Chemistry and Physics of Lipids 153 (2008) 34–46

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Chemistry and Physics of Lipids

journa l homepage: www.e lsev ier .com/ locate /chemphys l ip

Biochemical and biological functions of docosahexaenoic acid in thenervous system: modulation by ethanol

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inadells. Td moare d

Hee-Yong Kim ∗

Laboratory of Molecular Signaling, NIAAA, NIH, 5625 Fishers Lane, Room 3N07, MSC941

a r t i c l e i n f o

Article history:Available online 2 March 2008

Keywords:Docosahexaenoic acidNeuronal cellsSurvivalDevelopmentEthanoln-3 Fatty acid deficiencyPhosphatidylserineAktHippocampal neurons

a b s t r a c t

Docosahexaenoic acid (DHis essential for proper neuin neuronal membranes,loss of DHA in neuronal cebiological, biochemical ansurvival and development

1. Introduction

Polyunsaturated fatty acids, especially arachidonic acid (20:4n-

6, AA) and docosahexaenoic acid (22:6n-3, DHA), are highlyconcentrated in neuronal membranes within the brain and retina.Numerous studies have indicated that DHA is essential for properneuronal and retinal functions (Salem et al., 2001). It has beenshown that inadequate supply of n-3 fatty acids during prenataland postnatal development decreases DHA in the brain and recipro-cally increases docosapentaenoic acid (DPAn6, 22:5n-6), resultingin a variety of cognitive and/or behavioral deficits in animal mod-els (Moriguchi et al., 2000). In contrast, dietary supplementationof DHA during infancy has been shown to improve mental devel-opment in humans (Willatts et al., 1998). In addition, it has beenindicated that low levels of DHA in the brain are associated withneuro-degenerative diseases, such as generalized peroxisomal dis-orders (Martinez, 1990) and Alzheimer’s disease (Soderberg etal., 1991). Furthermore, DHA supplementation has been shown toalleviate symptoms in some patients with peroxisomal disorders(Martinez et al., 2000), prevent dendritic pathology observed ina mouse model of Alzheimer’s disease (Calon et al., 2004), andreduce neuronal injury in experimental brain ischemia (Okada etal., 1996). Although all this evidence indicated a beneficial effect of

∗ Tel.: +1 301 402 8746; fax: +1 301 594 0035.E-mail address: hykim@mail.nih.gov.

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hesda, MD 20892-9410, USA

:6n-3), an n-3 fatty acid highly concentrated in the central nervous system,l and retinal function. While a high level of DHA is generally maintainedquate supply of n-3 fatty acid or ethanol exposure leads to a significanthe roles of DHA in neuronal signaling have been emerging. In this review,lecular mechanisms supporting the essential function of DHA in neuronalescribed in relation to n-3 fatty acid depleting conditions.

Published by Elsevier Ireland Ltd

DHA and suggested a requirement of DHA accretion in neural cells,underlying mechanisms have not been well understood.

The research aim of our laboratory has been to elucidate thebiochemical and biological mechanisms supporting the needfor this particular fatty acid in the central nervous system. Thepolyunsaturated fatty acids are mostly esterified to membrane

phospholipids and mobilized upon activation of phospholipases. Asthe hydrolysis of AA and the production of its oxygenated metabo-lite (eicosanoids) have been implicated in signal transduction,DHA or eicosanoid-like DHA products may also play an importantrole in neuronal signaling. However, we found that DHA is neithereasily released nor enzymatically oxygenated in neuronal cellsunder a normal condition, but tenaciously retained in membranephospholipids (Kim et al., 1991, 1996, 1999; Sawazaki et al., 1994;Garcia and Kim, 1997; Kim and Edsall, 1999). In contrast, astrogliacells, which support neurons by providing trophic factors, havebeen shown to release this fatty acid readily (Garcia and Kim,1997; Kim et al., 1999). These findings suggested that DHA may betrophic and enrichment of this fatty acid in neuronal membranesmay be an important aspect of neuronal development and survival.Long-term ethanol exposure has been shown to decrease theastroglial release of DHA (Garcia et al., 1997), indicating effects ofethanol on the neuronal supply of DHA.

DHA, delivered from the blood stream or biosynthesized inthe brain, is uniquely processed in neural cells and incorpo-rated into neuronal membranes (Kim, 2007). DHA accumulatesparticularly in phosphatidylserine (PS) primarily as 1-stearoyl-2-

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docosahexaenoyl-PS (18:0,22:6-PS) in the brain. This lipid profileappears to be rigorously maintained and may be important fornormal brain function in view of the fact that the lipid environ-ment can affect functions of membrane proteins (Niu et al., 2004).Generally, it is difficult to alter fatty acyl composition in the neu-ronal membranes of adult mammals. However, ethanol can lowerthe DHA fatty acyl content in brain as has been indicated by sev-eral reports (Alling et al., 1982; Harris et al., 1984; Pawlosky andSalem, 1995), particularly from PS. Considering the fact that PS is themajor negatively charged phospholipid class in many mammaliancell membranes and many of the signaling proteins such as proteinkinases are influenced by PS, the accumulation of DHA in PS of neu-ral membranes and its depletion by ethanol may have a significantmodulatory role in signaling pathways in the nervous system. Withthis background, following hypotheses have been developed.

2. Hypotheses/propositions

1. DHA is neurotrophic.2. The incorporation of DHA in membrane phospholipids, espe-

cially accumulation in PS is an important aspect of its biologicalfunction in the nervous system.

3. Ethanol exerts its effect, at least in part, through alteration of theDHA supply and phospholipid remodeling processes modulatedby DHA.

To test these hypotheses we asked questions concerning theeffect of DHA and ethanol on PS regulation, neuronal survival anddifferentiation as well as underlying biochemical and biologicalmechanisms. This review will summarize our findings for thosequestions, which furthered our understanding for the mechanis-tic role of DHA and ethanol in neuronal function and the signalingprocesses involved.

