The formation of highly soluble oligomers of alpha-synuclein is regulated by fatty acids and enhanced in Parkinson's disease

Accumulation of misfolded proteins as insoluble aggregates occurs in several neurodegenerative diseases. In Parkinson's disease (PD) and dementia with Lewy bodies (DLB), alpha-synuclein (alpha S) accumulates in insoluble inclusions. To identify soluble alpha S oligomers that precede insoluble aggregates, we probed the cytosols of mesencephalic neuronal (MES) cells, normal and alpha S-transgenic mouse brains, and normal, PD, and DLB human brains. All contained highly soluble oligomers of alpha S whose detection was enhanced by delipidation. Exposure of living MES neurons to polyunsaturated fatty acids (PUFAs) increased alpha S oligomer levels, whereas saturated FAs decreased them. PUFAs directly promoted oligomerization of recombinant alphaS. Transgenic mice accumulated soluble oligomers with age. PD and DLB brains had elevated amounts of the soluble, lipid-dependent oligomers. We conclude that alpha S interacts with PUFAs in vivo to promote the formation of highly soluble oligomers that precede the insoluble alpha S aggregates associated with neurodegeneration.     

β-Synuclein occurs in vivo in lipid-associated oligomers and forms hetero-oligomers with α-synuclein

α-synuclein (αS) and β-synuclein (βS) are homologous proteins implicated in Parkinson's disease and related synucleinopathies. While αS is neurotoxic and its aggregation and deposition in Lewy bodies is related to neurodegeneration, βS is considered as a potent inhibitor of αS aggregation and toxicity. No mechanism for the neuroprotective role of βS has been described before. Here, we report that similar to αS, βS normally occurs in lipid-associated, soluble oligomers in wild-type (WT) mouse brains. We partially purified βS and αS proteins from whole mouse brain by size exclusion followed by ion exchange chromatography and found highly similar elution profiles. Using this technique, we were able to partially separate βS from αS and further separate βS monomer from its own oligomers. Importantly, we show that although αS and βS share high degree of similarities, βS oligomerization is not affected by increasing cellular levels of polyunsaturated fatty acids (PUFAs), while αS oligomerization is dramatically enhanced by PUFA. We show the in vivo occurrence of hetero-oligomers of αS and βS and suggest that βS expression inhibits PUFA-enhanced αS oligomerization by forming hetero-oligomers up to a quatramer that do not further propagate.     

Altered fatty acid composition of dopaminergic neurons expressing alpha-synuclein and human brains with alpha-synucleinopathies

Alpha-synuclein (alphaS) is an abundant neuronal protein that accumulates in insoluble inclusions in Parkinson's disease (PD) and the related disorder, dementia with Lewy bodies (DLB). A central question about the role of alphaS in the pathogenesis of PD and DLB concerns how this normally soluble protein assembles into insoluble aggregates associated with neuronal dysfunction. We recently detected highly soluble oligomers of alphaS in normal brain supernatants and observed their augmentation in PD and DLB brains. Further, we found that polyunsaturated fatty acids (PUFAs) enhanced alphaS oligomerization in intact mesencephalic neuronal cells. We now report the presence of elevated PUFA levels in PD and DLB brain soluble fractions. Higher PUFA levels were also detected in the supernatants and high-speed membrane fractions of neuronal cells over-expressing wild-type or PD-causing mutant alphaS. This increased PUFA content in the membrane fraction was accompanied by increased membrane fluidity in the alphaS overexpressing neurons. In accord, membrane fluidity and the levels of certain PUFAs were decreased in the brains of mice genetically deleted of alphaS. Together with our earlier observations, these results suggest that alphaS-PUFA interactions help regulate neuronal PUFA levels as well as the oligomerization state of alphaS, both normally and in human synucleinopathies.     

Effects of metabolic rate and sperm competition on the fatty‐acid composition of mammalian sperm

The sperm membrane is a key structure affecting sperm function and thus reproductive success. Spermatozoa are highly specialized and differentiated cells that undergo a long series of processes in the male and female reproductive tracts until they reach the site of fertilization. During this transit, the sperm membrane is prone to damage such as lipid peroxidation. The characteristics and performance of the sperm membrane are strongly determined by the fatty‐acid composition of membrane phospholipids. Polyunsaturated fatty‐acids (PUFAs) are the most prone to lipid peroxidation. Lipid peroxidation and other types of oxidative damage increase with higher metabolism and with higher levels of sperm competition due to the increased ATP production to fuel higher sperm velocities. Consequently, we hypothesized that, in order to avoid oxidative damage, and the ensuing impairment of sperm function, sperm cells exhibit a negative relationship between PUFA content and mass‐specific metabolic rate (MSMR). We also hypothesized that higher sperm competition leads to a reduction in the proportion of sperm PUFAs. We performed a comparative study in mammals and found that high MSMR and high levels of sperm competition both promote a decrease in the proportion of PUFAs that are more prone to lipid peroxidation. The negative relationship between MSMR and these PUFAs in sperm cells is surprising, because a positive relationship is found in all other cell types so far investigated. Our results support the idea that the effects of MSMR and sperm competition on sperm function can operate at very different levels.     

