Alpha-synuclein gene deletion decreases brain palmitate uptake and alters the palmitate metabolism in the absence of alpha-synuclein palmitate binding.

Alpha-synuclein is an abundant protein in the central nervous system that is associated with a number of neurodegenerative disorders, including Parkinson's disease. Its physiological function is poorly understood, although recently it was proposed to function as a fatty acid binding protein. To better define a role for alpha-synuclein in brain fatty acid uptake and metabolism, we infused awake, wild-type, or alpha-synuclein gene-ablated mice with [1-(14)C]palmitic acid (16:0) and assessed fatty acid uptake and turnover kinetics in brain phospholipids. Alpha-synuclein deficiency decreased brain 16:0 uptake 35% and reduced its targeting to the organic fraction. The incorporation coefficient for 16:0 entering the brain acyl-CoA pool was significantly decreased 36% in alpha-synuclein gene-ablated mice. Because incorporation coefficients alone are not predictive of fatty acid turnover in individual phospholipid classes, we calculated kinetic values for 16:0 entering brain phospholipid pools. Alpha-synuclein deficiency decreased the incorporation rate and fractional turnover of 16:0 in a number of phospholipid classes, but also increased the incorporation rate and fractional turnover of 16:0 in the choline glycerophospholipids. No differences in incorporation rate or turnover were observed in liver phospholipids, confirming that these changes in lipid metabolism were brain specific. Using titration microcalorimetry, we observed no binding of 16:0 or oleic acid to alpha-synuclein in vitro. Thus, alpha-synuclein has effects on 16:0 uptake and metabolism similar to those of an FABP, but unlike FABP, it does not directly bind 16:0; hence, the mechanism underlying these effects is different from that of a classical FABP.

[1]  F. Schroeder,et al.  Differential influence of rat liver fatty acid binding protein isoforms on phospholipid fatty acid composition: phosphatidic acid biosynthesis and phospholipid fatty acid remodeling. , 1998, Biochimica et biophysica acta.

[2]  S. Pelech,et al.  Plasticity of the Kinomes in Monkey and Rat Tissues , 2002, Science's STKE.

[3]  M. M. Bradford A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[4]  J. Folch,et al.  A simple method for the isolation and purification of total lipides from animal tissues. , 1957, The Journal of biological chemistry.

[5]  S. Rapoport,et al.  Effect of inhibition of β-oxidation on incorporation of [U-14C]palmitate and [1-14C]arachidonate into brain lipids , 1994, Brain Research.

[6]  R. Nussbaum,et al.  Lipid Droplet Binding and Oligomerization Properties of the Parkinson's Disease Protein α-Synuclein* , 2002, The Journal of Biological Chemistry.

[7]  S. Rapoport,et al.  Brain lipid metabolism in the cPLA2 knockout mouse Published, JLR Papers in Press, October 1, 2002. DOI 10.1194/jlr.M200298-JLR200 , 2003, Journal of Lipid Research.

[8]  E. Masliah,et al.  Abnormal distribution of the non-Abeta component of Alzheimer's disease amyloid precursor/alpha-synuclein in Lewy body disease as revealed by proteinase K and formic acid pretreatment. , 1998, Laboratory investigation; a journal of technical methods and pathology.

[9]  G. Gokel,et al.  Titration calorimetric analysis of AcylCoA recognition by myristoylCoA:protein N-myristoyltransferase. , 1997, Biochemistry.

[10]  B. Hyman,et al.  Subcellular localization of alpha-synuclein in primary neuronal cultures: effect of missense mutations. , 2000, Journal of neural transmission. Supplementum.

[11]  Charles N. Serhan,et al.  Altered Fatty Acid Composition of Dopaminergic Neurons Expressing α-Synuclein and Human Brains with α-Synucleinopathies* , 2003, Journal of Biological Chemistry.

[12]  F. Spener,et al.  Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein. , 1995, Biochimica et biophysica acta.

[13]  Nigel J. Cairns,et al.  Filamentous α-synuclein inclusions link multiple system atrophy with Parkinson's disease and dementia with Lewy bodies , 1998, Neuroscience Letters.

[14]  B. Kragelund,et al.  Thermodynamics of ligand binding to acyl-coenzyme A binding protein studied by titration calorimetry. , 1996, Biochemistry.

[15]  J. Hoenicka,et al.  The new mutation, E46K, of α‐synuclein causes parkinson and Lewy body dementia , 2004, Annals of neurology.

[16]  Noriko Kitanaka,et al.  Brain arachidonic acid incorporation is decreased in heart fatty acid binding protein gene-ablated mice. , 2005, Biochemistry.

[17]  E. Masliah,et al.  ALTERATIONS IN APOLIPOPROTEIN E EXPRESSION DURING AGING AND NEURODEGENERATION , 1996, Progress in Neurobiology.

[18]  G. Arvidson,et al.  Initial incorporation into rat liver glycerolipids of intraportally injected (3H)glycerol. , 1970, Biochimica et biophysica acta.

