Neurol Sci DOI 10.1007/s10072-014-1630-y
ORIGINAL ARTICLE
Betaine suppressed Ab generation by altering amyloid precursor protein processing Xiu-Ping Liu • Xiang Qian • Yue Xie • Yan Qi • Min-Feng Peng • Bi-Cui Zhan Zheng-Qing Lou
•
Received: 25 May 2013 / Accepted: 8 January 2014 Ó Springer-Verlag Italia 2014
Abstract Betaine was an endogenous catabolite of choline, which could be isolated from vegetables and marine products. Betaine could promote the metabolism of homocysteine in healthy subjects and was used for hyperlipidemia, coronary atherosclerosis, and fatty liver in clinic. Recent findings shown that Betaine rescued neuronal damage due to homocysteine induced Alzheimer’s disease (AD) like pathological cascade, including tau hyperphosphorylation and amyloid-b (Ab) deposition. Ab was derived from amyloid precursor protein (APP) processing, and was a triggering factor for AD pathological onset. Here, we demonstrated that Betaine reduced Ab levels by altering APP processing in N2a cells stably expressing Swedish mutant of APP. Betaine increased a-secretase activity, but decreased b-secretase activity. Our data indicate that Betaine might play a protective role in Ab production. Keywords Betaine Alzheimer’s disease Amyloid-b Amyloid precursor protein Introduction Alzheimer disease (AD) is one of the most prevalent neurodegenerative disorders worldwide, clinically characterized by X.-P. Liu (&) X. Qian Y. Xie Y. Qi M.-F. Peng B.-C. Zhan Z.-Q. Lou (&) Department of Clinical Laboratory, Hangzhou Hospital of Traditional Chinese Medicine, Hangzhou 310007, China e-mail: [email protected] Z.-Q. Lou e-mail: [email protected] X. Qian Zhejiang University School of Medicine, Hangzhou 310058, China
progressive impairments of cognitive and intellectual functions. Senile plaques (SP), neurofibrillary tangles (NFT), and synaptic loss within the affected brain regions especially the hippocampus are the specific pathological hallmarks of AD [1, 2]. Emerging evidence suggests that amyloid-b (Ab), the core component of SP, might play an essential and primary role in AD pathogenesis. Aggregation of extracellular Ab is a triggering event leading to a variety of neurotoxic cascades including activation of inflammatory factors, altered oxidative stress, neurotrophic signaling, Tau phosphorylation and synaptic dysfunction, which ultimately resulted in cognitive impairments and death in AD patients [3–5]. Ab was generated from amyloid precursor protein (APP) via sequential cleavage by b- and c-secretases. b-Secretase, known as beta-site APP cleaving enzyme (BACE), cleaved APP at the N-terminus, producing a soluble extracellular fragment (sAPPb) and a transmembrane C-terminal fragment (CTFb). CTFb was further cleaved by the c-secretase complex consisting of presenilins 1 and 2 (PS1 and PS2), Aph-1, Pen-2, nicastrin, and TMP21 to generate Ab40 or Ab42 [6]. Alternatively, APP was cleaved by a-secretase within the Ab domain, precluding the generation of Ab. Ab targeting treatment has been considered as a critical therapeutic strategy for the treatment of AD, and a variety of approaches were used to suppress Ab production or promote its clearance using b- or c-secretase inhibitors, as well as a-secretase agonist [7–9]. Betaine, known as trimethylglycine, is a methyl donor naturally occurring compound in vegetables and marine products, such as pretzels, shrimp, spinach, and wheat bran. In vivo, betaine is a catabolite of choline, and it acts as an effective methyl donor for homocysteine (Hcy) remethylation into methionine, as it contains three active methyl groups [10]. Epidemiology and clinical studies have shown
123
Neurol Sci
that the increased plasma Hcy was positively correlated with the onset of AD, and hyperhomocysteinemia was proposed to be an independent risk factor of AD [11]. Previous investigations reported that Betaine could promote the metabolism of Hcy in healthy subjects and was used for hyperlipidemia, coronary atherosclerosis, and fatty liver clinically. Recent findings also shown that Betaine could rescue the neuronal damage due to Hcy-induced Alzheimer’s disease (AD) like pathological cascades, including tau hyperphosphorylation and Ab deposition [12, 13]. Here, we investigated the role of Betaine in the modulation of APP processing and Ab production in N2a cells stably expressing Swedish mutant of APP (N2a/APP).