3. Findings

3.1. Does DHA have a specific biochemical function in PSbiosynthesis and accumulation?

It is well known that DHA is highly concentrated in aminophos-pholipids, PE and PS, in neuronal cells. Compositional studies haveindicated that DHA comprises 35–40% of total fatty acid in synap-tic membrane PS (Hitzemann, 1981), implying that probably more

than at least 60% of the PS molecular species contain DHA, basedon the fact that di-22:6-PS species is rather a minor species in neu-ronal membranes except the retina. Since PS biosynthesis in animalcells occurs exclusively through the serine base exchange reactionusing PC or PE as substrates, the high compositional profile of DHAin PS may be due to the specificity in PS biosynthesis and degrada-tion although the specificity in deacylation/reacylation reactionsmay also contribute. In such case, the PS content may in turn bemodulated according to the DHA status in substrate phospholipids.

The effect of the DHA compositional status on the synthesisof PS was investigated using two approaches: an n-3 fatty aciddeficiency animal model and a DHA-supplemented cell system(Garcia et al., 1998). Fatty acid composition of brain microsomesfrom the offspring of rats artificially reared on an n-3 deficientdiet showed a dramatic reduction of DHA (1.7 ± 0.1%) when com-pared to control animals (15.0 ± 0.2%). Instead, docosapentaenoicacid (DPAn-6, 22:5n-6) quantitatively replaced DHA. The molecularspecies analysis by HPLC/electrospray mass spectrometry revealedthat 18;0,22:6 was the major PS species which was replaced by18:0,22:5-PS by n-3 fatty acid deficiency. It was also revealed thatthe total polyunsaturated PS level decreased after depleting n-3

of Lipids 153 (2008) 34–46 35

fatty acids although the total amount of polyunsaturated phospho-lipids in brain microsomes remained unchanged. The decrease in PSwas mainly due to the incomplete replacement of 18:0:22:6 with18:0:22:5 species. This reduction of PS was also accompanied byan accumulation of 18:0:22:5-PC, suggesting a preferential PS syn-thesis from 18:0:22:6-PC in comparison to 18:0:22:5-PC. The serinebase exchange activity evaluated by the incorporation of [3H]serineinto the microsomal membranes was also decreased significantlyby n-3 fatty acid deficiency. These data suggested that the pres-ence of DHA phospholipid species in the membrane may promotethe serine base exchange reaction, either as a preferred substrateor as an enhancer of enzymatic activity. Similarly, Neuro 2A orC6 glioma cells cultured for 24–48 h in DHA-supplemented media(10–40 �M) significantly increased their PS content as well as thesynthesis of [3H]PS when compared with non-supplemented or AA-supplemented cells. The increase of total PS was primarily due toa dramatic increase in 18:0,22:6-PS in DHA supplemented cells.These data indicated that neuronal and glial PS synthesis was sen-sitive to the changes in DHA levels in phospholipids and suggestedthat DHA may be a positive modulator of PS synthesis.

3.2. How is PS accumulation regulated by DHA?

To elucidate the biochemical mechanisms underlying the con-centration of PS in neuronal membranes, where the DHA levelis high, we investigated substrate preference in PS biosynthesisand degradation in vitro. We also extended our investigation onthe effect of the DHA status on PS levels to various neuronal andnon-neuronal tissues and cell lines, to gain an insight on neu-ronal specificity of DHA function. In addition, we searched forother means to alter PS levels in biomembranes by manipulatingthe expression of PS synthetic enzymes. Our investigations pro-vided the following information; DHA increases the PS contentspecifically in neuronal cells, primarily due to the accumulationof 18:0,22:6-PS. The DHA-containing PS accumulation was notdue to a decrease in degradation, but due to the fact that PSsynthesis is most favored for DHA-containing substrates. Deple-tion of DHA by n-3 fatty acid deficiency has a profound effect onthe PS accumulation specifically in neuronal tissues where DHAis highly enriched, despite the compensatory replacement withDPAn6. Over-expressing or silencing genes responsible for PS syn-thesis did not alter PS accumulation by DHA. The modulation of thePS pool in neuronal membranes due to the DHA status appearedto be a unique mechanism allowing the disruption of membrane

homeostasis in living animal cells.

3.2.1. Selective decreases in neuronal accumulation ofphosphatidylserine by n-3 fatty acid deficiency

The data compiled over the course of our study indicated thatn-3 fatty acid deficiency decreases PS selectively from neuronalmembranes (Garcia et al., 1998; Hamilton et al., 2000; Murthy et al.,2002). We observed that rat brain cortex, hippocampus, retina andolfactory bulb, where DHA is highly concentrated, contain signifi-cantly higher levels of PS in comparison to liver and adrenal whereDHA is a rather minor component. Phospholipid molecular speciesanalysis revealed that in brain cortex, hippocampus and olfactorybulb, 18:0,22:6-PS was the most abundant PS species representing45–65% of the total PS. Dietary depletion of n-3 fatty acids dur-ing development decreased the brain DHA content by more than80% with a concomitant increase in DPAn6 in all tissues. Underthese conditions, an approximately 25–35% reduction in total phos-phatidylserine in all neuronal tissues was observed, while PS levelsin liver and adrenal were unchanged (Fig. 1). The observed reduc-tion of PS content in neuronal membranes was due to a dramaticreduction of 18:0,22:6-PS which was not fully compensated with

36 H.-Y. Kim / Chemistry and Physics of Lipids 153 (2008) 34–46

Fig. 1. Neuronal tissue-specific PS reduction by n-3 fatty acid deficienc

18:0,22:5n-6-PS. We also observed similar reduction of PS in thepineal gland (Zhang et al., 1998) and retina which are also of a neu-ronal origin. Collectively, it was concluded that DHA depletion ledto a selective decrease in PS accumulation in neuronal tissues wherethis fatty acid is highly concentrated.

