J Mol Neurosci (2007) 33:129–145 DOI 10.1007/s12031-007-0057-9

Abstracts of Poster Presentations International Workshop on Brain Uptake and Utilization of Fatty Acids, Lipids & Lipoproteins: Applications to Neurological Diseases Bethesda, MD, October 7–9, 2004

I-1 The Influence of Hydrocarbon Chain Length, Polyunsaturation, and of Lipid Headgroups on Lateral Diffusion Rates of Lipids Rachel Casas*, John Dustman*, Walter Teague, Klaus Gawrisch Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD 20852, USA

our understanding of biophysical membrane models and cellular processes within the human body. Lipid diffusion coefficients directly influence the kinetics of membrane protein–protein interactions, protein–ligand interactions, mixed lipid systems, and lipid barriers. 1. Gaede, H.C., Gawrisch, K. Biophys. J. 85 (2003) 1734–1740 2. Gaede, H.C., Gawrisch, K. Magn. Reson. Chem. 42 (2004) 115–122

*Both authors contributed equally to this work I-2 We measured the rate of lipid lateral diffusion in phospholipid bilayers using a novel experimental approach, magic angle spinning (MAS) nuclear magnetic resonance (NMR) with application of pulsed field gradients (1, 2). In MAS NMR the prepared sample is rapidly spun at an angle of 54.7° to the NMR magnetic field. MAS averages all angular-dependent magnetic interactions of the lipids that broaden NMR resonance lines resulting in spectral resolution close to high-resolution NMR. With application of magnetic field gradients, NMR is an effective and efficient means to monitor the lateral diffusion of lipids. The method measures the endogenous proton resonance signal of lipids, which makes the use of potentially perturbing labels unnecessary. Each lipid studied was monitored over four different diffusion times at a fixed temperature to estimate the liposome radius of curvature, and then at five different temperatures with a fixed diffusion time to determine the activation energy of diffusion. The samples consisted of purified lipids comparing the headgroups phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine; contrasting chain lengths of 14, 16, and 18 carbons; and comparing degrees of unsaturation from one to six double bonds along a chain. With increasing chain length, the activation energy increases and the diffusion coefficient decreases. With increasing unsaturation, the activation energy decreases and the diffusion coefficient increases. There is insignificant difference in the rates of lateral diffusion and activation energy of the lipids with different headgroups. Knowledge of lateral diffusion contributes to

Polyunsaturated Docosahexaenoic Acid vs Docosapentaenoic Acid—Differences in Lipid Matrix Properties from the Loss of One Double Bond Nadukkudy V. Eldho1, Scott E. Feller2, Stephanie TristramNagle3, Ivan V. Polozov1, Klaus Gawrisch1 1 Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, National Institutes of Health, Rockville, MD, USA 2 Department of Chemistry, Wabash College, Crawfordsville, IN 47933, USA 3 Department of Physics, Carnegie Mellon University, Pittsburgh, PA 15213, USA Insufficient supply of docosahexaenoic acid (DHA, 22: 6n−3) or its ω-3 fatty acid precursors during infancy results in replacement of DHA by docosapentaenoic acid (DPA, 22:5n−6), an ω-6 fatty acid with the same number of carbon atoms but one fewer double bond at the terminal methyl end of the chain. This substitution is generally nonlethal, but is associated with some loss of brain function. In this study, we studied if this insignificant difference in chain structure results in differences of membrane structure and dynamics. Experiments were conducted by NMR and x-ray diffraction, and results were compared with the outcome of molecular dynamics simulations. Both polyunsaturated chains are motionally disordered to the point that intermolecular choline headgroup-to-chain methyl group

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contacts occur with low but measurable probability. The correlation times of polyunsaturated chain motions decrease in steps from double bond to double bond. The chain motions near the carbonyl group have significant contributions from correlations times in the nanosecond range whereas the motions near the terminal methyl group are dominated by correlation times of picoseconds. The DHA chains with one additional double bond are more flexible at the methyl end and move with shorter correlation times. The stearic acid paired with the DHA in mixed-chain lipids has significantly lower order parameters from the center of the chains to the terminal methyl group, indicating differences in the packing near the center of the bilayer. Unsaturation affects the distribution of chain density along the bilayer normal with polyunsaturated chains having higher density near the lipid water interface whereas the density of saturated chains is higher in the bilayer center. The asymmetry of distribution is smaller for lipids with DPA compared to DHA. We propose that the function of integral membrane proteins like GPCR is altered by such differences in membrane structure and dynamics.

I-3 Monitoring Uptake of Fatty Acids and the Effects of Phloretin by BODIPY Fluorescence Wen Guo, James A. Hamilton Boston University School of Medicine, Boston, MA, USA BODIPY-fatty acid (FA) is used extensively as a fluorescent probe for the study of FA uptake into cells but without differentiation of transport and metabolism. We employed this probe to test if the two processes can be monitored separately and to test effects of the inhibitor phloretin on uptake and efflux of BODIPY-FA from cells. 3T3-L1 preadipocytes and differentiated 3T3-L1 adipocytes were incubated with BODIPY-FAC12 (20 μM) in DMEM/10% fetal bovine serum (FBS) and 7 μM bovine serum albumin (BSA) for 5 s to 30 min. Incubation was stopped by washing the cell monolayer with ice-cold KRB buffer with or without 0.1% BSA or 200 μM phloretin (×3), covered with 1 ml KRB and imaged using an inverted fluorescent microscope equipped with a Nikon Digital Camera. For selected experiments, cells were preincubated with phloretin at (250 μM) in DMEM/FBS for 1 h before BODIPY-FA was added. Preadipocytes and adipocytes exhibited high fluorescence after a 5-s incubation and washing with KRB without BSA. Adding BSA to the washing buffer removed the fluorescence from preadipocytes completely after shortterm incubations (5 s to 2 min); BSA washing became progressively less effective with longer incubation times and was completely ineffective after 30 min of incubation.

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In contrast, washing differentiated adipocytes failed to remove BODIPY-FA even when the incubation time was short (10 s). Preincubation with phloretin reduced the uptake of BODIPY-FA by preadipocytes, possibly because the very high concentrations of phloretin can saturate the plasma lipid bilayer with phloretin. Because these cells do not express CD36 or other putative FA transporters, previous findings that phloretin inhibits FA uptake in adipocytes might not result from a specific inhibition of CD36, as proposed by others. Adding phloretin to BSAcontaining washing buffer had no effect on the removal of BODIPY-FA from preadipocytes, but increased the efflux of BODIPY-FA from preadipocytes during the wash without BSA after short-term incubation periods (5 s to 2 min). After prolonged incubation (30 min), adding phloretin to the wash solution (±BSA) had no effect on cell fluorescence. BODIPY-FA is converted into esterified products slowly in preadipocytes and rapidly in adipocytes. Only nonmetabolized BODIPY-FA is removed by washing with BSA. Thus, the use of BODIPY-FA for measuring FA uptake into cells is complicated by the intracellular metabolism of this derivatized FA, and the transport of FA through the plasma membrane can be studied only under restricted conditions. Phloretin creates ambiguities in the interpretation of FA uptake data, and does not “block” the uptake of FA by inhibition of putative transport proteins.

I-4 Fatty Acid Transport Across the Plasma Membrane of Rat Adipocytes Treated with “Inhibitors” of Membrane Transport Proteins J. R. Simard1, N. Huang2, and J. A. Hamilton2 1 Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, MA, USA 2 Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA, USA The mechanism by which fatty acids (FA) pass through cell membranes is widely debated. Recent studies have shown that FA diffuse across the plasma membrane of cells such as adipocytes rapidly enough to support metabolism. Alternatively, FA uptake into cells may be mediated by one or more plasma membrane proteins (i.e., fatty acid transport protein, CD36, and FABPpm). These proteins have been suggested to catalyze the movement of FA through the lipid bilayer of membranes, an activity that can reportedly be inhibited by a number of chemicals and enzymes. In this study, we investigated the effects of several “inhibitors” on

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the binding and transmembrane movement of FA in the plasma membrane of rat adipocytes. We utilized entrapped BCECF, a pH-sensitive dye, to monitor movement of FA to the inner leaflet of the plasma membrane after their binding to the outer leaflet. Without inhibitors present, addition of oleic acid (OA), caused a rapid (<5 s) decrease in BCECF fluorescence (pH drop). Pretreatment of cells with trypsin, pronase, quercetin, phloretin, diisothiocyanatostilbenedisulfonic acid, and succinimidyl oleate did not affect the rate or extent of the pH decrease in cells in response to 10 μM OA. In contrast, pentachlorophenol (PCPL) and sulfosuccinimidyl oleate (SSO) decreased the extent of the pH drop. However, SSO and PCPL alone caused a large pH drop in protein-free phospholipid vesicles, and may decrease inward diffusion of FA by establishing an unfavorable pH gradient. These results support the hypothesis that FA diffuse rapidly across biological membranes without catalysis by protein transporters. The widely used “inhibitors” of FA transport are known to have broad effects, and most can enter the cell. Their effects on FA transport may therefore be indirect, including interference with lipid metabolism.

