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1,3-Capryloyl-2-arachidonoyl glycerol activates α-secretase activity and suppresses Aβ40 secretion in A172 cells
Neuroscience Letters 450 (2009) 324–326
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
1,3-Capryloyl-2-arachidonoyl glycerol activates ␣-secretase activity and suppresses A40 secretion in A172 cells Chiaki Tanabe a,1 , Maiko Ebina a,1 , Masashi Asai b , Eugene Futai a , Noboru Sasagawa a , Kenji Katano c , Harukazu Fukami d , Shoichi Ishiura a,∗ a
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan Department of Pharmacology, Faculty of Medicine, Saitama Medical University, 38 Moroyama-machi, Moro-hongo, Iruma-gun, Saitama 350-0495, Japan Health Care Science Center, Suntory Technological Development Center, Suntory, Ltd., 5-2-5 Yamazaki, Shimamoto-cho, Mishima-gun, Osaka 618-0001, Japan d Department of Bioscience and Biotechnology, Faculty of Bioenvironmental Science, Kyoto Gakuen University, 1-1 Nanjyo, Sogabe-cho, Kameoka-city, Kyoto 621-8555, Japan b c
a r t i c l e
i n f o
Article history: Received 8 October 2008 Received in revised form 12 November 2008 Accepted 14 November 2008 Keywords: Alzheimer’s disease Amyloid precursor protein (APP) Arachidonic acid Medium chain fatty acid ␣-Secretase -Secretase
a b s t r a c t Alzheimer’s disease (AD) is characterized by the deposition of amyloid -peptide (A), derived from amyloid precursor protein (APP). Membrane states, such as lipid components or membrane fluidity, are important for enzymes related to APP processing in meeting their substrates efficiently. We analyzed the effects of triglycerides combined with polyunsaturated fatty acids (PUFAs) and/or caprylic acids on APP proteolysis. Our results showed that 1,3-capryloyl-2-arachidonoyl glycerol (8A8) moderately increased ␣-secretase activity (18%) in A172 cells. -Secretase activity was not statistically significantly changed in HEK293 cells stably expressing BACE1. However, A40 secretion decreased by 22%. Thus, we conclude that 8A8 is a useful lipid compound for activating ␣-secretase activity and suppressing A40 secretion. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Alzheimer’s disease (AD) is characterized by the formation of senile plaques, composed of amyloid -peptide (A). A production is initialized by -secretase, also known as -site APP cleaving enzyme 1 (BACE1), which cleaves amyloid precursor protein (APP), and then the remaining membrane-associated fragment is cleaved by ␥-secretase. However, APP is usually cleaved within the A sequence by ␣-secretase, precluding the generation of A. Activation of a non-amyloidogenic pathway or suppression of an amyloidogenic pathway can preclude A deposition. Recently, there have been several reports on the relationship between lipids and APP proteolysis. ␣-Secretase cleavage occurs in non-lipid rafts, whereas -secretase cleavage occurs in lipid rafts [5]. We previously showed that an effective BACE1 inhibitor changed the localization of BACE1 from a lipid raft to a non-lipid raft [4]. In another report, A and presenilin regulated cholesterol and
∗ Corresponding author. Tel.: +81 3 5454 6739 fax: +81 3 5454 6739. E-mail address: [email protected] (S. Ishiura). 1 These authors equally contributed to this work. This work is dedicated to late Miss Maiko Ebina. 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.11.039
sphingomyelin metabolism [6]. These findings suggest that lipid components and membrane fluidity are important for enzymes and substrates to meet each other efficiently. Furthermore, polyunsaturated fatty acids (PUFAs), especially n3 PUFAs, such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), are necessary in the development of the prenatal brain and in maintaining brain function and vision in adults [13]. Deficiencies in DHA, evidenced by low serum levels, have been linked to cognitive impairment and AD. Low levels of DHA have been shown in brain membranes of AD patients [16]. It has also been reported that DHA and EPA treatment in cultured cells