Polyunsaturated fatty acids and their metabolites in brain function and disease

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1 Nature Reviews Neuroscience AOP, published online 12 November 2014; doi: /nrn3820 REVIEWS Polyunsaturated fatty acids and their metabolites in brain function and disease Richard P. Bazinet 1 and Sophie Layé 2,3 Abstract The brain is highly enriched with fatty acids. These include the polyunsaturated fatty acids (PUFAs) arachidonic acid and docosahexaenoic acid, which are largely esterified to the phospholipid cell membrane. Once PUFAs are released from the membrane, they can participate in signal transduction, either directly or after enzymatic conversion to a variety of bioactive derivatives ( mediators ). PUFAs and their mediators regulate several processes within the brain, such as neurotransmission, cell survival and neuroinflammation, and thereby mood and cognition. PUFA levels and the signalling pathways that they regulate are altered in various neurological disorders, including Alzheimer s disease and major depression. Diet and drugs targeting PUFAs may lead to novel therapeutic approaches for the prevention and treatment of brain disorders. 1 Department of Nutritional Sciences, University of Toronto, Toronto, Ontario M5S 3E2, Canada. 2 INRA, Nutrition et Neurobiologie Intégrée, UMR 1286, Bordeaux, France. 3 University of Bordeaux, Nutrition et Neurobiologie Intégrée, UMR 1286, Bordeaux, France. Correspondence to R.P.B. richard.bazinet@ utoronto.ca doi: /nrn3820 Published online 12 November 2014 In the brain, polyunsaturated fatty acids (PUFAs) regulate both the structure and the function of neurons, glial cells and endothelial cells. In the past decade, there have been major advances in our understanding of brain PUFA metabolism in health and disease. Mechanisms by which PUFAs enter and are regulated within the brain have been identified and characterized, as have a host of novel signalling molecules derived from PUFAs. In addition, studies have shown crucial roles for PUFAs in neuronal survival, neurogenesis, synaptic function and the regulation of brain inflammation. Thus, it is perhaps not surprising that altered dietary intake of PUFAs and altered PUFA metabolism have been reported in a range of neurological and psychiatric disorders. In this Review, we provide an update on our current understanding of the molecular and cellular targets of PUFAs and of PUFA metabolism in the healthy brain and in brain disorders. Fatty acids in the brain Fatty acid entry into the brain. Saturated and monounsaturated fatty acids can be synthesized de novo within the brain, but PUFAs are mainly supplied by the blood. The PUFAs linoleic acid (LNA) and α-linolenic acid (ALA) are obtained through the diet and act as precursors of arachidonic acid (ARA) and docosahexaenoic acid (DHA), which are also PUFAs (BOX 1; FIG. 1). The brain expresses the enzymes that are necessary for the synthesis of DHA and ARA. However, in rodents the synthesis rate of these PUFAs in the brain is much lower than the rate of PUFA uptake from the plasma. Furthermore, the brain levels of enzymes involved in the synthesis of ARA and DHA seem to be static 1 (in contrast to the liver, which regulates the expression of these enzymes in response to dietary supply). Collectively, these observations suggest that the brain relies on a constant supply of ARA and DHA from the blood (BOX 2; FIG. 2). There has been considerable debate about how PUFAs are delivered to the brain 2. They may be transported into the brain as unesterified (free) fatty acids or esterified to lipids (in the form of lysophospholipids and lipoproteins). In rat pups the main plasma pool of PUFAs for the brain may be DHA-containing lysophospholipids, but in adult rats the main plasma pool is thought to be unesterified fatty acids. Nevertheless, the orphan receptor MFSD2A (major facilitator superfamily domain-containing protein 2A) has recently been identified as a transporter for DHA esterified to lysophospholipids, confirming that lysophospholipids do indeed target the brain 3. The apparent discrepancy between these studies may be explained by differences in the plasma pool concentrations of esterified versus unesterified PUFAs and, more importantly, their half-lives in the plasma, but this remains to be investigated. Studies using mice lacking the low- and very-low-density lipoprotein receptors have suggested that these receptors are not necessary for maintaining brain PUFA levels (for a review, see REF. 4). However, according to a recent NATURE REVIEWS NEUROSCIENCE ADVANCE ONLINE PUBLICATION 1

2 Box 1 Fatty acid classification Fatty acids can be classified by their carbon chain length and by their number of double bonds (see the table). Long-chain fatty acids contain more than 12 carbon atoms, and fatty acids containing 22 or more carbon atoms are sometimes referred to as very-long-chain fatty acids. Within the brain, palmitic acid and stearic acid are the main saturated fatty acids (that is, fatty acids that contain no double bonds between their carbon atoms), and oleic acid is the main monounsaturated fatty acid (that is, a fatty acid that contains one double bond). The polyunsaturated fatty acids (PUFAs), which contain multiple double bonds between carbon atoms, can be classified into two families depending on the position of the double bond on the methyl terminal (ω; n-) end. The two predominant PUFAs in the brain are omega 6 arachidonic acid (ARA) and ω 3 docosahexaenoic acid (DHA). Other PUFAs, such as linoleic acid (LNA), eicosapentaenoic acid (EPA) and α-linolenic acid (ALA), are either not detectable in the brain or are orders of magnitude lower in concentration than ARA and DHA. Within the brain, fatty acids can be unesterified (free) or esterified to lipids such as triacylglycerol, cholesterol and phospholipids. In the brain, fatty acids are predominantly esterified to phospholipids in the plasma membrane. Phospholipids containing DHA are enriched in grey matter and the synaptosomal fraction, whereas other esterified fatty acids, such as palmitate and oleate, are enriched in myelin. DHA and ARA are esterified to phospholipids at a concentration of approximately 10,000 nmol per gram of brain tissue, whereas their unesterified levels are about 1 nmol per gram brain tissue. ARA and DHA each make up about just over 10% of the total fatty acids within brain