3.2.2. Substrate specificity in phosphatidylserine synthesis anddegradation

As it became clear that PS content is particularly elevated in neu-ronal cells due to the specific concentration of 18:0,22:6-PS, thequestion concerning the biochemical mechanism underlying theobserved effect of DHA on PS accumulation naturally followed. Inanimal cells, PS is synthesized in mitochondria associated mem-

Fig. 2. In vitro assay for PS biosynthesis using HPLC/MS and lip

y (Garcia et al., 1998; Hamilton et al., 2000; Murthy et al., 2002).

branes (MAM) and microsomes from pre-existing PC or PE byPS synthase1 (PSS1) and PSS2, respectively, which can then beconverted to PE by the PS decarboxylation (PSD) enzyme in mito-chondria (Vance and Vance, 2004). Therefore, substrate preferencein PS synthesis or decarboxylation can contribute to the accumu-lation of 18:0,22:6-PS species in neuronal membranes. To this end,an in vitro method has been developed to directly determine thesubstrate preference in PS synthesis and PSD, utilizing liposomescontaining deuterium labeled phospholipid substrate species andmass spectrometry. The conversion of PC (PE) or PS substrates tothe corresponding PS or PE species was monitored for PSS and PSDactivity, respectively, by using reversed-phase liquid chromatogra-phy by electrospray ionization mass spectrometry (Fig. 2).

osomes containing labeled substrates (Kim et al., 2004).

H.-Y. Kim / Chemistry and Physics of Lipids 153 (2008) 34–46 37

y (a)

Fig. 3. PE formation as a function of PS substrate concentration (�M) b

Fig. 4. PS formation by brain cortex microsomes as a functio

With this approach we were able to establish a tis-sue specific substrate preference in PSD in liver (18:0,18:1≥ 18:0,22:6 > 18:0,20:4-PS) and brain cortex (18:0,22:6 > 18:0,18:1 > 18:0,20:4-PS) as shown in Fig. 3 (Kevala and Kim,2001). Similarly, PS synthesis by brain (18:0,22:6 > 18:0,22:5 > 18:0,18:1 ≥ 18:0,20:4-PS) (Fig. 4) and liver microsomes(18:0,22:6 > 18:0,20:4 ≥ 18:0,18:1 > 18:0,22:5-PS) also showedtissue-specific substrate preference (Kim et al., 2004). While bothbrain and liver microsomes favored 18:0,22:6-PC, the overall PSSactivity in brain (normalized to the protein content) was more thantwice that of liver. The most striking difference observed was PSsynthesis from 18:0,22:5-PC which was efficiently converted to thecorresponding PS species only by brain microsomes as long as ser-ine was present in the medium. In the absence of serine, however,PS synthesis by brain microsomes from 18:0,20:4-, 18:0,18:1- and18:0,22:5-PC was negligible, and only 18:0,22:6-PC was converted

Fig. 5. Effect of silencing pss1 and pss2 on PS accum

liver or (b) brain cortex mitochondrial protein (Kevala and Kim, 2001).

n of serine concentration and time (Kim et al., 2004).

to PS species. These data suggested that the preferred synthesis of18:0,22:6-PS, rather than unfavored degradation of this PS speciesby decarboxylation, was primarily responsible for the observedaccumulation of 18:0,22:6-PS in neuronal membranes. In order tomaintain a high level of PS content in neuronal tissues during n-3fatty acid deficiency, an increase of DPAn6 in neuronal tissues maybe warranted since phospholipid species containing DPAn6 is thenext best substrate for PS biosynthesis.

3.2.3. Regulation of phosphatidylserine accumulation in culturedcells

The PS level in Neuro-2A cells was also increased by supple-menting cells with DHA and DPAn6 while oleic acid (OA, 18:1n-9)did not have any effect. Consistent with the brain microsomalPS biosynthetic activity shown above, DHA was more effectivein accumulating PS in comparison to DPAn6. Non-neuronal cell

ulation in Neuro 2A cells (Akbar et al., 2005).

38 H.-Y. Kim / Chemistry and Physics of Lipids 153 (2008) 34–46

ation, 2000).

The effect of AA was not affected by the cyclooxygenase inhibitorindomethacin or the lipoxygenase inhibitor NDGA, indicating thatthe observed effect was likely due to free AA (Kim et al., 2001). Theprotection by DHA, as evidenced by the reduced caspase-3 activityand DNA fragmentation, became evident only after at least 24 h ofenrichment prior to serum starvation. During this period, DHA wasgradually incorporated into membrane phoispholipids, and after48 h over 90% of the exogenously added DHA was found in phospho-lipids. The enrichment of cells with AA was also able to reduce theDNA fragmentation; however, the protective effect was sensitive tothe duration of serum starvation. After a prolonged serum starva-tion period (more than 24 h), the protective effect of AA was lostand only DHA remained protective as shown for the caspase-3 acti-vation time course and DNA ladder formation in Fig. 6. The Neuro2A cells enriched with DHA for 48 h showed much less fragmentednuclei after 48 h of serum starvation as evidenced by Hoechst stain-ing (Fig. 7), consistently indicating the protective effect of DHA.These results established that DHA as a membrane component pre-vented apoptotic cell death induced by serum starvation.

