Micron-scale phase segregation in lipid monolayers induced by myelin basic protein in the presence of a cholesterol analog

It was previously shown that myelin basic protein (MBP) can induce phase segregation in whole myelin monolayers and myelin lipid films, which leads to the accumulation of proteins into a separate phase, segregated from a cholesterol-enriched lipid phase. In this work we investigated some factors regulating the phase segregation induced by MBP using fluorescent microscopy of monolayers formed with binary and ternary lipid mixtures of dihydrocholesterol (a less-oxidable cholesterol analog) and phospholipids. The influence of the addition of salts to the subphase and of varying the lipid composition was analyzed. Our results show that MBP can induce a dihydrocholesterol-dependent segregation of phases that can be further regulated by the electrolyte concentration in the subphase and the composition (type and proportion) of non-sterol lipids. In this way, changes of the lipid composition of the film or the ionic strength in the aqueous media modify the local surface density of MBP and the properties (phase state and composition) of the protein environment.     

Effects of in vitro incorporation of cholesterol and cholesterol analogues into rat platelets

Cholesterol and analogues of cholesterol bearing shorter side chains were incorporated into rat platelet membranes by incubation with sterol-rich liposomes in vitro. Cholesterol-enriched platelets showed increased aggregability to collagen compared with controls. Platelets containing the cholesterol analogues pregn-5-en-3β-ol and chol-5-en-3β-ol were even more sensitive to aggregation and could aggregate spontaneously on stirring. The size of the platelets containing pregn-5-en-3β-ol was markedly reduced when compared with controls in the scanning electron microscope. The results suggest that the sterol content and structure of the platelet membrane can have a critical role in maintaining the normal function of the cell.     

iPSC-derived myelinoids to study myelin biology of humans

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Amperometric determination of serum total cholesterol with nanoparticles of cholesterol esterase and cholesterol oxidase

We describe the preparation of glutaraldehyde cross-linked and functionalized cholesterol esterase nanoparticles (ChENPs) and cholesterol oxidase nanoparticles (ChOxNPs) aggregates and their co-immobilization onto Au electrode for improved amperometric determination of serum total cholesterol. Transmission electron microscope (TEM) images of ChENPs and ChOxNPs showed their spherical shape and average size of 35.40 and 56.97 nm, respectively. Scanning electron microscope (SEM) studies of Au electrode confirmed the co-immobilization of enzyme nanoparticles (ENPs). The biosensor exhibited optimal response at pH 5.5 and 40 °C within 5 s when polarized at +0.25 V versus Ag/AgCl. The working/linear range of the biosensor was 10–700 mg/dl for cholesterol. The sensor showed high sensitivity and measured total cholesterol as low as 0.1 mg/dl. The biosensor was evaluated and employed for total cholesterol determination in sera of apparently healthy and diseased persons. The analytical recovery of added cholesterol was 90%, whereas the within-batch and between-batch coefficients of variation (CVs) were less than 2% and less than 3%. There was a good correlation (r = 0.99) between serum cholesterol values as measured by the standard enzymic colorimetric method and the current method. The initial activity of ENPs/working electrode was reduced by 50% during its regular use (200 times) over a period of 60 days when stored dry at 4 °C.     

Quantitative analysis of cholesterol nucleation with time in supersaturated model bile

The amount of cholesterol (Ch) crystals formed in supersaturated taurochenodeoxycholate (TCDC) - lecithin (L) solutions of the same Ch saturation index (CSI) but at different Ch thermodynamic activities (Ch A_T) was quantified at different time intervals. The initial Ch nucleation rate (i.e., amount of Ch crystals formed with respect to time) in a Ch A_T = 1.73 and TCDC to L molar ratio (TCDC:L) = 5.1 system was faster than that in a Ch A_T = 1.42 and TCDC:L = 3.4 system. Shaking could enhance the early appearance of Ch crystals and cause the fast initial Ch nucleation rates for the TCDC:L = 5.1 and the TCDC:L = 3.4 systems. The final Ch nucleation rates were faster than the initial Ch nucleation rates for the TCDC:L = 5.1 and the TCDC:L = 3.4 systems. According to a light scattering analysis of vesicle concentration in supersaturated TCDC–L solutions, vesicles provide nucleation sites only in the Ch nucleation process and the vesicle concentration may not be an important factor for the Ch nucleation rate. A model of a mixed TCDC–L micelle releasing Ch molecules together with the surface area of Ch crystals formed was used in the interpretation of the Ch nucleation.     