phospholipids. Within the phospholipids, there is further selectivity: DHA is largely esterified to ethanolamine glycerophospholipids and forms up to 35% of the fatty acids in the phosphatidylserine fraction, whereas ARA is esterified to choline glycerophospholipids and can also be up to 40% of the fatty acids within phosphatidylinositol 52. PUFAs are predominately esterified to the sn 2 position of phospholipid membranes, whereas saturated and monounsaturated fatty acids are mainly esterified to the sn 1 position. Upon activation of phospholipase A2, PUFAs are released from the membrane and can exert their effects either themselves or when converted into bioactive mediators, which are often present in picomol per gram levels (that is, about a billion times lower than phospholipid levels). Name Type Number of carbon atoms Number of double bonds Palmitic acid Saturated :0 Stearic acid Saturated :0 Symbol Oleic acid Monounsaturated :1n 9 α-linolenic acid (ALA) ω 3 polyunsaturated :3n 3 Eicosapentaenoic acid (EPA) ω 3 polyunsaturated :5n 3 Docosapentaenoic acid (DPA) n-3 ω 3 polyunsaturated :5n 3 Docosahexaenoic acid (DHA) ω 3 polyunsaturated :6n 3 Linoleic acid (LNA) ω 6 polyunsaturated :2n 6 DPA n-6 ω 6 polyunsaturated :5n 6 Arachidonic acid (ARA) ω 6 polyunsaturated :4n 6 proposal, the enzyme lipoprotein lipase may hydrolyse circulating plasma lipoproteins and release unesterified PUFAs and/or lysophospholipid-containing PUFAs, which are then taken up by the brain 5,6. Clearly, more work is needed to identify the plasma fatty acid pools that enter the brain and to quantify their uptake under normal and pathological conditions. Experiments in artificial membranes, which do not contain proteins, indicate that unesterified fatty acids can passively diffuse into the brain 7. Nevertheless, several candidate fatty acid transporters have been identified within the brain. Most fatty acid transporters have long-chain-fatty-acid-coa synthase (ACSL) activity and probably trap fatty acids (rather than transport them) 8, and thereby facilitate their targeting to specific lipid pools 9. Consistent with a lack of active transport, entry of unesterified fatty acids into the brain does not seem to be selective. For example, unesterified eicosapentaenoic acid (EPA) enters the brain at a similar rate as unesterified DHA, even though the brain concentration of unesterified EPA is fold lower than that of unesterified DHA. Fatty acid concentrations in the brain are further regulated through metabolism. For example, EPA is rapidly catabolized by β-oxidation, elongation and desaturation to docosapentaenoic acid n-3 (DPA n3; 22:5n 3) and DHA, and is not heavily recycled within brain phospholipids 4 ; as a result, brain EPA concentrations are very low. One candidate fatty acid transporter that does not seem to have ACSL activity is CD36 (also known as SRB1). However, recent studies have shown that CD36 is not a classical transporter but probably facilitates fatty acid metabolism 10. In fact, CD36 probably transports cholesteryl esters rather than fatty acids 11. Transport of fatty acids occurs on the millisecond timescale. The plasma half-life of unesterified fatty acids is approximately 30 seconds in vivo, and they can be taken up by the liver and secreted as lipoproteins or be esterified into other blood lipid pools within 30 minutes. Much confusion in the field regarding the uptake of PUFAs into the brain may have arisen because studies of brain PUFA levels in which fatty 2 ADVANCE ONLINE PUBLICATION

3 18:3n-3 (ALA) 18:4n-3 20:4n-3 20:5n-3 Figure 1 Synthesis of PUFAs in the liver. In the liver, the n-3 polyunsaturated fatty acid (PUFA) α-linolenic acid (ALA; 18:3n 3) and the n-6 PUFA linoleic acid (LNA; 18:2n 6) can be desaturated (which involves the addition of a double bond) and elongated to become longer-chain PUFAs. Of note, the enzyme Δ6 desaturase is considered to be the rate-limiting step for the synthesis of docosahexaenoic acid (DHA; 22:6n 3). Not only are both ALA and LNA substrates for Δ6 desaturase, but 24:5n 3 and 24:4n 6 recycle back and require Δ6 desaturation before β-oxidation in the peroxisome. It is thought that both the n-3 PUFAs and n-6 PUFAs share the same enzymes and thus compete for their desaturation and elongation. Synthesized PUFAs, including DHA and docosapentaenoic acid n-6 (22:5n-6), but also arachidonic acid (20:4n 6), can be exported into the blood as lipoproteins. Accretion Gradual accumulation. 6 desaturase Elongase 5 desaturase 22:6n-3 24:6n-3 24:5n-3 22:5n-3 Circulation Peroxisome β-oxidation Hepatocyte Elongase 22:5n-6 24:5n-6 24:4n-6 22:4n-6 6 desaturase Elongase 5 desaturase 18:2n-6 (LNA) 18:3n-6 20:3n-6 20:4n-6 acids were infused into the plasma and measured in the brain hours later often do not distinguish between the effects of transport, uptake and metabolism. Fatty acid accretion in the brain. During myelination, the brain accumulates fatty acids associated with the myelin sheath, especially oleic acid. In the last trimester of gestation in humans, the brain accelerates its accumulation of PUFAs, especially DHA 12. The turnover rate of DHA during development is not known, but estimates suggest that it is high, which could further increase the demand for DHA in the developing brain 13. The rapid accretion of DHA in the developing brain in combination with the presence of DHA in breast milk suggest that dietary DHA may be important for neurodevelopment. Post-mortem studies have shown that infants who had been fed formula lacking DHA had lower brain DHA levels than infants who had been fed breast milk. In addition, some, but not all, clinical trials have found higher neurodevelopmental scores in infants who had been fed formula containing DHA than those fed formula lacking DHA. In general, dietary DHA intake seems to benefit preterm infants more than healthy-term infants, possibly because preterm infants have not been exposed to the third trimester in utero increase in brain and adipose DHA accretion. The role of fatty acids in infant brain development has been reviewed elsewhere In the adult brain, ARA and DHA are no longer accreted 17, and plasma ARA and DHA only need to replace brain consumption. Current estimates of ARA and DHA uptake in the brain from the plasma unesterified pool are about 18 and 4 mg per day, respectively (BOX 1), and their half-lives in the brain are estimated to be 147 and 773 days, respectively 