[19]  J C Sacchettini,et al.  Crystal structure and thermodynamic analysis of human brain fatty acid-binding protein. , 2000, The Journal of biological chemistry.

[20]  D. Prows,et al.  Liver fatty acid-binding protein expression in transfected fibroblasts stimulates fatty acid uptake and metabolism. , 1996, Biochimica et biophysica acta.

[21]  A. Kleinfeld,et al.  Fatty acid binding proteins from different tissues show distinct patterns of fatty acid interactions. , 2000, Biochemistry.

[22]  J. Trojanowski,et al.  Synucleins Are Developmentally Expressed, and α-Synuclein Regulates the Size of the Presynaptic Vesicular Pool in Primary Hippocampal Neurons , 2000, The Journal of Neuroscience.

[23]  A. Kleinfeld,et al.  Equilibrium constants for the binding of fatty acids with fatty acid-binding proteins from adipocyte, intestine, heart, and liver measured with the fluorescent probe ADIFAB. , 1994, The Journal of biological chemistry.

[24]  M. L. Liu,et al.  Oleic acid distribution in small intestinal epithelial cells expressing intestinal-fatty acid binding protein. , 1998, Biochimica et biophysica acta.

[25]  R. Perrin,et al.  Exposure to Long Chain Polyunsaturated Fatty Acids Triggers Rapid Multimerization of Synucleins* , 2001, The Journal of Biological Chemistry.

[26]  L. Trombetta,et al.  The induction of amyloid precursor protein and alpha-synuclein in rat hippocampal astrocytes by diethyldithiocarbamate and copper with or without glutathione. , 2004, Toxicology letters.

[27]  G. J. van der Vusse,et al.  Impaired long-chain fatty acid utilization by cardiac myocytes isolated from mice lacking the heart-type fatty acid binding protein gene. , 1999, Circulation research.

[28]  Michel Goedert,et al.  Identification of two distinct synucleins from human brain , 1994, FEBS letters.

[29]  A. Morris,et al.  Regulation of phospholipase D2: selective inhibition of mammalian phospholipase D isoenzymes by alpha- and beta-synucleins. , 1998, Biochemistry.

[30]  S. Rhee,et al.  Inhibition of Phospholipase D by Amphiphysins* , 2000, The Journal of Biological Chemistry.

[31]  M. Golovko,et al.  An improved method for tissue long-chain acyl-CoA extraction and analysis Published, JLR Papers in Press, June 21, 2004. DOI 10.1194/jlr.D400004-JLR200 , 2004, Journal of Lipid Research.

[32]  T. Iwatsubo,et al.  Degenerative terminals of the perforant pathway are human α-synuclein-immunoreactive in the hippocampus of patients with diffuse Lewy body disease , 1998, Neuroscience Letters.

[33]  Rebecca A. Betensky,et al.  α-Synuclein occurs in lipid-rich high molecular weight complexes, binds fatty acids, and shows homology to the fatty acid-binding proteins , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[34]  S. Rapoport,et al.  A quantitative method for measuring regional in vivo fatty-acid incorporation into and turnover within brain phospholipids: review and critical analysis , 1992, Brain Research Reviews.

[35]  Robert L. Nussbaum,et al.  Mutation in the α-Synuclein Gene Identified in Families with Parkinson's Disease , 1997 .

[36]  P. S. St George-Hyslop,et al.  α-Synuclein Membrane Interactions and Lipid Specificity* , 2000, The Journal of Biological Chemistry.

[37]  S. Scarlata,et al.  Fluorescence studies suggest a role for alpha-synuclein in the phosphatidylinositol lipid signaling pathway. , 2005, Biochemistry.

[38]  N. Bazan,et al.  Quantitative Analysis of Acyl Group Composition of Brain Phospholipids, Neutral Lipids, and Free Fatty Acids , 1988 .

[39]  D. Prows,et al.  Effect of insulin on fatty acid uptake and esterification in L-cell fibroblasts. , 1996, Archives of biochemistry and biophysics.

[40]  B. Luxon,et al.  Sex differences in intracellular fatty acid transport: role of cytoplasmic binding proteins. , 1993, The American journal of physiology.

[41]  R. Scheller,et al.  Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal , 1988, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[42]  F. Schroeder,et al.  Recombinant liver fatty acid binding protein interacts with fatty acyl-coenzyme A. , 1994, Biochemistry.

[43]  Clemens R Scherzer,et al.  Gene expression changes presage neurodegeneration in a Drosophila model of Parkinson's disease. , 2003, Human molecular genetics.

[44]  Min Zhu,et al.  Lipid Binding Inhibits α-Synuclein Fibril Formation* , 2003, The Journal of Biological Chemistry.

[45]  J. Trojanowski,et al.  α‐synuclein is developmentally expressed in cultured rat brain oligodendrocytes , 2000, Journal of neuroscience research.

[46]  S. Rapoport,et al.  Utilization of Plasma Fatty Acid in Rat Brain: Distribution of [14C]Palmitate Between Oxidative and Synthetic Pathways , 1987, Journal of neurochemistry.