reduced sphingomyelin and cholesterol contents in lipid rafts [18]. Thus, it is supposed that n-3 PUFA affects APP processing via lipid raft alterations. The n-6 PUFA, arachidonic acid (ARA), is also related to memory. Long-term administration of ARA-supplemented diets to aged rats alleviated age-related deficits in spatial cognition [15]. In another report, ARA induced long-term activity-dependent enhancement of synaptic transmission [21]. Here, we analyzed the effects of triglycerides combined with caprylic acids or PUFAs, which were synthesized by Suntory Co. Ltd., on APP proteolysis. The lipid compounds used in this study are shown in Table 1. SUNTGA40S is an arachidonate-enriched triglyceride oil extracted from a biomass of submerged fermented
C. Tanabe et al. / Neuroscience Letters 450 (2009) 324–326
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Table 1 The abbreviations of lipid compounds used in this study. Abbreviations
Compounds
AAA 88A 8A8 SUN ARA-EE DHA-EE 8A8e 2AG
Triarachidonoyl glycerol 1,2-capryloyl-3-arachidonoyl glycerol 1,3-capryloyl-2-arachidonoyl glycerol SUNTGA40S Arachidonic acid ethyl ester Docosahexaenoic acid ethyl ester SUN8A8-E 2-Arachidonoyl glycerol
Mortierella alpina [11]. SUN8A8-E contains 8A8, prepared through enzymatic transesterification of triacyl glycerol with Rhizopus delemer lipase [7,14]. 2-Arachidonoyl glycerol (2AG) is an endogenous ligand of the cannabinoid CB1 receptor [3]. Cannabinoids blocked A-induced microglial activation and prevented neurodegenerative processes occurring in AD [17]. These lipid compounds were dissolved in DMSO as stock solutions. The concentration of the lipids was 40 mM, except for 2AG, which was 4 mM. First, we determined the effect on ␣-secretase activity. ␣Secretase belongs to the “a disintegrin and metalloprotease” (ADAM) family; ADAM9, ADAM10, ADAM17, and ADAM19 have been identified as putative ␣-secretases [2,8,9,20]. In previous reports, we showed internal activity of ADAM9, 10, 17, and 19 in A172 glioblastoma cells by RNAi [1,20], for which reason we used A172 cells in this study. A172 cells at 90% confluence in a 6-cm dish were washed twice with phosphate-buffered saline (PBS) and 1 mL of serum-free Dulbecco’s modified Eagle’s medium (DMEM) containing 1/1000 volume of lipid stock solution. Following incubation for 4 h, the cells and media were collected. The medium was concentrated by precipitation with 10% trichloroacetic acid (TCA) and Western blot analysis was performed with the 6E10 antibody (Fig. 1). DHA-EE increased ␣-secretase activity by 30% (P = 0.02). Intriguingly, 8A8 increased ␣-secretase activity by 18% (P < 0.05), although ARA-EE, AAA, and 88A did not. When comparing results between 8A8 and 88A, the arachidonic acid in the sn-2 position was apparently important. It is thought that 8A8 is hydrolyzed to 2AG after being taken into cells. However, 2AG itself did not affect ␣-secretase activity, the relative activity of which was 1.07 times that of the control (P = 0.42). However, because 2AG is highly susceptible to oxidization in the medium, these results may be attributable to the rapid oxidization of 2AG before being taken into cells. Among triacylglycerols used in this study, 88A and 8A8 are about three times more stable to oxidation than AAA (data not shown). In our data, SUN8A8-E did not change the activity, the relative activity of which was 1.14 times that of the control (P = 0.16),
Fig. 1. Influence of lipids on constitutive ␣-secretase activity in A172 cells. (A) A172 cells were incubated with 40 M of lipid compounds or 4 M 2AG, dissolved in DMSO in serum-free media. Each medium also contained 0.1% DMSO. After a 4h incubation, the medium was concentrated and sAPP␣ content was determined by Western blot analysis using the 6E10 antibody. (B) Relative ␣-secretase activity, compared with the control, was calculated from (A) and adjusted for the protein content of the cell lysate. *Statistical significance compared to the control, based on a two-tailed Student’s t-test and values of p < 0.05.