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capped linked peptides were observed. In the darkadapted rhodopsin, we detected crosslinking between K67 and K141 using BS3 and DSS but not DSG, indicating that these two residues were closely located in the dark and this proximity appears to be lost after the light exposure. Furthermore, in the absence of the corresponding crosslinked peak using shorter linker, DSG demonstrates that the distance between these two residues were between 7.7 and 11.4 Å. Two other throughspace crosslinking pairs were also observed between K67K339 and K245-K311, respectively, in both light-exposed and dark-adapted rhodopsin. The special distance constraint generated by the crosslinking was consistent with the solution structure of rhodopsin obtained by NMR. The majority of lysines in the sequence of rhodopsin are located in the cytosolic loops. Therefore, analysis of the mass spectrometric data offers insight to the possible differences in the conformation changes and the position of the cytosolic loops relative to one another in these two activation states.

I-6 I-5 Characterization of Intrahelical Arrangement of Rhodopsin by Site-specific Chemical Crosslinking and Mass Spectrometry Zohra Olumee-Shabon, Kirk Hines, Burton Litman, and Hee-Yong Kim Laboratory of Membrane Biophysics and Biochemistry, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD, USA It has been a great challenge to obtain high-resolution three-dimensional structure of rhodopsin, a G-proteincoupled receptor because of its size, hydrophobicity, and solubility. Rhodopsin is activated as the light is absorbed by its ligands, 11-cis-retinal, which is covalently attached to lysine 296. Upon activation, this molecule goes through a conformational change to all trans isoform particularly at the cytoplasmic loops. To address these conformational changes of rhodopsin at different activation states, we used chemical crosslinking in conjunction with mass spectrometry. Protein was modified with lysine specific crosslinkers, bis(sulfosuccinimidyl) suberate (BS3), disuccinimidyl suberate (DSS), and disuccinimidyl glutarate (DSG). The crosslinked protein was then digested with trypsin. Peptides involved in crosslinking were determined by nanospray using either an orthogonal Qq-TOF or LCMSD trap mass spectrometers. Six modified sites including three through space, one internal, and two end-

Conformational Changes of Human Serum Albumin Induced by Long-chain Polyunsaturated and Monounsaturated Fatty Acids Bill Huang, Hee-Yong Kim* Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 12420 Parklawn Drive, Bethesda, MD 20892, USA Human serum albumin (HSA) is the most abundant protein in plasma. Its primary function is to transport hydrophobic fatty acids that are otherwise insoluble in circulating system. Although x-ray crystallographic and NMR studies have demonstrated that HSA undergoes conformational changes upon interacting with fatty acids, the detailed mechanism and sequential physiological implication is still unclear. In this study, mass spectrometry with chemical crosslinking was used to probe the conformational changes of HSA in solution after interacting with monounsaturated oleic acid (OA), polyunsaturated arachidonic acid (AA), or docosahexaenoic acid (DHA). Free or fatty-acid-bound HSA was modified with lysine-specific crosslinkers and digested with trypsin. Crosslinked peptides were analyzed by nanoelectrospray ionization mass spectrometry to localize the sites of crosslinking. Our data indicated that a local conformational change involving movement of the side chains of K402 (IIIA) or K541 (IIIB) occurred upon binding of all three fatty acids, suggesting a common fatty acid interaction site in this region. Our data also indicated that the side chains of

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K205 (IIA) and K466 (IIIA) moved closer toward each other upon binding with AA or DHA, but not with OA, suggesting that conformation of HSA bound to mono- and polyunsaturated fatty acid is distinctively different. Whereas these observations agreed with previous x-ray crystallographic studies, the distance between ɛ-amino groups of some crosslinked lysine pairs were shorter than the crystal structure predicted, possibly reflecting discrepancy from the solution structure. This method can serve as useful complement to x-ray crystallography, particularly in probing the structure of a protein in solution.

I-7 What Can Molecular Genetics and Yeast Tell Us About Fatty Acid Transport and Trafficking? Paul N. Black1,2, Hong Li1, Dina Darwis1,2, Paul A. Watkins3, Johannes Berger4, Concetta C. DiRusso1,2 1 Center for Metabolic Diseases, Ordway Research Institute, Albany, NY, USA 2 Center for Cardiovascular Sciences, Albany Medical College, Albany, NY, USA 3 Kennedy Kreiger Research Institute, Baltimore, MD, USA 4 Brain Research Institute, University of Vienna, Vienna, Austria The transmembrane movement of exogenous fatty acids is intimately tied to downstream metabolism and therefore in whole cells it is difficult to distinguish protein-mediated transport from metabolism. Biophysical studies are consistent with the postulate that fatty acids bind to and flip between membrane surfaces. In contrast, biochemical and genetic studies have implicated distinct membrane-bound and membrane-associated proteins in the trafficking of exogenous fatty acids across the plasma membrane. These latter studies do not discount the diffusional component but rather are consistent with the hypothesis that proteinmediated processes govern the selective movement of fatty acids into discreet metabolic pools. The fatty acid transport protein (FATP) family is a group of proteins predicted to be components of specific fatty acid trafficking pathways, including those governing the import of exogenous longchain fatty acids. In mammalian systems, six isoforms have been identified as players in the import of exogenous fatty acids or in the activation of very long-chain fatty acids. This has led to controversy as to whether these proteins function as membrane-bound fatty acid transporters or as acyl-CoA synthetases (ACS), which activate long-chain fatty acids concomitant with transport. The yeast FATP orthologue, Fat1p, is a bifunctional protein and is required

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for both the import of long-chain fatty acids and the activation of very-long-chain fatty acids (VLCF); these activities intrinsic to Fat1p are separable functions. To more precisely define the roles of the different mammalian isoforms in fatty acid trafficking, the six murine proteins (mmFATP1-6) were expressed and characterized in a genetically defined yeast strain, which cannot transport long-chain fatty acids and has reduced long-chain ACS activity (fat1Δfaa1Δ). Each isoform was evaluated for fatty acid transport, fatty acid activation (using C18:1, C20:4, and C24:0 as substrates), and accumulation of very longchain fatty acids. mmFATP1, -2, and -4 complemented the defects in fatty acid transport and VLCF activation associated with a deletion of the yeast FAT1 gene; mmFATP3, -5, and -6 did not complement the transport function although each was localized to the yeast plasma membrane. mmFATP3 and -6 both activated C20:4 and C24:0, whereas the expression of mmFATP5 did not substantially increase ACS activities using the substrates tested. These data support the conclusion that the different mmFATP isoforms play unique roles in the fatty acid trafficking, including the transport of exogenous long-chain fatty acids.

I-8 The Fatty Acid Transport Protein (FATP) Family: VeryLong-Chain Acyl-CoA Synthetases or Solute Carriers? Zhengtong Pei, Dony Maiguel, Zhenzhen Jia, Cicely J. Toomer, Paul A. Watkins Kennedy Krieger Institute, Baltimore, MD, USA Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Cellular fatty acids arise either from de novo synthesis or uptake from the extracellular milieu. Extracellular fatty acids must traverse the plasma membrane, after which they are activated to their CoA thioesters for subsequent metabolism. Both uptake and metabolism are rapid processes, and there has been considerable debate as to whether transport of fatty acids across the lipid bilayer of the plasma membrane proceeds by diffusion or requires transport proteins. One group of proteins proposed to translocate fatty acids is the six-member fatty acid transport protein (FATP) family. These proteins were designated as such because when overexpressed, host cells exhibited higher rates of accretion of radioactive or fluorescent fatty acids. However, one member of this family, FATP2, is identical to an enzyme with very long-chain acyl-CoA synthetase (ACSVL) activity. This enzyme (ACSVL1 or FATP2) was isolated using

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classical protein purification techniques. In fact, the sixmember ACSVL protein family is identical to the sixmember FATP family. Others and we have established that all six proteins have acyl-CoA synthetase activity. It remains to be established whether they participate in the physical translocation process, or facilitate transport by trapping, as CoA derivatives, fatty acids that enter cells by diffusion. To characterize the biological functions of the ACSVLs, we are investigating the properties of the overexpressed proteins and the endogenous proteins. We observed that for many ACSVLs, the subcellular location of the overexpressed protein differs from that of the endogenous protein. Using RNA interference (siRNA), we knocked down expression of ACSVL3 (FATP3) in MA-10 cells and FATP4 in Neuro-2a cells. Activation of both long-chain (C16:0) and verylong-chain fatty acids (C24:0) was decreased when ACSVL3 was depleted, whereas only C24:0 activation was significantly decreased when cellular FATP4 was depleted. Despite decreased enzyme activity, initial rates of uptake of [14C] C16:0 were not decreased when either ACSVL3 or FATP4 were depleted. In contrast, COS-1 cells overexpressing FATP4 showed enhanced [14C]C16:0 uptake. Neither ACSVL3 nor FATP4 were localized to plasma membrane under routine cell culture conditions. We conclude that, in the cell lines studied, endogenous ACSVL3 and FATP4 are not functioning as plasma membrane fatty acid translocators. Supported by National Institutes of Health grants ND37355, HD10981, and HD24061.

