Polyunsaturated fatty acids and their metabolites in brain function and disease.

Nature Reviews Neuroscience | AOP, published online 12 November 2014; doi:10.1038/nrn3820

REVIEWS

Polyunsaturated fatty acids and their metabolites in brain function and disease Richard P. Bazinet1 and Sophie Layé2,3

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.

Department of Nutritional Sciences, University of Toronto, Toronto, Ontario M5S 3E2, Canada. 2 INRA, Nutrition et Neurobiologie Intégrée, UMR 1286, 33076 Bordeaux, France. 3 University of Bordeaux, Nutrition et Neurobiologie Intégrée, UMR 1286, 33076 Bordeaux, France. Correspondence to R.P.B. e-mail: richard.bazinet@ utoronto.ca doi:10.1038/nrn3820 Published online 12 November 2014 1

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 static1 (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 brain2. 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 brain3. 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

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REVIEWS 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 phosphatidylinositol52. 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

Symbol

Palmitic acid

Saturated

16

0

16:0

Stearic acid

Saturated

18

0

18:0

Oleic acid

Monounsaturated

18

1

18:1n‑9

α-linolenic acid (ALA)

ω‑3 polyunsaturated

18

3

18:3n‑3

Eicosapentaenoic acid (EPA)


20

5

20:5n‑3

Docosapentaenoic acid (DPA) n-3


22

5

22:5n‑3

Docosahexaenoic acid (DHA)


22

6

22:6n‑3

Linoleic acid (LNA)


18

2

18:2n‑6

DPA n-6


22

5

22:5n‑6

Arachidonic acid (ARA)


20

4

20:4n‑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 brain5,6. 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 brain7. 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 pools9. 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 200–500-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 phospholipids4; 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 metabolism10. In fact, CD36 probably transports cholesteryl esters rather than fatty acids11. 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

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REVIEWS 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.

Circulation Peroxisome 22:6n-3

22:5n-6 β-oxidation Hepatocyte

18:3n-3 (ALA)

24:6n-3

24:5n-6

24:5n-3

24:4n-6

∆6 desaturase 18:4n-3

∆6 desaturase 18:3n-6 Elongase

Elongase 22:5n-3

20:4n-3

18:2n-6 (LNA)

∆5 desaturase

22:4n-6 Elongase

20:5n-3

20:3n-6 ∆5 desaturase 20:4n-6

Figure 1 | Synthesis of PUFAs in the liver. In the liver, the n-3 polyunsaturated fatty Nature Reviews | Neuroscience 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.

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.

Accretion Gradual accumulation.

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 DHA12. 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 brain13. 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 elsewhere14–16. In the adult brain, ARA and DHA are no longer accreted17, and plasma ARA and DHA only need to replace brain consumption. Current estimates of ARA and DHA

Fatty acid metabolism in the brain. Upon entry into the brain, most PUFAs — especially DHA and ARA — are activated by an ACSL19 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-oxidation20. 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 metabolism22 (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 enzymes23. 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 areas24). 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 process25,26. Consistent with this theory, pharmacological inhibition of β-oxidation in the brain decreases the levels of auto-oxidative PUFA metabolites in the brain27. 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 ARA29, 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 cells30, further supporting the notion of selective release of fatty acids from the membrane (for a review, see REF. 31).



REVIEWS 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 tissue12. Adipose DHA is thought to supply the brain with a reserve of DHA193. 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 normal194. 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 brain195. 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 rates196. Furthermore, in rodents and humans, following dietary intake of ALA, a significant proportion is directed to the adipose tissue197, 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 DHA198. It is important to note that dietary DHA targets the brain more effectively than ALA converted to DHA199, 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 ATP32. 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 dopaminergic33, cholinergic34, serotonergic35 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 membrane37 (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 techniques41; rapid in vivo fixation techniques, including high-energy, head-focused microwave irradiation, can be used to avoid such postmortem release of fatty acids41. 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 tissue43,44. 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 tissue42,45. 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 fold47. 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‑38393 (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 raclopride40. Recently, increases in ARA-mediated signalling were observed in humans upon acute apomorphine administration with the use of 11C-labelled ARA and PET48. 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 details40.