Long chain‐polyunsaturated fatty acids modulate membrane phospholipid composition and protein localization in lipid rafts of neural stem cell cultures

Rat neural stem cells/neural progenitors (NSC/NP) are generally grown in serum‐free medium. In this study, NSC/NP were supplemented with the main long‐chain polyunsaturated fatty acids (PUFAs) present in the brain, arachidonic acid (AA), or docosahexaenoic acid (DHA), and were monitored for their growth. Lipid and fatty acid contents of the cells were also determined. Under standard conditions, the cells were characterized by phospholipids displaying a highly saturated profile, and very low levels of PUFAs. When cultured in the presence of PUFAs, the cells easily incorporated them into the phospholipid fraction. We also compared the presence of three membrane proteins in the lipid raft fractions: GFR and connexin 43 contents in the rafts were increased by DHA supplementation, whereas Gβ subunit content was not significantly modified. The restoration of DHA levels in the phospholipids could profoundly affect protein localization and, consequently, their functionalities. J. Cell. Biochem. 110: 1356–1364, 2010. © 2010 Wiley‐Liss, Inc.     

Gene transfer of the Caenorhabditis elegans n-3 fatty acid desaturase inhibits neuronal apoptosis

Previous studies have shown that n-3 polyunsaturated fatty acids (PUFAs) can exert an antiapoptotic effect on neurons. The present study was designed to investigate whether the Caenorhabditis elegans fat-1 gene encoding an n-3 fatty acid desaturase (an enzyme that converts n-6 PUFAs to corresponding n-3 PUFAs) can be expressed functionally in rat cortical neurons and whether its expression can change the ratio of n-6 : n-3 fatty acids in the cell membrane and exert an effect on neuronal apoptosis. Infection of primary rat cortical cultures with Ad-fat-1 resulted in high expression of the fat-1 gene. Lipid analysis indicated a decrease in the ratio of n-6 : n-3 PUFAs from 5.9 : 1 in control cells, to 1.45 : 1 in cells expressing the n-3 fatty acid desaturase. Accordingly, the levels of prostaglandin E2, an eicosanoid derived from n-6 PUFA, were significantly lower in cells infected with Ad-fat-1 when compared with control cells. Finally, there was a significant inhibition of growth factor withdrawal-induced apoptotic cell death in neurons expressing the fat-1 gene. These results demonstrate that expression of the fat-1 gene can inhibit apoptotic cell death in neurons and suggest that the change in the n-6 : n-3 fatty acid ratio may play a key role in this protective effect.     

Plasminogen Binds Specifically to α-Enolase on Rat Neuronal Plasma Membrane

Abstract: Plasminogen (PGn) that we identified in microglial-conditioned medium has a neurotrophic factor-like effect on cultured neurons. We have also shown that PGn binds specifically to a protein with a molecular mass of 45 kDa in the neuronal plasma membrane. As a candidate PGn receptor-like molecule on the neuronal surface, this 45-kDa protein was purified from the plasma membrane of embryonic rat brain. Amino acid sequence analysis of polypeptides derived from the cleavage of the protein with cyanogen bromide and V8 protease revealed that the 45-kDa protein is identical to rat α-enolase. In fact, PGn was found to bind to purified rat α-enolase and also to a synthetic peptide (30 residues) that corresponds to the carboxyl terminal region of rat α-enolase. Physical properties of the 45-kDa protein, such as molecular mass, isoelectric point, and the ability to form dimers, are quite similar to those of α-enolase. The 45-kDa PGn-binding protein in the plasma membrane was also recognized by anti-rat α-enolase antibody, and pretreatment with α-enolase antibody markedly diminished the PGn-binding to the plasma membrane. In addition, immunocytochemical staining of the cultured cells under the nonpermeable condition showed that α-enolase is present on the cell surface of a certain population of neurons. These results suggest that α-enolase may function as a PGn-binding molecule on the neuronal cell surface.     

