Metformin alleviates cryoinjuries in porcine oocytes by reducing membrane fluidity through the suppression of mitochondrial activity.

Plasma membrane damage in vitrified oocytes is closely linked to mitochondrial dysfunction. However, the mechanism underlying mitochondria-regulated membrane stability is not elucidated. A growing body of evidence indicates that mitochondrial activity plays a pivotal role in cell adaptation. Since mitochondria work at a higher temperature than the constant external temperature of the cell, we hypothesize that suppressing mitochondrial activity would protect oocytes from extreme stimuli during vitrification. Here we show that metformin suppresses mitochondrial activity by reducing mitochondrial temperature. In addition, metformin affects the developmental potential of oocytes and improves the survival rate after vitrification. Transmission electron microscopy results show that mitochondrial abnormalities are markedly reduced in vitrified oocytes pretreated with metformin. Moreover, we find that metformin transiently inhibits mitochondrial activity. Interestingly, metformin pretreatment decreases cell membrane fluidity after vitrification. Furthermore, transcriptome results demonstrate that metformin pretreatment modulates the expression levels of genes involved in fatty acid elongation process, which is further verified by the increased long-chain saturated fatty acid contents in metformin-pretreated vitrified oocytes by lipidomic profile analysis. In summary, our study indicates that metformin alleviates cryoinjuries by reducing membrane fluidity via mitochondrial activity regulation.

Activation of hTREK-1 by polyunsaturated fatty acids involves direct interaction.

TREK-1 is a mechanosensitive channel activated by polyunsaturated fatty acids (PUFAs). Its activation is supposed to be linked to changes in membrane tension following PUFAs insertion. Here, we compared the effect of 11 fatty acids and ML402 on TREK-1 channel activation using the whole cell and the inside-out configurations of the patch-clamp technique. Firstly, TREK-1 activation by PUFAs is variable and related to the variable constitutive activity of TREK-1. We observed no correlation between TREK-1 activation and acyl chain length or number of double bonds suggesting that the bilayer-couple hypothesis cannot explain by itself the activation of TREK-1 by PUFAs. The membrane fluidity measurement is not modified by PUFAs at 10 µM. The spectral shift analysis in TREK-1-enriched microsomes indicates a KD,TREK1 at 44 µM of C22:6 n-3. PUFAs display the same activation and reversible kinetics than the direct activator ML402 and activate TREK-1 in both whole-cell and inside-out configurations of patch-clamp suggesting that the binding site of PUFAs is accessible from both sides of the membrane, as for ML402. Finally, we proposed a two steps mechanism: first, insertion into the membrane, with no fluidity or curvature modifications at 10 µM, and then interaction with TREK-1 channel to open it.

Investigating the impact of 2-OHOA-embedded liposomes on biophysical properties of cancer cell membranes via Laurdan two-photon microscopy imaging.

2-Hydroxyoleic acid (2-OHOA) has gained attention as a membrane lipid therapy (MLT) anti-cancer drug. However, in the viewpoint of anti-cancer drug, 2-OHOA shows poor water solubility and its effectiveness still has space for improvement. Thus, this study aimed to overcome the problems by formulating 2-OHOA into liposome dosage form. Furthermore, in the context of MLT reagents, the influence of 2-OHOA on the biophysical properties of the cytoplasmic membrane remains largely unexplored. To bridge this gap, our study specifically focused the alterations in cancer cell membrane fluidity and lipid packing characteristics before and after treatment. By using a two-photon microscope and the Laurdan fluorescence probe, we noted that liposomes incorporating 2-OHOA induced a more significant reduction in cancer cell membrane fluidity, accompanied by a heightened rate of cellular apoptosis when compared to the non-formulated 2-OHOA. Importantly, the enhanced efficacy of 2-OHOA within the liposomal formulation demonstrated a correlation with its endocytic uptake mechanism. In conclusion, our findings underscore the significant influence of 2-OHOA on the biophysical properties of cancer plasma membranes, emphasizing the potential of liposomes as an optimized delivery system for 2-OHOA in anti-cancer therapy.

Acrolein Induces Changes in Cell Membrane and Cytosol Proteins of Erythrocytes.