I-9 Heart Fatty Acid-binding Protein Expression Differentially Increases Brain Fatty Acid Uptake E. J. Murphy1, J. F. C. Glatz2 1 Department of Pharmacology, Physiology, and Therapeutics, University of North Dakota, Grand Forks, ND 58203, USA 2 Department of Molecular Genetics, Cardiovascular Research Institute Maastricht, Maastricht, The Netherlands Heart fatty acid-binding protein (H-FABP) is expressed in neuronal cell bodies and preferentially binds n−6 fatty acids in vitro. We examined the effect of H-FABP gene ablation on modifying arachidonic (20:4n−6) or palmitic (16:0) acid uptake and targeting in the brain in vivo. All lipids were analyzed using standard analytical techniques. H-FABP gene ablation reduced (24%) the uptake of labeled 20:4n−6 into the brain, whereas labeled 16:0 uptake was unaffected. This reduction in uptake resulted in diminished incorporation into phospholipids. Within the phospholipids,

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there was a marked reduction in incorporation in the choline glycerophospholipids (ChoGpl). This resulted in an increase in targeting to the phosphatidylinositol fraction at the expense of the ChoGpl. Because FABP can alter phospholipid metabolism, the effects of H-FABP gene ablation on brain lipid mass and composition were also determined. Total phospholipid mass, individual phospholipid class mass, and composition were unaltered; however, brain cholesterol content was markedly reduced in the gene-ablated mice, decreasing the cholesterol to phospholipid ratio. The proportion of n−6 fatty acids was reduced in brain phospholipids in gene-ablated mice, increasing the n−3/n−6 ratio. Thus, H-FABP expression influences brain phospholipid acyl chain and cholesterol content. Hence, this is the first demonstration of a protein-dependent, fatty acid selective uptake mechanism in the brain and suggests that H-FABP expression is important for brain 20:4n−6 uptake and trafficking.

I-10 Docosahexaenoic Acid/Poly-L-Lysine Conjugates Bind to the Cerebrovascular Endothelium Robert Katz1,4, Maria Tomoaia-Cotisel2,4, Mario C. Rattazzi3,4, Paul Fishman5 1 Molecular/Structural Biotechnologies, Inc., Bethesda, MD, USA 2 Molecular/Structural Biotechnologies, Inc., Rockville, MD, USA 3 Molecular/Structural Biotechnologies, Inc., Long Island, NY, USA 4 Omega-3 Research Institute, Inc., Bethesda, MD, USA 5 University of Maryland School of Medicine, VA Medical Center, Baltimore, MD, USA Deca-L-lysine monodocosahexaenoate and deca-L-lysine didocosahexaenoate were prepared and tagged with TexasRed. Deca-L-lysine was labeled with fluorescein to serve as control. The conjugates were purified and identified by mass spectral analysis. Rats were anesthetized, carotid arteries were exposed, and a solution of the two conjugates and the control in calf serum was injected into the exposed artery. The rats were killed, their brains removed and frozen in dry ice. Thin sections from the frontal lobe were examined in a fluorescent microscope. Slides will be presented to demonstrate binding of the conjugates to the endothelium in sections of the frontal lobe. Prior work by others demonstrated limited uptake of DHA–polypeptide conjugates, such as growth factors, into the brain parenchyma. Given the ability of poly-L-

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lysine to also penetrate the blood–brain barrier, these conjugates could provide a new approach for delivery of large biomolecules to the brain. The approach would utilize a carrier composed of a relatively short-chained poly-L-lysine conjugated to one, two, or more long-chain omega-3 polyunsaturated fatty acid. The molecule(s) to be carried across the blood–brain barrier could be attached to these lipophilic-polycationic carriers. Thus, carriers could serve both as research tools and as therapeutic agents for brain cancers, Alzheimer’s disease, stroke, depression, schizophrenia, inherited metabolic diseases, and other disorders.

I-11 α-Synuclein Gene Ablation Decreases Astrocyte Fatty Acid Uptake and Alters Fatty Acid Trafficking P. I. Castagnet1, R. L. Nussbaum2, E. J. Murphy1 1 Department of Pharmacology, Physiology and Therapeutics, University of North Dakota, Grand Forks, ND, 58203 2 Genetic Diseases Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA α-Synuclein is a small, cytosolic protein that is abundant in the brain; however, the function of the protein remains unclear. We tested the hypothesis that α-synuclein behaves as a fatty acid-binding protein in cultured astrocytes isolated from wild-type and α-synuclein gene-ablated mice by measuring the uptake of [1-14C]16:0, [1-14C]20:4n−6, and [1-14C]22:6n−3 and subsequent targeting to individual lipid pools. α-Synuclein gene ablation decreased 16:0 and 20:4n−6 total uptake by 31 and 39%, respectively, whereas 22:6n−3 uptake was unaffected. In neutral lipids, gene ablation decreased 16:0 esterification 31%, did not affect 20:4n−6, and increased 22:6n−3 esterification 1.7-fold. The fractional distribution of 20:4n−6 and 22:6n−3 into the neutral lipid fraction was increased by 1.7- and 1.6-fold, respectively, with an increase predominantly in diacylglycerols and triacylglycerols. α-Synuclein gene ablation decreased 16:0 esterification by 39% and 20:4n−6 esterification by 43% into total phospholipids and decreased the distribution of these fatty acids into this lipid pool. More importantly, α-synuclein gene ablation significantly decreased the esterification of these fatty acids into and distribution to phosphatidylinositol. These results demonstrate that α-synuclein facilitates fatty acid uptake into and alters fatty acid targeting to cellular lipid pools, suggesting that α-synuclein has a function similar to fatty acid-binding proteins in astrocytes.

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α-Synuclein Expression Enhances Brain Palmitate Uptake and Differentially Affects Palmitate Incorporation and Turnover in Brain Phospholipids Mikhail Y. Golovko1, Paula I. Castagnet1, Robert L. Nussbaum2, Eric J. Murphy1 1 Department of Pharmacology, Physiology, and Therapeutics, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND 58202–9037, USA 2 Genetics Diseases Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892–4448, USA α-Synuclein is an abundant protein in the CNS that is associated with a number of neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease. Its physiological function is poorly understood, although recently it was proposed to function as fatty acid-binding protein. To better define a role for α-synuclein in brain fatty acid uptake and metabolism, we infused awake, wild-type, or αsynuclein gene-ablated mice with [1-14C]16:0 and assessed fatty acid uptake and turnover kinetics in brain phospholipid pools. α-Synuclein expression increased brain 16:0 uptake by 1.35-fold and increased its targeting to the brain organic fraction. Despite the changes in total 16:0 uptake, there were no changes in the coefficients of its incorporation into individual phospholipid classes. However, α-synuclein expression did increase the incorporation rate and fractional turnover of 16:0 in a number of phospholipid classes, but decreased the incorporation rate and fractional turnover of 16:0 in the choline glycerophospholipids. These data show for the first time that α-synuclein expression affects brain fatty acid uptake and turnover kinetics, indicating that it has a function in brain lipid metabolism consistent with that of a fatty acid-binding protein. II-1 Effects of Dietary n–3 Fatty Acid on Liver, Heart, and Brain Phospholipid Composition and Phospholipid Acyl Chain Mass G. Barceló-Coblijn1, M. Cinnamon1, C. A. Jolly2, E. J. Murphy1 1 Department of Pharmacology, Physiology and Therapeutics, University of North Dakota, Grand Forks, ND 58203, USA 2 University of Texas, Division of Nutritional Science, Austin, TX 78712, USA

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Fish oil-enriched diets increase n−3 fatty acid mass in tissue phospholipids; however, a similar effect by plantderived n−3 fatty acids is poorly defined. To address this question, we determined changes in phospholipid mass, phospholipid fatty acid composition, and cholesterol mass in livers, hearts, and brains of rats fed for 8 weeks of diets enriched in flax oil (18:3n−3, linolenic acid), fish oil (22:6n−3, docosahexaenoic acid), or safflower oil (18:2n− 6, linoleic acid). The fish oil group had decreased ethanolamine glycerophospholipid mass in liver and heart compared to the safflower oil group. In phospholipids from these tissues, 22:6n−3 levels were increased only in the fish oil group, although rats fed with flax oil accumulated 20:5n−3 and 22:5n−3, products derived from the 18:3n−3. In brain, both flax and fish oil diets increased 22:6n−3 levels in phospholipids and resulted in a concomitant reduction in brain arachidonic acid (20:4n−6) levels. In all tissues, both n−3-enriched groups decreased 20:4n−6 mass, although the effect was more marked in the fish oil than in the flax oil group. The different response of the brain to the dietary lipids compared to heart and liver might be because of differences in fatty acid uptake and trafficking. Although these data do not provide direct evidence for 18:3n−3 elongation by the brain, these data do demonstrate that 18:3n−3-enriched diets reduce tissue 20:4n−6 levels and increase cellular n−3 levels in a tissuedependent manner. We hypothesize, based on the lack of increased 22:6n−3 in liver and heart, but increased 18:3n−3, that the flax oil diet increases circulating 18:3n−3, thereby presenting tissue with this essential fatty acid for further elongation and desaturation. The ability to elongate and desaturate 18:3n−3 to 22:6n−3 appears to be highly tissuedependent, suggesting that the brain is uniquely positioned to carry out this process.

