2 activation and neuritogenesis via intracellular reactive oxygen species production in human neuroblastoma SH-SY5Y cells

Biochimica et Biophysica Acta 1791 (2009) 8–16

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a l i p

Docosahexaenoic acid induces ERK1/2 activation and neuritogenesis via intracellular reactive oxygen species production in human neuroblastoma SH-SY5Y cells Haitao Wu a,b,1, Sanae Ichikawa a,1, Chiharu Tani a, Beiwei Zhu b, Mikiro Tada c, Yasuaki Shimoishi a, Yoshiyuki Murata a, Yoshimasa Nakamura a,⁎ a b c

Department of Biofunctional Chemistry, Division of Bioscience, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan College of Bio and Food Technology, Dalian Polytechnic University, Dalian 116034, China Department of Human Nutrition, Faculty of Contemporary Life Science, Chugoku Gakuen University, Okayama 701-0197, Japan

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Article history: Received 7 May 2008 Received in revised form 19 August 2008 Accepted 3 October 2008 Available online 1 November 2008 Keywords: Docosahexaenoic acid Neuritogenesis Growth associated protein 43 Reactive oxygen species Extracellular signal-regulated kinase

a b s t r a c t Docosahexaenoic acid (22: 6n-3; DHA) is a long chain polyunsaturated fatty acid that exists highly enriched in fish oil, and it is one of the low molecular weight food chemicals which can pass a blood brain barrier. A preliminary survey of several fatty acids for expression of growth-associated protein-43 (GAP-43), a marker of axonal growth, identified DHA as one of the most potent inducers. The human neuroblastoma SH-SY5Y cells exposed to DHA showed significant and dose-dependent increases in the percentage of cells with longer neurites. To elucidate signaling mechanisms involved in DHA-enhanced basal neuritogenesis, we examined the role of extracellular signal-regulated kinase (ERK)1/2 and intracellular reactive oxygen species (ROS) production using SH-SY5Y cells. From immunoblotting experiments, we observed that DHA induced the ROS production, protein tyrosine phosphatase inhibition, mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) phosphorylation, and sequentially ERK1/2 phosphorylation, the last of which was significantly reduced by MEK inhibitor U0126. Both antioxidants and MEK inhibitor affected DHA-induced GAP-43 expression, whereas the specific PI3K inhibitor LY294002 did not. We found that total protein tyrosine phosphatase activity was also downregulated by DHA treatment, which was counteracted by antioxidant pretreatment. These results suggest that the ROS-dependent ERK pathway, rather than PI3K, plays an important role during DHA-enhanced neurite outgrowth. © 2008 Elsevier B.V. All rights reserved.

1. Introduction The growth of neurites is a critical step in neuronal development and requires generation of additional plasma membrane. The plasma membranes of neurons are enriched in phospholipids containing polyunsaturated fatty acids (PUFAs), especially arachidonic acid and docosahexaenoic acid (DHA) [1]. Previous studies have shown that PUFAs promoted basal and nerve growth factor (NGF)-induced neurite extension of the pheochromocytoma cell line PC12, one of the most widely used cell lines as a model for the study of neuronal differentiation [2–4]. Among the PUFAs, DHA (22: 6n-3) is predominantly found in the mammalian brain and is the most abundant n-3 PUFA in the neural membrane [5,6]. Docosahexaenoic acid delivery to the central nervous

Abbreviations: DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; ERK, extracellular-regulated kinase; GAP-43, growth-associated protein-43; H2DCF-DA, 2′,7′-dichlorofluorescin diacetate; PI3K, phosphatidyl inositol 3 kinase; PTPase, protein tyrosine phosphatase; PUFAs, polyunsaturated fatty acids; ROS, reactive oxygen species; (MAPK), mitogen-activated protein kinase; MEK, mitogen-activated protein kinase (MAPK)/ERK kinase; NAC, N-acetylcysteine ⁎ Corresponding author. Tel.: +81 86 251 8300; fax: +81 86 251 8388. E-mail address: [email protected] (Y. Nakamura). 1 These authors contributed equally to this work. 1388-1981/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2008.10.004