18. If plasma pools other than the pool of unesterified fatty acids are available to the brain, then the uptake of ARA and DHA in the brain may be higher, their turnover more rapid and their half-lives shorter. Fatty acid metabolism in the brain. Upon entry into the brain, most PUFAs especially DHA and ARA are activated by an ACSL 19 and then esterified to phospholipid membranes. Other PUFAs, such as LNA, ALA and EPA, are β-oxidized. Studies in peripheral organs have shown that ACSLs facilitate the transport of fatty acids to various metabolic fates, including esterification and β-oxidation 20. As mentioned above, some fatty acid transporters also have ACSL activity 21 and therefore probably facilitate in combination with fatty-acid-binding proteins (FABPs) in the brain the partitioning of fatty acids to either esterification or metabolism 22 (FIG. 2). The acyl-coa esterification of fatty acids to lysophospholipids is probably mediated by the 1 acylglycerol 3 phosphate-o acyltransferase (AGPAT) and the lysophosphatidic acid acyltransferase (LPAAT) family of enzymes 23. Members of the ACSL-activity-containing fatty acid transporter family and the AGPAT and LPAAT families seem to have moderate selectivity, but if they act sequentially with other proteins, this could explain the highly specific distribution of brain fatty acids: slight differences in selectivity could target fatty acids towards esterification to specific lysophospholipid species, towards β-oxidation or towards other metabolic pathways within the brain. In the adult brain, the amount of ATP generated from fatty acid β-oxidation is much less than the ATP generated by glucose oxidation (with the notable exception of the circumventricular areas 24 ). It has recently been hypothesized that the relatively low level of brain fatty acid β-oxidation is due in part to the excessive oxidative stress (and subsequent cell damage) generated during this process 25,26. Consistent with this theory, pharmacological inhibition of β-oxidation in the brain decreases the levels of auto-oxidative PUFA metabolites in the brain 27. In phospholipids, two fatty acids attach to the stereospecifically numbered first and second carbons (sn 1 and sn 2 positions) of the glycerol molecule. Upon esterification to the phospholipid plasma membrane, fatty acids at the sn 1 position can be de esterified and released from the membrane by phospholipase A1 (PLA1), whereas fatty acids at the sn 2 position (such as ARA and DHA) are de esterified by PLA2 (REF. 28). The selectivity of PLA2 for specific fatty acids varies several-fold in vitro. In vivo studies have shown that the turnover of DHA in brain phospholipids can be reduced without a decrease in the turnover of ARA 29, suggesting that the metabolism of these fatty acids is selectively regulated. With regard to DHA release, inhibition of calcium-independent PLA2 (ipla2) selectively decreased ATP-induced DHA release from neuronal cells 30, further supporting the notion of selective release of fatty acids from the membrane (for a review, see REF. 31). NATURE REVIEWS NEUROSCIENCE ADVANCE ONLINE PUBLICATION 3

4 Box 2 The adipose brain and adipose liver brain fatty acid axes Although polyunsaturated fatty acids (PUFAs) cannot be completely synthesized de novo, it has become apparent that adipose tissue can serve as a reservoir for PUFAs. During the last trimester of gestation in humans, the rate of fetal adipose tissue deposition increases, which may be an important source of energy in the newborn, as does the amount of docosahexaenoic acid (DHA) in the adipose tissue 12. Adipose DHA is thought to supply the brain with a reserve of DHA 193. Moreover, DHA released from adipose tissue is unesterified and therefore available to the brain, indicating the existence of an adipose brain fatty acid axis. A similar axis is thought to exist for arachidonic acid (ARA). Holman and colleagues were the first to report that a 6 year-old girl who received total parenteral nutrition with a solution containing the n-6 PUFA linoleic acid (LNA) but devoid of all n-3 PUFAs for at least 4 months developed neurological symptoms including distal numbness, paresthesias, visual blurring, decreased peripheral vibratory sensation and a mild tremor in the left upper extremity, although her blood chemistry was relatively normal 194. Not surprisingly, the patient also had very low plasma n-3 PUFA levels. The neurological symptoms disappeared rapidly after administration of a total parenteral nutrition solution containing α-linolenic acid (ALA). Subsequent to this case study, Scott and Bazan demonstrated that in the liver ALA is converted to DHA and exported to the blood, where it can eventually supply the brain 195. A recent kinetic modelling study in rats showed that the conversion of ALA to DHA in the liver is several-fold higher than brain DHA uptake rates 196. Furthermore, in rodents and humans, following dietary intake of ALA, a significant proportion is directed to the adipose tissue 197, where it may provide a reservoir and be converted to DHA to supply the brain. A study in which rats received only dietary stable isotope-labeled ALA as a source of n-3 PUFAs showed that most brain DHA accretion was not labelled, which suggests that previously stored ALA or DHA was used for the synthesis or accretion of brain DHA 198. It is important to note that dietary DHA targets the brain more effectively than ALA converted to DHA 199, but under conditions of chronic low dietary n 3 fatty acids, the liver upregulates its ability to synthesize DHA and presumably receives ALA from the adipose tissue. This suggests the existence of an adipose liver brain fatty acid axis. Paresthesias Sensations of tingling, tickling, prickling or burning of a person s skin. Lands cycle The process of deacylation and reacylation of fatty acids, sometimes referred to as recycling within membrane phospholipids. The pathway was discovered by William Lands. Mediators Derivatives of a fatty acid that are bioactive. The term lipid mediator is distinct from derivative as it implies that the molecule is bioactive. Specialized pro-resolving mediators Mediators that promote pro-resolution, which is an active process involving several lipids that turns off pro-inflammatory signalling and promotes the clearance of leukocytes and cellular debris, thereby returning the tissue to homeostasis. After release from the plasma membrane, most