Western blot Western blot was performed as previous reported [14]. In brief, equal amount of proteins was loaded onto 15 % SDSPAGE gel, and electrotransferred to nitrocellulose membranes immunoblotted for specific primary antibodies. Immunoblots were visualized using HRP-conjugated secondary antibodies and the ECL Western Blot Detection kit (GE, NJ, USA). Analysis of plasma Hcy
Materials and methods
Treated cells were harvested and centrifuged at 2,0009g for 10 min at 4 °C, and the supernatant was used for detection of plasma Hcy. Plasma Hcy level was detected using fluorescence high-performance liquid chromatograph (HPLC) as described previously [15].
Reagents and cell culture
Statistical analysis
Betaine and hydrogen peroxide were purchased from Sigma-Aldrich Inc. (MO, USA). N2a cells were obtained from ATCC, and N2a/APP cells stably expressing Swedish mutant of APP were generated in our lab. Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum, 100 lg/mL streptomycin, 150 lg/mL hygromycin B, and 100 U/mL penicillin (Invitrogen, CA, USA) at 37 °C with 5 % CO2.
Data were expressed as the mean ± SD. For comparison among groups, ANOVA was used. The significance level was set at P \ 0.05.
Cellular toxicity analysis
The cytotoxic effect of Betaine in N2a/APP cells was determined by detecting LDH release. Betaine showed no obvious effect on LDH release from 0.1 to 100 lM for 48 h of incubation compared with vehicle treatment (Fig. 1). Hydrogen peroxide was used as a positive control, which strongly led to cell death. Betaine had no effect on
Cells were plated on 24-well plates at a density of 1 9 104 cells per well. After 24 h of incubation, cells were treated with Betaine at different concentrations. 48 h later, lactate dehydrogenase (LDH) activity was measured to evaluate the cytotoxicity of Betaine using the Cytotoxicity Detection Kit (abcam, Cambridge, UK). Hydrogen peroxide was used as a positive control.
Results Effect of Betaine on cell viability
Sandwich ELISA 48 h after Betaine treatment, the cultured media were collected and extracellular Ab levels were determined using the Human Ab Assay Kit (Invitrogen, CA, USA), according to the manufacturer’s instructions. b-Secretase activity assay Activity of b-secretase was determined by b-secretase Activity Assay Kit purchased from Calbiochem (CA, USA), following the manufacturer’s guidelines. The fluorescent signal was captured and analyzed in a Bio-Rad microplate reader (PA, USA).
123
Fig. 1 Effect of Betaine on cell toxicity in N2a/APP cells. Amount of LDH released into the media from N2a/APP cells treated with/ without Betaine was measured, and Hydrogen peroxide was used as a positive control. Data are drawn from five separate experiments. **P \ 0.01, compared with the vehicle-treated group
Neurol Sci Fig. 2 Reduction of Ab production by Betaine in N2a/ APP cells. Amounts of Ab released into the medium from N2a/APP cells treated with/ without Betaine were measured by sandwich ELISA. Data represent the mean ± SD of three independent experiments. **P \ 0.01, compared with the vehicle-treated group
Fig. 3 Alteration of APP processing by Betaine in N2a/ APP cells. Amounts of APP fragments and GAPDH from N2a/APP cells treated with/ without Betaine were assayed by Western blot. The typical blots of APP, sAPP, sAPPa, sAPPb, and GAPDH are shown. Data represent the mean ± SD of four independent experiments. *P \ 0.05, **P \ 0.01, compared with the vehicle-treated group
cell morphology or the multiplication rate of N2a/APP cells (data not shown).
secretase by a commercial kit. Results showed that Betaine significantly inhibited the activity of b-secretase compared with the vehicle group (Fig. 4).
Reduction in Ab levels by Betaine Betaine decreased plasma Hcy of N2a/APP cells Extracellular Ab levels in the medium from N2a/APP cells were measured by sandwich ELISA. 50 lM Betaine significantly reduced levels of Ab40 and Ab42 (Fig. 2).
Plasma Hcy levels of N2a/APP cells treated by Betaine were analyzed by fluorescence HPLC. 50 lM Betaine significantly decreased plasma Hcy level (Fig. 5).