system (CNS) is most efficient at times prior to synaptogenesis when plasma DHA is primarily esterified in phospholipids (PLs) [7]. Current information from studies in animal and human models indicates that DHA is essential for optimal development and function of the brain [8]. Loss of brain DHA results in the loss of many behavioral and cognitive functions in animals [9,10]. It has been demonstrated that the intelligence quotients of children fed with breast milk (rich in DHA) exceed those of children fed with formula milk [11]. When human infants had DHA-deficient diets, impaired cortical development and neurodevelopmental quotient have been noted [12–14]. Most of these studies have required long-term treatment of whole animals or humans in order to study the effects of DHA on the structural and functional changes in neurons of the CNS because mature neurons will not survive for long periods in vitro. Investigation of the DHA on developing neurons can, however, be conducted in cell culture. Cell culture makes individual living neurons accessible and permits direct observation of growing neurons, their neurite formation and cell signaling alteration. The cell culture model can also be used to investigate the direct effects of DHA on special target neurons apart from endogenous endocrine or metabolic influences. Although it has been reported that DHA has positive effects on the neurite outgrowth of rat clonal pheochromocytoma PC12 cells [2],

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hippocampal cells in vitro [15], and rat primary cortical neuron cultures [16], little is known about the underlying molecular mechanism for DHA-enhanced basal neuritogenesis. In the present study, therefore, the human neuroblastoma cell line SH-SY5Y was selected as a neuronal cell model to examine the neurite outgrowth and the changes in the growth-associated protein-43 (GAP-43) mRNA expression. SH-SY5Y cells are a neuroblastic subclone of the neuroblastoma cell line SK–N–SH [17] and are able to differentiate into a functional and morphological neuronal phenotype when treated with retinoic acid (RA) [18], neurotrophic factors, or phorbol esters [19–22]. In this study, we found that DHA enhances basal neuritogenesis not only by inducing intracellular reactive oxygen species (ROS) generation but also by promoting the ERK-related proliferation pathways. This provides a novel molecular basis for the preventative effects of dietary DHA supplementation on neurodegeneration. 2. Materials and methods 2.1. Chemicals and antibodies DHA, retinoic acid (RA), fetal bovine serum (FBS), 2′,7′-dichlorofluorescin diacetate (H2DCF-DA), U0126 and LY294002 were purchased from Sigma (St. Louis, MO, USA). Anti-phospho-ERK1/2, antiERK1/2, anti-phospho-MEK, anti-MEK, anti-actin and anti-mouse and rabbit IgG horseradish peroxidase (HRP)-conjugated antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were purchased from Wako Pure Chemical Industries, Osaka, Japan. 2.2. Cell cultures The SH-SY5Y human neuroblastoma cells (a kind gift from Dr. S. Maeda of Setsunan University) were maintained in DMEM supplemented with 10% heat-inactivated FBS, penicillin (50 unit/ml), and streptomycin (50 µg/ml). The cells were grown in an atmosphere of 95% air and 5% CO2 at 37 °C. The DHA or other fatty acids were dissolved in ethanol as a stock and to reach ethanol concentrations (0.1%) demonstrated previously to be nontoxic (data not shown), fatty acids were freshly diluted further in culture medium.

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were conducted in each treatment and the results are expressed as mean ± SD. 2.5. Reverse transcription-polymerase chain reaction (RT-PCR) Total cellular RNA was isolated with Trizol reagent (Invitrogen, Tokyo, Japan) according to the manufacturer's recommendations and spectrophotometrically quantified. The RT reaction was performed with 5 µg of total RNA and an oligo (dT) primer at 42 °C for 1 h using M-MLV reverse transcriptase (Takara Bio, Inc., Kyoto, Japan). The reaction mixture was then subjected to brief incubation at 70 °C in order to inactivate the enzyme. PCR was performed with the resulting single-strand cDNA and specifically designed primers using rTaq DNA polymerase (Takara Bio, Inc., Kyoto, Japan). The reaction mixtures were heated at 94 °C for 3 min and then immediately cycled 30 times through a 1-min denaturing step at 94 °C, a 1-min annealing step at 60 °C, and a 1-min extension step at 72 °C. After the cycling procedure, a final 10-min elongation step at 72 °C was performed. The following primers were used: GAP-43, (F) 5′-CTG TCC TTT CCC ACC CAC TA-3′ and (R) 5′-GAA CGG AAC ATT GCA CAC AC-3′ (277 bp): β-actin 5′-GTC ACC CAC ACT GTG CCC ATC TA-3′ and 5′-GCA ATG CCA GGG TAC ATG GTG GT-3′ (455 bp). PCR products were analyzed by electrophoresis on 2% agarose gel and stained with ethidium bromide. The bands were imaged with an LAS3000 image-analyzer (Fuji Film, Tokyo, Japan). 2.6. Immunoblot analysis Whole cell lysates were prepared in lysis buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 2 mM DTT, 10 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 1% SDS and 1% Triton X-100) containing protease cocktail (Sigma) and left on ice for 20 min. After sonication lysates were clarified by centrifugation at 12000 rpm for 25 min and frozen at −80 °C until use. Whole cell lysates treated with or without DHA were incubated with the SDS-sample buffer for 5 min at 100 °C. These samples were then separated with a 10% SDS/PAGE. The gel was transblotted onto an Immobilon-P membrane (Millipore), incubated with 5% Skim Milk for blocking, then washed and incubated with antibody. This procedure was followed by the addition of horseradish peroxidase conjugated to IgG and Chemi-Lumi One reagent (Nacalai Tesque, Kyoto, Japan). The bands were imaged with an LAS3000 image-analyzer (Fuji Film, Tokyo, Japan).

