in CD14 and TLR4 activity and the significant impact exerted on that activity by endocytosis and intracellular trafficking of the both LPS acceptors, it seemed likely that the pro-inflammatory reaction could be modulated by flotillins. Flotillin-1 and -2 are scaffolding proteins associated with the plasma membrane and also with endo-membranes, affecting both the plasma membrane dynamics and intracellular protein trafficking. To verify the above hypothesis, a set of shRNA was used to down-regulate flotillin-2 in Raw264 cells, which were found to also become deficient in flotillin-1. The flotillin deficiency inhibited strongly the TRIF-dependent endosomal signaling of LPS-activated TLR4, and to a lower extent also the MyD88-dependent one, without affecting the cellular level of TLR4. The flotillin depletion also inhibited the pro-inflammatory activity of TLR2/TLR1 and TLR2/TLR6 but not TLR3. In agreement with those effects, the depletion of flotillins down-regulated the CD14 mRNA level and the cellular content of CD14 protein, and also inhibited constitutive CD14 endocytosis thereby facilitating its shedding. Ultimately, the cell-surface level of CD14 was markedly diminished. Concomitantly, CD14 recycling was enhanced via EEA1-positive early endosomes and golgin-97-positive trans-Golgi network, likely to compensate for the depletion of the cell-surface CD14. We propose that the paucity of surface CD14 is the reason for the down-regulated signaling of TLR4 and the other TLRs depending on CD14 for ligand binding.Graphical abstract

Lovastatin Treatment Inducing Apoptosis in Human Pancreatic Cancer Cells by Inhibiting Cholesterol Rafts in Plasma Membrane and Mitochondria

Resistance to anticancer drugs is a problem in the treatment of pancreatic ductal carcinoma (PDAC) and overcoming it is an important issue. Recently, it has been reported that statins induce apoptosis in cancer cells but the mechanism has not been completely elucidated. We investigated the antitumor mechanisms of statins against PDAC and their impact on resistance to gemcitabine (GEM). Lovastatin (LOVA) increased mitochondrial oxidative stress in PDAC cells, leading to apoptosis. LOVA reduced lipid rafts in the plasma membrane and mitochondria, suppressed the activation of epithelial growth factor receptor (EGFR) and AKT in plasma membrane rafts, and reduced B-cell lymphoma 2 (BCL2)-Bcl-2-associated X protein (BAX) binding and the translocation of F1F0 ATPase in mitochondrial rafts. In the three GEM-resistant cell lines derived from MIA and PANC1, the lipid rafts in the cell membrane and the mitochondria were increased to activate EGFR and AKT and to increase BCL2-BAX binding, which suppressed apoptosis. LOVA abrogated these anti-apoptotic effects by reducing the rafts in the resistant cells. By treating the resistant cells with LOVA, GEM sensitivity improved to the level of the parental cells. Therefore, cholesterol rafts contribute to drug resistance in PDAC. Further clinical research is warranted on overcoming anticancer drug resistance by statin-mediated intracellular cholesterol regulation.

HIV-1 Nef Changes the Proteome of T Cells Extracellular Vesicles Depleting IFITMs and Other Antiviral Factors

Extracellular vesicles (EVs) are biomolecule carriers for intercellular communication in health and disease. Nef is a HIV virulence factor that is released from cells within EVs and is present in plasma EVs of HIV-1 infected individuals. We performed a quantitative proteomic analysis to fully characterize the Nef-induced changes in protein composition of T cell-derived EVs and identify novel host targets of HIV. Several proteins with well-described roles in infection or not previously associated with HIV pathogenesis were specifically modulated by Nef in EVs. Among the downregulated proteins are the interferon-induced transmembrane 1, 2, and 3 (IFITM1-3) proteins, broad-spectrum antiviral factors known to be cell-to-cell transferable by EVs. We demonstrate that Nef depletes IFITM1-3 from EVs by excluding these proteins from the plasma membrane and lipid rafts, which are sites of EVs biogenesis in T cells. Our data establish Nef as a modulator of EVs' global protein content and as an HIV factor that antagonizes IFITMs.