although it contained 8A8. That may also be attributable to rapid oxidation. The susceptibility to oxygen of SUN8A8-E is more than that of SUNTGA40S [7]. Thus, chemically synthesized 8A8 was an effective arachidonyl-containing triglyceride to elevate ␣-secretase activity. Next, we analyzed the effects on -secretase activity. As secretase activity was not detected in A172 cells, we used HEK293 cells stably expressing BACE1 (BACE1-HEK) and treated them with lipids as described above. After incubation for 6 h, secreted APP (sAPP) in medium was detected with an antibody recognizing the C-terminal fragment of sAPP (Fig. 2). No significant effect was observed with any of the lipids. The relative activity of 8A8 was 96.7% compared to the control (P = 0.829). It is possible that the effects of the lipids may depend upon cell type and that the effect was too modest to be detected in the HEK system. Finally, we measured A40 contents in extracellular media by enzyme-linked immunosorbent assay (ELISA; Amersham Biosciences). A172 cells grown to 90% confluence in 3.5-cm dishes were washed twice with 1 mL of PBS, and 0.5 mL of serum-free DMEM containing 40 M lipid compounds was added. Following incubation for 4 h, the A40 content of the medium was measured by ELISA. Relative A40 generation is shown in Fig. 3. We used 30 nM phorbol 12-myristate 13-acetate (PMA; Wako), a PKC activator, as a positive control. PMA activates PKC-dependent ␣-
Fig. 2. Influence of lipids on -secretase activity in BACE1-HEK cells. BACE1-HEK cells were incubated with 40 M of lipid compounds or 4 M 2AG, as described in Fig. 1, for 6 h. BACE1 activity was determined according to the method described by Ebina et al. [4].
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References
Fig. 3. Effects of various lipids on A40 secretion in A172 cells. A172 cells were incubated with 40 M of lipid compounds dissolved in DMSO in serum-free medium (final DMSO conc., 0.1%). Relative A40 secretion, compared to the control, is presented. *Statistical significance compared to the control, based on a two-tailed Student’s t-test and values of p < 0.05.
secretase activity, competing with -secretase for the cleavage of APP in the trans-Golgi network [19]. PMA suppressed A40 production by 9% (P = 0.04). However, 8A8 showed much more suppression by 22% (P = 0.01). Contrary to our expectations, DHA-EE did not affect A40 secretion. The reason may be that the duration time of exposure of the cells to DHA-EE was too short. In a study using Tg2576 mice fed with high-DHA chow, there was a reduction in amyloid burden, but they had been fed for at least 3 months [10]. The report suggested that dietary DHA reduced both amyloidogenic and non-amyloidogenic pathways in those transgenic mice under those conditions. Our data (Fig. 1) differ from that report, but the elevation of ␣-secretase activity by DHA-EE seems to be at an early stage, at only 4 h after addition. Recently, the same group indicated that DHA increased the expression level of SorLA, which functions as a sorting protein that directs APP into the recycling endosome pathway, leading to a reduction in A production [12]. Thus, it is possible that DHA-mediated effects (through the regulation of SorLA expression) require more time to become apparent. However, 8A8 dramatically reduced A40 production at an early stage, by an unidentified mechanism. Since the effect is observed at 4 h, we suppose that 8A8 directly affects membrane lipids. To clarify the mechanism, we tried to isolate lipid rafts after 8A8 treatment. The content of lipid rafts, fractionated with 1% TritonX-100 lysis buffer (150 mM NaCl and 20 mM HEPES pH 7.5), decreased (data not shown). ADAM17 in lipid rafts also moved into non-lipid rafts. Thus, 8A8 may rapidly alter the membrane state and subsequent APP proteolysis. We conclude that 8A8 is a useful lipid compound activating ␣secretase activity and suppressing A40 secretion. Acknowledgments We thank Dr. Takaomi C. Saido (RIKEN) for providing the sAPP antibody and Dr. Yasuhiro Hashimoto (Fukushima Medical University) for providing BACE1-HEK cells. This work was supported by a grant from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and the Human Frontier Science Program.