II-2 Brain Conversion of Linoleic Acid to Arachidonic Acid is Not a Major Source of the Arachidonate Found in Brain Phospholipids of Adult Rats James C. DeMar*, Kaizong Ma, Lisa Chang, Jane Bell, Stanley I. Rapoport Brain Physiology and Metabolism Section, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892, USA Introduction: The extent to which the mammalian brain synthesizes arachidonic acid (AA, 20:4n−6) in vivo from linoleic acid (LA, 18:2n−6) is uncertain. Using a kinetic in vivo rat infusion model, we determined the degree to which LA entering brain from plasma is converted to

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AA. Methods: Adult rats (n=6) were infused i.v. over 5 min with [1-14C]-LA, and the brain was removed after microwaving. Brain and plasma lipids were extracted and analyzed using thin layer chromatography and highperformance liquid chromatography. Radioactivity was determined by scintillation counting. Results: Whole brain fractional uptake of [14C]-LA from plasma was 1.0±0.1%× ml plasma×min−1 ×g−1 brain. Most of the brain radioactivity, 61%, was in water-soluble β-oxidation products, whereas the remainder was in lipids. Of the total brain lipid radioactivity, 27% was [14C]-LA-esterified in phospholipids, whereas only 2% was present as [14C]-AA in phospholipids. Remaining total lipid radioactivity was in β-oxidation products, mostly recycled into cholesterol and saturated fatty acids. Some brain AA synthesis was implied by the presence of brain [14C]-LA-CoA and [14C]-AA-CoA at a concentration ratio of 16 to 1. From the unesterified LA concentration in plasma (281±85 nmol/ml), we determined that the incorporation rate of brain-synthesized AA into phospholipids is 0.022±0.009 nmol×min−1 ×g−1 brain, which is ∼6% of the published incorporation rate for plasma AA into brain phospholipids. Conclusion: LA that enters the brain from plasma is largely subjected to βoxidation, and is a negligible source of the AA found within brain phospholipids.

II-3 Role of Docosahexaenoic Acid and RXR Receptor on Neurite Growth in Hippocampal Neurons and Neuroblastoma Cells Frances Calderon, Hee-Yong Kim Section of Mass Spectrometry, Laboratory of Membrane Biochemistry and Biophysics, National Institutes of Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, USA Docosahexaenoic acid (DHA; 22:6n−3) is an essential fatty acid particularly enriched in brain. DHA-deficiency during development is associated with an impairment of learning and memory tasks, strongly suggesting that DHA may have an important role in neuronal development. In this study, we show that DHA has a remarkable effect on morphological differentiation in hippocampal neurons by increasing the population of neurons with higher number of branches and longer neurites in primary cultures grown in chemically defined medium. The effect was specific as no other polyunsaturated fatty acid had this effect. Because DHA, as well as 9-cis retinoic acid (9cRA), is a ligand of the retinoid X receptor (RXR, a ligand activated transcription factor), we evaluated the

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participation of RXR as the possible mechanism by which DHA promotes neurite growth. We first evaluated the expression of the RXR in hippocampal cells in different developmental stages, and in Neuro-2a cells. Secondly, we tested the effect of DHA and 9cRA on neurite growth in Neuro-2a cells. Our preliminary studies revealed that both hippocampal and Neuro-2a cells express the RXR. In addition, both DHA and 9cRA similarly increased the population of cells with longer neurites in comparison to control cells, although 9cRA had more profound effect. We suggest the involvement of an RXR-associated mechanism in the DHA promoted neurite growth in neuronal cells.

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gas chromatography/mass spectrometry analysis of the FA composition of Acsl6 overexpressing cells supplemented with AA and DHA revealed significant coincident increases in PUFAs and saturated FAs, but no change in monosaturated FAs. Taken together, these results support a role for Acsl6 in DHA metabolism, but do not support a role for Acsl6, nor Acsl1, in preferential partitioning of FA into TAG or PL. Acsl6 overexpression most likely increases DHA-supplemented neurite outgrowth by significantly increasing its internalization during the first 24 h of differentiation leading to increased PL synthesis.

II-5 II-4 Acsl6 Overexpression Preferentially Promotes DHA Metabolism in PC12 cells Joseph R. Marszalek1, Claire Kitidis1, Lori Materese3, Concetta DiRusso3, Harvey F. Lodish1,2 1 Whitehead Institute for Biomedical Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA 2 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA 3 Ordway Research Institute, Albany, NY, USA Supplementation of differentiating PC12 cells with the polyunsaturated fatty acids (PUFAs), arachidonic acid (AA) or docosahexaenoic acid (DHA), but not the monosaturated oleic acid (OA), increases neurite outgrowth. Overexpression of rat Acyl-CoA synthetase 6 (Acsl6) further augments PUFA-enhanced neurite outgrowth. In this study, Acsl6 overexpressing cells were supplemented with 5 μM of 14Clabeled OA, AA, or DHA and the metabolic fate of the fatty acids (FAs) were examined to determine whether altered kinetics of PUFA uptake or their metabolism are associated with enhanced neurite outgrowth. In support of this hypothesis, 14C DHA accumulation in Acsl6 overexpressing cells was increased by 90, 150, and 25% over control cells after 15 min, 24 h, and 72 h, respectively, whereas it was increased by only 30% in Acsl1 overexpressing cells after 15 min with no change after 24 or 72 h. In contrast, 14 C AA accumulation was increased by ∼50% in Acsl1 or Acsl6 overexpressing after 15 min, but was increased only by 30% in Acsl6 cells after 24 h and neither was increased after 72 h. For the conditions where 14C FAs were increased, both phospholipids (PLs) or triacylglyceride (TAG) levels were increased, suggesting that neither Acsl1 nor Acsl6 exclusively targets FAs into PLs or TAGs. Consistent with increased neurite length, the PL levels were increased by 11 and 14% in Acsl6 overexpressing cells supplemented with AA and DHA for 72 h. Interestingly,

EPA, But Not Oleate, Stimulates β-Oxidation in Adipocytes Wen Guo, James A. Hamilton Boston University School of Medicine, Boston, MA, USA The beneficial roles of dietary fish oil in lowering serum triglyceride (TAG) levels in animals and humans have been attributed in part to the high content of two ω-3 polyunsaturated very-long-chain fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Both EPA and DHA activate peroxisome proliferator-activated receptor alpha (PPARα), the nuclear transcription factor that regulates the expression of key enzymes in control of fatty acid β-oxidation (β-ox) in mitochondria and peroxisomes. Recent studies have shown that EPA rather than DHA is responsible for the TAG-lowering effect, and, mitochondria rather than peroxisomes are the principal target. A possible mechanism is increased expression of carnitine palmitoyl transferase I (CPT-I) in the hepatocytes. Whether EPA affects fatty acid storage or oxidation in adipocytes is not known. To investigate this possibility, 3T3-L1 adipocytes were incubated with EPA (200 μM) for 24 h and used for assays of β-ox, CPT-I enzyme activity, and mRNA expression of CPT-I and some of its relevant genes. In parallel experiments, cells were treated with oleate, octanoate, and clofibrate, a synthetic ligand for PPARα. β-ox was measured by the release of 3H2O from endogenous TAG prelabeled with [9, 10-3H] palmitate. CPT-I expression was measured by reverse transcriptase PCR, and CPT-I activity was measured by the formation of isotope labeled palmitoylcarnitine using palmitoyl-CoA and 3 H-L-carnitine as substrates. Mitochondria were isolated by differential centrifugation, and mitochondrial membrane acyl chain composition was measured by GLC after methylation of the membrane phospholipids. EPA increased the oxidation of endogenous palmitate but did not inhibit TAG synthesis in adipocytes. In the same

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assay, the common dietary long-chain fatty acid, oleate, did not affect fatty acid oxidation. The medium-chain fatty acid, octanoate, which bypasses the carnitine pathway, suppressed the oxidation of palmitate, possibly because its oxidation contributed to the total oxidation. Increased β-ox by EPA was associated with increased CPT-1 activity without changing the mRNA and protein expression levels of this enzyme. EPA treatment increased the percentage of this fatty acid in the mitochondrial membrane lipids. Hence, we suggest that EPA increased the activity of CPT-1 and βoxidation in adipocytes by altering the structure or dynamics of mitochondrial membranes.

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characterization of ACADH9 as an enzyme involved in the mitochondrial catabolism of unsaturated long-chain substrates, the finding of ACADH10 rewrites our view of long-chain fatty acid β-oxidation and raises new questions about the interrelationship of mitochondria and peroxisomes in long-chain fat catabolism.