Cholesterol-Depletion-Induced Membrane Repair Carries a Raft Conformer of P-Glycoprotein to the Cell Surface, Indicating Enhanced Cholesterol Trafficking in MDR Cells, Which Makes Them Resistant to Cholesterol Modifications.

The human P-glycoprotein (P-gp), a transporter responsible for multidrug resistance, is present in the plasma membrane's raft and non-raft domains. One specific conformation of P-gp that binds to the monoclonal antibody UIC2 is primarily associated with raft domains and displays heightened internalization in cells overexpressing P-gp, such as in NIH-3T3 MDR1 cells. Our primary objective was to investigate whether the trafficking of this particular P-gp conformer is dependent on cholesterol levels. Surprisingly, depleting cholesterol using cyclodextrin resulted in an unexpected increase in the proportion of raft-associated P-gp within the cell membrane, as determined by UIC2-reactive P-gp. This increase appears to be a compensatory response to cholesterol loss from the plasma membrane, whereby cholesterol-rich raft micro-domains are delivered to the cell surface through an augmented exocytosis process. Furthermore, this exocytotic event is found to be part of a complex trafficking mechanism involving lysosomal exocytosis, which contributes to membrane repair after cholesterol reduction induced by cyclodextrin treatment. Notably, cells overexpressing P-gp demonstrated higher total cellular cholesterol levels, an increased abundance of stable lysosomes, and more effective membrane repair following cholesterol modifications. These modifications encompassed exocytotic events that involved the transport of P-gp-carrying rafts. Importantly, the enhanced membrane repair capability resulted in a durable phenotype for MDR1 expressing cells, as evidenced by significantly improved viabilities of multidrug-resistant Pgp-overexpressing immortal NIH-3T3 MDR1 and MDCK-MDR1 cells compared to their parents when subjected to cholesterol alterations.

Phase Separation Driven by Active Flows.

We extend the continuum theories of active nematohydrodynamics to model a two-fluid mixture with separate velocity fields for each fluid component, coupled through a viscous drag. The model is used to study an active nematic fluid mixed with an isotropic fluid. We find microphase separation, and argue that this results from an interplay between active anchoring and active flows driven by concentration gradients. The results may be relevant to cell sorting and the formation of lipid rafts in cell membranes.

Involvement of Lipid Rafts in the Invasion of Opportunistic Bacteria Serratia into Eukaryotic Cells.

Cell membrane rafts form signaling platforms on the cell surface, controlling numerous protein-protein and lipid-protein interactions. Bacteria invading eukaryotic cells trigger cell signaling to induce their own uptake by non-phagocytic cells. The aim of this work was to reveal the involvement of membrane rafts in the penetration of the bacteria Serratia grimesii and Serratia proteamaculans into eukaryotic cells. Our results show that the disruption of membrane rafts by MβCD in the three cell lines tested, M-HeLa, MCF-7 and Caco-2, resulted in a time-dependent decrease in the intensity of Serratia invasion. MβCD treatment produced a more rapid effect on the bacterial susceptibility of M-HeLa cells compared to other cell lines. This effect correlated with a faster assembly of the actin cytoskeleton upon treatment with MβCD in M-HeLa cells in contrast to that in Caco-2 cells. Moreover, the 30 min treatment of Caco-2 cells with MβCD produced an increase in the intensity of S. proteamaculans invasion. This effect correlated with an increase in EGFR expression. Together with the evidence that EGFR is involved in S. proteamaculans invasion but not in S. grimesii invasion, these results led to the conclusion that an increase in EGFR amount on the plasma membrane with the undisassembled rafts of Caco-2 cells after 30 min of treatment with MβCD may increase the intensity of S. proteamaculans but not of S. grimesii invasion. Thus, the MβCD-dependent degradation of lipid rafts, which enhances actin polymerization and disrupts signaling pathways from receptors on the host cell's surface, reduces Serratia invasion.