Alteration of APP processing by Betaine The effects of Betaine on a- and/or b-secretase activities in N2a/APP cells were assayed by Western blot. Betaine did not significantly change APP expression in N2a/APP cells or sAPP secreted into the media. In contrast, Betaine increased sAPPa while downregulated sAPPb expression, produced by a- and b-secretase cleavage, respectively (Fig. 3). These data suggested that Betaine suppressed Ab production partially by stimulating a-secretase, while inhibiting b-secretase activity. Inhibition of b-secretase activity by Betaine To further confirm the hypothesis that Betaine might inhibit b-secretase activity, we detect the activity of b-
Discussion AD, characterized by numerous senile plaques and NFT in the brain, is a specific and the most common form of dementia in the elderly. The major component of senile plaques is Ab, a fragment of APP [1]. Up to now, the upstream factors leading to the formation of plaques were not fully illustrated. Accumulating epidemiologic and clinical data, as reviewed by Boldyrev AA [16], and Van Dam F [17] suggest a positive correlation between Hcy level and the pathogenesis of AD. Patients of AD with high Hcy levels displayed more rapid and serious neural atrophy than those with normal Hcy levels [18]. Hcy has been widely used to induce AD-like pathologic model. Betaine,
123
Neurol Sci Fig. 4 Inhibition of b-Secretase activity by Betaine in N2a/APP cells. b-Secretase activity of N2a/APP cells treated with/ without Betaine was measured using a sensitive fluorogenic assay kit. Data represent the mean ± SD of four independent experiments. ** P \ 0.01, compared with the vehicletreated group
Fig. 5 Betaine decreased plasma Hcy of N2a/APP cells. Plasma Hcy of N2a/APP cells treated with/without Betaine was measured by fluorescence HPLC. Data represent the mean ± SD of five independent experiments. ** P \ 0.01, compared with the vehicle-treated group
an endogenous catabolite of choline, served as an effective methyl donor for Hcy remethylation. Previous studies have shown that Betaine could attenuate AD-like Ab accumulation, Tau hyperphosphorylation, and behavioral impairments in Hcy-induced rat model. In this study, we investigated the role of Betaine in the modulation of APP metabolism, and found that Betaine treatment could effectively stimulate a-secretase activity and inhibit bsecretase activity, thus decreasing Ab generation. APP processing was tightly regulated by several secretase complexes, and abnormal APP metabolism occurred in both AD patients and AD animal models [19, 20]. APP was
123
cleaved by b-secretase BACE1 into sAPPb and CTFb, which was further cleaved by the c-secretase to generate Ab. Alternatively, APP underwent a-secretase mediated cleavage within the Ab domain, precluding the generation of Ab. Chai et al. reported that Betaine supplement could decrease Tau phosphorylation and Ab production in an Hcy-induced AD-like rat model. In that model, they have shown that Betaine reversed PS1 expression which was increased by Hcy [13]. Here, we found that in N2a/APP cells Betaine could promote a-secretase activity as shown by increased sAPPa protein level determined by Western blot. Furthermore, Betaine repressed b-secretase activity as shown by decreased sAPPb protein level, as well as suppressed b-secretase activity determined by a commercial fluorogenic b-secretase activity assay kit. Moreover, Betaine treatment significantly decreased plasma Hcy level as well. Our work was consistent with Lin HC et al. They reported that Betaine could reverse S-Adenosylhomocysteine induced hypomethylation of BACE1 promoter in BV2 mouse microglial cells. Betaine supplement could regulate the methylation level of gene promoter has been also found in other models. Our work expanded the biological function of Betaine more than a methyl donor for Hcy remethylation. Together, we have found in this study that Betaine could decrease Ab generation in N2a cells stably expressing Swedish mutant of APP with possible mechanisms that might involve in the upregulation of a-secretase and downregulation of b-secretase activity.
Neurol Sci Acknowledgments This work was supported in part by the National Natural Science Foundation of China (31300932).