Neurite outgrowth stimulation by n-3 and n-6 PUFAs of phospholipids in apoE-containing lipoproteins secreted from glial cells

PUFAs, which account for 25–30% of the total fatty acids in the human brain, are important for normal brain development and cognitive function. However, it remains unclear how PUFAs are delivered to neurons and exert their effects. In this study, we demonstrated that n-3 and n-6 PUFAs added to the medium are incorporated into membrane phospholipids of primary glial cells from rat cortices, and then secreted as the fatty acid moiety of phospholipids in apoE-containing lipoproteins (LpEs). Tandem mass spectrometry analysis further showed that LpEs secreted from glial cells contain a variety of metabolites of PUFAs produced in glial cells by elongation and unsaturation. LpEs are absorbed by endocytosis into neurons via LDL receptor-related protein 1. LpE-containing n-3 and n-6 PUFAs exhibit a strong effect on neurite outgrowth of hippocampal neurons by increasing the number of branches. This study sheds light on the novel role of LpEs in the central nervous system and also a novel pathway in which PUFAs act on neurons.     

Polyunsaturated eicosapentaenoic acid displaces proteins from membrane rafts by altering raft lipid composition

Polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (20:5 (n-3)) inhibit T lymphocyte activation probably by displacing acylated signaling proteins from membrane lipid rafts. Under physiological conditions, saturated fatty acyl residues of such proteins partition into the cytoplasmic membrane lipid leaflet with high affinity for rafts that are enriched in saturated fatty acyl-containing lipids. However, the biochemical alteration causing displacement of acylated proteins from rafts in PUFA-treated T cells is still under debate but could principally be attributed to altered protein acylation or changes in raft lipid composition. We show that treatment of Jurkat T cells with polyunsaturated eicosapentaenoic acid (20:5 (n-3)) results in marked enrichment of PUFAs (20:5; 22:5) in lipids from isolated rafts. Moreover, PUFAs were significantly incorporated into phosphatidylethanolamine that predominantly resides in the cytoplasmic membrane lipid leaflet. Notably, palmitate-labeled Src family kinase Lck and the linker for activation of T cells (LAT) were both displaced from lipid rafts indicating that acylation by PUFAs is not required for protein displacement from rafts in PUFA-treated T cells. In conclusion, these data provide strong evidence that displacement of acylated proteins from rafts in PUFA-treated T cells is predominantly due to altered raft lipid composition.     

α - synuclein and Parkinson's disease: the first roadblock

α-synuclein gene mutations are major underlying genetic defects known in familial juvenile onset Parkinson's disease (PD), and α-synuclein is a major constituent of Lewy Bodies, the pathological hallmark of PD. The normal cellular function of α-synuclein has been elusive, and its exact etiological mechanism in causing dopaminergic neuronal death in PD is also not clearly understood. Very recent reports now indicate that mutant or simply over-expressed α-synuclein could cause damage by interfering with particular steps of neuronal membrane traffic. α-synuclein selectively blocks endoplamic reticulum-to-Golgi transport, thus causing ER stress. A screen in a yeast revealed that α-synuclein toxicity could be suppressed by over-expression of the small GTPase Ypt1/Rab1, and that over-expression of the latter rescues neuron loss in invertebrate and mammalian models of α-synuclein-induced neurodegeneration. α-synuclein may also serve a chaperone function for the proper folding of synaptic SNAREs that are important for neurotransmitter release. We discuss these recent results and the emerging pathophysiological interaction of α-synuclein with components of neuronal membrane traffic.     

Role of a new mammalian gene family in the biosynthesis of very long chain fatty acids and sphingolipids

Whereas the physiological significance of microsomal fatty acid elongation is generally appreciated, its molecular nature is poorly understood. Here, we describe tissue-specific regulation of a novel mouse gene family encoding components implicated in the synthesis of very long chain fatty acids. The Ssc1 gene appears to be ubiquitously expressed, whereas Ssc2 and Cig30 show a restricted expression pattern. Their translation products are all integral membrane proteins with five putative transmembrane domains. By complementing the homologous yeast mutants, we found that Ssc1 could rescue normal sphingolipid synthesis in the sur4/elo3 mutant lacking the ability to synthesize cerotic acid (C(26:0)). Similarly, Cig30 reverted the phenotype of the fen1/elo2 mutant that has reduced levels of fatty acids in the C(20)-C(24) range. Further, we show that Ssc1 mRNA levels were markedly decreased in the brains of myelin-deficient mouse mutants known to have very low fatty acid chain elongation activity. Conversely, the dramatic induction of Cig30 expression during brown fat recruitment coincided with elevated elongation activity. Our results strongly implicate this new mammalian gene family in tissue-specific synthesis of very long chain fatty acids and sphingolipids.     