Identification of GPI-anchored protein LYPD1 as an essential factor for odontoblast differentiation in tooth development

Lipid rafts are membrane microdomains rich in cholesterol, sphingolipids, glycosylphosphatidylinositol-anchored proteins (GPI-APs), and receptors. These lipid raft components are localized at the plasma membrane and are essential for signal transmission and organogenesis. However, few reports have been published on the specific effects of lipid rafts on tooth development. Using microarray and single-cell RNA sequencing methods, we found that a GPI-AP, lymphocyte antigen-6/Plaur domain-containing 1 (Lypd1), was specifically expressed in preodontoblasts. Depletion of Lypd1 in tooth germ using an exvivo organ culture system and in mouse dental pulp (mDP) cells resulted in the inhibition of odontoblast differentiation. Activation of bone morphogenetic protein (BMP) signaling by BMP2 treatment in mDP cells promoted odontoblast differentiation via phosphorylation of Smad1/5/8, while this BMP2-mediated odontoblast differentiation was inhibited by depletion of Lypd1. Furthermore, we created a deletion construct of the C terminus containing the omega site in LYPD1; this site is necessary for localizing GPI-APs to the plasma membrane and lipid rafts. We identified that this site is essential for odontoblast differentiation and morphological change of mDP cells. These findings demonstrated that LYPD1 is a novel marker of preodontoblasts in the developing tooth; in addition, they suggest that LYPD1 is important for tooth development and that it plays a pivotal role in odontoblast differentiation by regulating Smad1/5/8 phosphorylation through its effect as a GPI-AP in lipid rafts.

The Pathological Effects of Circulating Hydrophobic Bile Acids in Alzheimer's Disease.

Recent clinical studies have revealed that the serum levels of toxic hydrophobic bile acids (deoxy cholic acid, lithocholic acid [LCA], and glycoursodeoxycholic acid) are significantly higher in patients with Alzheimer's disease (AD) and amnestic mild cognitive impairment (aMCI) when compared to control subjects. The elevated serum bile acids may be the result of hepatic peroxisomal dysfunction. Circulating hydrophobic bile acids are able to disrupt the blood-brain barrier and promote the formation of amyloid-β plaques through enhancing the oxidation of docosahexaenoic acid. Hydrophobic bile acid may find their ways into the neurons via the apical sodium-dependent bile acid transporter. It has been shown that hydrophobic bile acids impose their pathological effects by activating farnesoid X receptor and suppressing bile acid synthesis in the brain, blocking NMDA receptors, lowering brain oxysterol levels, and interfering with 17β-estradiol actions such as LCA by binding to E2 receptors (molecular modelling data exclusive to this paper). Hydrophobic bile acids may interfere with the sonic hedgehog signaling through alteration of cell membrane rafts and reducing brain 24(S)-hydroxycholesterol. This article will 1) analyze the pathological roles of circulating hydrophobic bile acids in the brain, 2) propose therapeutic approaches, and 3) conclude that consideration be given to reducing/monitoring toxic bile acid levels in patients with AD or aMCI, prior/in combination with other treatments.

Impact of Experimental Conditions on Extracellular Vesicles' Proteome: A Comparative Study.

Extracellular vesicle (EV) research is a rapidly developing field, mainly due to the key role of EVs in intercellular communication and pathophysiological processes. However, the heterogeneity of EVs challenges their exploration and the establishment of gold-standard methods. Here, we aimed to reveal the influence of technical changes on EV biology and the reliability of experimental data. We used B16F1 melanoma cells as a model and applied nanoparticle tracking analysis, mass spectrometry (LC-MS/MS) and pathway enrichment analysis to analyze the quantity, size distribution, proteome and function of their small EVs (sEVs) produced in sEV-depleted fetal bovine serum (FBS)-containing medium or serum-free medium. Additionally, we investigated the effects of minor technical variances on the quality of sEV preparations. We found that storage of the isolates at -80 °C has no adverse effect on LC-MS/MS analysis, and an additional washing step after differential ultracentrifugation has a minor influence on the sEV proteome. In contrast, FBS starvation affects the production and proteome of sEVs; moreover, these vesicles may have a greater impact on protein metabolism, but a smaller impact on cell adhesion and membrane raft assembly, than the control sEVs. As we demonstrated that FBS starvation has a strong influence on sEV biology, applying serum-free conditions might be considered in in vitro sEV studies.