Fig. 6. Effect of fatty acid enrichment on caspase-3 activation and DNA ladder formstarved. DNA ladder formation was tested after 48 h of serum starvation (Kim et al.

lines including CHO-K1, NIH-3T3, HEK-293 and HELA did notincrease total PS in response to DHA or DPAn6, suggesting thatthe phospholipid profile is differently regulated in neuronal cells(Guo et al., 2007). The molecular species analysis by electrosprayHPLC/MS indicated that DHA was incorporated into PS mainly as18:0,22:6 species also in non-neuronal cells. However, the increaseof 18:0,22:6-PS was compensated by the decrease of other speciessuch as 18:0, 18:1- or 18:1,18:1-PS similar to the product inhibitionof PS synthesis observed in CHO cells (Nishijima et al., 1986; Kuge etal., 1998). In neuronal cells, the product feed back was not as promi-nent, resulting in the increase of the total PS content after DHAsupplementation. Silencing pss1 and pss2 genes for the enzymeresponsible for PS biosynthesis affected neither the PSS enzymelevel nor DHA-induced PS accumulation in Neuro-2A cells, althoughmRNA levels were successfully manipulated (Fig. 5). Only the cellsenriched with DHA in a serine depleted condition showed inhib-ited PS accumulation in agreement with the serine requirement forthe mammalian PS synthesis using the serine-base exchange reac-tion. Similarly, over-expressing pss1 and pss2 genes cloned from

the liver (Stone and Vance, 1999; Sturbois-Balcerzak et al., 2001)in non-neuronal cells did not increase the PS accumulation (Guo etal., 2007). These data suggest that PS levels are tightly regulated inliving cells and these highly unsaturated very long chain fatty acidsare uniquely capable of expanding the PS pool specifically in neu-ronal cells. This observation is consistent with the fact that neuronaltissues which are abundant in DHA generally contain higher levelsof PS in comparison to non-neuronal tissues (Ansell and Spanner,1982; Rouser et al., 1969). It is also in agreement with the PS reduc-tion observed selectively in neuronal tissues with n-3 fatty aciddeficiency.

3.3. Does DHA support neuronal survival?

To test the hypothesis that DHA provided by astroglia is neu-rotrophic, the effect of DHA on apoptotic neuronal cell death causedby serum starvation was evaluated in comparison to other fattyacids using PC-12 or Neuro 2A cells (Kim et al., 2000). Direct expo-sure of these cells to DHA during the 5 h serum starvation perioddid not influence cell death while AA decreased the DNA fragmen-tation in a dose-dependent manner under the same conditions.

. Neuro-2A cells were enriched with fatty acids for 48 h, and subsequently serum

Fig. 7. Effect of fatty acid enrichment on DNA fragmentation detected by Hoechststaining (Kim et al., 2000).

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Fig. 8. Differential effect of DHA and DPAn6 on PS ac

3.4. Is PS responsible for the anti-apoptotic effect of DHA?

3.4.1. Involvement of PS in the anti-apoptotic effect of DHATo provide a mechanistic basis supporting the beneficial effects

of DHA, the involvement of PS in the anti-apoptotic effect of DHAwas first determined by evaluating the cell death in relation tothe PS content modulated by varying the enrichment period orby depleting the serine supply needed for the PS biosynthesis.The anti-apoptotic effect of DHA was potentiated as a function ofenrichment period. During enrichment, DHA incorporated into PSsteadily, resulting in a significant increase in the total PS content.Similar treatment with oleic acid (OA, 18:1n-9) neither altered PScontent nor resulted in a protective effect. Hindering PS accumu-

lation by enriching cells in a serine-free medium diminished theprotective effect of DHA concomitantly inhibiting PS accumulation,strongly suggesting that PS mediates the anti-apoptotic effect ofDHA (Kim et al., 2000).

3.4.2. Differential effect of DHA and DPAn6 on neuronal survivalTo gain an insight into the essential role of DHA and the adverse

effects of n-3 fatty acid deficiency, we also examined the differ-ential effect of DHA and DPAn6 on survival in vitro and extendedthe investigation to a primary cell culture model in conjunctionwith in vivo depletion of DHA. Neuro 2A cells contained DHAand DPAn6 as minor components, each less than 1% of the totalfatty acid. By enriching the cells with 25 �M DHA for 48 h, theDHA level was raised to 15–16%, the approximate level commonlyfound in neuronal membranes. Since DPAn6 replaces DHA in n-3fatty acid deficiency, the cells were supplemented with DHA andDPAn6 at various ratios to model the varying extent of n-3 fattyacid deficiency. DHA and DPAn6 differentially affected the PSaccumulation and neuronal survival under a serum starvationcondition (Fig. 8). The cells enriched with DHA or DPAn6 aloneshowed significantly higher levels of the total PS in comparison

Fig. 9. Effect of in vivo depletion of n-3 fatty acids on apoptotic cell death and the

of Lipids 153 (2008) 34–46 39

lation and caspase-3 activation (Akbar et al., 2005).

to non-enriched control cells. However, the level of PS attained byDPAn6 was less than 80% of that observed with DHA enrichment. Asthe proportion of DHA with respect to DPAn6 increased, a gradualenhancement in the total PS content was observed, in agreementwith our finding that DPAn6-phospholipids are less effective thanDHA species as substrates in PS biosynthesis (Kim et al., 2004).The protective effect against apoptotic cell death as evaluated bycaspase-3 activity gradually increased with an increasing propor-tion of DHA, indicating that the PS content and anti-apoptoticeffects were well correlated. The apoptotic cell death evaluatedby in situ DNA fragmentation showed consistent results. DHAenrichment significantly decreased the serum starvation-inducedTUNEL positive cells in comparison to non-enriched control. Cells

enriched with DPAn6 also contained a reduced number of TUNELpositive cells, however, to a lesser extent, again confirming thecorrelation between the PS content and an anti-apoptotic effect.When PS accumulation was inhibited by depleting serine from theenrichment media, the extent of protection as well as the differ-ential effect of these polyunsaturated fatty acids was diminished,indicating that PS accumulation is primarily responsible for theprotective effect of both DHA and DPAn6 (Akbar et al., 2005).