References 1. Honjo K, Black SE, Verhoeff NP (2012) Alzheimer’s disease, cerebrovascular disease, and the beta-amyloid cascade. Can J Neurol Sci 39(6):712–728 2. Singh S, Kushwah AS, Singh R, Farswan M, Kaur R (2012) Current therapeutic strategy in Alzheimer’s disease. Eur Rev Med Pharmacol Sci 16(12):1651–1664 3. Lane RF, Shineman DW, Steele JW, Lee LB, Fillit HM (2012) Beyond amyloid: the future of therapeutics for Alzheimer’s disease. Adv Pharmacol 64:213–271. doi:10.1016/B978-0-12394816-8.00007-6 4. Takahashi RH, Capetillo-Zarate E, Lin MT, Milner TA, Gouras GK (2013) Accumulation of intraneuronal beta-amyloid 42 peptides is associated with early changes in microtubule-associated protein 2 in neurites and synapses. PLoS One 8(1):e51965. doi:10.1371/journal.pone.0051965 5. Seward ME, Swanson E, Norambuena A, Reimann A, Cochran JN, Li R, Roberson ED, Bloom GS (2013) Amyloid-beta signals through tau to drive ectopic neuronal cell cycle re-entry in Alzheimer’s disease. J Cell Sci. doi:10.1242/jcs.1125880 6. St George-Hyslop P, Fraser PE (2012) Assembly of the presenilin gamma-/epsilon-secretase complex. J Neurochem 120(Suppl 1):84–88. doi:10.1111/j.1471-4159.2011.07505.x 7. Wolfe MS (2012) gamma-Secretase as a target for Alzheimer’s disease. Adv Pharmacol 64:127–153. doi:10.1016/B978-0-12394816-8.00004-0 8. Ly PT, Wu Y, Zou H, Wang R, Zhou W, Kinoshita A, Zhang M, Yang Y, Cai F, Woodgett J, Song W (2013) Inhibition of GSK3beta-mediated BACE1 expression reduces Alzheimerassociated phenotypes. J Clin Invest 123(1):224–235. doi:10. 1172/JCI64516 9. Jeppsson F, Eketjall S, Janson J, Karlstrom S, Gustavsson S, Olsson LL, Radesater AC, Ploeger B, Cebers G, Kolmodin K, Swahn BM, von Berg S, Bueters T, Falting J (2012) Discovery of AZD3839, a potent and selective BACE1 inhibitor clinical candidate for the treatment of Alzheimer disease. J Biol Chem 287(49):41245–41257. doi:10.1074/jbc.M112.409110 10. Zeisel SH, Mar MH, Howe JC, Holden JM (2003) Concentrations of choline-containing compounds and betaine in common foods. J Nutr 133(5):1302–1307
11. Hooshmand B, Solomon A, Kareholt I, Leiviska J, Rusanen M, Ahtiluoto S, Winblad B, Laatikainen T, Soininen H, Kivipelto M (2010) Homocysteine and holotranscobalamin and the risk of Alzheimer disease: a longitudinal study. Neurology 75(16):1408–1414. doi:10.1212/WNL.0b013e3181f8816275/16/ 1408 12. Kharbanda KK, Mailliard ME, Baldwin CR, Beckenhauer HC, Sorrell MF, Tuma DJ (2007) Betaine attenuates alcoholic steatosis by restoring phosphatidylcholine generation via the phosphatidylethanolamine methyltransferase pathway. J Hepatol 46(2):314–321. doi:10.1016/j.jhep.2006.08.024 13. Chai GS, Jiang X, Ni ZF, Ma ZW, Xie AJ, Cheng XS, Wang Q, Wang JZ, Liu GP (2013) Betaine attenuates Alzheimer-like pathological changes and memory deficits induced by homocysteine. J Neurochem 124(3):388–396. doi:10.1111/jnc.12094 14. Liu XP, Zheng HY, Qu M, Zhang Y, Cao FY, Wang Q, Ke D, Liu GP, Wang JZ (2012) Upregulation of astrocytes protein phosphatase-2A stimulates astrocytes migration via inhibiting p38 MAPK in tg2576 mice. Glia 60(9):1279–1288. doi:10.1002/glia. 22347 15. Lu YQ, Luo Y, He ZF, Chen J, Yan BL, Wang Y, Yu Q (2013) Hydroxysafflor Yellow A ameliorates homocysteine induced Alzheimer-like pathologic dysfunction and memory/synaptic disorder. Rejuvenation Res. doi:10.1089/rej.2013.1451 16. Boldyrev AA, Johnson P (2007) Homocysteine and its derivatives as possible modulators of neuronal and non-neuronal cell glutamate receptors in Alzheimer’s disease. J Alzheimers Dis 11(2):219–228 17. Van Dam F, Van Gool WA (2009) Hyperhomocysteinemia and Alzheimer’s disease: a systematic review. Arch Gerontol Geriatr 48(3):425–430. doi:10.1016/j.archger.2008.03.009 18. Clarke R, Smith AD, Jobst KA, Refsum H, Sutton L, Ueland PM (1998) Folate, vitamin B12, and serum total homocysteine levels in confirmed Alzheimer disease. Arch Neurol 55(11):1449–1455 19. Zhang C (2012) Natural compounds that modulate BACE1-processing of amyloid-beta precursor protein in Alzheimer’s disease. Discov Med 14(76):189–197 20. Ferretti MT, Allard S, Partridge V, Ducatenzeiler A, Cuello AC (2012) Minocycline corrects early, pre-plaque neuroinflammation and inhibits BACE-1 in a transgenic model of Alzheimer’s disease-like amyloid pathology. J Neuroinflammation 9:62. doi:10. 1186/1742-2094-9-62