Mechanism of Tripterygium wilfordii Hook.F.- Trichosanthes kirilowii Maxim decoction in treatment of diabetic kidney disease based on network pharmacology and molecular docking.

Background: Diabetic kidney disease (DKD) is the most common cause of end-stage renal disease. The effective treatment of DKD would rely on the incorporation of a multi-disciplinary. Studies have shown that Tripterygium wilfordii Hook.F. and Trichosanthes kirilowii Maxim have remarkable curative effects in treating DKD, but their combination mechanism has not been fully elucidated. Methods: We explored the mechanism of Tripterygium wilfordii Hook.F.-Trichosanthes kirilowii Maxim decoction (Leigongteng-Tianhuafen Decoction,LTD) in the treatment of DKD by network pharmacology and molecular docking. The main active components and action targets of LTD were collected from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database. The speculative targets of DKD were obtained from GeneCards, DisGeNET, and Online Mendelian Inheritance in Man (OMIM) databases. Then, an herb-component-target network was constructed based on the above analyses. The biological function of targets was subsequently investigated, and a protein-protein interaction (PPI) network was constructed to identify hub targets of DKD. The gene ontology (GO) function enrichment analysis and kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed by RStudio. Finally, molecular docking was performed by AutoDock Vina and PyMOL software to explore the interaction between compounds and targets. Furthermore, the DKD model of human renal tubular cells (HK-2) induced by high glucose (HG) was selected, and the predicted results were verified by western blot analysis and immunofluorescence. Results: A total of 31 active components of LTD were screened out, and 196 targets were identified based on the TCMSP database. A total of 3,481 DKD related targets were obtained based on GeneCards, DisGeNET, and OMIM databases. GO function enrichment analysis included 2,143, 50, and 167 GO terms for biological processes (BPs), cellular composition (CCs), and molecular functions (MFs), respectively. The top 10 enrichment items of BP annotations included response to lipopolysaccharide, response to molecule of bacterial origin, response to extracellular stimulus, etc. CC was mainly enriched in membrane raft, membrane microdomain, plasma membrane raft, etc. The MF of LTD analysis on DKD was predominately involved in nuclear receptor activity, ligand-activated transcription factor activity, RNA polymerase II-specific DNA-binding transcription factor binding, etc. The involvement signaling pathway of LTD in the treatment of DKD included AGE-RAGE signaling pathway in diabetic complications, IL-17 signaling pathway, insulin resistance, TNF signaling pathway, etc. Molecular docking results showed that kaempferol, triptolide, nobiletin, and schottenol had a strong binding ability to PTGS2 and RELA. Furthermore, the in vitro experiments confirmed that LTD effectively decreased the expression of PTGS2, NF-κB, JNK, and AKT in the HG-induced DKD model. Conclusion: The findings of this study revealed that the therapeutic efficacy of LTD on DKD might be achieved by decreasing the expression of PTGS2, NF-κB, JNK, and AKT, which might improve insulin resistance, inflammation, and oxidative stress. These findings can provide ideas and supply potential therapeutic targets for DKD.

Mechanism of Yangxin Tongmai Decoction in the Treatment of Coronary Heart Disease with Blood Stasis Syndrome Based on Network Pharmacology and Molecular Docking.