Myelin-associated glycoprotein and myelin galactolipids stabilize developing axo-glial interactions

We have analyzed mice that lack both the myelin-associated glycoprotein (MAG) and the myelin galactolipids, two glial components implicated in mediating axo-glial interactions during the myelination process. The single-mutant mice produce abnormal myelin containing similar ultrastructural abnormalities, suggesting that these molecules may play an overlapping role in myelin formation. Furthermore, the absence of the galactolipids results in a disruption in paranodal axo-glial interactions, and we show here that similar, albeit less severe, abnormalities exist in the developing MAG mutant. In the double-mutant mice, maintenance of axo-glial adhesion is significantly more affected than in the single mutants, supporting the overlapping function hypothesis. We also show that independently of MAG, galactolipids, and paranodal junctional components, immature nodes of Ranvier form normally, but rapidly destabilize in their absence. These data indicate that distinct molecular mechanisms are responsible for the formation and maintenance of axo-glial interactions.     

iRhom1 rescues cognitive dysfunction in multiple sclerosis via preventing myelin injury

Multiple sclerosis (MS) is characterized by myelin sheath injury. A disintegrin and metalloprotease-17 (ADAM17), a disintegrin and metalloproteinase, is essential in regulating oligodendrocyte (OL) regeneration and remyelination under demyelinating conditions. iRhom1, a highly conserved inactive protease that belongs to the rhomboid family, is one of key regulators for ADAM17 maturation. However, it is unknown whether iRhom1 also plays a role in central neuron system myelination under demyelinating conditions like MS. In this study, we investigated the function of iRhom1/ADAM17 in cognitive capability in MS by establishing the mice with iRhom1 overexpression in the hippocampus.     

Peripheral nervous system myelin assembly in vitro: perturbation by the ionophore monensin

Abstract: Sciatic nerves from 13-day-old rats were incubated in vitro with [³⁵S]methionine in the presence or absence of 0.22 μM monensin and total paniculate and myelin fractions prepared. The total particulate was further subfractionated by continuous density gradient centrifugation, after which the maximal specific activities of three marker enzymes, 2′,3′-cyclic nucleotide phospho-diesterase (myelin), 5′-nucleotidase (plasma membrane), and cerebroside sulphotransferase were recovered at 0.72, 0.82, and 0.92 M sucrose, respectively. The radiolabelled proteins present in the gradient subtractions were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography, and bands corresponding to the P₀ and myelin basic proteins were identified by co-migration with unlabelled myelin marker proteins on both one-dimensional SDS-PAGE and two-dimensional nonequilibrium isoelectric focussing/SDS-PAGE systems. Following a 90-min incubation with [³⁵S]methionine, newly synthesized myelin basic proteins were recovered in fractions between 0.5 and 0.7 M sucrose; this distribution was unaltered by monensin. In contrast, the distribution of newly synthesized P₀ protein across the gradients was influenced by monensin: a bimodal distribution across the control gradients with peaks of recovery of 0.60 and 0.82 M sucrose was altered to give a single peak at an intermediate density of 0.72 M sucrose. The total proportions of newly synthesized P₀ and myelin basic proteins (MBP) present across the entire gradients were calculated from the fluorograms, and the ratio was found to be 2.8 P₀: (LBP + SBP), in both the presence and absence of the ionophore. However, only 70% and 50% of the control levels of MBP and P₀ were recovered with a purified myelin fraction after incubation with monensin. The results are discussed with reference to different intracellular transport processes for the P₀ glycoprotein and the MBP within the Schwann cell, and also to the differential compartmentation of the sites of synthesis and membrane export within the Golgi body.     