of the ARA and DHA (>90% under basal conditions) is immediately re esterified into brain phospholipids via ACSL through to the Lands cycle. ACSL uses two high-energy phosphates from ATP and, on the basis of the relatively high rate of fatty acid release from PLA2 and recycling into the membrane (about 100% per day in rodents), it has been calculated that total fatty acid recycling consumes up to 5% of brain ATP 32. The importance of this high-energy consumption pathway is not known, but it may serve, in part, to conserve brain PUFA levels. PUFA release and conversion in the brain As mentioned above, the PUFAs ARA and DHA within brain phospholipid membranes can be de esterified by PLA2, which is coupled to many receptors in the brain, including dopaminergic 33, cholinergic 34, serotonergic 35 and N methyl d aspartate (NMDA) 36 receptors. Upon activation of these receptors, PLA2 probably calcium-dependent cytosolic PLA2 (cpla2) triggers the release of ARA from the synaptic membrane 37 (FIG. 3). ARA is also released from the membrane by PLA2 activation in response to ischaemia, excitotoxicity and inflammation. The release of DHA is less-well studied but seems to occur upon stimulation of cholinergic and serotonergic receptors, ischaemia and in response to ATP and bradykinin (for reviews, see REFS 31,38 40). Importantly, ARA and DHA are also released during brain removal using standard laboratory techniques 41 ; rapid in vivo fixation techniques, including high-energy, head-focused microwave irradiation, can be used to avoid such postmortem release of fatty acids 41. The signalling pathways in which PLA2 released fatty acids participate are not clear and remain an active area of research. PUFAs are converted into bioactive derivatives (which are often referred to as mediators ) upon activation of receptors that are coupled to PLA2. The main enzymes involved in the synthesis of PUFA mediators are cyclooxygenases (COX), lipoxygenases (LOX) and cytochrome P450 (REF. 42). Unlike in many other tissues, basal COX2 expression is high in neurons and facilitates the conversion of ARA to prostaglandin E2 (PGE2), which is a potent signalling molecule in the brain. So far, numerous derivatives of ARA have been identified within the brain (FIG. 3), and several excellent reviews have described ARA metabolism in this tissue 43,44. To date, only a few DHA-derived mediators, including 17S hydroxy-dha (17 HDHA), neuroprotectin D1 (NPD1), resolvin D5 (RvD5), 14 HDHA and maresin 1 (MaR1), have been identified within brain tissue 42,45. NPD1, RvD5 and MaR1 in the brain are bioactive and are produced through the LOX pathway; they have been coined the specialized pro-resolving mediators (for a recent review, see REF. 42). The brain cell types involved in the synthesis of PUFA mediators, especially those synthesized from DHA, have not been fully elucidated, but glial cells have been shown to produce NPD1 (REF. 46). Furthermore, many lipid derivatives may only be produced upon brain injury, ischaemia, inflammation or its resolution. For example, upon reperfusion after medial cerebral artery occlusion, levels of the DHA mediators NPD1, 17R HDHA, 22 hydroxy,14,17s-dihdha and 7,8,17R trihdha increase several fold 47. A more thorough investigation is warranted to identify the specialized pro-resolving mediators and PUFA derivatives that are present in the brain and their bioactive roles. The consumption and replacement of PUFAs in the brain can be imaged in animals with autoradiography and in humans with positron emission tomography (PET) 40. For instance, upon injection of quinpirole an agonist of the dopamine D2 receptor (which is coupled to PLA2) in rats that are being infused with unesterified radiolabelled ARA, the radiolabelled ARA replaces the PLA2 mediated release of ARA in brain regions enriched in dopamine D2 receptors. This increase does not occur after injection of the dopamine D1 receptor agonist SKF (REF. 40), which suggests that dopamine D1 activation is not involved in PLA2 mediated release. Similarly, acute administration of apomorphine and amphetamine also induces PLA2 mediated ARA release, and this is blocked with pre-administration of the dopamine D2 like receptor antagonist raclopride 40. Recently, increases in ARA-mediated signalling were observed in humans upon acute apomorphine administration with the use of 11 C-labelled ARA and PET 48. The release of fatty acids by PLA2 is considered to be a marker of neuroreceptor activity, and we refer the reader to a recent review on fatty acid imaging in the brain for more details ADVANCE ONLINE PUBLICATION

5 Plasma Endothelial cell Brain 1 LDLR Vesicle Lands cycle Unesterified fatty acid 4 5 LPL Albumin LDL particle 2 3 MFSD2A 6 CD36 Passive diffusion Hydrolysis 7 8 Lipoprotein lipase FABP 9 Passive diffusion CoA ACSL FATP 12 Mediator synthesis Elongation/desaturation Peroxisome Mitochondrion Figure 2 Fatty acid entry from the plasma into the brain. Fatty acids that enter the brain can come from several candidate pools in the plasma, including lipoproteins, lysophospholipids (LPLs) or unesterified fatty acids. Lipoproteins, such as low-density lipoprotein (LDL), can bind to their respective lipoprotein receptors, such as the LDL receptor (LDLR), inducing endocytosis (1). Alternatively, lipoprotein lipase can interact with lipoproteins (2) to produce a fatty-acid-containing LPL, which is taken up by MFSD2A (major facilitator superfamily domain-containing protein 2A) (3), and unesterified fatty acids (4). Unesterified fatty acids, which may be associated with albumin (5), can subsequently be taken up via a candidate transporter (such as CD36) (6) or passively diffuse into the endothelial membrane (7). Within endothelial cells, which make up the blood brain barrier, lipoproteins can be hydrolysed to release polyunsaturated fatty acids (PUFAs) (8), which associate with fatty-acid-binding proteins (FABPs) (9) and are subsequently transported across the neuronal membrane, either passively (10) or by fatty acid transporter proteins (FATPs) (11). Upon entering the neuronal membrane, fatty acids are converted to CoA thioesters by a family of proteins with long-chain-fatty-acid-coa synthase (ACSL) activity this family includes FATPs (12). This partitions them towards esterification and de-esterification via the Lands cycle and towards β-oxidation in mitochondria and peroxisomes. Reprinted from Prostaglandins