This study aimed to explore the mechanism of Yangxin Tongmai decoction (YXTMD) in the treatment of coronary heart disease (CHD) with blood stasis syndrome (BSS) using network pharmacology and molecular docking, and to verify these results through clinical trials. The active compounds of YXTMD were identified using the Traditional Chinese Medicine Systems Pharmacology database, and the targets of the active compounds were predicted using the SwissTarget Prediction database. The targets of CHD and BSS were predicted using the GeneCards, OMIM, PharmGKB, TTD, and DrugBank databases. The common targets of “herb-disease-phenotype” were obtained using a Venn diagram, then used Cytoscape software 3.8.2 and its plug-in CytoNCA and STRING database to construct the “herb active compounds-common target” and protein–protein interaction networks. R language software and bioconductor plug-in were used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. AutoDock was used for the molecular docking analysis. Finally, clinical trials were conducted to confirm the results of network pharmacology. Eighty-three active components were obtained, and the core active components were 5,7,4′-trimethoxyflavone, tetramethoxyluteolin, isosinensetin, sinensetin, and 5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one. A total of 140 common targets were identified, and the core targets were EGFR, VEGFA, AKT1, STAT3, TP53, ERBB2, and PIK3CA. Biological processes identified by the GO analysis primarily involved wound healing, regulation of body fluid levels, and vascular process in circulatory system. The cellular components were primarily located in the membrane raft, membrane microdomain, and plasma membrane raft. The primary molecular functions were activity of transmembrane receptor protein kinase, transmembrane receptor protein tyrosine kinase, and protein tyrosine kinase. KEGG analysis showed that the PI3K-Akt signaling pathway was closely related to the treatment of CHD with BSS by YXTMD. Molecular docking results showed that the core active components had a good binding activity with the core targets. The clinical trial results showed that YXTMD improved the BSS scores and decreased the serum levels of total cholesterol and low-density lipoprotein cholesterol. Moreover, the levels of PI3k and AKt mRNA were upregulated and the levels of GSK-3β mRNA were downregulated. YXTMD has multicomponent, multitarget, and multipathway effects in the treatment of CHD with BSS, and its mechanism of action may involve activation of the PI3K-AKt signaling pathway, downregulation of GSK-3β, and mediation of in vivo lipid metabolism-based metabolic processes.

Neurological Consequences of Sphingosine Phosphate Lyase Insufficiency.

In 2017, an inborn error of metabolism caused by recessive mutations in SGPL1 was discovered. The disease features steroid-resistant nephrotic syndrome, adrenal insufficiency, and neurological defects. The latter can include sensorineural hearing loss, cranial nerve defects, peripheral neuropathy, abnormal brain development, seizures and/or neurodegeneration. SGPL1 encodes the pyridoxal-5’-phosphate (PLP) dependent enzyme sphingosine phosphate lyase (SPL), and the condition is now referred to as SPL insufficiency syndrome (SPLIS). SPL catalyzes the final step in the degradative pathway of sphingolipids in which the bioactive sphingolipid sphingosine-1-phosphate (S1P) is irreversibly degraded to a long chain aldehyde and phosphoethanolamine (PE). SPL guards the only exit point for sphingolipid metabolism, and its inactivation leads to accumulation of various types of sphingolipids which have biophysical roles in plasma membrane rafts and myelin, and signaling roles in cell cycle progression, vesicular trafficking, cell migration, and programmed cell death. In addition, the products of the SPL reaction have biological functions including regulation of autophagic flux, which is important in axonal and neuronal integrity. In this review, the neurological manifestations of SPLIS will be described, and insights regarding the neurological consequences of SPL insufficiency from the study of brain-specific SPL knockout mice and Drosophila SPL mutants will be summarized.

Effect of Sublethal Copper Overload on Cholesterol De Novo Synthesis in Undifferentiated Neuronal Cells.