High concentrations of acrolein (2-propenal) are found in polluted air and cigarette smoke, and may also be generated endogenously. Acrolein is also associated with the induction and progression of many diseases. The high reactivity of acrolein towards the thiol and amino groups of amino acids may cause damage to cell proteins. Acrolein may be responsible for the induction of oxidative stress in cells. We hypothesized that acrolein may contribute to the protein damage in erythrocytes, leading to the disruption of the structure of cell membranes. The lipid membrane fluidity, membrane cytoskeleton, and osmotic fragility were measured for erythrocytes incubated with acrolein for 24 h. The levels of thiol, amino, and carbonyl groups were determined in cell membrane and cytosol proteins. The level of non-enzymatic antioxidant potential (NEAC) and TBARS was also measured. The obtained research results showed that the exposure of erythrocytes to acrolein causes changes in the cell membrane and cytosol proteins. Acrolein stiffens the cell membrane of erythrocytes and increases their osmotic sensitivity. Moreover, it has been shown that erythrocytes treated with acrolein significantly reduce the non-enzymatic antioxidant potential of the cytosol compared to the control.

Lipid phase separation impairs membrane thickness sensing by the Bacillus subtilis sensor kinase DesK.

Membrane fluidity and thickness have emerged as crucial factors for the activity of and resistance to several antimicrobials. However, the lack of tools to study membrane fluidity and, in particular, thickness in living bacteria limits our understanding of this interplay. The Bacillus subtilis histidine kinase/phosphatase DesK is a molecular sensor that directly detects membrane thickness. It controls activity of DesR, which regulates expression of the lipid desaturase Des, known for its role in cold adaptation and daptomycin susceptibility. We hypothesized that this property could be exploited to develop biosensors and reporters for antibiotic-induced changes in membrane fluidity and thickness. To test this, we designed three assays based on the des system: activation of the Pdes promoter as reporter for membrane thickening, localization of DesK-GFP(green-fluorescent protein) as proxy for rigidified membrane domains, and antibiotic sensitivity of des, desK, and desR deletion mutants as readout for the importance of membrane rigidification/thickening under the tested condition. While we could not confirm the suitability of the des system as reporter for antibiotic-induced changes in membrane thickness, we did observe that des expression is only activated by mild temperature shocks, likely due to partitioning of the sensor DesK into fluid membrane domains upon phase separation, precluding effective thickness sensing under harsh cold shock and antibiotic stress conditions. Similarly, we did not observe any sensitivity of the deletion mutants to either temperature or antibiotic stress, raising the question to what extent the des system contributes to fluidity adaptation under these conditions. IMPORTANCE: The B. subtilis des system is a prime model for direct molecular membrane thickness sensor and, as such, has been well studied in vitro. Our study shows that our understanding of its function in vivo and its importance under temperature and antibiotic stress is still very limited. Specifically, our results suggest that (i) the des system senses very subtle membrane fluidity changes that escape detection by established fluidity reporters like laurdan; (ii) membrane thickness sensing by DesK is impaired by phase separation due to partitioning of the protein into the fluid phase; and (iii) fluidity adaptations by Des are too subtle to elicit growth defects under rigidifying conditions, raising the question of how much the des system contributes to adaptation of overall membrane fluidity.

Defective pgsA contributes to increased membrane fluidity and cell wall thickening in Staphylococcus aureus with high-level daptomycin resistance.

Daptomycin is a membrane-targeting last-resort antimicrobial therapeutic for the treatment of infections caused by methicillin- and/or vancomycin-resistant Staphylococcus aureus. In the rare event of failed daptomycin therapy, the source of resistance is often attributable to mutations directly within the membrane phospholipid biosynthetic pathway of S. aureus or in the regulatory systems that control cell envelope response and membrane homeostasis. Here we describe the structural changes to the cell envelope in a daptomycin-resistant isolate of S. aureus strain N315 that has acquired mutations in the genes most commonly reported associated with daptomycin resistance: mprF, yycG, and pgsA. In addition to the decreased phosphatidylglycerol (PG) levels that are the hallmark of daptomycin resistance, the mutant with high-level daptomycin resistance had increased branched-chain fatty acids (BCFAs) in its membrane lipids, increased membrane fluidity, and increased cell wall thickness. However, the successful utilization of isotope-labeled straight-chain fatty acids (SCFAs) in lipid synthesis suggested that the aberrant BCFA:SCFA ratio arose from upstream alteration in fatty acid synthesis rather than a structural preference in PgsA. Transcriptomics studies revealed that expression of pyruvate dehydrogenase (pdhB) was suppressed in the daptomycin-resistant isolate, which is known to increase BCFA levels. While complementation with an additional copy of pdhB had no effect, complementation of the pgsA mutation resulted in increased PG formation, reduction in cell wall thickness, restoration of normal BCFA levels, and increased daptomycin susceptibility. Collectively, these results demonstrate that pgsA contributes to daptomycin resistance through its influence on membrane fluidity and cell wall thickness, in addition to phosphatidylglycerol levels. IMPORTANCE: The cationic lipopeptide antimicrobial daptomycin has become an essential tool for combating infections with Staphylococcus aureus that display reduced susceptibility to ß-lactams or vancomycin. Since daptomycin's activity is based on interaction with the negatively charged membrane of S. aureus, routes to daptomycin-resistance occur through mutations in the lipid biosynthetic pathway surrounding phosphatidylglycerols and the regulatory systems that control cell envelope homeostasis. Therefore, there are many avenues to achieve daptomycin resistance and several different, and sometimes contradictory, phenotypes of daptomycin-resistant S. aureus, including both increased and decreased cell wall thickness and membrane fluidity. This study is significant because it demonstrates the unexpected influence of a lipid biosynthesis gene, pgsA, on membrane fluidity and cell wall thickness in S. aureus with high-level daptomycin resistance.