Leukot. Essent. Fatty Acids, Chen, C. T. & Bazinet, R. P., 2014, with permission from Elsevier. PUFA functions in the brain Fatty acids and their mediators have numerous functions in the CNS, including a role in hypothalamic regulation of hepatic glucose production 49 and food intake 50 and in analgesia 51. Here, we focus on more recently discovered functions of DHA and, to a lesser extent, ARA. Synaptic effects of PUFAs. PUFAs and their metabolites act in the brain through several potential mechanisms. One of these is the regulation of membrane dynamics, which has been discussed extensively in several reviews Another mechanism involves the activation of receptors and consequent activation of cell signalling pathways. For example, unesterified PUFAs and/or their mediators are agonists for the oxysterols receptor LXR, peroxisome proliferator-activated receptor (PPAR), hepatic nuclear factor 4A (HNF4A; also known as NR2A1), chemokinelike receptor 1 (also known as CHEMR23), G-proteincoupled receptor 32 (GPR32) and lipoxin receptor ALX/ FPR2, and they can activate protein kinase C (PKC) and inhibit nuclear factor κb (NF κb) 31,38,42,55. PUFAs can also influence brain function through modulation of the endocannabinoid system (FIG. 4), and, notably, endocannabinoids themselves are derived from PUFAs. The endocannabinoids include the fatty acid ethanolamides anandamide (AEA), synaptamide (also known as docosahexaenoyl ethanolamide), oleylethanolamide and palmitoylethanolamide, as well as 2 arachidonoylglycerol (2 AG) 56. The most abundant endocannabinoids in the brain are the ARA metabolites AEA and 2 AG, which bind to cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2). Neurons and glial cells, including astrocytes and microglia, produce endocannabinoids and express cannabinoid receptors 57. Endocannabinoids are important regulators of synaptic function; they suppress neurotransmitter release (including the release of glutamate, GABA, monoamine neurotransmitters, opioids and acetylcholine) by acting as retrograde messengers at presynaptic CB1s 58.Retrograde endocannabinoid signalling mediates short-term forms of synaptic plasticity as well as presynaptic forms of longterm depression (LTD) at both excitatory and inhibitory synapses 58. Recent studies have shown that endocannabinoids can also modulate synaptic transmission through TRPV1 (transient receptor potential cation channel subfamily V member 1), which is located postsynaptically, and through CBs expressed on astrocytes 59. PUFA-mediated regulation of endocannabinoid signalling in the brain (FIG. 4) is an emerging area of research. One study demonstrated an increase in AEA and 2 AG levels in the brains of piglets consuming a milk formula enriched with ARA and DHA 60. Being kept on a highfat diet increases endocannabinoid levels in the mouse NATURE REVIEWS NEUROSCIENCE ADVANCE ONLINE PUBLICATION 5

6 cpla2 ARA Neurotransmitter Neurotransmitter receptor ARA CoA AGPAT ACSL CoA PGD2 5-HETE 12-HETE 15-HETE HETEs PGE2 8-HETE ETEs PGF2α TXA2 TXB2 PGF1α LTB4 LXA4 LXB4 ipla2 RvD2 NPD1 DHA COX LOX Cyt P450 LOX LOX Phospholipid DHA MaR1 AGPAT ACSL Figure 3 Fatty acid release and conversion to mediators upon receptor-mediated signal transduction. a In response to activation of dopaminergic, cholinergic, serotonergic or N methyl d aspartate (NMDA) receptors, esterified arachidonic acid (ARA) in the form of a phospholipid (shown in yellow) is released from the membrane (that is, is de esterified) by phospholipase A2 (PLA2), probably specifically by calcium-dependent cytosolic PLA2 (cpla2). A large proportion of ARA is subsequently re esterified into the membrane by long-chain-fatty-acid-coa synthase (ACSL) and 1 acylglycerol 3 phosphate-o acyltransferase (AGPAT). A small proportion of the ARA can be converted to eicosanoids (shown in beige) via cyclooxygenases (COX), lipoxygenases (LOX) and cytochrome P450 (Cyt P450). b A similar pathway exists for docosahexaenoic acid (DHA), which is released upon cholinergic or serotonergic receptor activation and can be converted to lipid mediators via LOX. The schematic here represents a neuron, but polyunsaturated fatty acids (PUFAs) are also released from glial cells in response to different signals, including excitotoxicity, inflammation and ischaemia. ETE, eicosatetraenoic acid; HETE, hydroxyeicosatetraenoic acid; ipla2, calcium-independent PLA2; LTB4, leukotriene B4; LXA4, lipoxin A4; MaR1, maresin 1; NPD1, neuroprotectin D1; PD1, protectin D1; PDG2, prostaglandin D2; PGF, prostaglandin F; PGE2, prostaglandin E2; RvD2, resolvin D2; TXA2, thromboxane A2; TXB2, thromboxane B2. CoA CoA brain, whereas endocannabinoid levels are reduced upon DHA consumption 61. Mice chronically fed a diet low in n-3 PUFAs have increased brain levels of 2 AG, whereas those fed a diet that is high in DHA have decreased 2 AG levels 62 without changes in ARA levels, suggesting that DHA can regulate the synthesis of 2 AG independently of changes in ARA. AEA and 2 AG are derived from ARA through conversion by N acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) and diacylglycerol lipase (DAGL), respectively. Interestingly, EPA (but not DHA) inhibits NAPE-PLD, further shunting ARA towards endocannabinoid synthesis 63. In addition to regulating endocannabinoid levels and metabolism, in mice, n-3 PUFAs also regulate CB1 activity and CB1 associated signalling pathways in the prefrontal cortex and nucleus accumbens 64,65. For example, as a consequence of chronic exposure to low levels of dietary n-3 PUFAs, which leads to low DHA levels in the brain, endocannabinoid-dependent LTD is impaired in these brain regions and mice develop depression-like and anxiety-like behaviour 64,65. DHA, which is particularly enriched in synapses, also has a role in synaptic integrity and the assembly of the SNARE complex. DHA has been shown to attenuate the altered expression of postsynaptic dendritic proteins (including drebrin, postsynaptic density 95 (PSD95), NMDA receptors and Ca 2+ calmodulindependent protein kinase 2 (CAMK2)) in a mouse model of Alzheimer s disease 66,67. DHA also regulates the SNARE fusion machinery involved in presynaptic exocytosis and endocytosis 68. Specifically, the consumption of a low n-3 PUFA diet increases the expression of SNARE complex proteins in the rat hippocampus, but not the expression of the SNARE proteins synaptosomal-associated protein 25 (SNAP25), syntaxin, synaptophysin and complex II in mouse cortex and rat hippocampus 67,69. In addition, S nitrosylation of syntaxin is decreased 69 in animals with low brain DHA levels. Furthermore, syntaxin 3 is activated by both ARA and DHA in vitro 70, and DHA promotes syntaxin 3 incorporation into SNARE complexes and, thereby, regulates rod photoreceptor biogenesis in the retina 71. DHA also impairs syntaxin binding to the SNARE complex in PC12 cells 70, but has no effect on synaptobrevin mrna and protein expression in a SH SY5Y neuroblastoma cell line 72. Together, these data suggest that DHA influences SNARE protein assembly rather than the expression of SNARE proteins. The role of PUFAs in neurogenesis and neuroprotection. Studies have indicated that DHA is involved in learning and memory, but the cellular and molecular mechanisms underlying these effects are poorly understood 73. One of the first described protective effects of DHA in the brain was its promotion of neuronal survival 74 and neurogenesis 75 (FIG. 5). DHA, the main PUFA in phosphatidylserine, enhances phosphatidylserine synthesis in vitro 76, and depletion of DHA from the membrane impairs phosphatidylserine-mediated AKT and RAF1 translocation and activation, which are important for promoting neurogenesis. The DHA mediator synaptamide 77 is sufficient to promote neuronal differentiation and is a much more potent promoter of neurite growth, synaptogenesis and synaptic function than DHA itself The brains of mice that are fed high levels of DHA have increased synaptamide levels and show increased neuronal differentiation of neural stem cells 79. The LOX-synthesized DHA mediator NPD1 may promote neuronal survival by upregulating genes encoding the anti-apoptotic proteins B cell lymphoma 2 (BCL2), BCLXL and BCL2 related protein A1 (BCL2A1; also known and BFL1), and downregulating genes encoding the pro-apoptotic proteins BCL2 associated agonist of cell death (BAD), BAX, BH3 interacting domain death agonist (BID) and BCL2 interacting killer (BIK) in vitro and in the brain in vivo 80,81. Furthermore, increased brain DHA levels have been shown to normalize brainderived neurotrophic factor (BDNF) levels in rats exposed to traumatic brain injury 82 ; consistent with this, addition of DHA increased BDNF levels in astrocytes in vitro, and DHA deprivation decreased BDNF levels in the rodent brain ADVANCE ONLINE PUBLICATION

7 ecb-ltd Phospholipid Neurotransmitter Ca 2+ + PLA2 Presynaptic neuron Neurotransmitter receptor PUFA Postsynaptic neuron ecb CB1 ecb Figure 4 Dietary PUFAs influence endocannabinoidmediated synaptic plasticity. Endocannabinoids (ecbs) are signalling lipids that are produced from polyunsaturated fatty acids (PUFAs) present in phospholipids in the neuronal cell membrane. PUFAs are released from the postsynaptic membrane by phospholipase A2 (PLA2) in response to neuronal activity. ecbs are released into the synaptic space and can then bind the presynaptic cannabinoid receptor type 1 (CB1) on the presynaptic neuron. The activation of CB1 results in inhibition of neurotransmitter release and synaptic activity. In rodents that are fed a diet that includes n-3 PUFAs, such ecb-mediated long-term depression (ecb LTD) is induced at excitatory synapses. This form of ecb-mediated synaptic plasticity is specifically ablated at synapses in the brains of rodents that are chronically fed a diet low in n-3 PUFAs. This effect is due to a reduction in the coupling of presynaptic CB1s to their effector Gi/o protein, but is not associated with reduced CB1 expression (not shown). The above-mentioned effects of DHA and its mediators on cell survival and neurogenesis have been further investigated in preclinical models of neurological diseases, including Parkinson s disease (for a recent review, see REF. 84). In addition, during ischaemia, unesterified DHA is released from the phospholipid membrane and is converted to NPD1 (REF. 47), which may promote antiapoptotic signalling. Increasing brain DHA levels through dietary supplementation, intravenous infusion or intracerebroventricular injection augments antiapoptotic and anti-inflammatory signalling in rodent brains 39,85. Increasing DHA supply to the brain also decreases infarct size in ischaemia reperfusion models 39. Consistent with these observations, elevated brain DHA or intravenous administration of unesterified DHA 3 5 hours after the induction of experimental stroke in mice have been shown to decrease infarct volume and pro-inflammatory signalling in the brain and to improve functional outcomes 39. Interestingly, an aspirin-induced stereoisomer of NPD1 is produced in the brain upon ischaemia followed by reperfusion, and co administration of both DHA and aspirin decreases lesion volume and improves functional outcomes after experimental stroke in rats 86. Intracerebroventricular administration of DHA or NPD1 also decreases stroke volume and attenuates the induction of the pro-inflammatory signalling proteins NF κb and COX2 (REF. 47). Thus, targeting the brain with DHA and its mediators is not only therapeutic when done before ischaemia, but can also have beneficial effects after ischaemic injury. The role of PUFAs in inflammation in the brain. DHA and its mediators have potent anti-inflammatory and pro-resolving properties in peripheral tissues 42. In humans, higher dietary intakes of DHA (from consuming fish) are associated with a lower risk of neurological disorders that have an inflammatory component, including Alzheimer s disease, Parkinson s disease and major depression 87. This has led to the hypothesis that DHA may have anti-inflammatory effects in the brain as well 88. Indeed, the expression of pro-inflammatory cytokines in the brain following systemic lipopolysaccharide (LPS) administration 89,90, brain ischaemia reperfusion 91, spinal cord injury 92 and ageing 93 is reduced in rodents with high levels of brain DHA 85. Furthermore, n-3 PUFAs can improve behaviour and neurophysiological systems affected by neuroinflammation. For example, shortterm exposure to EPA through the diet reduced spatial memory deficits and anxiolytic behaviour induced by central administration of interleukin 1β (IL 1β) 94 and improved inhibition of long-term potentiation (LTP) by LPS and amyloid-β in rats 93. In addition, ageingassociated microglia activation, and the associated production of IL 1β and alterations in hippocampal LTP, could be