2.3. Inhibitor treatments 2.7. Intracellular oxidative products determination A 10 mM stock solution each of U0126 and LY294002 were prepared in DMSO, stored in −20 °C in the dark, and diluted with medium just before use. A 1 M stock solution of N-acetylcysteine (NAC) was prepared in 1 M NaOH and 1 M HCl to adjust the pH 7.0. A 1000 unit/ml catalase was prepared with medium freshly. For experiments using the MEK inhibitor U0126 and PI3K inhibitor LY294002, SH-SY5Y cells either were pretreated with or without the inhibitors for 30 min, and then stimulated with 10 µM DHA in continuous presence of the inhibitors. As for antioxidant, SH-SY5Y cells were pretreated with or without NAC or catalase for 30 min, and stimulated with 20 µM DHA (ERK phosphorylation, GAP-43 expression) or directly treated with DHA in the presence or absence of NAC or catalase (intracellular ROS production, total PTP activity). 2.4. Measurement of neurite outgrowth For the analysis of neurite outgrowth, SH-SY5Y cells were plated in 60-mm dishes at a density of 3 × 105/dish. The cells from control and DHA-treated groups after 24 h were examined for neurite formation. A neurite was identified as a process greater than 2 × cell body diameter in length and possessing a terminal growth cone. The percentage of cells with neurites was calculated by counting 200 cells per well in triplicate wells. At least three independent experiments

Intracellular ROS production was detected by H2DCF-DA as an intracellular fluorescence probe [23]. Briefly, the cells were treated with H2DCF-DA (50 µM) for 30 min at 37 °C. Briefly, the cells were treated with H2DCF-DA (50 µM) for 30 min at 37 °C, and DHA was added to the complete medium. A flow cytometer (Beckman Coulter, Tokyo, Japan) was used to detect DCF formed by the reaction of H2DCF with the intracellular oxidative products. 2.8. Determination of protein tyrosine phosphatase (PTP) activity SH-SY5Y cells were snap-frozen in liquid N2, and deoxygenated homogenization buffer (150 mM NaCl, 5 mM EDTA, 5 mM EGTA, in 50 mM Hepes, pH 7.5, containing a protease inhibitor mixture (Sigma), 1% (v/v) Triton X-100). The whole cell lysate was cleared by centrifugation at 15,000 ×g for 20 min. Total PTP activity was measured by the hydrolysis of p-nitrophenyl phosphate (pNPP; Wako) in total cell lysates. Briefly, 25 µl of cell lysates (containing 25 µg of protein) were incubated in a final volume of 125 µl at 37 °C for 30 min in reaction buffer containing 10 mM pNPP and 2 mM EDTA in 20 mM MES at pH 6.0. The reaction was stopped by the addition of 200 µl of 1 M NaOH, and the absorption was determined at 410 nm [24].