St. John's wort extract Ze 117 alters the membrane fluidity of C6 glioma cells by influencing cellular cholesterol metabolism.

Chronic stress is associated with major depressive disorder (MDD). Increased glucocorticoid levels caused by uncontrolled release through the hypothalamic‒pituitary‒adrenal (HPA) axis can cause changes in the lipid content of the cellular plasma membrane. These changes are suspected to be involved in the development of depressive disorders. St. John's wort extract (SJW) Ze 117 has long been used as an alternative to synthetic antidepressants. Part of its effect may be due to an effect on the cellular lipid composition and thus on the properties of plasma membranes and receptor systems embedded therein. In this study, we investigated the effect of Ze 117 on that of dexamethasone and simvastatin. Dexamethasone increases the fluidity of C6 cell plasma membranes. This effect is counteracted by administration of Ze 117. Here we demonstrate that this is not due to a change in C16:1/16:0 and C18:1/18:0 ratios in C6 cell fatty acids. On the other hand, Ze 117 increased the cellular cholesterol content by 42.5%, whereas dexamethasone reduced cholesterol levels similarly to simvastatin. Lowering cholesterol levels by dexamethasone or simvastatin resulted in decreased ß-arrestin 2 recruitment to the 5-HT1a receptor. This effect was counterbalanced by Ze 117, whereas the SJW extract had little effect on ß-arrestin 2 recruitment in non-stressed cells. Taken together, in C6 cells, Ze 117 induces changes in membrane fluidity through its effect on cellular cholesterol metabolism rather than by affecting fatty acid saturation. This effect is reflected in an altered signal transduction of the 5-HT1a receptor under Ze 117 administration. The current in vitro results support the hypothesis that Ze 117 addresses relevant parts of the cellular lipid metabolism, possibly explaining some of the antidepressant actions of Ze 117.

Erythrocytes membrane fluidity changes induced by adenylyl cyclase cascade activation: study using fluorescence recovery after photobleaching.

In this study, fluorescence recovery after photobleaching (FRAP) experiments were performed on RBC labeled by lipophilic fluorescent dye CM-DiI to evaluate the role of adenylyl cyclase cascade activation in changes of lateral diffusion of erythrocytes membrane lipids. Stimulation of adrenergic receptors with epinephrine (adrenaline) or metaproterenol led to the significant acceleration of the FRAP recovery, thus indicating an elevated membrane fluidity. The effect of the stimulation of protein kinase A with membrane-permeable analog of cAMP followed the same trend but was less significant. The observed effects are assumed to be driven by increased mobility of phospholipids resulting from the weakened interaction between the intermembrane proteins and RBC cytoskeleton due to activation of adenylyl cyclase signaling cascade.

Plasma membrane rigidity effects of 4-hydroxy-2-nonenal in Leishmania, erythrocyte and macrophage.