Although copper (Cu) is an essential trace metal for cells, it can induce harmful effects as it participates in the Fenton reaction. Involuntary exposure to Cu overload is much more common than expected and has been linked with neurodegeneration, particularly with Alzheimer’s disease (AD) evidenced by a positive correlation between free Cu in plasma and the severity of the disease. It has been suggested that Cu imbalance alters cholesterol (Chol) homeostasis and that high membrane Chol promotes the amyloidogenic processing of the amyloid precursor protein (APP) secreting the β-amyloid (Aβ) peptide. Despite the wide knowledge on the effects of Cu in mature brain metabolism, the consequence of its overload on immature neurons remains unknown. Therefore, we used an undifferentiated human neuroblastoma cell line (SH-SY5Y) to analyze the effect of sublethal concentrations of Cu on 1— de novo Chol synthesis and membrane distribution; 2—APP levels in cells and its distribution in membrane rafts; 3—the levels of Aβ in the culture medium. Our results demonstrated that Cu increases reactive oxygen species (ROS) and favors Chol de novo synthesis in both ROS-dependent and independent manners. Also, at least part of these effects was due to the activation of 3-hydroxy-3-methyl glutaryl CoA reductase (HMGCR). In addition, Cu increases the Chol/PL ratio in the cellular membranes, specifically Chol content in membrane rafts. We found no changes in total APP cell levels; however, its presence in membrane rafts increases with the consequent increase of Aβ in the culture medium. We conclude that Cu overload favors Chol de novo synthesis in both ROS-dependent and independent manners, being at least in part, responsible for the high Chol levels found in the cell membrane and membrane rafts. These may promote the redistribution of APP into the rafts, favoring the amyloidogenic processing of this protein and increasing the levels of Aβ.

Decreased GM3 correlates with proteinuria inminimal change nephrotic syndrome and focal segmental glomerulosclerosis.

Glycolipids on cell membrane rafts play various roles by interacting with glycoproteins. Recently, it was reported that the glycolipid GM3 is expressed in podocytes and may play a role in podocyte protection. In this report, we describe the correlation between changes in GM3 expression in glomeruli and proteinuria in minimal change nephrotic syndrome (MCNS) and focal segmental glomerulosclerosis (FSGS) patients. We performed a case-control study of the correlation between nephrin/GM3 expression levels and proteinuria in MCNS and FSGS patients who underwent renal biopsy at our institution between 2009 and 2014. Normal renal tissue sites were used from patients who had undergone nephrectomy at our institution and gave informed consent. Both MCNS and FSGS had decreased GM3 and Nephrin expression compared with the normal (normal vs. MCNS, FSGS; all p < 0.01). Furthermore, in both MCNS and FSGS, GM3 expression was negatively correlated with proteinuria (MCNS: r = -0.61, p < 0.01, FSGS: r = -0.56, p < 0.05). However, nephrin expression had a trend to correlate with proteinuria in FSGS (MCNS: r = 0.19, p = 0.58, FSGS: r = -0.48, p = 0.06). Furthermore, in a simple linear regression analysis, GM3 expression also correlated with proteinuric change after 12months of treatment (MCNS: r = 0.40, p = 0.38, FSGS: r = 0. 68, p < 0.05). We showed for the first time that decreased GM3 expression correlates with proteinuria in MCNS and FSGS patients. Further studies are needed on the podocyte-protective effects of GM3.

Heat-induced increases in body temperature in lactating dairy cows: impact on the cumulus and granulosa cell transcriptome of the periovulatory follicle.

Cows acutely heat stressed after a pharmacologically induced luteinizing hormone (LH) surge had periovulatory changes in the follicular fluid proteome that may potentiate ovulation and impact oocyte developmental competence. Because the cellular origins of differentially abundant proteins were not known, we have examined the cumulus and granulosa cell transcriptomes from the periovulatory follicle in cows exhibiting varying levels of hyperthermia when occurring after the LH surge. After pharmacological induction of a dominant follicle, lactating dairy cows were administered gonadotropin releasing hormone (GnRH) and maintained in thermoneutral conditions (~67 temperature-humidity index [THI]) or heat stress conditions where THI was steadily increased for ~12 h (71 to 86 THI) and was sufficient to steadily elevate rectal temperatures. Cumulus-oocyte complexes and mural granulosa cells were recovered by transvaginal aspiration of dominant follicle content ~16 h after GnRH. Rectal temperature was used as a continuous, independent variable to identify differentially expressed genes (DEGs) increased or decreased per each 1 °C change in temperature. Cumulus (n = 9 samples) and granulosa (n = 8 samples) cells differentially expressed (false discovery rate [FDR] < 0.05) 25 and 87 genes, respectively. The majority of DEGs were upregulated by hyperthermia. Steady increases in THI are more like the "turning of a dial" than the "flipping of a switch." The moderate but impactful increases in rectal temperature induced modest fold changes in gene expression (<2-fold per 1 °C change in rectal temperature). Identification of cumulus DEGs involved in cell junctions, plasma membrane rafts, and cell-cycle regulation are consistent with marked changes in the interconnectedness and function of cumulus after the LH surge. Depending on the extent to which impacts may be occurring at the junctional level, cumulus changes may have indirect but impactful consequences on the oocyte as it undergoes meiotic maturation. Two granulosa cell DEGs have been reported by others to promote ovulation. Based on what is known, several other DEGs are suggestive of impacts on collagen formation or angiogenesis. Collectively these and other findings provide important insight regarding the extent to which the transcriptomes of the components of the periovulatory follicle (cumulus and mural granulosa cells) are affected by varying degrees of hyperthermia.