3.4.3. Neuronal cell death promoted by in vivo DHA depletionBased on the PS-dependent protective effect observed in trans-

formed cells, the implication of an in vivo reduction in PS, resultingfrom the depletion of DHA under n-3 fatty acid deficient condi-tions, in neuronal survival was examined using E-18 hippocampalprimary cultures (Fig. 9). When pregnant rats were fed with an n-3 fatty acid deficient diet from day 2 of pregnancy, a reciprocalreplacement of DHA by DPAn6 was observed in E-18 fetal hip-pocampi although the DHA accretion was low (3.3%) at this stage ofdevelopment. Consistently, the PS content in the fetal hippocampifrom the n-3 fatty acid deficient group was lower by 15% in compari-son to the n-3 fatty acid adequate group. The spontaneous apoptosis

fatty acid and PS profile in E18 hippocampal cultures (Akbar et al., 2005).

40 H.-Y. Kim / Chemistry and Physics of Lipids 153 (2008) 34–46

Akt tr

stimulation with IGF, Neuro 2A cells enriched with DHA and DPAn6showed considerably faster translocation in comparison to the non-supplemented control (Fig. 10). While translocation of GFP-AktPHto the plasma membrane reached a plateau at around 5.5 min incontrol cells, cells enriched with DHA or DPAn6 GFP-AktPH showedsuch translocation within 2.5 or 3.5 min, respectively. The fastertranslocation observed in DHA enriched cells was due to the PSconcentration, since the inhibition of PS accumulation by deplet-ing serine from the enrichment media prevented the facilitatedtranslocation observed in these cells. Therefore, the differentialability to raise the PS concentration by DPAn6 with respect toDHA is likely responsible for the slower translocation observedin DPAn6-enriched cells in comparison to DHA-enriched cells. Wealso found that PS is highly localized in the cytosolic face of theplasma membrane (Calderon and Kim, 2008), further supporting

Fig. 10. Effect of DHA enrichment on IGF-stimulated

under basal conditions was slightly but significantly higher in hip-pocampal primary culture from n-3 fatty acid deficient animals incomparison to the n-3 fatty acid adequate culture (32% vs. 25%).Under trophic factor removed conditions (−TF), a dramatic increasein cell death (42%) was observed in n-3 fatty acid deficient cultureswhile n-3 fatty acid adequate cultures did not show a significantincrease from the basal level. The increased susceptibility to celldeath in DHA-deficient neurons, where PS was reduced, is consis-tent with the PS-dependent protection by DHA against neuronalapoptosis observed in Neuro 2A cells. Similarly, it has been demon-strated that DHA supplementation prevented hippocampal celldeath induced by ischemia-reperfusion in an animal model (Caoet al., 2004). However, it still remains to be tested whether this invivo protection against hippocampal cell death by DHA is consis-tent with the PS-dependent protective effect of DHA on neuronalsurvival demonstrated in our study using cells in culture.

3.5. What are the signaling mechanisms responsible for theanti-apoptotic effect of DHA?

3.5.1. PI(3) kinase-dependent signaling mechanism for protectiveeffects of DHA: membrane translocation of Akt

In addition to the apoptotic cell death induced by serum starva-tion, neuronal apoptosis induced by staurosporine (ST) treatmentwas also inhibited by DHA in a PS-dependent manner (Akbarand Kim, 2002). These findings suggested a unique role of DHAin the membrane-related signaling events involved in cell sur-vival through modulating PS levels in neuronal membranes. Tounderstand the signaling mechanisms underlying the observed PS-dependent anti-apoptotic effect of DHA, effects of specific kinaseinhibitors were examined. The protective effect of DHA enrichmentwas abolished when the apoptosis was induced in the presenceof PI(3)K inhibitors such as wortmanin (WM) and LY-294002 (LY),indicating that PI(3)K pathway is required for the protection byDHA. Concurrently, phosphorylation of Akt at Thr-308 and Ser-473, the consequence of PI(3)K activation, was significantly reducedby serum starvation and enrichment of cells with DHA prior toserum starvation partially prevented the reduction of Akt phos-phorylation at Thr-308 and Ser-473. In vitro kinase assay usingimmuno-precipitated Akt consistently revealed the reduction ofAkt activity under serum starvation, which was partially rescued byDHA-enrichment. These data indicate that the anti-apoptotic effectof DHA was mediated at least partly via the PI(3)K/Akt pathway.

anslocation and phosphorylation (Akbar et al., 2005).

To determine the target of the PS- and PI(3)K/Akt pathway-dependent protection by DHA, we examined the translocation ofAkt, the prerequisite step for the phosphorylation and activationof Akt. Akt translocation, resulting from the interaction betweenthe PH domain of Akt and the plasma membrane PIP3 generatedby PI(3)K activation, was evaluated using the PH domain taggedwith the enhanced green fluorescence protein (GFP-AktPH). Upon

the involvement of PS in the modulation of the translocation of Aktto the plasma membrane.

The high resolution crystal structure has indicated that besidesthe binding pocket for PIP3, the PH domain of Akt contains basicresidues, Arg15, Lys20, Arg67 and Arg69, which form a flat surfacefor a possible interaction with acidic membrane phospholipids

Fig. 11. Interaction of Raf-1 with PS evaluated in vitro using biomolecular interactionanalysis (Kim et al., 2000).

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(Thomas et al., 2002). We observed that the mutation of anyone of these residues resulted in the abolition of or incompleteGFP-AktPH translocation in response to IGF at concentrations upto 1 �g/mL. DHA supplementation did not produce any significanteffect in these mutants, supporting the suggested interaction ofthese basic residues with acidic membrane components, in thiscase PS, and its importance for the observed acceleration of Akttranslocation by DHA.