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2.9. Densitometric and statistical analysis Densitometric measurement of the immunoblot or RT-PCR was performed using an LAS3000 image-analyzer, and Win MDI 2.9 Software (Fuji Film, Tokyo, Japan). Quantifications of the protein or PCR product levels were estimated by comparing the intensity of the specific band from the control to those of the DHA treatment conditions. All experimental data were compensated using each intensity of total protein (for ERK and MEK) or actin (RT-PCR) as an internal standard. When applicable, mean ± SD are shown. Statistical significance was assessed by Student's paired two-tailed t-test or analysis of variance on untransformed data, followed by comparison of group averages by contrast analysis, using the Super ANOVA statistical program (Abacus Concepts, Berkeley, CA). A P value of b0.05 was considered to be statistically significant. 3. Results 3.1. DHA is the most potent inducer of a neurite outgrowth To investigate if saturated and unsaturated fatty acids, including PUFAs, induce neurite outgrowth in human neuroblastoma SH-SY5Y cells, we carried out RT-PCR analysis using a primer for the outgrowth marker, GAP-43, which is targeted to neuronal growth cones via fast axonal transport and mainly localized in neurites [25]. SH-SY5Y cells were incubated for 15 h with 10 µM of behenic acid (22: 0), erucic acid (22: 1n-9), linoleic acid (18: 2n-6), α-linolenic acid (18: 3n-3), arachidonic acid (AA; 20: 4n-6), eicosapentaenoic acid (EPA; 20: 5n3) and DHA (22: 6n-3) in the presence of serum, and the GAP-43 mRNA level was determined. Under this condition, PUFAs including EPA and DHA significantly upregulated GAP-43 expression: the effect of DHA was maximal at among these fatty acids (Fig. 1). Conversely, saturated behenic acid, monounsaturated erucic acid and polyunsaturated α-linolenic acid as well as AA did not affect GAP-43 expression at the concentration of 10 µM. Next SH-SY5Y cells were incubated for 24 h with various concentrations of DHA, and neurite outgrowth was determined by

Fig. 2. DHA dose-dependently enhances neurite outgrowth in SH-SY5Y cells. SH-SY5Y cells were treated with or without DHA at the indicated concentrations for 24 h, and neurite outgrowth was evaluated. (A) Phase-contrast micrographs of SH-SY5Y cells treated with or without DHA (10 µM). (B) The percentage of cells with neurites was calculated by counting 200 cells per well in triplicate wells. Cells treated with 10 µM retinoic acid were used as the positive control. At least three independent experiments were conducted in each treatment, and the results are expressed as mean ± SD. ⁎, P b 0.05 versus control group.

the percentage of cells with neurites. DHA time-dependently (data not shown) and dose-dependently enhanced neurite outgrowth in SHSY5Y cells. As shown in Fig. 2A and B, a significant increase in the numbers of cells with neurites was found in the 5 µM DHA group (P b 0.05). The effect reached a plateau at 10 µM DHA, which is comparable to that of the positive control, retinoic acid (RA) [18,26]. These results suggested that DHA alone enhances neurite outgrowth in human neuroblastoma SH-SY5Y cells. 3.2. DHA activates the MEK/ERK pathway in SH-SY5Y cells

Fig. 1. DHA and EPA enhance GAP-43 mRNA expression in SH-SY5Y cells. SH-SY5Y cells were exposed to behenic acid (22: 0), erucic acid (22: 1n-9), linoleic acid (18: 2n-6), αlinolenic acid (18: 3n-3), AA (20: 4n-6), EPA (20: 5n-3) and DHA (22: 6n-3) at 10 µM for 15 h, and the GAP-43 mRNA level was determined. Data are means of three experiments. ⁎, P b 0.05 versus ethanol treatment control group.

In nervous tissue, ERKs play an important role in regulating typical neuronal functions such as synaptic plasticity, memory learning, and cell survival [27]. The ERK requirement in neuronal differentiation seems dependent on the kind of stimulus that induces the differentiation [20,28–30]. Knowledge about signaling pathways involved in DHA-induced neuronal growth and differentiation is of great interest considering the role of DHA as a physiologic effector of normal brain development and, under pathologic conditions, as a possible preventive agent of stem cells used for treatment of neurodegenerative diseases. To investigate if DHA induces ERK1/2 activation in human neuroblastoma SH-SY5Y cells, we carried out immunoblotting analysis using an antibody that specifically recognizes the phosphorylated forms of ERK1/2. SH-SY5Y cells exposed to DHA at the concentration that significantly extended neuritic outgrowth (10 µM; Fig. 2), and cell lysates were prepared at different times of DHA stimulation (from 30 min to 360 min). As shown in Fig. 3, at 30 min, a dramatic increase in phosphorylation of ERK1/2 over the