A Network Pharmacology Approach to Explore the Potential Mechanism of Ficus religiosa against Alzheimer’s Disease

Background: Ficus religiosa, also known as the peepal tree, is used to treat multiple diseases. It is proved to be effective in treating cognitive impairment. However, the potential targets and pharmacological and molecular mechanisms of its action on the management of Alzheimer’s Disease (AD) are not entirely clear. Therefore, a network pharmacology approach is required to further study and explore its treatment mechanism. Methods: ChEBI database was used to retrieve the active constituents. AD genes were retrieved from the DisGeNet database. SMILES of phytoconstituents were retrieved from PubChem, traced for the positive drug-likeness score, targets were predicted and regulated proteins were enriched to trace probable modulated proteins, pathways, and GO terms. Cytoscape ver. 3.7.2 was used to construct the active ingredient-target-pathway interaction of F. religiosa and network analysis. Molecular docking was also performed. Results: A total of 7 bioactive out of 25 were predicted to possess a positive drug likeliness score. Also, PIK3CA, PIK3RA, AKT1, MAP2K1 and RAF1 were predicted as key gene targets. The results of the GO analysis demonstrated that the somatodendritic compartment, cytoplasm, plasma membrane, and membrane raft are the main cellular components, its molecular functions are mainly catalytic activity, ion binding, protein kinase activity and, identical protein binding. The biological process is focused on biological quality, response to oxygen-contain-compound sound, and metabolic process. Conclusions: This study holistically illuminated possible pharmacological mechanisms of F. religiosa that might be strongly associated with multi-targeting compounds and act on many AD pathways at the same time.

Unconventional Protein Secretion Dependent on Two Extracellular Vesicles: Exosomes and Ectosomes.

In addition to conventional protein secretion, dependent on the specific cleavage of signal sequences, proteins are secreted by other processes, all together called unconventional. Among the mechanisms operative in unconventional secretion, some are based on two families of extracellular vesicle (EVs), expressed by all types of cells: the exosomes (before secretion called ILVs) and ectosomes (average diameters ∼70 and ∼250 nm). The two types of EVs have been largely characterized by extensive studies. ILVs are assembled within endocytic vacuoles by inward budding of small membrane microdomains associated to cytosolic cargos including unconventional secretory proteins. The vacuoles containing ILVs are called multivesicular bodies (MVBs). Upon their possible molecular exchange with autophagosomes, MVBs undergo two alternative forms of fusion: 1. with lysosomes, followed by large digestion of their cargo molecules; and 2. with plasma membrane (called exocytosis), followed by extracellular diffusion of exosomes. The vesicles of the other type, the ectosomes, are differently assembled. Distinct plasma membrane rafts undergo rapid outward budding accompanied by accumulation of cytosolic/secretory cargo molecules, up to their sewing and pinching off. Both types of EV, released to the extracellular fluid in their complete forms including both membrane and cargo, start navigation for various times and distances, until their fusion with target cells. Release/navigation/fusion of EVs establish continuous tridimensional networks exchanging molecules, signals and information among cells. The proteins unconventionally secreted via EVs are a few hundreds. Some of them are functionally relevant (examples FADD, TNF, TACE), governing physiological processes and important diseases. Such proteins, at present intensely investigated, predict future discoveries and innovative developments, relevant for basic research and clinical practice.