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New Acyl-CoA Dehydrogenases: Redefining Long-Chain Fatty Acid Catabolism in Humans M. He, J. Vockley. Children’s Hospital of Pittsburgh, University of Pittsburgh, School of Medicine, Pittsburgh, PA, USA

Reduced Brain DHA Content After a Single Reproductive Cycle in Female Rats on an α-Linolenic Acid-deficient Diet Beth Levant1, Jeffrey D. Radel2, Susan E. Carlson3 1 Department of Pharmacology, University of Kansas Medical Center, Kansas City, KS 66160, USA 2 Department of Occupational Therapy Education, University of Kansas Medical Center, Kansas City, KS 66160, USA 3 Department of Dietetics and Nutrition, University of Kansas Medical Center, Kansas City, KS 66160, USA

Very-long-chain fatty acids have traditionally been thought to be degraded exclusively by peroxisomal βoxidation, although mitochondrial malfunction has been reported in patients with peroxisomal defects. The AcylCoA dehydrogenases (ACDs) are a family of flavoenzymes involved in mitochondrial β-oxidation of fatty acid. Nine members of this family had been identified in the past, but none of them utilize acyl-CoAs with carbon chain >20. Recently, we identified two new potential ACD family members, ACDX1 and ACDX2, which are widely conserved across evolution. They are 20–30% identical to the other ACDs, whereas human ACDX1 and ACDX2 share 46% homology with each other. We have now definitively identified ACDX2 as a long chain ACD, and name it ACADH10. The ACADH10 locus is a complicated one with multiple predicted coding domains. The transcript from the ACD portion of this gene is efficiently imported into mitochondria when translated in vitro and processed to a mature form. Expression of the mature intramitochondrial form of human ACADH10 in Escherichia coli produces an enzyme with maximal ACD activity toward C22-CoA. It has relative activities toward C23-, C24-, and C26-CoA of 60, 15, and 15%, respectively, compared to C22-CoA. Computer modeling of the structures of ACADH10 and ACDX1 predicts that their catalytic base is an aspartic acid instead of the glutamic acid present in other ACDs. The distribution of EST fragments suggests that ACACH10 mostly expressed in human liver whereas ACDX1 mostly expressed in brain. The substrate specificity of ACDX1 remains under study. In conjunction with our recent

Low levels of n−3 polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA, 22:3n−6), are implicated in postpartum depression, nonpuerperal depression, and schizophrenia and affect the dopaminergic and serotonergic systems in animals. This study investigated the effects of pregnancy and lactation on brain DHA content in female Long–Evans rats maintained on purified diets containing sufficient (control) or negligible (deficient) αlinolenic acid (18:3n−3), the dietary precursor of DHA, produced by formulation with soy or sunflower oil, respectively (7% by wt.). Rats (14 weeks old) were mated and placed on the control or deficient diets at the time conception was noted. Litters were culled to eight pups on postnatal day 1 (P1), six pups on P7, and four pups on P14. At weaning (P21), dams were killed and phospholipid fatty acid levels were measured in whole brain by GC. Brain DHA content of postweaning dams maintained on the control diet was not significantly different than that of agematched virgin females. In contrast, in rats maintained on the deficient diet, pregnancy and lactation resulted in a decrease in brain DHA content to 66% of that observed in virgin females (P<0.01) and 75% of that observed in postweaning females maintained on the control diet (P< 0.01). This decrease in DHA content was accompanied by a compensatory increase in brain docosapentaenoic acid (22:5n−6) content to 300% of that of virgin females (P< 0.01). Thus, under dietary conditions that supply inadequate n − 3 PUFAs, maternal brain DHA content is substantially depleted by a single cycle of reproductive activity. This depletion of maternal brain DHA may affect

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the sensitivity of the postpartum organism to stress, suggesting the plausibility of a role for n−3 PUFAs in postpartum neuropsychiatric disorders in humans. This study was supported by The Roberts’ Family Foundation, the Lied Endowment, and National Institutes of Health Center grant HD02528.

II-8 Decreased brain DHA during development alters sucrose drinking in adult male rats Cheryl C. Miller3, Beth Levant1, Jeffrey D. Radel2, Susan E. Carlson3 1 Department of Pharmacology, University of Kansas Medical Center, Kansas City, KS 66160, USA 2 Department of Occupational Therapy Education, University of Kansas Medical Center, Kansas City, KS 66160, USA 3 Department of Dietetics and Nutrition, University of Kansas Medical Center, Kansas City, KS 66160, USA Decreases in brain docosahexaenoic acid (DHA) content during development have been shown to alter the mesocortical and mesolimbic dopaminergic systems in rats. Alterations in the mesolimbic dopamine system have been shown to influence behavioral responses to rewarding stimuli such as sucrose in rats. In these studies, intake of palatable food (sucrose) and unpalatable food (chow) were evaluated in adult male Long–Evans rats that were raised from conception on a deficient diet containing negligible amounts of α-linolenic acid (the dietary precursor of DHA) (n=12) or a control diet containing α-linolenic acid (n=11). The deficient diet reduces brain DHA content by roughly 20% compared to controls (Levant et al. 2004). Body weights did not differ between the treatment groups. Consumption of a 15% sucrose solution was measured at 5-min intervals for 30 min using calibrated drinking tubes. Sucrose intake was significantly increased at 5 and 10 min after presentation in rats fed the deficient diet although the total volume consumed over 30 min was similar for both groups. Intake of chow did not differ between the treatment groups. Early time point differences in the amount of sucrose consumed indicate that the dietary modulation produces alterations in neural pathways related to reward rather than an impairment of gastric feedback mechanisms that signal satiety. These results also suggest that a modest reduction in brain DHA content may contribute to increased motivation to consume sucrose in rats. This study was supported by The Roberts Family Foundation, the Lied Endowment, and National Institutes of Health Center grant HD02528.

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II-9 Cholesterol Synthesis is Upregulated in the Brains of Transgenic Mice Expressing Human ApoE4 or ApoE3 Specifically in Astrocytes P. Jansen1*, D. Lutjohann2, K. Thelen2, K. Von Bergmann2, F. Ramaekers1, M. Mulder1 1 Department of Molecular Cell Biology, University of Maastricht, Maastricht, The Netherlands 2 Department of Clinical Pharmacology, University of Bonn, Bonn, Germany [email protected] Accumulating evidence indicates a link between cholesterol and the deposition of b-amyloid in Alzheimer’s disease (AD). A major risk factor for the development of AD is apolipoprotein E4 (apoE4), one of the common human isoforms of apoE (E2, E3, and E4). ApoE is known to be a key protein in the transport of cholesterol in the circulation. Therefore, we hypothesized that apoE affects cholesterol homeostasis in the brain. We determined levels of cholesterol, its precursors (lathosterol, lanosterol, and desmosterol), and the metabolite 24-OH-cholesterol using gas chromatography/mass spectrometry in brain and serum of homozygous and heterozygous apoE knockout mice (mEko and mE±) and mice deficient for mouse apoE that express either human apoE3 (mEko/hE3) or human apoE4 (mEko/hE4) specifically in astrocytes. ApoE knockout mice display sevenfold increased serum levels of cholesterol and cholesterol precursors compared to heterozygous mice. Levels of cholesterol (-precursors) in serum of mEko/hE4 mice are about 2.5-fold higher than those of mE± mice, whereas no difference was observed between mEko/hE3 and mE± mice. The total amount of cholesterol in the brain of mEko mice is 9% higher than in mE± mice (mEko 73±4 mg/mg, mE± 67±2 mg/mg). Levels of cholesterol precursors were comparable in the brains of mEko (316±13 ng/mg brain) and mE± mice (341±20 ng/mg). Surprisingly, increased levels of cholesterol are observed in the brains of mEko/hE4 (78±3 mg/mg) and mEko/hE3 (84±4 mg/mg) mice. Also, the ratio of precursors/cholesterol in the brains of mEko/hE4 and mEko/hE3 mice are increased in comparison with the mE± mice. The ratio of desmosterol/cholesterol in mEko/hE4 (6.5±0.2 mg/mg) is 28% higher than mE± mice (5.1±0.2 μg/mg) (p=0.0018) and mEko/hE3 mice (6.0±0.3 μg/mg, p=0.09) have an 18% higher ratio of desmosterol/cholesterol. This suggests that the higher levels of cholesterol in the brains of the mice expressing human apoE can be explained by a higher rate of cholesterol synthesis. No differences were found in

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levels of 24-OH-cholesterol in the brains between the four groups of mice. The results from this study indicate that human apoE does affect cholesterol homeostasis in the brain, most likely through an effect on the synthesis rate. This study was supported by a grant from the Marie Curie Fellowship Organisation; Quality of Life and Management of Living Resources: contract number: QLK6-CT-2000-60042, fellow reference number: QLK6-GH-00-60042-20; and by the Hersenstichting Nederland.