REVIEWS Plasma

Endothelial cell

Brain

LDLR

Lands cycle

Vesicle 1 8 LDL particle

Hydrolysis

ACSL

Lipoprotein lipase

CoA

11

2

Unesterified fatty acid

Peroxisome 3

4 LPL

MFSD2A

CD36

6 Albumin

FATP

FABP

5

• Mediator synthesis • Elongation/desaturation

12

9 7

Passive diffusion

Mitochondrion

10

Passive diffusion

Nature | Neuroscience Figure 2 | Fatty acid entry from the plasma into the brain. Fatty acids that enter the brain canReviews 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, http://dx.doi.org/10.1016/j.plefa.2014.05.007, 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 production49 and food intake50 and in analgesia51. 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 reviews52–54. 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 receptors57. 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 CB1s58.Retrograde endocannabinoid signalling mediates short-term forms of synaptic plasticity as well as presynaptic forms of longterm depression (LTD) at both excitatory and inhibitory synapses58. 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 astrocytes59. 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 DHA60. Being kept on a highfat diet increases endocannabinoid levels in the mouse



REVIEWS Neurotransmitter Neurotransmitter receptor ARA cPLA2

CoA AGPAT

Phospholipid DHA CoA

iPLA2

AGPAT

ACSL ARA

DHA

CoA

CoA

LOX

Cyt P450

LOX

LOX

5-HETE 12-HETE 15-HETE 8-HETE

HETEs ETEs

RvD2 NPD1

MaR1

COX

PGD2 PGE2 PGF2α TXA2 TXB2 PGF1α

ACSL

LTB4

LXA4 LXB4

Figure 3 | Fatty acid release and conversion to mediators upon receptor-mediated Nature Reviews | Neuroscience 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.

brain, whereas endocannabinoid levels are reduced upon DHA consumption61. 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 levels62 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 synthesis63. 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 accumbens64,65. 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 disease66,67. DHA also regulates the SNARE fusion machinery involved in presynaptic exocytosis and endocytosis68. 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 hippocampus67,69. In addition, S‑nitrosylation of syntaxin is decreased69 in animals with low brain DHA levels. Furthermore, syntaxin 3 is activated by both ARA and DHA in vitro70, and DHA promotes syntaxin 3 incorporation into SNARE complexes and, thereby, regulates rod photoreceptor biogenesis in the retina71. DHA also impairs syntaxin binding to the SNARE complex in PC12 cells70, but has no effect on synaptobrevin mRNA and protein expression in a SH‑SY5Y neuroblastoma cell line72. 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 understood73. One of the first described protective effects of DHA in the brain was its promotion of neuronal survival74 and neurogenesis75 (FIG. 5). DHA, the main PUFA in phosphatidylserine, enhances phosphatidylserine synthesis in vitro76, 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 synaptamide77 is sufficient to promote neuronal differentiation and is a much more potent promoter of neurite growth, synaptogenesis and synaptic function than DHA itself 77–79. The brains of mice that are fed high levels of DHA have increased synaptamide levels and show increased neuronal differentiation of neural stem cells79. 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 vivo80,81. 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 brain83.



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and improves functional outcomes after experimental stroke in rats86. 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.

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Figure 4 | Dietary PUFAs influence endocannabinoidmediated synaptic plasticity. Endocannabinoids (eCBs) Nature Reviews | Neuroscience 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 brains39,85. Increasing DHA supply to the brain also decreases infarct size in ischaemia–reperfusion models39. 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 outcomes39. 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

The role of PUFAs in inflammation in the brain. DHA and its mediators have potent anti-inflammatory and pro-resolving properties in peripheral tissues42. 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 depression87. This has led to the hypothesis that DHA may have anti-inflammatory effects in the brain as well88. Indeed, the expression of pro-inflammatory cytokines in the brain following systemic lipopolysaccharide (LPS) administration89,90, brain ischaemia–reperfusion91, spinal cord injury 92 and ageing 93 is reduced in rodents with high levels of brain DHA85. 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 rats93. 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 rats95,96. 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 mice97. 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 neuroinflammation98,99. Moreover, DHA promotes the switching of microglia to an anti-inflammatory M2 phenotype that shows increased phagocytosis of amyloid-β isoform 42 (Aβ42) in vitro100. 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 response43,44,102; 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 tissue42, 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