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as an intermediate species and can be mimicked, at least partially, by exogenous hydrogen peroxide [34,35]. Thus we examined whether DHA enhances the intracellular ROS level by flow cytometry using H2DCF-DA, the most general fluorescent probe for detecting intracellular ROS. As shown in Fig. 5A and B, a dose-dependent ROS production was observed after treatment with DHA for 30 min in human neuroblastoma SH-SY5Y cells. When the cells were treated

Fig. 3. DHA time-dependently induces phosphorylation of ERK1/2 and MEK1/2 in SHSY5Y cells. SH-SY5Y cells were treated with 10 µM DHA for the indicated times. Lane 0 shows cells under the starting condition of each experiment. Cell lysates (25 µg/lane) were analyzed by immunoblotting using antibodies that specifically recognize phosphorylated ERK1/2 (p-ERK), ERK1/2 (ERK), phosphorylated MEK1/2 (p-MEK), and MEK1/2 (MEK). Results were reproduced at least four times for each point. Results are representative of 3 independent experiments.

control (0 min) was observed. DHA-induced ERK1/2 phosphorylation remained elevated up to 180 min and declined after 360 min (Fig. 3), whereas the phosphorylation of Akt was transiently and slightly increased only 30 min after DHA treatment (data not shown). The amount of ERK1/2 (Fig. 3) and actin (data not shown) was unchanged during the entire time of DHA stimulation. In addition, no significant changes in phosphorylation of other kinases including p38 MAPK and JNK were observed (data not shown). ERK1/2 are phosphorylated and activated generally by MEK, a specific threonine/tyrosine kinase [31,32]. Favata et al. previously reported the MEK involvement in ERK1/2 phosphorylation in SH-SY5Y cells using U0126, an inhibitor that selectively blocks MEK activation without affecting other known serine/threonine and tyrosine kinases [33]. This was supported in the present study by the data showing that MEK phosphorylation was significantly increased by 10 µM DHA treatment in the same timedependent manner as p-ERK1/2 (Fig. 3). In addition, as shown in Fig. 4A, the selective MEK inhibitor, U0126, significantly inhibited DHAinduced ERK1/2 phosphorylation. These results indicated that DHA activates the MEK/ERK pathway in SH-SY5Y cells. 3.3. ERK activation is necessary for neurite outgrowth We next examined the ERK1/2 requirement in DHA-induced neurite outgrowth. SH-SY5Y cells exposed to 10 µM DHA required for extending neuritic processes positive for neuron-specific markers (Fig. 2). We evaluated the percentage of neurite-bearing cells and the neurite outgrowth marker (GAP-43 mRNA) in the presence of 10 µM DHA with or without U0126 at the concentrations that significantly inhibited ERK phosphorylation (10 µM). Inhibition of ERK phosphorylation by U0126 impaired DHA-induced neurite outgrowth. In fact, in SH-SY5Y cells treated with DHA and 10 µM U0126, the decrease in GAP-43 expression reached significance (Fig. 4B), and similarly, the neurite-bearing cells were significantly reduced with respect to cells treated with DHA alone (Fig. 4C). Conversely, an inhibitor of PI3K/Akt pathway, LY294002, did not inhibit DHA-induced ERK1/2 phosphorylation, GAP-43 expression and neurite outgrowth. These results suggest that prolonged MEK/ERK phosphorylation, rather than the PI3K/Akt pathway, was involved in DHA-stimulated neuritogenesis. 3.4. Involvement of reactive oxygen species in the DHA-induced ERK signaling pathway It has been reported that ERK activation and neurite outgrowth elicited by neurotrophins, including NGF, require hydrogen peroxide

Fig. 4. A MEK/ERK pathway is involved in the DHA-induced GAP-43 expression and neurite outgrowth in SH-SY5Y cells. U0126, but not LY294002, inhibits DHA-induced ERK phosphorylation (A), GAP-43 expression (B), and neurite outgrowth (C). SH-SY5Y cells were treated with 10 µM U0126 or 10 µM LY294002 for 30 min and then were stimulated with 10 µM DHA for 30 min (ERK phosphorylation), 15 h (GAP-43) or 24 h (neuritogenesis) in continuous presence of the inhibitor. Quantifications of the protein or PCR product levels were estimated by comparing the intensity of the specific band from the control to those of the DHA treatment conditions and compensated using each intensity of the internal standard. The percentage of cells with neurites was calculated by counting 200 cells per well in triplicate wells. The graph shows the amounts of each parameter by taking the control level as 100%. At least three independent experiments were conducted in each treatment, and the results are expressed as mean ± SD. a, P b 0.05 versus control group; b, P b 0.05 versus DHA (no inhibitor) group.