123
ORIGINAL ARTICLE
Betaine suppressed Ab generation by altering amyloid precursor protein processing Xiu-Ping Liu • Xiang Qian • Yue Xie • Yan Qi • Min-Feng Peng • Bi-Cui Zhan Zheng-Qing Lou
•
Received: 25 May 2013 / Accepted: 8 January 2014 Ó Springer-Verlag Italia 2014
Abstract Betaine was an endogenous catabolite of choline, which could be isolated from vegetables and marine products. Betaine could promote the metabolism of homocysteine in healthy subjects and was used for hyperlipidemia, coronary atherosclerosis, and fatty liver in clinic. Recent findings shown that Betaine rescued neuronal damage due to homocysteine induced Alzheimer’s disease (AD) like pathological cascade, including tau hyperphosphorylation and amyloid-b (Ab) deposition. Ab was derived from amyloid precursor protein (APP) processing, and was a triggering factor for AD pathological onset. Here, we demonstrated that Betaine reduced Ab levels by altering APP processing in N2a cells stably expressing Swedish mutant of APP. Betaine increased a-secretase activity, but decreased b-secretase activity. Our data indicate that Betaine might play a protective role in Ab production. Keywords Betaine Alzheimer’s disease Amyloid-b Amyloid precursor protein Introduction Alzheimer disease (AD) is one of the most prevalent neurodegenerative disorders worldwide, clinically characterized by X.-P. Liu (&) X. Qian Y. Xie Y. Qi M.-F. Peng B.-C. Zhan Z.-Q. Lou (&) Department of Clinical Laboratory, Hangzhou Hospital of Traditional Chinese Medicine, Hangzhou 310007, China e-mail: [email protected] Z.-Q. Lou e-mail: [email protected] X. Qian Zhejiang University School of Medicine, Hangzhou 310058, China
progressive impairments of cognitive and intellectual functions. Senile plaques (SP), neurofibrillary tangles (NFT), and synaptic loss within the affected brain regions especially the hippocampus are the specific pathological hallmarks of AD [1, 2]. Emerging evidence suggests that amyloid-b (Ab), the core component of SP, might play an essential and primary role in AD pathogenesis. Aggregation of extracellular Ab is a triggering event leading to a variety of neurotoxic cascades including activation of inflammatory factors, altered oxidative stress, neurotrophic signaling, Tau phosphorylation and synaptic dysfunction, which ultimately resulted in cognitive impairments and death in AD patients [3–5]. Ab was generated from amyloid precursor protein (APP) via sequential cleavage by b- and c-secretases. b-Secretase, known as beta-site APP cleaving enzyme (BACE), cleaved APP at the N-terminus, producing a soluble extracellular fragment (sAPPb) and a transmembrane C-terminal fragment (CTFb). CTFb was further cleaved by the c-secretase complex consisting of presenilins 1 and 2 (PS1 and PS2), Aph-1, Pen-2, nicastrin, and TMP21 to generate Ab40 or Ab42 [6]. Alternatively, APP was cleaved by a-secretase within the Ab domain, precluding the generation of Ab. Ab targeting treatment has been considered as a critical therapeutic strategy for the treatment of AD, and a variety of approaches were used to suppress Ab production or promote its clearance using b- or c-secretase inhibitors, as well as a-secretase agonist [7–9]. Betaine, known as trimethylglycine, is a methyl donor naturally occurring compound in vegetables and marine products, such as pretzels, shrimp, spinach, and wheat bran. In vivo, betaine is a catabolite of choline, and it acts as an effective methyl donor for homocysteine (Hcy) remethylation into methionine, as it contains three active methyl groups [10]. Epidemiology and clinical studies have shown
123
Neurol Sci
that the increased plasma Hcy was positively correlated with the onset of AD, and hyperhomocysteinemia was proposed to be an independent risk factor of AD [11]. Previous investigations reported that Betaine could promote the metabolism of Hcy in healthy subjects and was used for hyperlipidemia, coronary atherosclerosis, and fatty liver clinically. Recent findings also shown that Betaine could rescue the neuronal damage due to Hcy-induced Alzheimer’s disease (AD) like pathological cascades, including tau hyperphosphorylation and Ab deposition [12, 13]. Here, we investigated the role of Betaine in the modulation of APP processing and Ab production in N2a cells stably expressing Swedish mutant of APP (N2a/APP).