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Reviews | Neuroscience Figure 5 | Roles of PUFAs in the brain. Docosahexaenoic acid (DHA) and arachidonicNature 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 103–105). 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 neuroinflammation85. It is, therefore, perhaps not surprising that increasing DHA levels attenuates neuroinflammation, although the precise role of DHA in this process needs further investigation85. 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 ARA106,107. 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 disease85. Cell culture studies show that acute administration of DHA or its mediators attenuates markers of neuroinflammation85,108 and pro-inflammatory signalling. In addition, in vivo attenuation of neuroinflammation correlates with unesterified, but not phospholipid, DHA levels45, 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 stroke47 and in a mouse model of LPS-induced neuroinflammation45, and both DHA and its mediator NPD1 downregulate pro-inflammatory cytokine signalling and decrease the activation of microglia109. 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 uptake110. Moreover, DHA supplementation can rescue decreased GLUT1 levels induced by low DHA and increase GLUT1 density in rat brain endothelial cells111. 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 catabolism52. 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



REVIEWS 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 PUFA83,112 or n-6 PUFA deprivation113, 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 ARA112, presumably as a mechanism to conserve brain DHA levels115. 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 disease118. One study reported that the unesterified pool of DHA was lower in the hippocampus of patients with mild Alzheimer’s disease compared with controls81, 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 disease81. 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 disease39. Brain levels of DHA as well as other PUFAs were also decreased in some post-mortem studies in patients with major depression119 or bipolar disorder 120. The fact that the decreases often apply to several PUFAs in addition to DHA suggests that increased oxidative stress121 — 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 subjects122. Lower DHA levels have also been reported in the post-mortem orbitofrontal cortex (OFC) of patients with major depression119 or bipolar disorder 123. Of note, no differences in DHA or ARA levels were found in the frontal cortex of patients with bipolar disorders124. Furthermore, drug-free patients with bipolar disorder showed higher decreases in the levels

of DHA and ARA than those treated with lithium125, 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 disorders127. Some therapeutic benefits of fish oil supplementation have been reported in depression, although not in all studies128. 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 symptoms122,127. 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 effective128. However, publication bias could account for the reported beneficial n-3 PUFA effect on major depressive disorder in meta-analyses129–131. 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 monkeys65,83,132, and these behaviours are associated with decreased brain DHA levels, including in the prefrontal cortex and the hippocampus65. Long-term dietary n-3 PUFA deprivation also impairs brain monoamine systems in rats133 and piglets134. 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 PUFAs135. 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 supplementation136. Furthermore, dietary supplementation with n-3 PUFAs to increase brain DHA levels in rats also increased brain 5‑HT and dopamine levels133. 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