4-hydroxy-2-nonenal (HNE) is a reactive aldehyde produced by cells under conditions of oxidative stress, which has been shown to react with proteins and phosphatidylethanolamine in biological membranes. Using electron paramagnetic resonance (EPR) spectroscopy of a spin label it was demonstrated that 2 h of treatment with HNE causes membrane rigidity in promastigotes of Leishmania (L.) amazonensis, J774.A1 macrophages and erythrocytes. Remarkable fluidity-reducing effects on the parasite membrane were observed at HNE concentrations approximately 4-fold lower than in the case of erythrocyte and macrophage membranes. Autofluorescence of the parasites in PBS suspension (1 × 107 cell/mL) with excitation at 354 nm showed a linear increase of intensity in the range of 400 to 600 nm over 3 h after treatment with 30 µM HNE. Parasite ghosts prepared after this period of HNE treatment showed a high degree of membrane rigidity. Bovine serum albumin (BSA) in PBS treated with HNE for 2 h showed an increase in molecular dynamics and suffered a decrease in its ability to bind a lipid probe. In addition, the antiproliferative activity of L. amazonensis promastigotes, macrophage cytotoxicity and hemolytic potential were assessed for HNE. An IC50 of 24 µM was found, which was a concentration > 10 times lower than the cytotoxic and hemolytic concentrations of HNE. These results indicate that the action of HNE has high selectivity indices for the parasite as opposed to the macrophage and erythrocyte.

Enhancing the Endocytosis of Phosphatidylserine-Containing Liposomes through Tim4 by Modulation of Membrane Fluidity.

Phosphatidylserine (PS) is a unique lipid that is recognized by the endogenetic receptor, T-cell immunoglobulin mucin protein 4 (Tim4), and PS-containing liposomes have potential use in therapeutic applications. We prepared PS-containing liposomes of various lipid compositions and examined how lipid membrane fluidity affects PS recognition by Tim4 and the resulting endocytosis efficiency into Hela cells. Surface plasmon resonance and laurdan studies showed that increasing lipid membrane fluidity increased the stability of the PS-Tim4 interaction but hampered the entry of liposomes into cells. These results show that endocytosis efficiency is determined by balancing opposing forces induced by membrane fluidity. We found that inclusion of the zwitterionic helper lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, into liposomes ensured efficient cellular internalization because the presence of this lipid provides an ideal balance of lipid fluidity and Tim4 affinity. The results showed that PS recognition by Tim4 and the resulting endocytosis efficiency can be maximized by modulating the membrane fluidity of liposomes by selecting a zwitterionic helper lipid. This study improves our understanding of how to rationally optimize nanotechnology for targeted drug delivery.

Electron Spin Resonance Evaluation of Buccal Membrane Fluidity Alterations by Sodium Caprylate and L-Menthol.

The ability of sodium caprylate and l-menthol to fluidize phospholipid bilayers composed of lipids simulating the buccal epithelium was investigated using electron spin resonance (ESR) to evaluate the action of these agents as permeation enhancers. 5-Doxyl stearic acid (5-DSA) and 16-doxyl stearic acid (16-DSA) were used as spin labels to identify alterations in membrane fluidity near the polar head groups or inner acyl regions of the lipid bilayer, respectively. The molecular motion of both 5-DSA and 16-DSA showed increased disorder near the polar and inner hydrophobic regions of the bilayer in the presence of sodium caprylate suggesting fluidization in both the regions, which contributes to its permeation enhancing effects. L-menthol decreased the order parameter for 16-DSA, showing membrane fluidization only in the inner acyl regions of the bilayer, which also corresponded to its weaker permeation enhancing effects. The rapid evaluation of changes in fluidity of the bilayer in the presence of potential permeation enhancers using ESR enables improved selection of effective permeation enhancers and enhancer combinations based on their effect on membrane fluidization.

The Impact of Short-Term Shark Liver Oil Supplementation on the Fatty Acid Composition of Erythrocyte Membranes.

Fatty acid (FA) balance is strictly related to human health. The composition of fatty acids in lipid membranes seems to be influenced by diet. Shark liver oil (SLO) supplementation has been widely used recently in the prevention and treatment of human diseases. We analyzed the impact of short-term SLO supplementation on certain biochemical parameters and erythrocyte FA composition in a group of young healthy women. Our results showed that 6 weeks of SLO supplementation led to a significant decrease in C-reactive protein levels in sera and intracellular cholesterol levels in peripheral blood mononuclear cells. SLO supplementation caused a significant increase in the content of the polyunsaturated omega-3 FAs: docosahexaenoic acid, docosapentaenoic acid and α-linolenic acid. In the group of omega-6 FAs, we observed a significant elevation of arachidonic and dihomo-gamma-linoleic acid content. Due to these alterations, the omega-3 index increased significantly from 3.6% (before) to 4.2% (after supplementation). We also observed the impact of SLO supplementation on the membrane fluidity index. The ratio between saturated and unsaturated FAs decreased significantly from 13.1 to 9.9. In conclusion, our results show that even short-term SLO supplementation can improve human erythrocyte fatty acid composition and other parameters that may have health-promoting consequences.