Western blot Western blot was performed as previous reported [14]. In brief, equal amount of proteins was loaded onto 15 % SDSPAGE gel, and electrotransferred to nitrocellulose membranes immunoblotted for specific primary antibodies. Immunoblots were visualized using HRP-conjugated secondary antibodies and the ECL Western Blot Detection kit (GE, NJ, USA). Analysis of plasma Hcy
Materials and methods
Treated cells were harvested and centrifuged at 2,0009g for 10 min at 4 °C, and the supernatant was used for detection of plasma Hcy. Plasma Hcy level was detected using fluorescence high-performance liquid chromatograph (HPLC) as described previously [15].
Reagents and cell culture
Statistical analysis
Betaine and hydrogen peroxide were purchased from Sigma-Aldrich Inc. (MO, USA). N2a cells were obtained from ATCC, and N2a/APP cells stably expressing Swedish mutant of APP were generated in our lab. Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum, 100 lg/mL streptomycin, 150 lg/mL hygromycin B, and 100 U/mL penicillin (Invitrogen, CA, USA) at 37 °C with 5 % CO2.
Data were expressed as the mean ± SD. For comparison among groups, ANOVA was used. The significance level was set at P \ 0.05.
Cellular toxicity analysis
The cytotoxic effect of Betaine in N2a/APP cells was determined by detecting LDH release. Betaine showed no obvious effect on LDH release from 0.1 to 100 lM for 48 h of incubation compared with vehicle treatment (Fig. 1). Hydrogen peroxide was used as a positive control, which strongly led to cell death. Betaine had no effect on
Cells were plated on 24-well plates at a density of 1 9 104 cells per well. After 24 h of incubation, cells were treated with Betaine at different concentrations. 48 h later, lactate dehydrogenase (LDH) activity was measured to evaluate the cytotoxicity of Betaine using the Cytotoxicity Detection Kit (abcam, Cambridge, UK). Hydrogen peroxide was used as a positive control.
Results Effect of Betaine on cell viability
Sandwich ELISA 48 h after Betaine treatment, the cultured media were collected and extracellular Ab levels were determined using the Human Ab Assay Kit (Invitrogen, CA, USA), according to the manufacturer’s instructions. b-Secretase activity assay Activity of b-secretase was determined by b-secretase Activity Assay Kit purchased from Calbiochem (CA, USA), following the manufacturer’s guidelines. The fluorescent signal was captured and analyzed in a Bio-Rad microplate reader (PA, USA).
123
Fig. 1 Effect of Betaine on cell toxicity in N2a/APP cells. Amount of LDH released into the media from N2a/APP cells treated with/ without Betaine was measured, and Hydrogen peroxide was used as a positive control. Data are drawn from five separate experiments. **P \ 0.01, compared with the vehicle-treated group
Neurol Sci Fig. 2 Reduction of Ab production by Betaine in N2a/ APP cells. Amounts of Ab released into the medium from N2a/APP cells treated with/ without Betaine were measured by sandwich ELISA. Data represent the mean ± SD of three independent experiments. **P \ 0.01, compared with the vehicle-treated group
Fig. 3 Alteration of APP processing by Betaine in N2a/ APP cells. Amounts of APP fragments and GAPDH from N2a/APP cells treated with/ without Betaine were assayed by Western blot. The typical blots of APP, sAPP, sAPPa, sAPPb, and GAPDH are shown. Data represent the mean ± SD of four independent experiments. *P \ 0.05, **P \ 0.01, compared with the vehicle-treated group
cell morphology or the multiplication rate of N2a/APP cells (data not shown).
secretase by a commercial kit. Results showed that Betaine significantly inhibited the activity of b-secretase compared with the vehicle group (Fig. 4).
Reduction in Ab levels by Betaine Betaine decreased plasma Hcy of N2a/APP cells Extracellular Ab levels in the medium from N2a/APP cells were measured by sandwich ELISA. 50 lM Betaine significantly reduced levels of Ab40 and Ab42 (Fig. 2).
Plasma Hcy levels of N2a/APP cells treated by Betaine were analyzed by fluorescence HPLC. 50 lM Betaine significantly decreased plasma Hcy level (Fig. 5).