REVIEWS major depression 137. Healthy individuals receiving n-3 PUFA supplementation display lower anxiety symptoms 138 and a blunted cortisol response to a psychological stressor 139 or LPS140. The link between dysregulation of the hypothalamus–pituitary–adrenal axis, mood disorders and n-3 PUFA levels has yet to be elucidated. Nevertheless, rats subjected to chronic low dietary n-3 PUFA have an exaggerated hypothalamus–pituitary–adrenal axis response to acute stress141, whereas n-3 PUFA supplementation prevents chronic stress-induced increases in plasma corticosterone levels142,143. In addition, in the olfactory bulbectomy rat model of depression, exposure to a diet enriched with EPA increased performance in emotional and cognitive tests, reduced plasma corticotropin-releasing factor and corticosterone levels and increased nerve growth factor expression in the hippocampus144. Thus, it is possible that n-3 PUFAs exert an antidepressant effect via multiple mechanisms, including the modulation the hypothalamus–pituitary–adrenal axis, neuro inflammation and endocannabinoid metabolism, all of which are thought to be involved in depression. However, the use of n-3 PUFAs to treat mood disorders requires a better understanding of their exact mechanisms of action and pharmacokinetics in mood disorders. The role of PUFAs in cognition. The effect of DHA on neurodevelopment has been extensively studied in infants145, but little is known about its effect in early childhood and adolescence. Post-mortem brain studies revealed that brain DHA levels increase during childhood and adolescence and reach a plateau by 20 years of age145. Animal studies using diets that are lacking or are enriched in n-3 PUFAs have indicated that there is a critical period for DHA accretion in the brain, normal brain development and cognition. In children, low blood n-3 PUFA levels have been associated with an increased risk of developing cognitive deficits, attention deficit hyperactivity disorder (ADHD) and autism146. Children with ADHD have lower blood levels of n-3 PUFAs than healthy controls, but whether these differences are due to poor nutritional intake or to altered PUFA metabolism is not clear 147,148. In a large sample of healthy children aged 7 to 9 with reading difficulties, low blood EPA and DHA levels were associated with low reading scores, low working memory performance and ADHD-like symptoms149. Higher blood levels of DHA and EPA may be due to fish consumption but could also be due to other nutrients found in fish or to confounding factors associated with a diet rich in fish. The possible role of n-3 PUFAs in improving cognition in children and adolescents has been reviewed elsewhere145,150. Of relevance to memory, rodents with lower brain DHA levels have decreased performance in learning tasks151,152. Dietary supplementation of n-3 PUFAs in rodents facilitated LTP in the hippocampus in some studies153,154. However, most studies do not report a benefit of EPA and/or DHA supplementation on memory, cognition or mood in healthy humans145. Chronic

n-3 PUFA supplementation increased synaptic plasticity in the hippocampus and improved long-term memory in rodents under stress and in ageing 145, but it may show little effect under normal conditions. A potential role for n-3 PUFAs in cognitive deficits — which, in animals, are associated with structural and functional alterations of synaptic connections, including abnormal density and morphology of dendritic spines, synapse loss and aberrant signalling and plasticity of neuronal networks — in the elderly and in patients with Alzheimer’s disease has also been explored. As previously mentioned, some studies have reported decreased DHA levels in plasma and post-mortem brains from patients with Alzheimer’s disease145, whereas others have found that DHA levels remain unchanged155. It is possible that the changes may be due to membrane remodelling (which may result in the redistribution of DHA in phospholipids) rather than a decrease in n-3 PUFA levels156. Similarly, some studies have found that elderly subjects with low levels of circulating EPA157 or low levels of DHA in red blood cells158 are more likely to develop dementia, amygdala atrophy 159 and a smaller brain volume158. However, this did not apply in a study of aged Swedish subjects160, and an 18‑month-long randomized controlled trial of DHA supplementation in patients with Alzheimer’s disease reported no effect on brain volume161. Observational studies suggest that diets rich in n-3 PUFAs offer decreased risk of depression and dementia in elderly individuals162. However, studies involving the administration of dietary supplements, such as fish oils or relatively pure DHA supplements, to patients with Alzheimer’s disease have not yielded positive results161,163, especially in terms of the studies’ pre-registered primary endpoints. Secondary and subgroup analyses suggest some benefit of n-3 PUFA supplementation in individuals with mild cognitive dysfunction164. Although a positive response of DHA supplementation on the rate of cognitive decline in patients with Alzheimer’s disease who do not carry the APOE4 allele is often cited, this finding was not statistically significant upon correction for multiple comparisons161. Nevertheless, several epidemiological studies suggest a protective effect of fish consumption on the risk of dementia in non‑APOE4 carriers165. In addition, the half-life of plasma DHA seems to be increased in subjects with the APOE4 allele166, and mice expressing APOE4 have a decreased uptake of unesterified DHA into the brain167. Thus, the APOE status of patients should be considered in future research assessing the effect of DHA supplementation in patients with Alzheimer’s disease. DHA has also been implicated in the formation of amyloid plaques, a key characteristic of Alzheimer’s disease. In mouse models of Alzheimer’s disease, increased brain DHA levels are associated with a reduction in the formation of amyloid plaques66,168,169, a protective effect that could be due to the anti-amyloidogenic activity of DHA or of its mediator NPD1 (REFS 67,170). Low brain DHA levels impaired behaviour and augmented pathology in a mouse model of Alzheimer’s disease by