Alterations of cholesterol precursor levels in Alzheimer's disease

Cerebral and extracerebral cholesterol metabolism are altered in Alzheimer's disease (AD) as indicated by reduced plasma levels of the cholesterol elimination products 24S-hydroxycholesterol, which is of cerebral origin, and of 27-hydroxycholesterol, which is formed extracerebrally. However, it has to be evaluated, if changes of cholesterol metabolism in the whole body or in the CNS are exclusively due to the altered elimination of cholesterol or are also due to altered de novo synthesis in AD. We investigated CSF and plasma levels of cholesterol and of its precursors lanosterol, lathosterol and desmosterol in AD patients and non-demented controls. We found CSF levels of cholesterol (p = 0.011), absolute levels of all investigated cholesterol precursors (each p < 0.001) and ratios of cholesterol precursors/cholesterol (each < 0.01) to be lower in AD patients as compared to controls. In plasma, the absolute levels of lanosterol (p = 0.026) and lathosterol (p < 0.001) and the ratio of lathosterol/cholesterol (p = 0.002) but none of the other investigated parameters were reduced in AD patients (p > 0.1). Furthermore, ratios of desmosterol/lathosterol in CSF (p = 0.023) and plasma (p = 0.009) were higher in AD patients as compared to controls. Our data support the hypothesis that cholesterol metabolism is altered in AD and further suggest that especially cholesterol de novo synthesis within the CNS of AD patients might be reduced. These findings raise doubt on a beneficial effect of cholesterol lowering treatment in manifest AD.     

Evidence for a water-soluble intermediate in exchange of cholesterol between membranes

The mechanism of inter-membrane cholesterol exchange has been a matter of some debate. Evidence from kinetic studies indicates that cholesterol must transfer to and from membranes in a water-soluble form. In this study attempts have been made to demonstrate that this occurs using either dialysis membranes or a barrierless multiphase polymer system to physically separate the membranes. In both systems small amounts of cholesterol were seen to transfer from one membrane pool to another using both liposomes and erythrocyte membranes as donors or acceptors. The cholesterol transfer was shown to be independent of the movement of other membrane components. The amount of transfer observed was limited by the physical properties of the systems employed. The barrier to cholesterol transfer in the dialysis membrane system is primarily the pore size of the membrane, while in the multiphase polymer system the transfer was limited by the viscosity of the medium and the distance between the lower and upper phases containing the membranes. Nevertheless, the results provide evidence that cholesterol transfer is by a dissociation of molecules from membranes into the aqueous medium and does not require the formation of a collision complex between the membranes.     

Galectin-3 drives oligodendrocyte differentiation to control myelin integrity and function

Galectins control critical pathophysiological processes, including the progression and resolution of central nervous system (CNS) inflammation. In spite of considerable progress in dissecting their role within lymphoid organs, their functions within the inflamed CNS remain elusive. Here, we investigated the role of galectin-glycan interactions in the control of oligodendrocyte (OLG) differentiation, myelin integrity and function. Both galectin-1 and -3 were abundant in astrocytes and microglia. Although galectin-1 was abundant in immature but not in differentiated OLGs, galectin-3 was upregulated during OLG differentiation. Biochemical analysis revealed increased activity of metalloproteinases responsible for cleaving galectin-3 during OLG differentiation and modulating its biological activity. Exposure to galectin-3 promoted OLG differentiation in a dose- and carbohydrate-dependent fashion consistent with the 'glycosylation signature' of immature versus differentiated OLG. Accordingly, conditioned media from galectin-3-expressing, but not galectin-3-deficient (Lgals3(-/-)) microglia, successfully promoted OLG differentiation. Supporting these findings, morphometric analysis showed a significant decrease in the frequency of myelinated axons, myelin turns (lamellae) and g-ratio in the corpus callosum and striatum of Lgals3(-/-) compared with wild-type (WT) mice. Moreover, the myelin structure was loosely wrapped around the axons and less smooth in Lgals3(-/-) mice versus WT mice. Behavior analysis revealed decreased anxiety in Lgals3(-/-) mice similar to that observed during early demyelination induced by cuprizone intoxication. Finally, commitment toward the oligodendroglial fate was favored in neurospheres isolated from WT but not Lgals3(-/-) mice. Hence, glial-derived galectin-3, but not galectin-1, promotes OLG differentiation, thus contributing to myelin integrity and function with critical implications in the recovery of inflammatory demyelinating disorders.     

The effects of cholesterol on the development of Heliothis zea

Heliothis zea was reared on an artificial diet, which lacked supplementation with plant materials, in order to determine the effects of cholesterol on the development of this insect. A number of parameters of larval development were found to be dependent upon the concentration of dietary sterol including: the number of moults which the larvae completed within a particular time interval, the ability of the larvae to pupate and the survival of the larvae. The number of moults which a larva completed prior to pupation, though, was independent of the concentration of sterol.     