Clotrimazole Fluidizes Phospholipid Membranes and Localizes at the Hydrophobic Part near the Polar Part of the Membrane.

Clotrimazole (1-[(2-chlorophenyl)-diphenylmethyl]-imidazole) is an azole antifungal drug belonging to the imidazole subclass that is widely used in pharmacology and that can be incorporated in membranes. We studied its interaction with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) phospholipid vesicles by using differential scanning calorimetry and found that the transition temperature decreases progressively as the concentration of clotrimazole increases. However, the temperature of completion of the transition remained constant despite the increase of clotrimazole concentration, suggesting the formation of fluid immiscibility. 1H-NMR and 1H NOESY MAS-NMR were employed to investigate the location of clotrimazole in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) phospholipid membranes. In the presence of clotrimazole, all the resonances originating from POPC were shifted upfield, but mainly those corresponding to C2 and C3 of the fatty acyl, chains suggesting that clotrimazole aromatic rings preferentially locate near these carbons. In the same way, 2D-NOESY measurements showed that the highest cross-relaxation rates between protons of clotrimazole and POPC were with those bound to the C2 and C3 carbons of the fatty acyl chains. Molecular dynamics simulations indicated that clotrimazole is located near the top of the hydrocarbon-chain phase, with the nitrogen atoms of the imidazole ring of clotrimazole being closest to the polar group of the carbonyl moiety. These results are in close agreement with the NMR and the conclusion is that clotrimazole is located near the water-lipid interface and in the upper part of the hydrophobic bilayer.

An In Vitro Comparative Study of the Effects of Tetrabromobisphenol A and Tetrabromobisphenol S on Human Erythrocyte Membranes-Changes in ATP Level, Perturbations in Membrane Fluidity, Alterations in Conformational State and Damage to Proteins.

Brominated flame retardants (BFRs) are substances used to reduce the flammability of plastics. Among this group, tetrabormobisphenol A (TBBPA) is currently produced and used on the greatest scale, but due to the emerging reports on its potential toxicity, tetrabromobisphenol S (TBBPS)-a compound with a very similar structure-is used as an alternative. Due to the fact that the compounds in question are found in the environment and in biological samples from living organisms, including humans, and due to the insufficient toxicological knowledge about them, it is necessary to assess their impacts on living organisms and verify the validity of TBBPA replacement by TBBPS. The RBC membrane was chosen as the research model. This is a widely accepted research model for assessing the toxicity of xenobiotics, and it is the first barrier to compounds entering circulation. It was found that TBBPA and TBBPS caused increases in the fluidity of the erythrocyte membrane in their hydrophilic layer, and conformational changes to membrane proteins. They also caused thiol group elevation, an increase in lipid peroxidation (TBBPS only) and decreases in the level of ATP in cells. They also caused changes in the size and shape of RBCs. TBBPA caused changes in the erythrocyte membrane at lower concentrations compared to TBBPS at an occupational exposure level.

Surfactin cyclic lipopeptides change the plasma membrane composition and lateral organization in mammalian cells.

The specific structure and composition of the cell plasma membrane (PM) is crucial for many cellular processes and can be targeted by various substances with potential medical applications. In this context, biosurfactants (BS) constitute a promising group of natural compounds that possess several biological functions, including anticancer activity. Despite the efficiency of BS, their mode of action had never been elucidated before. Here, we demonstrate the influence of cyclic lipopeptide surfactin (SU) on the PM of CHO-K1 cells. Both FLIM and svFCS experiments show that even a low concentration of SU causes significant changes in the membrane fluidity and dynamic molecular organization. Further, we demonstrate that SU causes a relevant dose-dependent reduction of cellular cholesterol by extracting it from the PM. Finally, we show that CHO-25RA cells characterized by increased cholesterol levels are more sensitive to SU treatment than CHO-K1 cells. We propose that sterols organizing the PM raft nanodomains, constitute a potential target for SU and other biosurfactants. In our opinion, the anticancer activity of biosurfactants is directly related with the higher cholesterol content found in many cancer cells.

