Nicolai A. Avdulov

Increasing Age Alters Transbilayer Fluidity and Cholesterol Asymmetry in Synaptic Plasma Membranes of Mice

Abstract: Previous studies examining age differences in membrane fluidity and cholesterol content have reported on the average or total change in membrane structure, respectively. However, a membrane consists of an exofacial leaflet and a cytofacial leaflet that differ in fluidity and cholesterol distribution. The purpose of the present experiments was to determine fluidity and cholesterol distribution of the exofacial and cytofacial leaflets of brain synaptic plasma membranes (SPMs) from 3–4‐, 14–15‐, and 24–25‐month‐old C57BL/6NNIA mice by using trinitrobenzenesulfonic acid (TNBS)‐quenching techniques and fluorescent probes. The exofacial leaflet of SPMs from young mice was significantly more fluid compared with the cytofacial leaflet. The large difference in fluidity between the two leaflets was abolished in SPMs of the oldest age group. Total SPM cholesterol and the cholesterol‐to‐phospholipid molar ratio did not differ among the three different age groups of mice. However, considerable differences were observed in the distribution of cholesterol in the two SPM leaflets. The exofacial leaflet contained substantially less cholesterol than did the cytofacial leaflet (13 vs. 87%, respectively) in SPMs of young mice. This asymmetric distribution of cholesterol was significantly modified with increasing age. There was an approximately twofold increase in exofacial leaflet cholesterol in the oldest group compared with the youngest age group. Transbilayer fluidity and cholesterol asymmetry were altered in SPMs of older mice. This approach is a new and different way of viewing how aging modifies membrane structure. Age differences in SPM leaflet structure may be an important factor regulating activity of certain membrane proteins.

Brain membrane cholesterol domains, aging and amyloid beta-peptides

Lipids are essential for the structural and functional integrity of membranes. Membrane lipids are not randomly distributed but are localized in different domains. These domains consist of the exofacial and cytofacial leaflets, cholesterol pools, annular lipids, and lipid rafts. Membrane lipid domains have been proposed to be involved in a variety of different functions including e.g. signal transduction, lipid transport and metabolism, and cell growth. Membrane lipid domains have been identified in brain and can be modified by different experimental conditions, aging and certain neurodegenerative diseases. Recent data reveal the very interesting possibility that membrane lipid domains may be a target of Alzheimers disease. There is a growing body of evidence showing an association between cholesterol and Alzheimers disease, and cholesterol is a major component of membrane lipid domains. Here we discuss recent data on brain membrane lipid domains emphasizing the structural and functional role of cholesterol. In addition, lipid domains and aging, and the potential interaction of lipid domains and amyloid beta-peptides (Abeta) that are a major component of senile plaques in brains of Alzheimers patients are considered. We propose that age changes in the asymmetric distribution of cholesterol in contrast to total or bulk cholesterol in neuronal plasma membranes provides a cooperative environment for accumulation of Abeta in plasma membranes and the accumulation of Abeta is due in part to a direct physico-chemical interaction with cholesterol in the membrane exofacial or outer leaflet.

Amyloid β‐Peptides Increase Annular and Bulk Fluidity and Induce Lipid Peroxidation in Brain Synaptic Plasma Membranes

Abstract: Amyloid β‐peptides (Aβ) may alter the neuronal membrane lipid environment by changing fluidity and inducing free radical lipid peroxidation. The effects of Aβ1–40 and Aβ25–35 on the fluidity of lipids adjacent to proteins (annular fluidity), bulk lipid fluidity, and lipid peroxidation were determined in rat synaptic plasma membranes (SPM). A fluorescent method based on radiationless energy transfer from tryptophan of SPM proteins to pyrene and pyrene monomer‐eximer formation was used to determine SPM annular fluidity and bulk fluidity, respectively. Lipid peroxidation was determined by the thiobarbituric acid assay. Annular fluidity and bulk fluidity of SPM were increased significantly (p≤ 0.02) by Aβ1–40. Similar effects on fluidity were observed for Aβ25–35 (p≤ 0.002). Increased fluidity was associated with lipid peroxidation. Both Aβ peptides significantly increased (p≤ 0.006) the amount of malondialdehyde in SPM. The addition of a water‐soluble analogue of vitamin E (Trolox) inhibited effects of Aβ on lipid peroxidation and fluidity in SPM. The fluidizing action of Aβ peptides on SPM may be due to the induction of lipid peroxidation by those peptides. Aβ‐induced changes in neuronal function, such as ion flux and enzyme activity, that have been reported previously may result from the combined effects of lipid peroxidation and increased membrane fluidity.