REVIEWS increasing oxidative stress, decreasing the levels of the p85α subunit of phosphatidylinositol 3‑kinase (PI3K) and reducing phosphorylation of the pro-apoptotic protein BAD66,171. NPD1 has been shown to alter the processing of amyloid precursor protein-β (APPβ), shifting it 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). This is another potential mechanism by which DHA could decrease amyloid-β levels in the brain170 (FIG. 5). There has been considerable discussion about the discrepancy between the lack of efficacy of DHA dietary supplements in patients with Alzheimer’s disease and the apparent beneficial effect in animal models of the disease172. It is important to consider that many of these animal studies compared animals receiving dietary DHA or n-3 PUFAs with animals exposed to an n-3 PUFAdeficient diet, which might not be a clinically relevant control group. However, because lipids cannot be silenced or knocked out (unlike genes), diet-based animal models may be the best way to study the role of lipids in health and disease. For a recent review on the role of DHA in cognition, see REF. 145. PUFAs in schizophrenia. Changes in brain PUFA levels have also been observed in patients with schizophrenia. Specifically, these patients, including both those treated with antipsychotic medication and antipsychotic-naive patients, have reduced levels of n-3 PUFAs in plasma and red blood cells173,174. Furthermore, DHA levels are 20% lower in post-mortem hippocampal samples of patients with schizophrenia than samples from controls175; however, this decrease is not seen in the prefrontal cortex 176. Of note, the levels of n-6 PUFAs (specifically, ARA and DPA n-6) have also been shown to be decreased in the brains of patients with schizophrenia. Because the reduction in brain PUFA levels is not limited to DHA, this suggests that oxidative stress or other aspects of degradative fatty acid metabolism may contribute to the low PUFA levels177. One study reported an increase in the mRNA levels of Δ6‑desaturase (the rate-limiting enzyme in the synthesis of DHA from ALA) in the frontal cortex of patients with schizophrenia178, which suggests that the brain may attempt to increase its synthesis of DHA to compensate for the low DHA levels. In humans, the gene encoding FABP7 (which is involved in the transport of PUFAs in neurons) has been identified as a susceptibility gene for schizophrenia, and Fabp7‑knockout mice have abnormal sensory gating response in pre-pulse inhibition, which is thought to model an aspect of schizophrenia179. The link between altered brain PUFA levels and schizophrenia is poorly understood, despite the relatively strong data indicating that n-3 PUFA supplementation in subjects with sub-threshold psychosis can decrease the rate of progression to first-episode psychotic disorder and reduce the severity of positive and negative symptoms180.