Caffeic acid phenolipids in the protection of cell membranes from oxidative injuries. Interaction with the membrane phospholipid bilayer.

Caffeic acid (CA) has demonstrated a strong intracellular antioxidant ability by scavenging ROS, contributing to the maintenance of cell membrane structural integrity and to reduce oxidative injuries in other cell components. Nevertheless, caffeic acid has limited usage, due to its hydrophilic character. In this work, the introduction of alkyl chains in the caffeic acid molecule by esterification (methyl - C1, ethyl - C2, butyl - C4, hexyl - C6, octyl - C8 and hexadecyl - C16), significantly increased its lipophilicity. All caffeates tested showed a much higher protective activity than caffeic acid against red blood cells (RBCs) AAPH-induced oxidative stress; this protection was heavily dependent on the length of the alkyl chain of the esters, and on their concentration. At 2.5 and 5 µM, the more lipophilic compounds (C8 and C16) showed a remarkable antioxidant activity, inhibiting hemolysis; probably, their better location within the membrane leads to a better antioxidative protection; however, at 50 µM, the more hydrophilic compounds (C1-C4) showed a better activity against hemolysis than the more lipophilic ones (C8-C16). At this higher concentration, the better interaction of the more lipophilic compounds with the membrane seems to cause changes in RBC membrane fluidity, disturbing membrane integrity. Our data show that the antioxidant activity of these compounds could play an important role for the protection of different tissues and organs, by protecting cell membranes from oxidative injuries.

Staphyloxanthin inhibitory potential of thymol impairs antioxidant fitness, enhances neutrophil mediated killing and alters membrane fluidity of methicillin resistant Staphylococcus aureus.

Staphylococcus aureus is a leading pathogen responsible for mild to severe invasive infections in humans. Especially, methicillin resistant Staphylococcus aureus (MRSA) is prevalent in hospital and community associated infections. Staphyloxanthin is a golden yellow color eponymous pigment produced by S. aureus and provides resistance to reactive oxygen species (ROS) and host neutrophil-based killing. In addition, this membrane pigment contributes to membrane rigidity and helps MRSA to survive under stress conditions. Targeting virulence of pathogen without exerting selection pressure is the recent approach to fight bacterial infections without developing drug resistance. The present study for the first time evaluated the staphyloxanthin inhibitory potential of thymol against MRSA. Qualitative and quantitative analyses demonstrated 90% of staphyloxanthin inhibition at 100 µg/mL concentration of thymol without alteration in growth. Molecular docking analysis and in vitro measurement of metabolic intermediates of staphyloxanthin revealed that thymol could possibly interact with CrtM to inhibit staphyloxanthin. Absorbance and infra red spectra further validated the inhibition of staphyloxanthin by thymol. In addition, thymol treatment significantly reduced the resistance of MRSA to ROS and neutrophil-based killing as exhibited by oxidant susceptibility assays and ex vivo innate immune clearance assay using human whole blood and neutrophils. Further, reduction in staphyloxanthin by thymol treatment increased the membrane fluidity and made MRSA cells more susceptible to membrane targeting antibiotic polymyxin B. Especially, thymol was found to be non-cytotoxic to human peripheral blood mononuclear cells. Our study validated the antivirulence potential of thymol against MRSA by inhibiting staphyloxanthin and suggests the prospective therapeutic role of thymol to combat MRSA infections.

N-(4-Hydroxyphenyl) Retinamide Suppresses SARS-CoV-2 Spike Protein-Mediated Cell-Cell Fusion by a Dihydroceramide Δ4-Desaturase 1-Independent Mechanism.