II-10 Regional Effects of Aging on the Fatty Acid Concentration of Various Phospholipids in the Rhesus Monkey Brain Daniel Maoz, Jana Mayette, Henry N. Nguyen, Ho-Joo Lee, Stanley I. Rapoport, Abesh K. Bhattacharjee, Richard P. Bazinet Brain Physiology and Metabolism Section, National Institute on Aging, National Institutes of Health, Bethesda, MD 20852, USA [email protected] Brain fatty acids are involved in membrane structure and transport; they are a key component in both extracellular and intracellular signaling processes; they help regulate enzyme activity, act as an important energy source and are the precursors to a variety of prostaglandins. Thus, changes in fatty acid metabolism because of aging may influence the pathogenesis of conditions known to be related to fatty acid metabolism, such as brain trauma, ischemia, neuroinflammation, and neurodegenerative diseases such as Alzheimer’s and Parkinson. To study the effects of aging we measured the brain fatty acid concentration in phospholipids from 5-month-old and 5- and 15-year-old Rhesus monkeys. Total lipids were extracted according to the Folch method, phospholipid classes were separated via thin layer chromatography and fatty acid methyl esters were analyzed using GC. Different brain regions (frontal cortex, cerebellum, and brain stem) were studied under the assumption of an existing regional variation. Frontal cortex arachidonic acid levels decreased within phosphatidyl serine (5-month-old vs 5- and 15-year-old), whereas arachidonic acid decreased steadily over time within cerebellar phosphatidyl ethanolamine. The frontal cortex concentration of linoleic acid within cardiolipin decreased in the 15-year-old age group compared to the 5-month-old and 5-year-old groups. The results of the

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brain stem are currently in progress and will be presented. Some of the fatty acids within phospholipids demonstrated very high concentration diversities in different brain regions. Thus far, the rhesus monkey brain shows regional variations in fatty acid concentration and the response to aging.

II-11 The Effects of Carbamazepine on the Metabolism of Arachidonic and Docosahexaenoic Acid in the Brain of the Awake Rat Ho-Joo Lee, Jagadeesh Rao, Lisa Chang, Stanley I. Rapoport, Richard P. Bazinet Brain Physiology and Metabolism Section, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892, USA [email protected] Several drugs shown to be effective in the treatment of bipolar disorder such as lithium and valproic acid also decrease the turnover of arachidonic acid (AA) in the brain of the awake rat. These drugs are specific for AA as they do not alter the turnover of docosahexaenoic acid (DHA). Evidence from our laboratory has shown that chronic carbamazepine (CBZ) treatment decreases the activity of a key enzyme regulating the turnover of AA (cytosolic phospholipase A2), similar to what has been observed with chronic lithium treatment. We therefore hypothesized that CBZ would decrease the turnover of AA but not DHA in the brain of the awake rat. To see if decreased turnover of AA is a specific target of CBZ, rats were chronically treated with either 25 mg/kg CBZ, i.p., for 30 days or vehicle and then infused with [1-14C] AA or DHA. We then we applied our in vivo fatty acid model to examine the turnover of AA and DHA in the brain of the awake rat. Lipid classes were separated by thin layer chromatography, whereas GC and scintillation counting were used to measure AA and DHA concentrations and radioactivities, respectively, in the lipid classes. CBZtreated rats had no change in the unidirectional incorporation coefficient and the rate of AA and DHA incorporation from plasma into brain phospholipids compared to controls. Also, CBZ treatment did not change the net incorporation rate of DHA from brain DHA-CoA into phospholipids or the turnover of DHA in brain phospholipids. Results of AA turnover are in progress and will be presented. Thus far, similar to lithium and valproic acid, CBZ treatment does not alter the brain metabolism of DHA.

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II-12 Docosahexaenoic Acid: A Positive Cross-talk Mediator Between Membrane Phosphatidylserine and Akt kinase Mohammed Akbar, Hee-Yong Kim Section of Mass Spectrometry, Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD 20852, USA n−3 fatty acid-deficiency depletes docosahexaenoic acid (DHA, 22:6n−3), an omega-3 fatty acid, causing many neurological disorders. We have previously demonstrated that n−3 fatty acid deficiency decreases the total phosphatidylserine (PS) levels, and DHA enrichment accumulates PS and inhibit apoptotic cell death. To demonstrate the mechanism underlying the protective effect of DHA by PS accumulation, neuronal cells were either enriched with DHA, 16:0,18:1-PS (POPS) or 16:0,16:0-PS (DPPS), and the survival signal was evaluated. After 48 h of enrichment, cells were harvested for phospholipid analysis by reversed phase high-performance liquid chromatography/electrospray mass spectrometry, treated with staurosporine or serum deprived for 48 h to induce apoptosis. The cells enriched with DHA alone showed a decrease in caspase-3 activity, and increase the total PS levels in comparison to nonenriched control cells. In contrast, POPS and DPPS were either ineffective or less effective in inhibiting caspase-3 activity or PS accumulation. In addition, PI3 kinase inhibitors abolished the protective effect of DHA, suggesting that DHA through PS accumulation may influence Akt activation. Indeed, enhanced Akt translocation and activation in DHA-enriched cells was demonstrated by real time translocation of GFP-tagged PH domain of Akt upon IGF stimulation. Treatment with wortmannin (a PI3 kinase inhibitor) or mutations in basic residues of PH domain of Akt interfered with Akt phosphorylation and translocation. In conclusion, enrichment of neuronal cells with DHA positively modulates the accumulation of PS and prevents apoptosis. These studies demonstrate an antiapoptotic effect of DHA in neuronal cells by promoting PS-dependent Akt translocation.

II-13 Neuronal Phosphatidylserine Accumulation Modulated by Docosahexaenoic Acid and PSS1 and PSS2 Gene Silencing Lyubov Stockert, Mingquan Guo, Mohammed Akbar, Hee-Yong Kim Section of Mass Spectrometry, Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892, USA

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Biosynthesis of phosphatidylserine (PS), major acidic phospholipid class in eukaryotic biomembranes, plays an important role in various signaling pathways. We have previously demonstrated that docosahexaenoic acid (DHA) promotes PS biosynthesis and inhibits apoptosis in neuronal cells. We have also demonstrated that n−3 fatty aciddeficiency decreases the total PS levels, adversely influencing neuronal survival. To study the role of PS synthase (PSS) enzymes in PS accumulation promoted by DHA, Neuro-2a cells were transfected with small interfering RNA (siRNA) against PSS1 and PSS2 enzymes or serine-depleted before the enrichment with DHA. After 48 h of enrichment, cells were harvested and mRNA and phospholipid levels were determined by reverse transcriptase PCR and LC/MS analysis, respectively. We found that, siRNA transfection considerably inhibited the mRNA levels both in control and DHA-enriched cells. DHA treatment significantly increased the total PS level in all cells, transfected or nontransfected. The DHA-induced PS increase was significantly inhibited only in serine-depleted cells. Although mRNA was successfully suppressed by siRNA transfection, the PS levels did not change by either pss1 or pss2 and their cotransfection. This suggests that although PSS1 and PSS2 are inhibited at the mRNA level by siRNA, protein levels may remain unchanged. Alternatively, concurrent adaptation of other phospholipid remodeling pathways such as PS decarboxylase may accompany to maintain the levels of PS.

II-14 n–3 Fatty Acid Deficiency Leads to Reduced G-Protein-Coupled Signaling Efficiency in Retinal Rod Outer Segments Shui-Lin Niu, Drake C. Mitchell, Sun-Young Lim, ZhiMing Wen, Hee-Yong Kim, Norman J. Salem, Burton J. Litman Laboratory of Membrane Biophysics and Biochemistry, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD, USA Dietary n−3 fatty acid (FA) deficiency is associated with suboptimal functions in learning, memory, olfactory-based discrimination, and visual acuity. A general observation in n−3 FA deficiency is the replacement of docosahexaenoic acid (DHA, 22:6n−3), by an n−6 FA, docosapentaenoic acid (DPA, 22:5n−6). DHA is a long-chain polyunsaturated FA highly enriched in membrane phospholipids of the retina and central nervous system. Because G-proteincoupled receptor (GPCR)-mediated signal transduction is a common signaling motif in these neuronal pathways, we postulated that reduced GPCR signaling caused by the

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replacement of DHA with DPA may contribute to the suboptimal neuronal functions. In this study, we raised a group of rats on n−3 FA-deficient and adequate diets for two generations and studied the visual signaling, which is in isolated retinal rod outer segment (ROS) membranes. In the n−3 FA-deficient rats ∼80% of the DHA in their ROS membranes was replaced by DPA. The loss of DHA in n− 3-deficient ROS membranes led to reduced membrane acyl chain packing free volume, fv, lower level of rhodopsin activation, slower rates of metarhodopsin II (MII) formation, delayed MII–G protein coupling, and an overall ∼3-fold reduction of light-activated PDE activity at physiological light stimulus. These findings are consistent with the reduced amplitude and delayed response of the retinal ERG a-wave observed in vivo in n−3 FA deficiency. These findings are also consistent with in vitro studies using reconstituted membranes demonstrating that DHA phospholipids promote rhodopsin activation and rhodopsin–G protein coupling. Members of the GPCR family are ubiquitous in signaling pathways in the nervous system, thus the effect of reduced GPCR signaling because of the loss of membrane DHA may explain the suboptimal signaling observed in a number of neural processes associated with dietary n−3 FA deficiency.