attenuated by dietary EPA supplementation in rats 95,96. Moreover, diet supplementation with EPA and DHA increased brain DHA levels, attenuated proinflammatory cytokine expression and astrogliosis and restored spatial memory deficits and FOS expression upon memory test exposure in the hippocampus of aged mice 97. The anti-inflammatory effects of DHA could be due to a direct effect of DHA on invading macrophages or microglia. Indeed, in vitro and in vivo data have shown that DHA blocks macrophage- and microglia-induced activation of NF κb in the CNS of rodents with neuroinflammation 98,99. Moreover, DHA promotes the switching of microglia to an anti-inflammatory M2 phenotype that shows increased phagocytosis of amyloid-β isoform 42 (Aβ42) in vitro 100. In vivo, low dietary consumption of n-3 PUFAs leads to microglia activation and the production of pro-inflammatory cytokines in the hippocampus of mice at weaning 101. Many elegant reviews have discussed how ARA metabolites contribute to the early pro-inflammatory response 43,44,102 ; so, here, we only briefly discuss the emerging area of research on the role of ARA in the resolution of brain inflammation. Similar to their effects in peripheral tissue 42, COX2 and its products may resolve inflammation in the brain. The importance of COX2 products in triggering inflammation resolution circuits may explain why the neuroinflammatory response to intracerebroventricular LPS administration is increased NATURE REVIEWS NEUROSCIENCE ADVANCE ONLINE PUBLICATION 7

8 a Microglia b Neuron c Non-amyloidogenic Amyloidogenic sappα sappβ COX ARA Prostaglandins PLA2 LOX + DHA Pro-resolving mediators RAF P RAF AKT Activation and translocation P AKT DHA NPD1 DHA NPD1 + ADAM10 β-secretase1 Aβ γ-secretase Pro-inflammatory factors Neuronal survival and differentiation C83 APP APP C99 AICD Figure 5 Roles of PUFAs in the brain. Docosahexaenoic acid (DHA) and arachidonic acid (ARA) can be consumed through the diet or synthesized from their dietary fatty acid precursors in the liver. Fatty acids are then transported to the brain, where they are incorporated into cell membranes, including those of neurons and glial cells. a In microglia, upon its release from the membrane by phospholipase A2 (PLA2), ARA can be converted by cyclooxygenases (COX) to prostaglandins, which promote pro-inflammatory signalling. Prostaglandins may also trigger the production of lipoxygenase (LOX) by other cells, which produces specialized pro-resolving mediators from DHA. b In neurons, DHA is present in high levels in phosphatidylserine, and here it promotes the translocation and subsequent activation of AKT and RAF1, which have a role in neurogenesis. c In neurons, DHA and/or its mediator neuroprotectin D1 (NPD1) shift the processing of amyloid precursor protein-β (APPβ) from the amyloidogenic to the non-amyloidogenic pathway by downregulating β-secretase 1 (BACE1) and activating the α secretase ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10) and soluble APPα (sappα). Aβ, amyloid-β; AICD, amyloid precursor protein intracellular domain. in COX2 knockout mice and after pharmacological inhibition of COX2 (REFS ). DHA mediators are also crucial components of inflammation resolution, and DHA downregulates the expression of several enzymes of the ARA cascade (including COX2) in the brain, both under basal conditions and in response to neuroinflammation 85. It is, therefore, perhaps not surprising that increasing DHA levels attenuates neuroinflammation, although the precise role of DHA in this process needs further investigation 85. DHA and/or its mediators also have many targets in anti-neuroinflammatory and/or pro-resolving signalling pathways (FIG. 5). However, it is difficult to untangle the effects of DHA-containing phospholipids and the effects of unesterified DHA in vivo through studies using dietary DHA manipulation. Importantly, many studies investigating the biological effects of ARA or DHA in the brain have not considered the potential of these PUFAs to be converted into their mediators. For example, it has long been recognized that ARA mediators (many of which have been detected in the brain) are responsible for many of the actions that were initially ascribed to ARA 106,107. Furthermore, because DHA has been shown to decrease infarct volume, it is difficult to separate the neuroprotective effects of DHA from the anti-inflammatory effects in animal models of disease 85. Cell culture studies show that acute administration of DHA or its mediators attenuates markers of neuroinflammation 85,108 and pro-inflammatory signalling. In addition, in vivo attenuation of neuroinflammation correlates with unesterified, but not phospholipid, DHA levels 45, suggesting that unesterified DHA may be the biologically active pool of DHA. Furthermore, unesterified DHA infused into the ventricle is protective in an ischaemia reperfusion model of stroke 47 and in a mouse model of LPS-induced neuroinflammation 45, and both DHA and its mediator NPD1 downregulate pro-inflammatory cytokine signalling and decrease the activation of microglia 109. Collectively, these studies suggest that unesterified DHA and/or its mediators are responsible for at least some of the anti-inflammatory effects that have been attributed to DHA. The emerging role PUFAs in the regulation of brain glucose uptake. DHA may also have a role in the regulation of brain glucose uptake. In rodents, low brain levels of DHA are associated with decreased cytochrome oxidase activity and decreased endothelial glucose transporter 1 (GLUT1)-mediated glucose uptake 110. Moreover, DHA supplementation can rescue decreased GLUT1 levels induced by low DHA and increase GLUT1 density in rat brain endothelial cells 111. These findings suggest that DHA may have a direct effect on brain glucose uptake. The effect of diet and disease on brain PUFA composition. Low brain levels of DHA and ARA can be due to a lack of supply in the diet or due to increased catabolism 52. Current technologies cannot measure the effects of dietary DHA manipulation on brain DHA levels in humans, and so assumptions about the role of dietary DHA and brain DHA levels come from post-mortem studies and animal models. Numerous animal studies have reported that brain DHA levels can be increased by 8 ADVANCE ONLINE PUBLICATION