The membrane fusion between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host cells is essential for the initial step of infection; therefore, the host cell membrane components, including sphingolipids, influence the viral infection. We assessed several inhibitors of the enzymes pertaining to sphingolipid metabolism, against SARS-CoV-2 spike protein (S)-mediated cell-cell fusion and viral infection. N-(4-Hydroxyphenyl) retinamide (4-HPR), an inhibitor of dihydroceramide Δ4-desaturase 1 (DES1), suppressed cell-cell fusion and viral infection. The analysis of sphingolipid levels revealed that the inhibition efficiencies of cell-cell fusion and viral infection in 4-HPR-treated cells were consistent with an increased ratio of saturated sphinganine-based lipids to total sphingolipids. We investigated the relationship of DES1 with the inhibition efficiencies of cell-cell fusion. The changes in the sphingolipid profile induced by 4-HPR were mitigated by the supplementation with exogenous cell-permeative ceramide; however, the reduced cell-cell fusion could not be reversed. The efficiency of cell-cell fusion in DES1 knockout (KO) cells was at a level comparable to that in wild-type (WT) cells; however, the ratio of saturated sphinganine-based lipids to the total sphingolipids was higher in DES1 KO cells than in WT cells. 4-HPR reduced cell membrane fluidity without any significant effects on the expression or localization of angiotensin-converting enzyme 2, the SARS-CoV-2 receptor. Therefore, 4-HPR suppresses SARS-CoV-2 S-mediated membrane fusion through a DES1-independent mechanism, and this decrease in membrane fluidity induced by 4-HPR could be the major cause for the inhibition of SARS-CoV-2 infection. IMPORTANCE Sphingolipids could play an important role in SARS-CoV-2 S-mediated membrane fusion with host cells. We studied the cell-cell fusion using SARS-CoV-2 S-expressing cells and sphingolipid-manipulated target cells, with an inhibitor of the sphingolipid metabolism. 4-HPR (also known as fenretinide) is an inhibitor of DES1, and it exhibits antitumor activity and suppresses cell-cell fusion and viral infection. 4-HPR suppresses membrane fusion through a decrease in membrane fluidity, which could possibly be the cause for the inhibition of SARS-CoV-2 infection. There is accumulating clinical data on the safety of 4-HPR. Therefore, it could be a potential candidate drug against COVID-19.

Therapeutic lithium alters polar head-group region of lipid bilayer and prevents lipid peroxidation in forebrain cortex of sleep-deprived rats.

Lithium is regarded as a unique therapeutic agent for the management of bipolar disorder (BD). In efforts to explain the favourable effects of lithium in BD, a wide range of mechanisms was suggested. Among those, the effect of clinically relevant concentrations of lithium on the plasma membrane was extensively studied. However, the biophysical properties of brain membranes isolated from experimental animals exposed to acute, short-term and chronic lithium have not been performed to-date. In this study, we compared the biophysical parameters and level of lipid peroxidation in membranes isolated from forebrain cortex (FBC) of therapeutic lithium-treated and/or sleep-deprived rats. Lithium interaction with FBC membranes was characterized by appropriate fluorescent probes. DPH (1,6-diphenyl-1,3,5-hexatriene) and TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulphonate) were used for characterization of the hydrophobic lipid core and Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) for the membrane-water interface. Lipid peroxidation was determined by immunoblot analysis of 4-HNE-(4-hydroxynonenal)-protein adducts. The organization of polar head-group region of FBC membranes, measured by Laurdan generalized polarization, was substantially altered by sleep deprivation and augmented by lithium treatment. Hydrophobic membrane interior characterized by steady-state anisotropy of DPH and TMA-DPH fluorescence was unchanged. Chronic lithium had a protective effect against peroxidative damage of membrane lipids in FBC. In summary, lithium administration at a therapeutic level and/or sleep deprivation as an animal model of mania resulted in changes in rat FBC membrane properties.

Hydroxyurea-induced membrane fluidity decreasing as a characterization of neuronal membrane aging in Alzheimer's disease.

Aging is one of the significant risk factors for Alzheimer's disease (AD). Therefore, this study aimed to propose a new hypothesis "membrane aging" as a critical pathogenesis of AD. The concept of "membrane aging" was reviewed, and the possible mechanisms of membrane aging as the primary culprit of AD were clarified. To further prove this hypothesis, a hydroxyurea-induced "membrane aging" model was established in vitro and in vivo. First, neuronal aging was validated by immunocytochemistry with age-related markers, and membrane aging phenotypes were confirmed. The alterations of membrane fluidity within APP/PS1 mice were re-proved by intracerebroventricular injection of hydroxyurea. Decreased membrane fluidity was found in vitro and in vivo, accompanied by increased total cholesterol concentration in neurons but decreased cholesterol levels within membrane fractions. The Aß level increased considerably after hydroxyurea treatment both in vitro and in vivo. DHA co-treatment ameliorated membrane aging phenotypes and Aß aggregation. The study revealed the AMP-activated protein kinase/acetyl CoA carboxylase/carnitine palmitoyl transferase 1 pathway involved in membrane aging processes. These results strongly supported the idea that membrane aging was a pathogenesis of AD and might serve as a new therapeutic target for AD.