II-15 Oxygen-induced Vascular Pathology Influenced by Dietary n–3 Fatty Acids in Neonatal Rat Retina J. D. Radel, T. Raghuveer, P. Mahtosh University of Kansas Medical Center, Kansas City, KS, USA Purpose: Newborn rats raised in alternating hyperoxic/ hypoxic conditions exhibit abnormal retinal vascular patterns similar to human retinopathies. Rats fed diets deficient in α-linolenic acid (ALA) possess low levels of docosahexaenoic acid (DHA). This study explores interaction of oxygen and diet as factors in determining severity of vascular pathology. Methods: Sprague–Dawley dams and their litters were maintained from embryonic day 16 (E16) to weaning (postnatal day 21 [P21]) on one of three nutritionally complete, semisynthetic diets identical except for fat source: (1) sufficient ALA (soybean oil), (2) ALA-deficient (sunflower oil), or (3) containing 4% lipids as DHA (Martek DHASCO oil). Litters were culled (n=8) at birth (E22/P0) and raised in either room air (21% O2) from P0 to P21 or in cycling oxygen conditions from P0– P14, then room air until P21. The cycling oxygen condition exposed rats to 80% O2 and 20% N2 for 12 h then 10% O2 and 90% N2 for 12 h daily. All rats were maintained on a 12 h/12 h light/dark cycle with lighting for cycling rats 6 h

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out of phase with changes in oxygen level. At P21, rats were perfused with India ink, the eyes fixed overnight, then retinas whole-mounted and photographed. Vascular patterns were quantified and the results compared across diets and atmospheric conditions. Results: Ratios of straight line vs actual length of retinal arteries increased (p<0.0001) (e.g., became tortuous, suggesting angiogenesis) in rats raised under cycling oxygen conditions, but diet-related effects weren’t observed. Analyzing the same retinas by fractal analysis demonstrated a reduction in density of smalldiameter vessels in rats raised on a 4% DHA diet in cycling oxygen condition relative to rats raised in room air (p< 0.05). Conclusions: This approach may, with refinement, be suitable for study of diet effects on retinal angiogenesis as a step toward evaluating interaction of multiple factors (diet, trace metals, antioxidants) contributing to retinal pathology and visual impairment.

II-16 Effect of Dietary DHA Depletion on Plasma Lipoprotein Content Alla Polozova, William Mook, Norman Salem Jr Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, USA Dietary deficiency in omega-3 polyunsaturated fatty acids (PUFA) during critical developmental periods or for prolonged periods of time results in DHA depletion of neural and heart tissues with deleterious health consequences. Insufficient intake of omega-3 PUFA is usually manifested in a low plasma status of docosahexaenoic acid (DHA). Plasma distribution of omega-3 PUFA is not uniform across the different subfractions. Under the n−3 dietary deficiency conditions, plasma fractions primarily responsible for omega-3 PUFA transport to tissues may become depleted in these fatty acids first. We compared composition of plasma lipoprotein fractions in animals raised on an omega-3-deficient diet and animals raised on an omega-3-adequate diet. Plasma levels of DHA in deficient animals were more than tenfold lower compared to animals on adequate diet. The overall proportions of fatty acids in different plasma pools were similar in both groups of animals, except for the very low-density lipoprotein (VLDL) fraction. The total amount of fat associated with the VLDL in omega-3-deficient animals appeared to be higher. In addition, VLDL and LDL fractions in deficient animals were almost completely depleted in DHA. The small amount of DHA detected in plasma was distributed between HDL and nonesterified (NEFA) fractions. Strikingly, the proportion of DHA in NEFA fractions was not

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affected by diet and was similar in omega-3-adequate and -deficient animals. We concluded that DHA, present in HDL in NEFA pool, is relatively inert, whereas VLDL and LDL fractions are likely to be a primary source of DHA supply to organs.

III-1 The Relationship of ω-3 Long-chain Polyunsaturated Fatty Acid Intake and Regular Aspirin Use with Neovascular Age-related Macular Degeneration J. P. SanGiovanni*, E. Y. Chew, R. D. Sperduto, F. L. Ferris, AREDS Research Group *Division of Epidemiology and Clinical Research, National Eye Institute, National Institutes of Health, Bethesda, MD, USA Purpose: To investigate the relationship of ω-3 long-chain polyunsaturated fatty acid (LCPUFA) intake and aspirin (ASA) use with neovascular age-related macular degeneration (NVAMD). Background: The likelihood of having NV AMD is lowest among people reporting highest consumption of ω3 LCPUFAs. Docosahexaenoic acid (DHA) is the major ω-3 LCPUFA of the neural and vascular retina. Eicosapentaenoic acid (EPA) is the primary ω-3 substrate of cyclooxygenase (COX) and lipoxygenase (LOX) pathways that yield eicosanoids capable of modulating inflammatory and vascular processes implicated in AMD pathogenesis. LCPUFAs of the retina are also capable of reacting through ASA-driven pathways to generate a family of bioactive molecules (lipoxins) with potent immunoregulatory properties. Lipoxins (LXs) are a family of LCPUFA-derived hydroxytetraenecontaining small molecules demonstrating modulatory capacity in inflammatory- and hypoxia-reperfusion-based physiologic responses. LXs may affect immune response and host defense processes in the eye via modulation of key signaling pathways that integrate multicellular communication and biosynthetic networks. ASA acetylates COX-2 to produce a compound that may subsequently act upon DHAand EPA-based substrates to yield a series of “aspirintriggered” LXs with potent anti-inflammatory properties. Methods: In this case control study of 657 people with NV AMD and 1,112 people with no clinical signs of AMD, we administered a validated food frequency questionnaire and a standardized drug use questionnaire to obtain estimates of habitual LCPUFA intake and ASA use. Results: Multivariable logistic regression analyses yielded relationships demonstrating that groups reporting highest ω-3 LCPUFA

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intake and the longest duration of aspirin use were also the least likely to have NV AMD. The likelihood of NV AMD associated with highest vs lowest reported levels of DHA intake was reduced by 40% among subjects who reported never regularly using ASA. The likelihood was reduced by 60 and 80% among those who reported ever regularly using ASA for at least 3 months or 5 years, respectively. For EPA, there were significant reductions among the regular ASA users only; respective values were 0, 60, and 80%. All decreases in the likelihood of NV AMD were statistically significant. Conclusion: These novel findings provide a reasonable basis for investigating putative mechanisms driving LCPUFA/ASA/NV AMD relationships.

IV-1 Flumazenil Binding in Synaptosomes Varies Between Female and Male Rats Fed an n–3–deficient Diet Brian W. Bailey, Jennifer Lewis, Drake C. Mitchell Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, USA Brain synaptosomes containing GABA(A) were obtained from the cerebra of 7- and 10-week-old Sprague–Dawley rats raised on a DHA-deficient (n−3-deficient) or a DHAadequate (n−3-adequate) control diet from 2 days postconception. Synaptosomes were examined for gender differences in binding of flumazenil, a benzodiazepine antagonist, by creating saturation curves with a dilution series of tritiated flumazenil bound to filters. Binding affinity (Kd) and number of available binding sites (Bmax) were determined for each rat group. n−3-deficient males had a larger increase between 7 and 12 weeks of age in both Kd and Bmax compared to n−3deficient females and n−3-adequate males. Female rats on the deficient diet only had a small deviation from adequate diet females in flumazenil binding as they aged. The differences in flumazenil binding between n−3-deficient female and male rats may be explained by metabolic differences between the genders. Females are more capable of ameliorating the negative consequences of an n−3deficient diet. In fact, gas chromatography analysis of synaptosomal lipid composition indicated that female rats were able to partially compensate for the n−3 deficiency as they aged from 7 to 12 weeks, increasing from 65 to 73% of the total n−3 fatty acid present in similarly aged adequate diet females. In contrast, deficient diet male rat

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synaptosome lipid compositions diverged from the male group on the n−3 adequate diet with age, falling from 73 to 62% of total n−3 fatty acids found in similarly aged adequate diet males. These gender differences in age-related lipid composition changes may explain the discrepancies seen between females and males for flumazenil binding and may thus also help to explain why male rats fed an n−3deficient diet perform worse in cognitive studies than females and exhibit signs of depression.

IV-2 Phospholipid Therapy as a Treatment Protocol in ALS P. C. Kane, D. Braccia, E. Kane Haverford Wellness Center, Havertown, PA, USA We have embarked on a clinical treatment plan for the past 2 years of therapy including both oral and intravenous lipid therapy to address the accumulation of ceramides and renegade fatty acids. Oral and IV lipid therapy may modify the motor neuron membrane distortion by displacing the subsequent early expression of sphingomyelin, which follows the rise of ceramides synthesis. Twelve weekly infusions of phosphatidylcholine by phospholipid exchange followed by rGSH fast push are administered. Oral therapy includes highly unsaturated fatty acids as a 4:1 omega-6 to omega-3 oil, evening primrose oil, fatty alcohols, butyrate, or sodium phenylbutyrate, and phosphatidylcholine (PC). Targeted treatment protocols are utilized after red cell lipid analysis has been completed. Application of phospholipid. exchange with rGSH fast push has been successfully utilized in clinical settings in patients with both familial and sporadic ALS presentations. Male patient age 48 was diagnosed with lower motor ALS in October 2002. Patient presented in a wheelchair unable to walk or move his arms, hands were clawed, and speech was slurred. Patient received IV lipid exchange with GSH therapy twice daily. On the third day of therapy, the patient could walk the length of the hallway in our clinic. Therapy has been intensified to three infusions daily. After 4 weeks of therapy, the patient was able to roll over in bed, cross his legs, grip objects, and walk with assistance. At present, the patient can stand and walk unassisted. We have noted dramatic and sustained clinical improvement within the first few weeks after initiation of treatment in our patient population. Continued IVand oral lipid therapy yields further improvement. These results demonstrate that lipid therapy may reverse prevalent symptoms in individuals with ALS and should be considered for more formal studies.