Alteration of APP processing by Betaine The effects of Betaine on a- and/or b-secretase activities in N2a/APP cells were assayed by Western blot. Betaine did not significantly change APP expression in N2a/APP cells or sAPP secreted into the media. In contrast, Betaine increased sAPPa while downregulated sAPPb expression, produced by a- and b-secretase cleavage, respectively (Fig. 3). These data suggested that Betaine suppressed Ab production partially by stimulating a-secretase, while inhibiting b-secretase activity. Inhibition of b-secretase activity by Betaine To further confirm the hypothesis that Betaine might inhibit b-secretase activity, we detect the activity of b-
Discussion AD, characterized by numerous senile plaques and NFT in the brain, is a specific and the most common form of dementia in the elderly. The major component of senile plaques is Ab, a fragment of APP [1]. Up to now, the upstream factors leading to the formation of plaques were not fully illustrated. Accumulating epidemiologic and clinical data, as reviewed by Boldyrev AA [16], and Van Dam F [17] suggest a positive correlation between Hcy level and the pathogenesis of AD. Patients of AD with high Hcy levels displayed more rapid and serious neural atrophy than those with normal Hcy levels [18]. Hcy has been widely used to induce AD-like pathologic model. Betaine,
123
Neurol Sci Fig. 4 Inhibition of b-Secretase activity by Betaine in N2a/APP cells. b-Secretase activity of N2a/APP cells treated with/ without Betaine was measured using a sensitive fluorogenic assay kit. Data represent the mean ± SD of four independent experiments. ** P \ 0.01, compared with the vehicletreated group
Fig. 5 Betaine decreased plasma Hcy of N2a/APP cells. Plasma Hcy of N2a/APP cells treated with/without Betaine was measured by fluorescence HPLC. Data represent the mean ± SD of five independent experiments. ** P \ 0.01, compared with the vehicle-treated group
an endogenous catabolite of choline, served as an effective methyl donor for Hcy remethylation. Previous studies have shown that Betaine could attenuate AD-like Ab accumulation, Tau hyperphosphorylation, and behavioral impairments in Hcy-induced rat model. In this study, we investigated the role of Betaine in the modulation of APP metabolism, and found that Betaine treatment could effectively stimulate a-secretase activity and inhibit bsecretase activity, thus decreasing Ab generation. APP processing was tightly regulated by several secretase complexes, and abnormal APP metabolism occurred in both AD patients and AD animal models [19, 20]. APP was
123
cleaved by b-secretase BACE1 into sAPPb and CTFb, which was further cleaved by the c-secretase to generate Ab. Alternatively, APP underwent a-secretase mediated cleavage within the Ab domain, precluding the generation of Ab. Chai et al. reported that Betaine supplement could decrease Tau phosphorylation and Ab production in an Hcy-induced AD-like rat model. In that model, they have shown that Betaine reversed PS1 expression which was increased by Hcy [13]. Here, we found that in N2a/APP cells Betaine could promote a-secretase activity as shown by increased sAPPa protein level determined by Western blot. Furthermore, Betaine repressed b-secretase activity as shown by decreased sAPPb protein level, as well as suppressed b-secretase activity determined by a commercial fluorogenic b-secretase activity assay kit. Moreover, Betaine treatment significantly decreased plasma Hcy level as well. Our work was consistent with Lin HC et al. They reported that Betaine could reverse S-Adenosylhomocysteine induced hypomethylation of BACE1 promoter in BV2 mouse microglial cells. Betaine supplement could regulate the methylation level of gene promoter has been also found in other models. Our work expanded the biological function of Betaine more than a methyl donor for Hcy remethylation. Together, we have found in this study that Betaine could decrease Ab generation in N2a cells stably expressing Swedish mutant of APP with possible mechanisms that might involve in the upregulation of a-secretase and downregulation of b-secretase activity.
Neurol Sci Acknowledgments This work was supported in part by the National Natural Science Foundation of China (31300932).