9 chronic consumption of n-3 PUFAs. However, with the exception of developmental studies, most of these studies compared the effects of dietary n-3 PUFA supplementation to the effects of diets containing very low or no n-3 PUFAs. Thus, one should be aware that any group differences in these studies could be driven by the effects of increased DHA levels in the experimental group and/or by the effects of low brain DHA levels in the reference group. In general, brain DHA or ARA levels are reduced by about 30% within 3 4 months of dietary n-3 PUFA 83,112 or n-6 PUFA deprivation 113, respectively, upon weaning. In rats, deprivation of n-3 PUFA decreases the cortical levels of ipla2 (REF. 114) and increases the half-life of DHA, but not ARA 112, presumably as a mechanism to conserve brain DHA levels 115. As brain phospholipid DHA levels begin to drop, DHA is replaced with esterified DPA n 6 (REF. 116). Because DPA n-6 levels are inversely related to DHA levels in the brain, it is important to consider DPA n-6 as a confounding factor in studies that have examined outcomes of manipulating brain DHA levels (for an excellent example of such a study, see REF. 117). Brain DHA levels have been reported to be decreased in some, but not all post-mortem studies of patients with Alzheimer s disease 118. One study reported that the unesterified pool of DHA was lower in the hippocampus of patients with mild Alzheimer s disease compared with controls 81, but not in the thalamus or occipital lobes. This indicates that it is important to distinguish between different pools of DHA within the brain. Furthermore, levels of LOX and NPD1 were also decreased, whereas levels of COX2 were increased in the hippocampus of patients with Alzheimer s disease 81. Thus, it has been hypothesized that a reduction in anti-inflammatory, neuroprotective DHA signalling may be an early feature of the development of Alzheimer s disease 39. Brain levels of DHA as well as other PUFAs were also decreased in some post-mortem studies in patients with major depression 119 or bipolar disorder 120. The fact that the decreases often apply to several PUFAs in addition to DHA suggests that increased oxidative stress 121 which degrades PUFAs into non-enzymatic oxidative products may contribute to the decreased levels of PUFAs in these patients. However, the mechanism underlying the lower PUFA levels is unclear, and the possible contribution of low dietary intake of PUFAs on brain PUFA levels in psychiatric disorders has not been extensively examined (see below). The role of PUFAs in mood. Findings from clinical and observational studies suggest that PUFAs have a role in the regulation of mood. For example, subjects with depressive symptoms or social anxiety disorders have lower levels of the n-3 PUFAs EPA and DHA and/or higher levels of the n-6 PUFA ARA in the blood compared with control subjects 122. Lower DHA levels have also been reported in the post-mortem orbitofrontal cortex (OFC) of patients with major depression 119 or bipolar disorder 123. Of note, no differences in DHA or ARA levels were found in the frontal cortex of patients with bipolar disorders 124. Furthermore, drug-free patients with bipolar disorder showed higher decreases in the levels of DHA and ARA than those treated with lithium 125, suggesting that lithium may attenuate changes in brain fatty acid metabolism. It remains to be determined whether decreased levels of DHA in the blood or brain of patients with major depression or bipolar disorder are due to altered dietary habits or altered fatty acid metabolism. Recent studies in patients with hepatitis C treated with interferon (IFN) showed that low blood DHA levels were associated with an increased vulnerability to developing major depressive disorder in response to IFN treatment 126. Interestingly, patients with polymorphisms in the genes encoding cpla2 or COX2 were more likely to develop IFN-induced somatic symptoms of depression and had lower plasma EPA and DHA levels before the start of IFN treatment 126, suggesting a role for PUFA metabolism in the risk of depression. In support of these clinical observations, several epidemiological studies have shown that a low dietary intake of n-3 PUFAs is linked to increased risk or prevalence of mood disorders 127. Some therapeutic benefits of fish oil supplementation have been reported in depression, although not in all studies 128. The discrepancy seems to be partly due to the inclusion criteria used and partly due to the PUFA composition of the fish oil. Indeed, a meta-analysis of randomized controlled trials carried out on patients with rigorously diagnosed major depression revealed that n-3 PUFA supplementation reduces symptoms in patients with severe depression, but not in those with less-severe symptoms 122,127. Several meta-analyses using the same criteria revealed that the therapeutic effect depends on the EPA content of the supplementation; that is, EPA+DHA supplements with a higher than 60% EPA content are more likely to be effective 128. However, publication bias could account for the reported beneficial n-3 PUFA effect on major depressive disorder in meta-analyses The involvement of brain DHA levels in the development of depression has also been assessed in animal studies. Single- or multi-generation exposure to dietary n-3 PUFA deprivation induces depressive and anxietylike behaviours in rats, mice and monkeys 65,83,132, and these behaviours are associated with decreased brain DHA levels, including in the prefrontal cortex and the hippocampus 65. Long-term dietary n-3 PUFA deprivation also impairs brain monoamine systems in rats 133 and piglets 134. For example, n-3 PUFA deprivation in rats increased basal synaptic 5 hydroxytryptamine (5 HT) levels, but decreased stimulated 5 HT release, and this effect could be reversed by early supplementation with n-3 PUFAs 135. In addition, tyrosine hydroxylase expression was increased in the brains of adolescent rats that were chronically subjected to a low n-3 PUFA diet, whereas it was decreased and vesicular monoamine transporter 2 expression was increased in the brains of adults rodents receiving dietary n-3 PUFA supplementation 136. Furthermore, dietary supplementation with n-3 PUFAs to increase brain DHA levels in rats also increased brain 5 HT and dopamine levels 133. Interestingly, blood levels of ARA and DHA are inversely related to cortisol levels, and lower ARA and DHA levels have been reported in patients with NATURE REVIEWS NEUROSCIENCE ADVANCE ONLINE PUBLICATION 9