[47]  S. Rapoport,et al.  Rapid synthesis and turnover of brain microsomal ether phospholipids in the adult rat. , 2002, Journal of lipid research.

[48]  Richard Paylor,et al.  Synaptic Vesicle Depletion Correlates with Attenuated Synaptic Responses to Prolonged Repetitive Stimulation in Mice Lacking α-Synuclein , 2002, The Journal of Neuroscience.

[49]  D. Prows,et al.  Intestinal and liver fatty acid binding proteins differentially affect fatty acid uptake and esterification in L-cells , 1995, Lipids.

[50]  S. Lindquist,et al.  Yeast Cells Provide Insight into Alpha-Synuclein Biology and Pathobiology , 2003, Science.

[51]  S. Rapoport,et al.  Cerebral metabolism of plasma [14C]palmitate in awake, adult rat: Subcellular localization , 2004, Neurochemical Research.

[52]  F. Schroeder,et al.  Isoforms of rat liver fatty acid binding protein differ in structure and affinity for fatty acids and fatty acyl CoAs. , 1997, Biochemistry.

[53]  S. Rapoport,et al.  Intravenously injected [1-14C]arachidonic acid targets phospholipids, and [1-14C]palmitic acid targets neutral lipids in hearts of awake rats , 2000, Lipids.

[54]  H. Brockerhoff Determination of the positional distribution of fatty acids in glycerolipids. , 1975, Methods in enzymology.

[55]  M. Goedert,et al.  Binding of α-Synuclein to Brain Vesicles Is Abolished by Familial Parkinson’s Disease Mutation* , 1998, The Journal of Biological Chemistry.

[56]  C. Jolly,et al.  Role of FABP in Cellular Phospholipid Metabolism , 2004 .

[57]  F. Schroeder,et al.  Fatty acid binding protein: stimulation of microsomal phosphatidic acid formation. , 1997, Archives of biochemistry and biophysics.

[58]  G. Barceló-Coblijn,et al.  Heart Fatty Acid Uptake Is Decreased in Heart Fatty Acid-binding Protein Gene-ablated Mice* , 2004, Journal of Biological Chemistry.

[59]  D. Bundle,et al.  Sensitive titration microcalorimetric study of the binding of Salmonella O-antigenic oligosaccharides by a monoclonal antibody. , 1991, European journal of biochemistry.

[60]  J. Trojanowski,et al.  Antibodies to α‐synuclein detect Lewy bodies in many Down's syndrome brains with Alzheimer's disease , 1999, Annals of neurology.

[61]  B. Atshaves,et al.  Ablation of the liver fatty acid binding protein gene decreases fatty acyl CoA binding capacity and alters fatty acyl CoA pool distribution in mouse liver. , 2003, Biochemistry.

[62]  Takeshi Iwatsubo,et al.  Fatal attractions: abnormal protein aggregation and neuron death in Parkinson's disease and Lewy body dementia , 1998, Cell Death and Differentiation.

[63]  A. Jonas,et al.  Stabilization of α-Synuclein Secondary Structure upon Binding to Synthetic Membranes* , 1998, The Journal of Biological Chemistry.

[64]  J. Mcwhir,et al.  Requirement for the heart‐type fatty acid binding protein in cardiac fatty acid utilization , 1999, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[65]  R. Nussbaum,et al.  Metal-catalyzed oxidation of alpha-synuclein: helping to define the relationship between oligomers, protofibrils, and filaments. , 2005, The Journal of biological chemistry.

[66]  Matthew P Frosch,et al.  The Formation of Highly Soluble Oligomers of α-Synuclein Is Regulated by Fatty Acids and Enhanced in Parkinson's Disease , 2003, Neuron.

[67]  Janel O. Johnson,et al.  α-Synuclein Locus Triplication Causes Parkinson's Disease , 2003, Science.

[68]  R. Chanderbhan,et al.  Sterol carrier protein 2 and fatty acid-binding protein. Separate and distinct physiological functions. , 1985, The Journal of biological chemistry.

[69]  S. Rapoport,et al.  Specific Activity of Brain Palmitoyl‐CoA Pool Provides Rates of Incorporation of Palmitate in Brain Phospholipids in Awake Rats , 1995, Journal of neurochemistry.

[70]  J. Payton,et al.  Structural determinants of PLD2 inhibition by α-synuclein , 2004 .

[71]  D. Prows,et al.  Liver and intestinal fatty acid-binding protein expression increases phospholipid content and alters phospholipid fatty acid composition in L-cell fibroblasts , 2000, Lipids.

[72]  B. Atshaves,et al.  Sterol carrier protein-2 localization in endoplasmic reticulum and role in phospholipid formation. , 2000, American journal of physiology. Cell physiology.

[73]  D. Prows,et al.  Intestinal fatty acid-binding protein expression stimulates fibroblast fatty acid esterification. , 1996, Chemistry and physics of lipids.

[74]  R. Nussbaum,et al.  Fatty acid incorporation is decreased in astrocytes cultured from α‐synuclein gene‐ablated mice , 2005 .

[75]  C. Lavedan The synuclein family. , 1998, Genome research.