[1] M. Asai, C. Hattori, B. Szabo, N. Sasagawa, K. Maruyama, S. Tanuma, S. Ishiura, Putative function of ADAM9, ADAM10, and ADAM17 as APP ␣-secretase, Biochem. Biophys. Res. Commun. 301 (2003) 231–235. [2] J.D. Buxbaum, K.N. Liu, Y. Luo, J.L. Slack, K.L. Stocking, J.J. Peschon, R.S. Johnson, B.J. Castner, D.P. Cerretti, R.A. Black, Evidence that tumor necrosis factor ␣ converting enzyme is involved in regulated ␣-secretase cleavage of the Alzheimer amyloid protein precursor, J. Biol. Chem. 273 (1998) 27765–27767. [3] V. Di Marzo, D. Melck, T. Bisogno, L. De Petrocellis, Endocannabinoids: endogenous cannabinoid receptor ligands with neuromodulatory action, Trends Neurosci. 21 (1998) 521–528. [4] M. Ebina, E. Futai, C. Tanabe, N. Sasagawa, Y. Kiso, S. Ishiura, Inhibition by KMI574 leads to dislocalization of BACE1 from lipid rafts, J. Neurosci. Res., in press. [5] R. Ehehalt, P. Keller, C. Haass, C. Thiele, K. Simons, Amyloidogenic processing of the Alzheimer -amyloid precursor protein depends on lipid rafts, J. Cell. Biol. 160 (2003) 113–123. [6] M.O. Grimm, H.S. Grimm, A.J. Pätzold, E.G. Zinser, R. Halonen, M. Duering, J.A. Tschäpe, B. De Strooper, U. Müller, J. Shen, T. Hartmann, Regulation of cholesterol and sphingomyelin metabolism by amyloid- and presenilin, Nat. Cell. Biol. 7 (2005) 1118–1123. [7] S. Kikuchi, X. Fang, M. Shima, K. Katana, H. Fukami, S. Adachi, Oxidation of arachidonoyl glycerols encapsulated with saccharides, Food Sci. Technol. Res. 12 (2006) 247–251. [8] H. Koike, S. Tomioka, H. Sorimachi, T.C. Saido, K. Maruyama, A. Okuyama, A. Fujisawa-Sehara, S. Ohno, K. Suzuki, S. Ishiura, Membrane-anchored metalloprotease MDC9 has an ␣-secretase activity responsible for processing the amyloid precursor protein, Biochem. J. 343 (1999) 371–375. [9] S. Lammich, E. Kojro, R. Postina, S. Gilbert, R. Pfeiffer, M. Jasionowski, C. Haass, F. Fahrenholz, Constitutive and regulated ␣-secretase cleavage of Alzheimer’s amyloid precursor protein by a disintegrin metalloprotease, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 3922–3927. [10] G.P. Lim, F. Calon, T. Morihara, F. Yang, B. Teter, O. Ubeda, N. Salem Jr., S.A. Frautschy, G.M. Cole, A diet enriched with the omega-3 fatty acid docosahexaenoic acid reduces amyloid burden in an aged Alzheimer mouse model, J. Neurosci. 25 (2005) 3032–3040. [11] B.A. Lina, A.P. Wolterbeek, Y. Suwa, S. Fujikawa, Y. Ishikura, S. Tsuda, M. Dohnalek, Subchronic (13-week) oral toxicity study, preceded by an in utero exposure phase, with arachidonate-enriched triglyceride oil (SUNTGA40S) in rats, Food Chem. Toxicol. 44 (2006) 326–335. [12] Q.L. Ma, B. Teter, O.J. Ubeda, T. Morihara, D. Dhoot, M.D. Nyby, M.L. Tuck, S.A. Frautschy, G.M. Cole, Omega-3 fatty acid docosahexaenoic acid increases SorLA/LR11, a sorting protein with reduced expression in sporadic Alzheimer’s disease (AD): relevance to AD prevention, J. Neurosci. 27 (2007) 14299–14307. [13] D.C. Mitchell, K. Gawrisch, B.J. Litman, N. Salem Jr., Why is docosahexaenoic acid essential for nervous system function? Biochem. Soc. Trans. 26 (1998) 365–370. [14] T. Nagao, A. Kawashima, M. Sumida, Y. Watanabe, K. Akimoto, H. Fukami, A. Sugihara, Y. Shimada, Production of structured TAG rich in 1,3-capryloyl-2arachidonoyl glycerol from Mortierella single-cell oil, J. Am. Oil Chem. Soc. 80 (2003) 867–872. [15] Y. Okaichi, Y. Ishikura, K. Akimoto, H. Kawashima, Y. Toyoda-Ono, Y. Kiso, H. Okaichi, Arachidonic acid improves aged rats’ spatial cognition, Physiol. Behav. 84 (2005) 617–623. [16] M.R. Prasad, M.A. Lovell, M. Yatin, H. Dhillon, W.R. Markesbery, Regional membrane phospholipids alterations in Alzheimer’s disease, Neurochem. Res. 23 (1998) 81–88. [17] B.G. Ramírez, C. Blázquez, T. Gómez del Pulgar, M. Guzmán, M.L. de Ceballos, Prevention of Alzheimer’s disease pathology by cannabinoids: neuroprotection mediated by blockade of microglial activation, J. Neurosci. 25 (2005) 1904–1913. [18] P.C. Schley, D.N. Brindely, C.J. Field, (n-3) PUFA alter raft lipid composition and decrease epidermal growth factor receptor levels in lipid rafts of human breast cancer cells, J. Nutr. 137 (2007) 548–553. [19] D.M. Skovronsky, D.B. Moore, M.E. Milla, R.W. Doms, V.M. Lee, Protein kinase C-dependent ␣-secretase competes with -secretase for cleavage of amyloid precursor protein in the trans-golgi network, J. Biol. Chem. 275 (2000) 2568–2575. [20] C. Tanabe, N. Hotoda, N. Sasagawa, A. Sehara-Fujisawa, K. Maruyama, S. Ishiura, ADAM19 is tightly associated with constitutive Alzheimer’s disease APP ␣secretase in A172 cells, Biochem. Biophys. Res. Commun. 352 (2007) 111–117. [21] J.H. Williams, M.L. Errington, M.A. Lynch, T.V. Bliss, Arachidonic acid induces a long-term activity-dependent enhancement of synaptic transmission in the hippocampus, Nature 341 (1989) 739–742.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