Altered membrane free unsaturated fatty acid composition in human colorectal cancer tissue

Polyunsaturated free fatty acids (PUFAs) participate in normal functioning of the cell, particularly in control intracellular cell signalling. As nutritional components they compose a human diet with an indirect promoting influence on tumourogenesis. The PUFAs level depends on the functional state of the membrane. This work is focused on changes only of free unsaturated fatty acids amount (AA – arachidonic acid, LA – linoleic acid, ALA – α-linolenic acid, palmitoleic acid (PA) and oleic acid) in cell membranes of colorectal cancer of pT3 stage, G2 grade without metastasis. Qualitative and quantitative composition of free unsaturated fatty acids in the membrane was determined by high-performance liquid chromatography. It was shown that the malignant transformation was accompanied by a decrease in amount of LA and ALA while arachidonic and oleic acids increased. It is of interest that free AA levels are elevated in colon cancer, as AA is the precursor to biologically active eicosanoids.     

Fatty acid unsaturation,mobilization, and regulation in the response of plants to stress

Stress acclimating plants respond to abiotic and biotic stress by remodeling membrane fluidity and by releasing α-linolenic (18:3) from membrane lipids. The modification of membrane fluidity is mediated by changes in unsaturated fatty acid levels, a function provided in part by the regulated activity of fatty acid desaturases. Adjustment of membrane fluidity maintains an environment suitable for the function of critical integral proteins during stress. α-Linolenic acid, released from membrane lipid by regulated lipase activity, is the precursor molecule for phyto-oxylipin biosynthesis. The modulation of chloroplast oleic acid (18:1) levels is central to the normal expression of defense responses to pathogens in Arabidopsis. Oleic (18:1) and linolenic (18:2) acid levels, in part, regulate development, seed colonization, and mycotoxin production by Aspergillus spp.     

Chronic Sodium Valproate Selectively Decreases Protein Kinase C α and ε In Vitro

Abstract: Valproic acid (VPA) is a fatty acid antiepileptic with demonstrated antimanic properties, but the molecular mechanism or mechanisms underlying its therapeutic efficacy remain to be elucidated. In view of the increasing evidence demonstrating effects of the first-line antimanic drug, lithium, on protein kinase C (PKC), we investigated the effects of VPA on various aspects of this enzyme. Chronic exposure (6–7 days) of rat C6 glioma cells to "therapeutic" concentrations (0.6 m M ) of VPA resulted in decreased PKC activity in both membrane and cytosolic fractions and increased the cytosol/membrane ratio of PKC activity. Western blot analysis revealed isozyme-selective decreases in the levels of PKC α and ε (but not δ or ζ) in both the membrane and cytosolic fractions after chronic VPA exposure; VPA added to reaction mixtures did not alter PKC activity or ³H-phorbol ester binding. Together, these data suggest that chronic VPA indirectly lowers the levels of specific isozymes of PKC in C6 cells. Given the pivotal role of PKC in regulating neuronal signal transduction and modulating intracellular cross-talk between neurotransmitter systems, the specific decreases in PKC α and ε may play a role in the antimanic effects of VPA.     

Modulation of adipocyte differentiation by omega‐3 polyunsaturated fatty acids involves the ubiquitin‐proteasome system

We have evaluated the effects of three different omega‐3 polyunsaturated fatty acids (ω‐3 PUFAs) – docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPA) on fat accumulation and expression of adipogenic and inflammatory markers using both 3T3‐L1 pre‐adipocytes and differentiated 3T3‐L1 adipocytes. Our results indicate that ω‐3 PUFAs induce the degradation of fatty acid synthase through the ubiquitin‐proteasome system, which is likely to have beneficial metabolic effect on adipose cells. Omega‐3 PUFAs also increase overall levels of polyubiquitinated proteins, at least in part through decreasing the expression of proteasome subunits. Moreover, adipocytes are resistant to proteasome inhibition, which induces adipophilin while decreasing perilipin expression. On the other hand, ω‐3 PUFAs decrease expression of SREBP1 while inducing expression of adipophilin and GLUT4. Moreover, all three ω‐3 PUFAs appear to induce tumour necrosis factor‐α without affecting NFκB levels. All three ω‐3 PUFAs appear to have overall similar effects. Further research is needed to elucidate their mechanism of action.     