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used 20 µM DHA, even though the required concentration to enhance neurite outgrowth was relatively lower (Fig. 2). We evaluated the ERK phosphorylation and neurite outgrowth marker (GAP-43 mRNA) in the presence of 20 µM DHA with or without NAC (1 mM) or catalase (100 unit/ml) at the concentrations that significantly inhibited ROS level enhancement (Fig. 6A). The effectiveness of exogenously added catalase suggested that DHA-induced ROS is at least partly mediated by hydrogen peroxide. Inhibition of ROS enhancement by both antioxidants impaired DHA-induced ERK phosphorylation (Fig. 6B). In fact, in SH-SY5Y cells treated with DHA and the antioxidants, the GAP-43 expression was significantly reduced with respect to cells treated with DHA alone (Fig. 6C). These results suggest that transient enhancement of the intracellular ROS level was involved in DHAstimulated, ERK-dependent neurite outgrowth. Additionally, treatment with 20 µM DHA for 5 min significantly reduced the total PTP activity, which is also inhibitable by NAC treatment (Fig. 6D). This is consistent with the hypothesis that DHA modulates signal transduction, at least in part through the redox regulation of PTP activities, still to be identified. 4. Discussion

Fig. 5. Intracellular production of ROS induced by DHA in SH-SY5Y cells. (A) Representative cytograms of three independent experiments treated with 20 µM or 50 µM DHA. (B) Summarizing the dose-dependent effect of DHA on the ROS level. After incubation with H2DCF-DA (50 µM) for 30 min, SH-SY5Y cells were treated with DHA at the indicated concentration for 30 min. After DHA stimulation, the cells were harvested, washed, and then analyzed by a flow cytometer. Each experiment was repeated at least twice for reproducibility. Values are mean ± SD (n = 3). ⁎, P b 0.05 versus control group.

with 50 µM DHA for 30 min, the intracellular ROS level was increased 2-fold compared to the control (0 h). We next examined the ROS requirement in DHA-induced neurite outgrowth. Concurrently, to see the effect of DHA clearly, we mainly

Although DHA possesses neuroprotective and neurotrophic-like activities in a number of in vitro and in vivo models as mentioned above, the mechanism by which DHA mediates these effects is not entirely clear. DHA was shown to promote phosphatidylserine accumulation in the cell membranes of Neuro2A cells, thereby enhancing Raf-1 and Akt translocation/activation as well as the associated survival pathways [36,37]. DHA also was reported not only to prevent the soluble amyloid β-mediated disappearance of the phosphorylated forms of ERK [38] but also to activate the ERK pathway to promote photoreceptor survival during early development [39]. However, the key molecules that intermediate the neurite outgrowth are not fully known. Therefore, the main goal of this study was to elucidate the mechanism by which DHA potentiates neurite outgrowth in SH-SY5Y cells by using the antioxidants and specific pharmacological inhibitors of signaling molecules downstream of DHA stimulation. We confirmed in the present study that DHA enhances not only the GAP-43 expression but also neurite growth in SH-SY5Y cells. Neurite growth requires newly synthesized membrane components, such as phospholipids and proteins [40]. GAP-43, also known as B-50, is a neuron-specific protein associated with axon growth and growth cone formation that also modulates neurite outgrowth [41,42]. In neuronal tissue culture, GAP-43 has been shown to be concentrated in the growth cones, the axons and the somal plasma membrane, and to be expressed concomitantly with neurite outgrowth [41,43]. Our data are consistent with the previous study demonstrating that DHA significantly increased the cellular GAP-43 immunoactivity or GAP-43 content in rat cortical neurons in primary cultures. The morphological changes observed in our study might be due to the increase in GAP-43 content or neurons by DHA treatment. It has been reported that DHA supplementation greatly increases DHA concentration in neuron-like cells at the expense of decreased AA, and combined DHA and AA supplementation results in enhanced neurite outgrowth [44]. However, the present result showed that AA itself was less effective on neurite outgrowth than DHA, which is consistent with previous reports using hippocampal neurons [15,45]. It is uncertain whether DHA synthesis from n-3 fatty acid precursors such as α-linolenic acid can occur in cultured neurons. Studies in animal models indicated that neurons cultured from the forebrain gray matter of 2- to 7-day-old rat pups took up DHA but did not synthesize DHA from α-linolenic acid [46]. However, more recently, it has been reported that rat hippocampal neuron cultures can synthesize DHA from n-3 fatty acid precursors [47]. The present result showed that α-linolenic acid did not up-regulate GAP-43 mRNA expression (Fig. 1). Taken together, it is suggested that the level of DHA