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IV-3 Cholesterol Homeostasis in Niemann–Pick Type C Disease Barbara Karten1, Hideki Hayashi1, Gordon Francis1, Robert B. Campenot2, Dennis E. Vance3, Jean E. Vance1 1 CIHR Group in Molecular and Cell Biology of Lipids, Department of Medicine, University of Alberta, Edmonton, Canada 2 Department Cell Biology, University of Alberta, Edmonton, Canada 3 Department of Biochemistry, University of Alberta, Edmonton, Canada Niemann–Pick type C (NPC) disease is a fatal, autosomal recessive disorder caused in 95% of cases by loss of function of the NPC1 protein leading to progressive neurodegeneration in the cerebellum. NPC1 is a late endosomal protein ubiquitously expressed in all cells, and is assumed to play a key role in intracellular cholesterol trafficking from late endosomes. Although all cells lacking NPC1 accumulate lipids in the endosomal pathway, neurons seem to be more vulnerable to loss of NPC1 than other cell types. Two possibilities that might explain this observation are that lipoprotein formation by glia cells lacking NPC1 is impaired and/or that NPC1 has an additional, neuron-specific function in addition to its function in peripheral cells. Using a murine model of NPC disease we isolated primary astrocytes from wild-type and npc1−/− cerebellum. Analysis of conditioned medium from these glia showed no difference in either cholesterol or apoE concentration. In addition, lipoproteins isolated from glial conditioned medium supported growth of retinal ganglion neurons to the same extent, indicating that lipoproteins from npc1−/− cerebellar glia are functional. In relation to a potential neuronal function of NPC1, we found that NPC1 was abundant in axons of sympathetic neurons and that it colocalized with the synaptic vesicle protein synaptophysin. We also demonstrated the presence of NPC1 in synaptosomes. Preliminary data indicate that synaptic vesicles isolated from the cerebellum of npc1−/− mice have a similar cholesterol to protein ratio as synaptic vesicles from wild-type mice. Electron microscopy of isolated synaptosomes indicated, however, the presence of a population of synaptic vesicles with an aberrantly large diameter. In summary, we have shown that lipoprotein production by glia cells lacking NPC1 is not compromised but that NPC1 might play a role in synaptic vesicles recycling and thus have a specific neuronal function.

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Treatment of Neurodegenerative Diseases by Antioxidant-loaded Sterically Stabilized Liposomes Pablo Kizelsztein, Haim Ovadia, Yechezkel Barenholz Laboratory of Membrane and Liposome Research, Department of Biochemistry, Hebrew University, Jerusalem, Israel Department of Neurology, Hadassah Medical School, Hadassah University Hospital, Jerusalem, Israel [email protected]

Despite Inducing Only Transient Ketosis, a Very High Fat Ketogenic Diet Raises Brain Content of Long-chain Polyunsaturates (PUFA) and Alters 13 C-PUFA Metabolism in Rats A. Y. Taha, M. A. Ryan*, and S. C. Cunnane* Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Canada *Present address: Research Center on Aging, Sherbrooke University Geriatric Institute, Sherbrooke, Québec, Canada

There is significant evidence that the pathogenesis of several neurodegenerative diseases, including Parkinson’s disease, multiple sclerosis (MS), Alzheimer’s disease, Friedreich’s ataxia, amyotrophic lateral sclerosis (ALS), and Huntington’s disease may involve oxidative damage because of the generation of reactive oxygen species (ROS) and mitochondrial dysfunction. Accumulating data indicate that oxidative stress plays a major role in the pathogenesis of MS through the generation of ROS primarily by macrophages, causing demyelination and axonal damage in both MS and experimental autoimmune encephalomyelitis, it’s animal model. Our work is intended to develop novel strategies to protect and repair the brain as a consequence of oxidative damage using low molecular weight antioxidants, which can cross the blood–brain barrier and reach the brain in a controlled manner to give favorable pharmacokinetics. To demonstrate feasibility we used an antioxidant that is encapsulated in 80 nm of sterically stabilized liposomes (SSL) having long circulation time using remote loading by ammonium sulfate gradient reaching high drug to lipid ratio. This approach enables the controlled release of the drug. Our studies, conducted in a mice model of MS, demonstrate the feasibility of our approach. MS treatments started 6 to 9 days after the disease was initiated, until day 16. In this studies we compared two mice groups (control and treated) for therapeutic efficacy using −SSL containing the antioxidant. This treatment significantly affects the incidence and duration of the disease. Our findings when combined with biodistribution profile may lead to a novel approach in treatment of MS and other neurodegenerative disorders, which involve oxidative damage.

A very high fat ketogenic diet is commonly used in the treatment of intractable childhood epilepsy. A better understanding of how this diet works might lead to a better tolerated alternative treatment of epilepsy. However, in contrast to humans, rats on a very high fat ketogenic diet normally seem incapable of maintaining plasma β-hydroxy butyrate >1 mM for more than a few days. Children on a ketogenic diet have elevated plasma polyunsaturated (PUFA), so our goal was to see whether changes in PUFA metabolism were occurring in rats despite the absence of sustained ketosis. Liver, adipose tissue, and brain fatty acid profiles were measured as was the tissue content of 13C-labeled PUFA after dosing with 13C-α-linolenic acid. Despite loss of ketosis by the time tissues were collected, the ketogenic diet alone reduced some PUFA by up to 90% in adipose tissue and plasma, but raised liver PUFA by up to 25-fold, and raised brain arachidonic and docosahexaenoic (DHA) acids by 15% each. With the single exception of raised 13C-DHA in the brain, 13C-labeled n − 3 PUFA were 34–66% lower while on the ketogenic diet. We confirm that growing rats cannot sustain ketosis while consuming a high fat ketogenic diet. Plasma PUFA in ketotic rats are reduced rather than increased as they are in humans. Nevertheless, marked changes in PUFA metabolism resulting in an increase in brain arachidonate and DHA might contribute to seizure protection by the ketogenic diet. This study was funded by National Suborbital Education and Research Center.

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IV-6 Response in Alzheimer’s Disease Patients of 24S Hydroxycholesterol to Statins: Effects of Gender, CYP46, and ApoE Polymorphisms Gloria Lena Vega, Ph.D., Myron Weiner, M.D. University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA Background: Treatment of dyslipidemia with statin drugs is associated with a reduced risk for Alzheimer’s disease (AD). The mechanism of action is unknown, but might be related to reducing cholesterol synthesis in brain, lowering levels of cholesterol-transporting proteins or lowering the production of β-amyloid. Cholesterol from damaged/dying neurons is converted by cholesterol 24-hydroxylase (CYP46) to 24S-hydroxycholesterol, which passes the blood–brain barrier, is transported to the liver by lowdensity lipoproteins (LDLs) and excreted as bile acids. Because most of plasma 24S-hydroxycholesterol is derived from brain, plasma levels parallel brain cholesterol catabolism. Objectives: To examine in AD patients the effect of three statins on plasma levels of 24S-hydroxycholesterol and apolipoprotein E (apoE) and to examine the effect of gender and CYP46 polymorphisms on plasma 24Shydroxycholesterol and variability of response to statin

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treatment. Methods: A group of 24 female and 17 male AD patients completed 6 weeks of treatment with 40 mg of lovastatin, simvastatin, or pravastatin per day. Blood samples were drawn after a 12-h fast for measurement of plasma sterols, oxysterols, lipoprotein cholesterol, and levels of apoE. Measurements were made at baseline and at 6 weeks. Results: Plasma lathosterol was reduced by 49.5%, 24S-hydroxycholesterol by 21.4%, LDL cholesterol by 34.9%, and total cholesterol by 25%. Plasma apoE levels were unchanged. Baseline 24S-hydroxycholesterol was 14% higher in women than men. There was a positive relationship between plasma oxysterol levels and total plasma cholesterol and non-HDL cholesterol (stronger in women than men), but not HDL cholesterol levels. There was no relationship between CYP46 or apoE polymorphisms with pre- or posttreatment levels of lathosterol, 24S-hydroxycholesterol, non-HDL, or LDL cholesterol, but subjects with apoE ɛ4/ɛ4 had less reduction in the ratio of 24Shydroxycholesterol to LDL cholesterol. Conclusions: The stain effect on plasma 24S-hydroxycholesterol in AD patients is not related to an effect on apoE concentration. The more pronounced effect on LDL cholesterol partly explains the reduction in oxysterol level. CYP46 and apoE polymorphisms did not explain levels of oxysterol or non-HDL cholesterol or responsiveness to statin treatment.