1,3-Capryloyl-2-arachidonoyl glycerol activates ␣-secretase activity and suppresses A40 secretion in A172 cells Chiaki Tanabe a,1 , Maiko Ebina a,1 , Masashi Asai b , Eugene Futai a , Noboru Sasagawa a , Kenji Katano c , Harukazu Fukami d , Shoichi Ishiura a,∗ a
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan Department of Pharmacology, Faculty of Medicine, Saitama Medical University, 38 Moroyama-machi, Moro-hongo, Iruma-gun, Saitama 350-0495, Japan Health Care Science Center, Suntory Technological Development Center, Suntory, Ltd., 5-2-5 Yamazaki, Shimamoto-cho, Mishima-gun, Osaka 618-0001, Japan d Department of Bioscience and Biotechnology, Faculty of Bioenvironmental Science, Kyoto Gakuen University, 1-1 Nanjyo, Sogabe-cho, Kameoka-city, Kyoto 621-8555, Japan b c
a r t i c l e
i n f o
Article history: Received 8 October 2008 Received in revised form 12 November 2008 Accepted 14 November 2008 Keywords: Alzheimer’s disease Amyloid precursor protein (APP) Arachidonic acid Medium chain fatty acid ␣-Secretase -Secretase
a b s t r a c t Alzheimer’s disease (AD) is characterized by the deposition of amyloid -peptide (A), derived from amyloid precursor protein (APP). Membrane states, such as lipid components or membrane fluidity, are important for enzymes related to APP processing in meeting their substrates efficiently. We analyzed the effects of triglycerides combined with polyunsaturated fatty acids (PUFAs) and/or caprylic acids on APP proteolysis. Our results showed that 1,3-capryloyl-2-arachidonoyl glycerol (8A8) moderately increased ␣-secretase activity (18%) in A172 cells. -Secretase activity was not statistically significantly changed in HEK293 cells stably expressing BACE1. However, A40 secretion decreased by 22%. Thus, we conclude that 8A8 is a useful lipid compound for activating ␣-secretase activity and suppressing A40 secretion. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Alzheimer’s disease (AD) is characterized by the formation of senile plaques, composed of amyloid -peptide (A). A production is initialized by -secretase, also known as -site APP cleaving enzyme 1 (BACE1), which cleaves amyloid precursor protein (APP), and then the remaining membrane-associated fragment is cleaved by ␥-secretase. However, APP is usually cleaved within the A sequence by ␣-secretase, precluding the generation of A. Activation of a non-amyloidogenic pathway or suppression of an amyloidogenic pathway can preclude A deposition. Recently, there have been several reports on the relationship between lipids and APP proteolysis. ␣-Secretase cleavage occurs in non-lipid rafts, whereas -secretase cleavage occurs in lipid rafts [5]. We previously showed that an effective BACE1 inhibitor changed the localization of BACE1 from a lipid raft to a non-lipid raft [4]. In another report, A and presenilin regulated cholesterol and
∗ Corresponding author. Tel.: +81 3 5454 6739 fax: +81 3 5454 6739. E-mail address: [email protected] (S. Ishiura). 1 These authors equally contributed to this work. This work is dedicated to late Miss Maiko Ebina. 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.11.039