The N-terminal domain of NPC1L1 protein binds cholesterol and plays essential roles in cholesterol uptake

Niemann-Pick C1-like 1 (NPC1L1) is a multitransmembrane protein playing a crucial role in dietary and biliary cholesterol absorption. Cholesterol promotes the formation and endocytosis of NPC1L1-flotillin-cholesterol membrane microdomains, which is an early step in cholesterol uptake. How cholesterol is sensed in this step is unknown. Here, we find that the N-terminal domain (NTD) of NPC1L1 binds cholesterol. Mutation of residue Leu-216 in NPC1L1-NTD eliminates cholesterol binding, decreases the formation of NPC1L1-flotillin-cholesterol membrane microdomains, and prevents NPC1L1-mediated cholesterol uptake in culture cells and mice livers. NPC1L1-NTD specifically binds cholesterol but not plant sterols, which may account for the selective cholesterol absorption in intestine. Furthermore, 25- or 27-hydroxycholesterol competes with cholesterol to bind NPC1L1-NTD and inhibits the cholesterol induced endocytosis of NPC1L1. Together, these results demonstrate that plasma membrane-localized NPC1L1 binds exogenous cholesterol via its NTD, and facilitates the formation of NPC1L1-flotillin-cholesterol membrane microdomains that are then internalized into cells through the clathrin-AP2 pathway. Our study uncovers the mechanism of cholesterol sensing by NPC1L1 and proposes a mechanism for selective cholesterol absorption.     

Isocoumarin-based inhibitors of pancreatic cholesterol esterase

Pancreatic cholesterol esterase (CEase), which is secreted from the exocrine pancreas, is a serine hydrolase that aids in the bile salt-dependent hydrolysis of dietary cholesteryl esters and contributes to the hydrolysis of triglycerides and phospholipids. Additional roles for CEase in intestinal micelle formation and in transport of free cholesterol to the enterocyte have been suggested. There also are studies that point to a pathological role(s) for CEase in the circulation where CEase accumulates in atherosclerotic lesions and triggers proliferation of smooth muscle cells. Thus, there is interest in CEase as a potential drug target. 4-Chloro-3-alkoxyisocoumarins are a class of haloenol lactones that inhibit serine hydrolases and serine proteases and have the potential to be suicide inhibitors. In the present study, we have developed 3-alkoxychloroisocoumarins that are potent inhibitors of CEase. These inhibitors were designed to have a saturated cycloalkane ring incorporated into a 3-alkoxy substituent. The size of the ring as well as the length of the tether holding the ring was found to be important contributors to binding to CEase. 4-Chloro-3-(4-cyclohexylbutoxy)isocoumarin and 4-chloro-3-(3-cyclopentylpropoxy)isocoumarin were demonstrated to be potent reversible inhibitors of CEase, with dissociation constants of 11 nM and 19 nM, respectively. The kinetic results are consistent with predictions from molecular modeling.     

Analogous standard motifs in myelin basic protein and in MARCKS

Myelin basic protein (MBP) and myristoylated alanine-rich C-kinase substrate (MARCKS) are similar in terms of having extended conformations regulated by their environment (i.e., solubilised or lipid-associated), N-terminal modifications, a dual nature of interactions with lipids, binding to actin and Ca²⁺-calmodulin, and being substrates for different kinds of protein kinases. The further sequence similarities of segments of MBP with lipid effector regions of MARCKS, and numerous reports in the literature, support the thesis that some developmental isoform of MBP functions in signal transduction.     

Expression of streptomycete cholesterol oxidase inEscherichia coli

Summary A streptomycete gene coding for extracellular cholesterol oxidase (choA) was subcloned and expressed inEscherichia coli. The pUCO series recombinants were obtained by inserting thechoA gene into the uniqueKpnI site of pUC19 vector. Expression was observed with pUCO192A and pUCO193 constructs in which the cloned gene(s) were aligned with the upstreamlacZ promoter. Isopropyl -d-thioglucopyranoside (IPTG) enhanced this expression up to 2.5-fold. Specific Cho activity in the cell extracts of the stable pUCO193 transformant were 0.004 U and 0.007 U per mg protein without and with IPTG induction, respectively. Cho activity was detected in the spent medium of this culture, suggesting possible secretion of the enzyme.     

Enhancing myelin renewal reverses cognitive dysfunction in a murine model of Alzheimer’s disease

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