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Fig. 6. ROS production requires DHA-induced ERK phosphorylation and GAP-43 expression. Antioxidants inhibit DHA-induced DCF oxidation (A), ERK phosphorylation (B), GAP-43 expression (C), and total PTP activity inhibition (D). A&D, SH-SY5Y cells were treated with 20 µM DHA for 30 min (DCF oxidation) or 5 min (total PTP activity) in the presence or absence of 1 mM NAC or 100 unit/ml catalase; B&C, SH-SY5Y cells were pretreated with 1 mM NAC or 100 unit/ml catalase for 30 min and stimulated with 20 µM DHA for 1 h (ERK phosphorylation) or 15 h (GAP-43 expression). Cells treated with 100 µM hydrogen peroxide were used as the positive control (total PTP activity). Quantifications of the protein or PCR product levels were estimated by comparing the intensity of the specific band from the control to those of the DHA treatment conditions and compensated using each intensity of the internal standard. The graph shows the amounts of each parameter by taking the control level as 1 or 100%. At least three independent experiments were conducted in each treatment and the results are expressed as mean ± SD except for B, C. The GAP-43 data are the means of three experiments, and the maximal standard deviation for the experiments was 5%. a, P b 0.05 versus control group; b, P b 0.05 versus DHA (no inhibitor) group.

synthesized from LA in SH-SY5Y cells is not enough for neurite outgrowth. ERK1/2 are key signaling molecules involved in cell proliferation and differentiation. In neuronal differentiation, ERKs represent an important convergence point for signaling information generated by axon growth-promoting molecules, but their specific involvement in neuritogenesis remains controversial. Generally, prolonged ERK activation is associated with cell differentiation [48]. Moreover, the requirement of ERK activation seems dependent on the kind of stimulus that induces differentiation and on the cellular model used. In rat pheochromocytoma PC12 cells, sustained ERK activation plays an essential role in neuronal differentiation induced by NGF [28,49] but not when N-acetyl-Leu-Leu-norleucinal is used as a morphogen [29]. In human neuroblastoma SH-SY5Y cells, persistent ERK activation is required in neuronal differentiation induced by insulin-like growth factor I (IGF-I) or brain-derived neurotrophic factor (BDNF) [20,21] but not when 12-O-tetradecanoylprobol 13-acetate (TPA) or RA is used [30]. In addition, DHA exclusively activates the ERK pathway in retina photoreceptors [39] but not in human T cells, and inhibits MEK/ ERK activation in response to phorbol-12-myristate-13-acetate (PMA)

in Jurkat T cells [50,51]. We demonstrate that in SH-SY5Y cells, DHA induces sustained ERK1/2 phosphorylation, and thus this activation is necessary for DHA-mediated neuritogenesis (Fig. 4) using the MEKselective inhibitor U0126. The recent study demonstrated that the MEK inhibitor U0126 causes more extensive and irreversible inhibition of ERK phosphorylation than PD98059, which caused only a reversible and time-dependent inhibition [26]. Even though we only analyzed an initial phase of neurite outgrowth (24 h after DHA stimulation), the observed ERK1/2 phosphorylation that occurs up to 6 h after DHA treatment may be necessary, at least, in the initial phase of neurite outgrowth examined. This idea is supported by the finding of Enarsson et al. [52], who monitored morphologic maturation of neurons and demonstrated that ERK activation seems dispensable for earlier steps of central nervous system (CNS) stem cell differentiation. It has been demonstrated that a variety of external signals such as cytokines, peptide growth factors, and agonists of seven-pass transmembrane receptors transiently increased the levels of ROS, especially hydrogen peroxide [53–56]. This growth factor-mediated increase in ROS, in turn, controls downstream signaling by oxidizing and inactivating protein-tyrosine phosphatases (PTPs) in vivo [57,58],