sphingomyelin metabolism [6]. These findings suggest that lipid components and membrane fluidity are important for enzymes and substrates to meet each other efficiently. Furthermore, polyunsaturated fatty acids (PUFAs), especially n3 PUFAs, such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), are necessary in the development of the prenatal brain and in maintaining brain function and vision in adults [13]. Deficiencies in DHA, evidenced by low serum levels, have been linked to cognitive impairment and AD. Low levels of DHA have been shown in brain membranes of AD patients [16]. It has also been reported that DHA and EPA treatment in cultured cells reduced sphingomyelin and cholesterol contents in lipid rafts [18]. Thus, it is supposed that n-3 PUFA affects APP processing via lipid raft alterations. The n-6 PUFA, arachidonic acid (ARA), is also related to memory. Long-term administration of ARA-supplemented diets to aged rats alleviated age-related deficits in spatial cognition [15]. In another report, ARA induced long-term activity-dependent enhancement of synaptic transmission [21]. Here, we analyzed the effects of triglycerides combined with caprylic acids or PUFAs, which were synthesized by Suntory Co. Ltd., on APP proteolysis. The lipid compounds used in this study are shown in Table 1. SUNTGA40S is an arachidonate-enriched triglyceride oil extracted from a biomass of submerged fermented
C. Tanabe et al. / Neuroscience Letters 450 (2009) 324–326
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Table 1 The abbreviations of lipid compounds used in this study. Abbreviations
Compounds
AAA 88A 8A8 SUN ARA-EE DHA-EE 8A8e 2AG
Triarachidonoyl glycerol 1,2-capryloyl-3-arachidonoyl glycerol 1,3-capryloyl-2-arachidonoyl glycerol SUNTGA40S Arachidonic acid ethyl ester Docosahexaenoic acid ethyl ester SUN8A8-E 2-Arachidonoyl glycerol
Mortierella alpina [11]. SUN8A8-E contains 8A8, prepared through enzymatic transesterification of triacyl glycerol with Rhizopus delemer lipase [7,14]. 2-Arachidonoyl glycerol (2AG) is an endogenous ligand of the cannabinoid CB1 receptor [3]. Cannabinoids blocked A-induced microglial activation and prevented neurodegenerative processes occurring in AD [17]. These lipid compounds were dissolved in DMSO as stock solutions. The concentration of the lipids was 40 mM, except for 2AG, which was 4 mM. First, we determined the effect on ␣-secretase activity. ␣Secretase belongs to the “a disintegrin and metalloprotease” (ADAM) family; ADAM9, ADAM10, ADAM17, and ADAM19 have been identified as putative ␣-secretases [2,8,9,20]. In previous reports, we showed internal activity of ADAM9, 10, 17, and 19 in A172 glioblastoma cells by RNAi [1,20], for which reason we used A172 cells in this study. A172 cells at 90% confluence in a 6-cm dish were washed twice with phosphate-buffered saline (PBS) and 1 mL of serum-free Dulbecco’s modified Eagle’s medium (DMEM) containing 1/1000 volume of lipid stock solution. Following incubation for 4 h, the cells and media were collected. The medium was concentrated by precipitation with 10% trichloroacetic acid (TCA) and Western blot analysis was performed with the 6E10 antibody (Fig. 1). DHA-EE increased ␣-secretase activity by 30% (P = 0.02). Intriguingly, 8A8 increased ␣-secretase activity by 18% (P < 0.05), although ARA-EE, AAA, and 88A did not. When comparing results between 8A8 and 88A, the arachidonic acid in the sn-2 position was apparently important. It is thought that 8A8 is hydrolyzed to 2AG after being taken into cells. However, 2AG itself did not affect ␣-secretase activity, the relative activity of which was 1.07 times that of the control (P = 0.42). However, because 2AG is highly susceptible to oxidization in the medium, these results may be attributable to the rapid oxidization of 2AG before being taken into cells. Among triacylglycerols used in this study, 88A and 8A8 are about three times more stable to oxidation than AAA (data not shown). In our data, SUN8A8-E did not change the activity, the relative activity of which was 1.14 times that of the control (P = 0.16),
Fig. 1. Influence of lipids on constitutive ␣-secretase activity in A172 cells. (A) A172 cells were incubated with 40 M of lipid compounds or 4 M 2AG, dissolved in DMSO in serum-free media. Each medium also contained 0.1% DMSO. After a 4h incubation, the medium was concentrated and sAPP␣ content was determined by Western blot analysis using the 6E10 antibody. (B) Relative ␣-secretase activity, compared with the control, was calculated from (A) and adjusted for the protein content of the cell lysate. *Statistical significance compared to the control, based on a two-tailed Student’s t-test and values of p < 0.05.