In agreement with the observed faster translocation of GFP-AktPH, Akt phosphorylation, the consequence of Akt translocationfrom cytosol to plasma membrane, was considerably faster in DHAenriched cells in comparison to non-enriched control cells. Thefaster translocation and phosphorylation of Akt observed in DHAenriched cells was not due to the DHA effect on the signaling eventswhich are upstream of Akt translocation, as in vitro PI(3)K activityas well as endogenous PIP3 formation monitored by metabolic 32Plabeling followed virtually the same time course for both controland DHA-enriched cells. In summary, we found that membranemodification by the DHA status affects Akt signaling, impactingneuronal survival. DHA promoted neuronal survival by facilitat-ing Akt translocation/activation through its capacity to increase

PS in neuronal membranes. DPAn6 was not as effective as DHA inaccumulating PS and translocating Akt, and thus, less effective inpreventing apoptosis. Consistently, in vivo reduction of DHA by n-3fatty acid deficiency increased neuronal susceptibility to apoptoticcell death tested in culture.

3.5.2. Raf-1 kinase dependent mechanism for DHA effectsIn addition to PI(3)K/Akt, Raf-1 kinase has been suggested to

be involved in cell survival (Neshat et al., 2000). It has beenshown that membrane translocation is required for the activationof Raf-1 (Stokoe et al., 1994) and the regulatory domain interact-ing with PS plays an important role in this process (Improta-Brearset al., 1999). We found that Raf-1 membrane translocation inresponse to BDNF was significantly enhanced by DHA enrichmentin Neuro 2A cells (Kim et al., 2000). Both anti-apoptotic effect ofDHA and Raf-1 translocation was sensitive to the DHA-induced PSaccumulation. Moreover, in vitro biomolecular interaction analysisby surface plasmon resonance indicated that the Raf-1 interac-tion with PS/PE/PC liposomes was PS-dependent as shown inFig. 11. These data suggested that Raf-1 activation facilitated bythe increased PS after DHA enrichment may also contribute to

Fig. 12. Effect of ethanol on total PS and PS molecular species

of Lipids 153 (2008) 34–46 41

the down-regulation of caspase-3 and the prevention of neuronalapoptosis. As the Raf-1 activation has been implicated in cell sur-vival, proliferation, differentiation and migration depending onthe type of cells (Galabova-Kovacs et al., 2006), modulation ofRaf-1 activation by DHA may affect neuronal cells with similarimplications.

3.6. Does ethanol exposure in vivo affect DHA-dependent PSaccumulation and neuronal survival?

Long-term ethanol exposure has been shown to interferewith PS accumulation induced by DHA in cultured cells (Kimand Hamilton, 2000), and this finding has been extended to ananimal model (Wen and Kim, 2004). It has been suggested thathippocampus-related cognitive processes are especially sensitiveto ethanol (Matthews and Morrow, 2000; Silvers et al., 2003).To provide an insight for the biochemical basis supporting thehippocampus-related functional deficits associated with pre-natal ethanol exposure, we investigated the effects of chronicethanol exposure on the phospholipid profile in developing rathippocampi. Using reversed phase HPLC-electrospray ionization

mass spectrometry (ESI-MS), we found that ethanol exposureduring the prenatal and developmental period lowered the totalPS level by 15–20% at postnatal day 0 (P0) and 21 (P21), primarilydue to the reduction of 18:0,22:6-PS species (Fig. 12).

An in vitro bioassay employing reversed phase HPLC-ESI-MS andbrain microsomes incubated with deuterium labeled exogenoussubstrates revealed that 18:0,22:6 species is the best substrate forbrain microsomal PS synthesis from both PC and PE, most likely rep-resenting PSS1 and PSS2 activities. The PS biosynthetic activity inthe brain, especially for 18:0,22:6-PS production from both PC andPE, was significantly hampered by maternal exposure to ethanolas shown in Fig. 13 (Wen and Kim, 2007). Under our experimentalconditions, the expression of PSS1 and PSS2 was not reduced bythe ethanol exposure based on the mRNA level. Similarly, the PSS1enzyme level did not change after ethanol exposure although PSS2could not be probed since the proper antibody is not currently avail-able. Degradation of PS by mitochondrial PS decarboxylation wasnot enhanced but also inhibited. These data indicated that atten-uated PS biosynthetic activity was largely responsible for the PSreduction observed in developing rat brains after maternal expo-sure to ethanol.

in rat hippocampus at P0 and P21 (Wen and Kim, 2004).

42 H.-Y. Kim / Chemistry and Physics of Lipids 153 (2008) 34–46

Fig. 13. Effect of ethanol on brain microsomal PS biosynt

Based on our findings that ethanol inhibited the accumulationof PS, especially DHA-containing PS species, and DHA preventedneuronal apoptosis through promoting PS accumulation, we exam-ined the effect of ethanol on DHA-dependent neuronal survivalin Neuro 2A cells. Also, the effect of in vivo exposure to ethanolon the hippocampal neuronal survival in culture was examined inrelation to the DHA and PS status. We found that Neuro 2A cellsexposed to ethanol accumulated considerably less PS in responseto DHA enrichment, and were less effective in phosphorylatingAkt and suppressing caspase-3 activity under serum starved orstaurosporine-treated conditions (Akbar et al., 2006). The in vivoparadigm correlated well with the in vitro findings. As was the casefor P0 and P21 hippocampi, the total PS content in the E-18 fetalhippocampus was slightly (14%) but significantly (p < 0.02) loweredby the prenatal ethanol exposure, primarily due to the reductionin the 18:0,22:6-PS species. Fetal hippocampal neuronal cultures

Fig. 14. Effect of prenatal ethanol exposure on hippocampal neuronal survival under bastaining (Akbar et al., 2006).

hetic activities at P0 and P21 (Wen and Kim, 2007).

obtained at E-18 from ethanol-treated pregnant rats contained sig-nificantly higher apoptotic cells after 7 days in vitro under basalconditions. These ethanol-exposed neuronal cultures were partic-ularly susceptible to apoptotic cell death induced by overnighttrophic factor removal in comparison to the pair-fed control group(Fig. 14). From these data it may be suggested that the loss of PS andthe resulting neuronal cell death inappropriately escalated duringdevelopment can contribute to the defects in brain function oftenobserved in fetal alcohol syndrome.