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because tyrosine phosphorylation events are controlled by the balance of activation of protein tyrosine kinases (PTKs) and PTPs. For instance, the insulin-mediated increase in ROS enhances early and later signaling cascades by inhibiting phosphatase PTP1B as well as general PTPs [59,60]. As mentioned before, in PC12 cells, ERK activation elicited by neurotrophins, including NGF, requires hydrogen peroxide as an intermediate species [35]. We demonstrate in this study that DHA induces a dose-dependent ROS production in human neuroblastma SH-SY5Y cells, consistent with the previous report [61]. Also, ROS are suggested to be required for the DHA-dependent activation of ERK, PTP activity inhibition, and GAP-43 expression. The leukocyte common antigen-related (LAR) receptor, a receptor-type PTP expressed by neurons [62] has been shown to play an important role in neurite outgrowth or promoting neurogenesis [63,64]. Moreover, LAR wedge domain peptides which exhibit homophilic binding to LAR and inhibit LAR function, augmented NGF-induced activation of ERK signaling pathway [65]. Therefore, although further study to reveal the target molecule is required, it appears certain that PTPs including LAR are key determinants to regulate neurite growth induced by DHA. The catalase-pretreatment experiment indicated that hydrogen peroxide is involved in the DHA-dependent activation of ERK and thus neurite outgrowth. The source(s) of ROS required for DHA-dependent activation of neurite outgrowth in SH-SY5Y cells is an open question. Previously, in cultured hippocampal neurons mitochondria have been implicated as a source of superoxide that is necessary for activitydependent increases in the phosphorylation of camp response element binding protein (CREB; [66]), a transcription factor known to be a downstream effector of ERK [27]. NADPH oxidase is another source of superoxide that could regulate ERK in the neurons. In phagocytic cells, NADPH oxidase is a heterotetramer consisting of two cytosolic components (p47phox and p67phox) and two membrane-associated proteins (p22phox and gp91phox or NOX2) that can be activated by the

small G protein Rac. Recent evidence indicates that components of the NADPH oxidase complex are localized to hippocampal neurons [67] and that the plasma membrane-bound NADPH oxidase complex including NOX2 plays an essential role in the glutamate-induced production of ROS in SH-SY5Y cells [68]. In addition, DHA has been more recently reported to modulate NOX4 anion superoxide production in human fibroblasts [69]. Interestingly, ERK is an effector of NADPH oxidase-dependent ROS signaling during LTP and hippocampus-dependent memory. The patients with chronic granulomatous disease (CGD), which is characterized by a deficient phagocytic NADPH oxidase [70], have been reported to suffer from cognitive deficits [71]. Thus, the critical task for DHA-induced neurogenesis will be to identify the specific molecular source that produce ROS and to find a possibility that such a signaling can occur in vivo. In conclusion, the results from this study provided biological evidence that DHA has the potential to enhance neuritogenesis in a redox-sensitive, ERK1/2-dependent manner (Fig. 7). DHA significantly enhanced the viable cell numbers at 10 µM and 20 µM, whereas DHA at 50 µM slightly induced cell death and DNA fragmentation in SHSY5Y cells (unpublished data). Therefore, neurite outgrowth could be a pathological response to ROS rather than a normal developing process. Because DHA possesses a high degree of unsaturation, it can increase lipid peroxidation, providing a variety of lipid peroxides and aldehydic breakdown products with pro-oxidant property [72]. Numerous in vivo and in vitro studies on the incidence of DHA supplementation at high doses have reported higher peroxidation and oxidative damage [73,74]. A concomitant supplementation with the antioxidant vitamin E has been proposed to minimize this deleterious side effect [75]. Although highly unsaturated fatty acids are believed to be easy targets of lipid peroxidation, their contribution to lipid peroxidation should be based on a complicated and not yet fully clarified oxidant/antioxidant balance. Although a further detailed

Fig. 7. Schematic representation of molecular events involved in DHA-induced neuritogenesis in SH-SY5Y cells. DHA increases ROS level in both extracellular and intracellular spaces. The resultant oxidative stress triggers MEK/ERK phosphorylation, inhibits PTPs activity, and potentiates activation of MEK/ERK signaling pathway, leading to transcription of the GAP43 gene and thus neurite outgrowth. Antioxidants such as catalase and NAC, as well as MEK/ERK inhibitor (U0126) have a potential to block or attenuate these DHA-induced molecular events.

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