although it contained 8A8. That may also be attributable to rapid oxidation. The susceptibility to oxygen of SUN8A8-E is more than that of SUNTGA40S [7]. Thus, chemically synthesized 8A8 was an effective arachidonyl-containing triglyceride to elevate ␣-secretase activity. Next, we analyzed the effects on -secretase activity. As secretase activity was not detected in A172 cells, we used HEK293 cells stably expressing BACE1 (BACE1-HEK) and treated them with lipids as described above. After incubation for 6 h, secreted APP (sAPP) in medium was detected with an antibody recognizing the C-terminal fragment of sAPP (Fig. 2). No significant effect was observed with any of the lipids. The relative activity of 8A8 was 96.7% compared to the control (P = 0.829). It is possible that the effects of the lipids may depend upon cell type and that the effect was too modest to be detected in the HEK system. Finally, we measured A40 contents in extracellular media by enzyme-linked immunosorbent assay (ELISA; Amersham Biosciences). A172 cells grown to 90% confluence in 3.5-cm dishes were washed twice with 1 mL of PBS, and 0.5 mL of serum-free DMEM containing 40 M lipid compounds was added. Following incubation for 4 h, the A40 content of the medium was measured by ELISA. Relative A40 generation is shown in Fig. 3. We used 30 nM phorbol 12-myristate 13-acetate (PMA; Wako), a PKC activator, as a positive control. PMA activates PKC-dependent ␣-
Fig. 2. Influence of lipids on -secretase activity in BACE1-HEK cells. BACE1-HEK cells were incubated with 40 M of lipid compounds or 4 M 2AG, as described in Fig. 1, for 6 h. BACE1 activity was determined according to the method described by Ebina et al. [4].
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References
Fig. 3. Effects of various lipids on A40 secretion in A172 cells. A172 cells were incubated with 40 M of lipid compounds dissolved in DMSO in serum-free medium (final DMSO conc., 0.1%). Relative A40 secretion, compared to the control, is presented. *Statistical significance compared to the control, based on a two-tailed Student’s t-test and values of p < 0.05.
secretase activity, competing with -secretase for the cleavage of APP in the trans-Golgi network [19]. PMA suppressed A40 production by 9% (P = 0.04). However, 8A8 showed much more suppression by 22% (P = 0.01). Contrary to our expectations, DHA-EE did not affect A40 secretion. The reason may be that the duration time of exposure of the cells to DHA-EE was too short. In a study using Tg2576 mice fed with high-DHA chow, there was a reduction in amyloid burden, but they had been fed for at least 3 months [10]. The report suggested that dietary DHA reduced both amyloidogenic and non-amyloidogenic pathways in those transgenic mice under those conditions. Our data (Fig. 1) differ from that report, but the elevation of ␣-secretase activity by DHA-EE seems to be at an early stage, at only 4 h after addition. Recently, the same group indicated that DHA increased the expression level of SorLA, which functions as a sorting protein that directs APP into the recycling endosome pathway, leading to a reduction in A production [12]. Thus, it is possible that DHA-mediated effects (through the regulation of SorLA expression) require more time to become apparent. However, 8A8 dramatically reduced A40 production at an early stage, by an unidentified mechanism. Since the effect is observed at 4 h, we suppose that 8A8 directly affects membrane lipids. To clarify the mechanism, we tried to isolate lipid rafts after 8A8 treatment. The content of lipid rafts, fractionated with 1% TritonX-100 lysis buffer (150 mM NaCl and 20 mM HEPES pH 7.5), decreased (data not shown). ADAM17 in lipid rafts also moved into non-lipid rafts. Thus, 8A8 may rapidly alter the membrane state and subsequent APP proteolysis. We conclude that 8A8 is a useful lipid compound activating ␣secretase activity and suppressing A40 secretion. Acknowledgments We thank Dr. Takaomi C. Saido (RIKEN) for providing the sAPP antibody and Dr. Yasuhiro Hashimoto (Fukushima Medical University) for providing BACE1-HEK cells. This work was supported by a grant from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and the Human Frontier Science Program.
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