3.7. Does DHA have other neurotrophic functions?

Although the precise molecular mechanisms of DHA actions inthe brain are still unknown, several studies have shown that DHAdeficiency during development is associated with impairment ofhippocampus-dependent neuronal function such as learning and

sal and trophic factor removed conditions (−TF). Cell death is indicated by TUNEL

H.-Y. Kim / Chemistry and Physics of Lipids 153 (2008) 34–46 43

by D

Fig. 15. Hippocamplal neurite growth promoted

memory (Yoshida et al., 1997; Moriguchi et al., 2000), suggest-ing another important trophic control by DHA in hippocampaldevelopment. Therefore, we examined the effect of DHA on neu-ronal differentiation, more specifically neurite development inhippocampal neurons.

A natural deficiency of highly polyunsaturated fatty acids hasbeen shown to occur in brain-derived cells when they are trans-ferred from the tissue to cell culture in chemically defined medium(Bourre et al., 1983), serving as a convenient in vitro model of n-3fatty acid deficiency. This paradigm together with in vitro supple-mentation of DHA, allowed us to modulate the DHA content inhippocampal neurons in a controlled manner without affecting thecontent of other polyunsaturated fatty acids. DHA supplementationfor 6 days in vitro enhanced the neurite growth in hippocam-pal neuronal cells (Fig. 15). Control and DHA treated hippocampalneurons were stained with MAP2 and traced using camera Lucidaand the individual neurite lengths were measured with the NIHImage Software. The cultures treated with DHA showed a signifi-cant increase in the population of neurons with longer total neuritelengths and a remarkable decrease in the number of neurons with

shorter total neurite lengths in comparison to the control culture.The observed increase in total neurite length was found to be due toboth longer individual neurites and more dendritic branches. DHAsupplemented neurons showed a tendency to contain more neu-rites with longer length. More dramatically, DHA supplementationincreased the number of branches per neuron as the population ofneurons with more branches increased with DHA. With DHA sup-plementation, the highest frequency was observed for the groupof neurons with 13–15 branches/neuron. In contrast, without DHA,the population of neurons with less number of branches was morefrequent as the highest frequency was observed for the group ofneurons with 4–6 branches.

Supplementation of hippocampal neurons in culture witharachidonic (AA), oleic (OA), or docosapentaenoic acid (DPAn6) didnot have any effect, indicating specificity of the DHA action on neu-rite growth. It is particularly interesting to note that DPAn6, whichreplaces DHA during n-3 fatty acid deficiency, was not effective inpromoting neurite outgrowth. The in vivo reduction of DHA in fetalhippocampi induced by n-3 fatty acid deficiency during prenatalperiod also produced similar results. Although accretion of DHA atE18 was not significant (3.3 ± 0.3%), an n-3 fatty acid deficient diet

HA supplementation (Calderon and Kim, 2004).

from day 2 of pregnancy caused reciprocal replacement of DHAwith DPAn6 (4.1 ± 0.5%) in fetal hippocampi. The culture obtainedfrom the deficient hippocampi contained a greater number of neu-rons with considerably shorter total neurite length per neuron,which was contributed by both shorter individual neurite lengthand fewer branches per neuron. Supplementation of the deficientculture with DHA at 1.5 �M reversed the effect of n-3 fatty aciddeficiency, increasing the number of neurons with longer neuritescomparable to the control culture from the animals fed the n-3 fattyacid adequate diet. These data suggested that DHA has a unique rolein promoting hippocampal development. According to our results,depletion of DHA in fetal hippocampus by n-3 fatty acid deficiencyhas an adverse effect on neurite growth in culture and this effect canbe reversed by DHA supplementation. It is possible that n-3 fattyacid deficiency may similarly decrease the dendritic length andnumber of branches in vivo, which in turn would affect the num-ber and quality of synaptic connections during development and inadulthood. As in the case with in vitro situation, DHA supplementa-tion may promote differentiation in hippocampal neurons in vivo.

The trophic action of DHA on neuronal differentiation may be

derived from the facilitated membrane interaction and activationof Raf-1 or Akt due to PS increase, as observed for neuronal sur-vival. Alternatively, the role of DHA as a free fatty acid may also beimportant as DHA has been indicated to be a ligand for the retinoidX receptor (RXR), a nuclear receptor that acts as a ligand-activatedtranscription factor (Mata de Urquiza et al., 2000). In fact, four dif-ferent isoforms of RXR were found to be present in hippocampalneurons with distinctive intracellular localization (Calderon andKim, 2007). Our data using the luciferase reporter assay indicatedthat DHA can activate the RXR� but at 10–30 �M concentrationswhile a similar activation can be achieved with 9-cis-retinoic acidat low nM concentrations (Calderon and Kim, 2007). The increase ofneurite outgrowth in hippocampal culture was observed at 1.5 �M,suggesting that RXR� activation by DHA may not be directly respon-sible for the observed effect of DHA on neurite growth. However,the involvement of other RXR isoforms has not been tested andmay need to be considered. It is also possible that a derivative(or derivatives) of DHA rather than DHA itself may mediate theobserved effect of DHA on promoting neurite growth, as noveldocosatrienes produced from DHA have been shown to exert vari-ety of biological actions at nM concentrations (Hong et al., 2003;

44 H.-Y. Kim / Chemistry and Physics of Lipids 153 (2008) 34–46

t diffe

Fig. 16. Nano-ESI-QqTOF mass spectra from cross-linked Akt a

Mukherjee et al., 2004). The capacity to produce 10,17-dihydroxy

docosatriene has been demonstrated in glial cells (Hong et al.,2003).