PUFAs as drug targets It is well recognized that lipids contribute to the pathogenesis of diseases and they are therefore actively investigated as drug targets181. Targeting PUFA metabolism within the brain with drugs, however, is a relatively new phenomenon. In 1996, studies by Rapoport and colleagues demonstrated that the mood stabilizer lithium selectively decreases the turnover of ARA, probably through downregulation of cPLA2 (for reviews, see REFS 38,55). This was accompanied by a decrease in the levels of COX2 and the ARA mediator PGE2. They subsequently showed that the mood stabilizers valproate and carbamazepine had the same effects. Recent studies examining the atypical antipsychotic olanzapine showed that it also decreases ARA turnover, COX2 expression and PGE2 levels; however, unlike mood stabilizers, olanzapine seems to decrease unesterified ARA availability to the brain. It is not clear whether any of these targets are integral for the therapeutic action of the drugs. One study showed that the COX2 inhibitor celecoxib has rapid-onset antidepressant effects in patients with bipolar disorder experiencing depressive or mixed episodes182, suggesting that COX2 or its products might be therapeutically relevant targets in this context. A post-mortem study demonstrated that levels of enzymes in the ARA cascade, including cPLA2 and COX2, were increased in the brains of patients with bipolar disorder 183. Interestingly, the drug topiramate (which was initially thought to be effective in bipolar disorder on the basis of open label trials but was later found to be not effective184) did not decrease ARA turnover, nor COX2 or PGE2 levels. This suggests that the brain ARA cascade may have predictive validity in screening drugs that are therapeutic in managing bipolar disorder. Future studies should assess whether targeting the brain ARA cascade is a sufficient or necessary component of mood stabilizers. Fish oils and supplements with n-3 PUFAs are also being used to target PUFAs to the brain in clinical trials. The brain uptake of ARA and DHA is 18 and 4 mg per day, respectively, in healthy subjects. Thus, if brain DHA content (about 5 grams per brain) is decreased by 30% — and assuming no changes in the brain uptake rate and no need to replace DHA that is consumed in the brain— it would take more than a year to restore brain DHA levels back to normal. Because the uptake of DHA from the diet is relatively slow, and because it is not clear whether supplementation increases the uptake rate, there is also active research into ways to increase the bioavailability of DHA to the brain. One strategy involved the injection — including intravenous administration — of unesterified DHA or lysophosphatidylcholine-containing DHA. In a rat model of seizure, infusions of DHA or NPD1 attenuated kindling progression and hippocampal hyperexcitability 185. In another rat model of seizure, it took 3 months of dietary fish oil intake to reduce seizure scores186, whereas subcutaneous administration of unesterified DHA was protective within 1 hour 187. In preclinical models of stroke, DHA-containing lyso-phosphatidylcholine has shown robust protective effects188, and low-dose intravenous infusion of unesterified DHA was protective in models of spinal cord injury 92,189 and



REVIEWS stroke39. Thus, the relatively slow uptake of DHA by the brain could explain some of the discrepancies between findings from basic and clinical studies; developing novel methods to target DHA to the brain is an active area of research190. It is not clear whether DHA or its biologically active mediators are responsible for the protective effects of DHA in preclinical models. Because the rate-limiting step for the synthesis of the mediators might be enzymatic, it is also unclear whether dietary DHA will sufficiently increase the level of mediators to be therapeutic, especially after acute administration. A recent study in humans who received a fish oil supplement did not detect circulating pro-resolving mediators in blood or urine191, and we are not aware of any animal studies demonstrating effects of dietary PUFA supplementation on the levels of specialized pro-resolving mediators in the brain (see Note added in proof). The development of drugs that target PUFAs and their mediators — as well as the development of stable analogues that enter the brain — could lead to novel therapeutics for brain diseases. However, a large clinical trial with the CB1 antagonist rimonabant was prematurely terminated in part due to an increase in neuropsychiatric events and suicides192. This highlights a potential challenge in

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targeting brain fatty acid metabolism — namely, that fatty acids regulate numerous processes and that changing fatty acid levels in the brain may therefore have adverse side-effects — and it stresses the importance of understanding brain PUFA metabolism in health and disease.

Conclusions and future directions Numerous molecular and cellular signalling pathways that are regulated by PUFAs have been identified. We are now beginning to understand how PUFAs are converted to a series of mediators that regulate many functions within the brain. However, major questions and areas of contention remain regarding how to target brain PUFA metabolism, including the delivery of PUFAs to the brain. Future studies aimed at identifying how PUFA signalling is altered in brain disorders and at developing methods to return PUFA metabolism to homeostasis may provide novel therapeutic approaches for diseases of the brain. Note added in proof A study reporting the detection of pro-resolving mediators in human plasma after short-term n-3 fatty acid supplementation was recently published200.

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Acknowledgements

The authors apologize to those whose valuable work was not cited owing to space limitations. R.P.B. acknowledges funding from the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada and holds a Canada Research Chair in Brain Lipid Metabolism. S.L. is supported by Institut National de la Recherche Agronomique (INRA), Bordeaux University, Région Aquitaine and Agence Nationale de la Recherche (ANR).

Competing interests statement

The authors declare competing interests: see Web version for details.


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