Lipid binding to sterol carrier protein-2 is inhibited by ethanol

Sterol carrier protein-2 (SCP-2) is an intracellular lipid carrier protein that binds cholesterol, phospholipids, fatty acids and other ligands. It has been reported that expression of SCP-2 was increased in brain nerve endings or synaptosomes of chronic ethanol-treated mice and it was shown that cholesterol homeostasis was altered in brain membranes of chronic ethanol-treated animals. Ethanol may interfere with the capacity of SCP-2 to bind cholesterol as well as other lipids. This hypothesis was tested using recombinant SCP-2 and fluorescent-labeled cholesterol, phosphatidylcholine (PC), and stearic acid. The association constants (Ka) of the ligand-SCP-2 complex were in the following order: NBD-cholesterol>NBD-PC>NBD-stearic acid. Ethanol, beginning at a concentration of 25 mM, significantly reduced the affinity of NBD-cholesterol and NBD-PC for SCP-2. Effects of ethanol on the Ka of NBD-stearic acid was significant only at the highest concentration that was examined (200 mM). Ethanol significantly increased the Bmax of NBD-cholesterol for SCP-2 but did not have a significant effect on the Bmax of NBD-PC. Similar results were found for effects of ethanol on the Kas and Bmaxs using pyrene-labeled cholesterol and PC. In conclusion, ethanol beginning at a physiological concentration of 25 mM inhibited binding of cholesterol and PC to SCP-2. However, effects of ethanol on lipid binding to SCP-2 were dependent on the type of lipid. Ethanol in vivo may interfere with lipid binding to SCP-2 and disrupt lipid trafficking within cells.

Cholesterol oxidation reduces Ca2+ + Mg2+-ATPase activity, interdigitation, and increases fluidity of brain synaptic plasma membranes

These experiments examined effects of cholesterol oxidation on Ca(2+)+Mg(2+)-ATPase activity, Na(+)+K(+)-ATPase activity, and membrane structure of brain synaptic plasma membranes (SPM). Cholesterol oxidase [E.C.1.1.3.6 from Brevibacterium sp.] was used to oxidize cholesterol. Two cholesterol pools were identified in synaptosomal membranes based on their accessibility to cholesterol oxidase. A rapidly oxidized cholesterol pool was observed with a 1t1/2 of 1.19 +/- 0.09 min and a second pool with a 2t1/2 of 38.30 +/- 4.16 min. Activity of Ca(2+)+Mg(2+)-ATPase was inhibited by low levels of cholesterol oxidation. Ten percent cholesterol oxidation, for example, resulted in approximately 35% percent inhibition of Ca(2+)+Mg(2+)-ATPase activity. After 13% cholesterol oxidation, further inhibition of Ca(2+)+Mg(2+)-ATPase activity was not observed. Activity of Na(+)+K(+)-ATPase was not affected by different levels of cholesterol oxidation (5%-40%). SPM interdigitation was significantly reduced and fluidity was significantly increased by cholesterol oxidation. The relationship observed between SPM interdigitation and Ca(2+)+Mg(2+)-ATPase activity was consistent with studies using model membranes [7]. Brain SPM function and structure were altered by relatively low levels of cholesterol oxidation and is a new approach to understanding cholesterol dynamics and neuronal function. The sensitivity of brain SPM to cholesterol oxidation may be important with respect to the proposed association between oxygen free radicals and certain neurodegenerative diseases.

Specific Binding of Ethanol to Cholesterol in Organic Solvents

Although ethanol has been reported to affect cholesterol homeostasis in biological membranes, the molecular mechanism of action is unknown. Here, nuclear magnetic resonance (NMR) spectroscopic techniques have been used to investigate possible direct interactions between ethanol and cholesterol in various low dielectric solvents (acetone, methanol, isopropanol, DMF, DMSO, chloroform, and CCl(4)). Measurement of (13)C chemical shifts, spin-lattice and multiplet relaxation times, as well as self-diffusion coefficients, indicates that ethanol interacts weakly, yet specifically, with the HC-OH moiety and the two flanking methylenes in the cyclohexanol ring of cholesterol. This interaction is most strong in the least polar-solvent carbon tetrachloride where the ethanol-cholesterol equilibrium dissociation constant is estimated to be 2 x 10(-3) M. (13)C-NMR spin-lattice relaxation studies allow insight into the geometry of this complex, which is best modeled with the methyl group of ethanol sandwiched between the two methylenes in the cyclohexanol ring and the hydroxyl group of ethanol hydrogen bonded to the hydroxyl group of cholesterol.

Lipid carrier proteins and ethanol

Ethanol has a pronounced effect on lipid homeostasis. It is our overall hypothesis that certain lipid carrier proteins are targets of acute and chronic ethanol exposure and that perturbation of these proteins induces lipid dysfunction leading to cellular pathophysiology. These proteins include both intracellular proteins and lipoproteins. This paper examines recent data on the interaction of ethanol with these proteins. In addition, new data are presented on the stimulatory effects of ethanol on low-density-lipoprotein (LDL)-mediated cholesterol uptake into fibroblasts and direct perturbation of the LDL apolipoprotein, apolipoprotein B. A cell model is presented that outlines potential mechanisms thought to be involved in ethanol perturbation of cholesterol transport and distribution.