Oocyte signals derived from polyunsaturated fatty acids control sperm recruitment in vivo

A fundamental question in animal development is how motile cells find their correct target destinations. During mating in the nematode Caenorhabditis elegans, males inject sperm through the hermaphrodite vulva into the uterus. Amoeboid sperm crawl around fertilized eggs to the spermatheca--a convoluted tube where fertilization occurs. Here, we show that polyunsaturated fatty acids (PUFAs), the precursors of eicosanoid signalling molecules, function in oocytes to control directional sperm motility within the uterus. PUFAs are transported from the intestine, the site of fat metabolism, to the oocytes yolk, which is a lipoprotein complex. Loss of the RME-2 low-density lipoprotein (LDL) receptor, which mediates yolk endocytosis and fatty acid transport into oocytes, causes severe defects in sperm targeting. We used an RNAi screen to identify lipid regulators required for directional sperm motility. Our results support the hypothesis that PUFAs function in oocytes as precursors of signals that control sperm recruitment to the spermatheca. A common property of PUFAs in mammals and C. elegans is that these fats control local recruitment of motile cells to their target tissues.     

Cadmium Alters the Concentration of Fatty Acids in THP-1 Macrophages

Fatty acid composition of human immune cells influences their function. The aim of this study was to evaluate the effects of known toxicant and immunomodulator, cadmium, at low concentrations on levels of selected fatty acids (FAs) in THP-1 macrophages. The differentiation of THP-1 monocytes into macrophages was achieved by administration of phorbol myristate acetate. Macrophages were incubated with various cadmium chloride (CdCl₂) solutions for 48 h at final concentrations of 5 nM, 20 nM, 200 nM, and 2 μM CdCl₂. Fatty acids were extracted from samples according to the Folch method. The fatty acid levels were determined using gas chromatography. The following fatty acids were analyzed: long-chain saturated fatty acids (SFAs) palmitic acid and stearic acid, very long-chain saturated fatty acid (VLSFA) arachidic acid, monounsaturated fatty acids (MUFAs) palmitoleic acid, oleic acid and vaccenic acid, and n-6 polyunsaturated fatty acids (PUFAs) linoleic acid and arachidonic acid. Treatment of macrophages with very low concentrations of cadmium (5–200 nM) resulted in significant reduction in the levels of arachidic, palmitoleic, oleic, vaccenic, and linoleic acids and significant increase in arachidonic acid levels (following exposure to 5 nM Cd), without significant reduction of palmitic and stearic acid levels. Treatment of macrophages with the highest tested cadmium concentration (2 μM) produced significant reduction in the levels of all examined FAs: SFAs, VLSFA, MUFAs, and PUFAs. In conclusion, cadmium at tested concentrations caused significant alterations in THP-1 macrophage fatty acid levels, disrupting their composition, which might dysregulate fatty acid/lipid metabolism thus affecting macrophage behavior and inflammatory state.     

Effects of arachidonic acid analogs on FcepsilonRI-mediated activation of mast cells

Polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA) have been shown to modulate a number of inflammatory disorders. Mast cells play a critical role in the initiation and maintenance of inflammatory responses. However, the effects of PUFAs on mast cell functions have not been fully addressed. We here-in examined the effects of PUFAs on the high affinity IgE receptor (FcepsilonRI)-mediated mast cell activation using RBL-2H3 cells, a rat mast cell line, that were cultured in the medium containing palmitic acid (PA), AA, or the AA analogs mead acid (MA) and eicosapentaenoic acid (EPA). In AA-supplemented cells, the FcepsilonRI-mediated beta-hexosamidase and TNF-alpha release, calcium (Ca(2+)) influx, and some protein tyrosine phosphorylations including Syk and linker for activation of T cells (LAT) were enhanced, whereas, in MA- or PA-supplemented cells, they were not changed when compared with cells cultured in control medium. In EPA-supplemented cells, the enhancements of beta-hexosamidase release and protein tyrosine phosphorylations were observed. Furthermore, in AA- or EPA-supplemented cells, FcepsilonRI-mediated intracellular production of reactive oxygen species (ROS) that is required for the tyrosine phosphorylation of LAT and Ca(2+) influx were enhanced when compared with the other cells. Thus, preincubation of AA or EPA augmented FcepsilonRI-mediated degranulation in mast cells by affecting early events of FcepsilonRI signal transduction, which might be associated with the change of fatty acid composition of the cell membrane and enhanced production of ROS. The results suggest that some PUFAs can modulate FcepsilonRI-mediated mast cell activation and might affect FcepsilonRI/mast cell-mediated inflammation, such as allergic reaction.