3.8. How does membrane interaction affect the conformation ofsignaling proteins?

We have established that the protective action of DHA in neu-ronal survival is targeted at the membrane translocation of Akt(Akbar et al., 2005) and Raf-1 (Kim et al., 2000). The immediateconsequence of translocation is conformational changes of the sig-naling protein for subsequent activation steps. This conformationalchange, which is pivotal for proper function of many translocatingkinases, can be influenced by the nature of the membranes whichthese proteins interact with. Interaction of Akt with plasma mem-brane will also result in conformational changes of Akt requiredfor phosphorylation at T308 and S473 in the kinase and regulatorydomains, respectively.

Although partial crystal structures of Akt have been reported,neither the 3D structure of the entire molecule nor its confor-mational changes accompanying each activation step have beendemonstrated. We used the cross-linking strategy (Huang et al.,

Fig. 17. Proposed conformational changes during Akt activ

rent stages of Akt activation sequence (Huang and Kim, 2006).

2004, 2005) to probe the conformational changes of Akt in various

activation states. The inactive Akt molecule was cross-linked withlysine-specific cross-linkers, digested with trypsin and analyzedby mass spectrometry. Through-space cross-linked pairs were con-firmed using O18 labeling, by identifying the incorporation of fourO18 atoms from two H2O18 molecules to the two carboxy terminiduring tryptic hydrolysis. Characterization of cross-liked peptidesby tandem mass spectrometry revealed the presence of two inter-domain cross-linked pairs, K30 of the PH domain linked to K389 ofthe kinase domain and K284 of the kinase domain to K426 of theregulatory domain.

Monitoring these inter-domain cross-linked pairs, we wereable to monitor dramatic inter-domain conformational changesof Akt at different activation stages (Fig. 16). When inactiveAkt was interacted with liposomes containing phospholipids(PE/PC/PS/PIP3 50/18/30/2), typically found in the inner leaflet ofplasma membrane, and cross-linked with a lysine specific cross-linker, K30-K389 and K284-K426 were no longer observed withinthe 24 A distance constraint, suggesting that the PH domain andregulatory domain moved farther away from the kinase domain.Individual lysine residues were modified by cross-linkers, indicat-ing that these residues were still accessible. For active Akt which

ation and substrate binding (Huang and Kim, 2006).

H.-Y. Kim / Chemistry and Physics of Lipids 153 (2008) 34–46 45

of DHA

Fig. 18. Positive effect

is phosphorylated at both T308 and S473, K30-K389 cross-linkingwas observed but K284-K426 cross-linking was still missing. Whenactive Akt was bound to substrate, GSK-3� peptide and an ATPanalogue, K284-K426 cross-linking reappeared in the active form.

Our results strongly suggested that Akt undergoes distinctiveconformational changes at each successive activation step. Basedon our data, the following activation scheme was proposed (Fig. 17).Inactive Akt exists as a folded structure in cytoplasm with the PHand regulatory domains covering the kinase domain, at least in part.Membrane translocation causes conformational changes of Akt sothat both PH and regulatory domains become open to expose T308and S473 for phosphorylation. Phosphorylation of Akt presumablytriggers the separation of the PH domain from the membrane,releasing Akt to the cytoplasm. The open conformation of the regu-latory domain in the activated form of cytosolic Akt allows substrateentry, which is an important determinant for the activated stateof Akt. Our results provided the first demonstration for the dra-matic inter-domain conformational change in the full-length Aktstructure in solution during activation and substrate binding. More

importantly, our results set up a stage for the next investigation onAkt-membrane and/or Akt-protein interaction which is the key tounderstand molecular mechanisms involved in survival signalingmodulated by DHA and ethanol.

4. Conclusions

Our studies established specific biochemical and biologicalfunctions of DHA, i.e., promoting PS accumulation, neuronal dif-ferentiation and survival. Preferred microsomal PS synthesis ofDHA-containing species appears to be a mechanism for accumu-lating PS. Depletion of DHA by n-3 fatty acid deficiency depletes PSselectively from neuronal membranes due to the fact that DPAn6,the substitute for DHA under this condition, cannot fully sup-port the original level of PS biosynthesis. Long-term exposure toethanol also interferes with PS accumulation in neuronal tissues,primarily due to impaired PS biosynthetic activities rather thandepletion of preferred substrates. The biochemical function of DHAto promote PS accumulation in the central nervous system is animportant underpinning of the maintenance of neuronal survival.The protection by DHA is PS- and PI(3) kinase-dependent. Specif-

on neuronal survival.

ically, facilitated PS-dependent Akt translocation is a target eventwhere the protective effect of DHA is derived. The PS-dependentacceleration of Akt translocation is particularly vital under sub-optimal conditions where normal survival signal is compromised.In addition, Raf-1 translocation which is also facilitated by DHAmay also contribute to the neuronal survival and differentiation.In this regard, the loss of PS either by n-3 fatty acid deficiencyor by ethanol exposure and thus increased susceptibility to celldeath may have significant implications in neuronal dysfunction.Our current understanding of DHA action is depicted in Fig. 18.

Although the effects of DHA on neuronal survival and differen-tiation have been repeatedly demonstrated in cultured cells, thecurrent understanding has yet to be verified in vivo. In addition,the molecular mechanism involved in the membrane interactionof the signaling proteins and subsequent modulation by DHA andethanol has not been fully investigated. As the PS accumulation is animportant aspect for the observed effects of DHA, continued charac-terization of the PS regulation in the neuronal system is warranted.The observation that DHA increases PS specifically in neuronal cells

may suggest the existence of a unique PSS isoform in neuronalcells. Alternatively, specific post-translational modification of PSSenzymes may confer the characteristics of PS synthetic activity inneuronal cells. Although the protective effect of DHA on neuronalsurvival observed in our study was PS-dependent, the involvementof DHA metabolites such as 10,17S-docosatriene (neuroprotectin D)may also be considered. The mechanism for the trophic action ofDHA on neuronal differentiation still remains an important topicfor future research.

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