References 1. Honjo K, Black SE, Verhoeff NP (2012) Alzheimer’s disease, cerebrovascular disease, and the beta-amyloid cascade. Can J Neurol Sci 39(6):712–728 2. Singh S, Kushwah AS, Singh R, Farswan M, Kaur R (2012) Current therapeutic strategy in Alzheimer’s disease. Eur Rev Med Pharmacol Sci 16(12):1651–1664 3. Lane RF, Shineman DW, Steele JW, Lee LB, Fillit HM (2012) Beyond amyloid: the future of therapeutics for Alzheimer’s disease. Adv Pharmacol 64:213–271. doi:10.1016/B978-0-12394816-8.00007-6 4. Takahashi RH, Capetillo-Zarate E, Lin MT, Milner TA, Gouras GK (2013) Accumulation of intraneuronal beta-amyloid 42 peptides is associated with early changes in microtubule-associated protein 2 in neurites and synapses. PLoS One 8(1):e51965. doi:10.1371/journal.pone.0051965 5. Seward ME, Swanson E, Norambuena A, Reimann A, Cochran JN, Li R, Roberson ED, Bloom GS (2013) Amyloid-beta signals through tau to drive ectopic neuronal cell cycle re-entry in Alzheimer’s disease. J Cell Sci. doi:10.1242/jcs.1125880 6. St George-Hyslop P, Fraser PE (2012) Assembly of the presenilin gamma-/epsilon-secretase complex. J Neurochem 120(Suppl 1):84–88. doi:10.1111/j.1471-4159.2011.07505.x 7. Wolfe MS (2012) gamma-Secretase as a target for Alzheimer’s disease. Adv Pharmacol 64:127–153. doi:10.1016/B978-0-12394816-8.00004-0 8. Ly PT, Wu Y, Zou H, Wang R, Zhou W, Kinoshita A, Zhang M, Yang Y, Cai F, Woodgett J, Song W (2013) Inhibition of GSK3beta-mediated BACE1 expression reduces Alzheimerassociated phenotypes. J Clin Invest 123(1):224–235. doi:10. 1172/JCI64516 9. Jeppsson F, Eketjall S, Janson J, Karlstrom S, Gustavsson S, Olsson LL, Radesater AC, Ploeger B, Cebers G, Kolmodin K, Swahn BM, von Berg S, Bueters T, Falting J (2012) Discovery of AZD3839, a potent and selective BACE1 inhibitor clinical candidate for the treatment of Alzheimer disease. J Biol Chem 287(49):41245–41257. doi:10.1074/jbc.M112.409110 10. Zeisel SH, Mar MH, Howe JC, Holden JM (2003) Concentrations of choline-containing compounds and betaine in common foods. J Nutr 133(5):1302–1307
11. Hooshmand B, Solomon A, Kareholt I, Leiviska J, Rusanen M, Ahtiluoto S, Winblad B, Laatikainen T, Soininen H, Kivipelto M (2010) Homocysteine and holotranscobalamin and the risk of Alzheimer disease: a longitudinal study. Neurology 75(16):1408–1414. doi:10.1212/WNL.0b013e3181f8816275/16/ 1408 12. Kharbanda KK, Mailliard ME, Baldwin CR, Beckenhauer HC, Sorrell MF, Tuma DJ (2007) Betaine attenuates alcoholic steatosis by restoring phosphatidylcholine generation via the phosphatidylethanolamine methyltransferase pathway. J Hepatol 46(2):314–321. doi:10.1016/j.jhep.2006.08.024 13. Chai GS, Jiang X, Ni ZF, Ma ZW, Xie AJ, Cheng XS, Wang Q, Wang JZ, Liu GP (2013) Betaine attenuates Alzheimer-like pathological changes and memory deficits induced by homocysteine. J Neurochem 124(3):388–396. doi:10.1111/jnc.12094 14. Liu XP, Zheng HY, Qu M, Zhang Y, Cao FY, Wang Q, Ke D, Liu GP, Wang JZ (2012) Upregulation of astrocytes protein phosphatase-2A stimulates astrocytes migration via inhibiting p38 MAPK in tg2576 mice. Glia 60(9):1279–1288. doi:10.1002/glia. 22347 15. Lu YQ, Luo Y, He ZF, Chen J, Yan BL, Wang Y, Yu Q (2013) Hydroxysafflor Yellow A ameliorates homocysteine induced Alzheimer-like pathologic dysfunction and memory/synaptic disorder. Rejuvenation Res. doi:10.1089/rej.2013.1451 16. Boldyrev AA, Johnson P (2007) Homocysteine and its derivatives as possible modulators of neuronal and non-neuronal cell glutamate receptors in Alzheimer’s disease. J Alzheimers Dis 11(2):219–228 17. Van Dam F, Van Gool WA (2009) Hyperhomocysteinemia and Alzheimer’s disease: a systematic review. Arch Gerontol Geriatr 48(3):425–430. doi:10.1016/j.archger.2008.03.009 18. Clarke R, Smith AD, Jobst KA, Refsum H, Sutton L, Ueland PM (1998) Folate, vitamin B12, and serum total homocysteine levels in confirmed Alzheimer disease. Arch Neurol 55(11):1449–1455 19. Zhang C (2012) Natural compounds that modulate BACE1-processing of amyloid-beta precursor protein in Alzheimer’s disease. Discov Med 14(76):189–197 20. Ferretti MT, Allard S, Partridge V, Ducatenzeiler A, Cuello AC (2012) Minocycline corrects early, pre-plaque neuroinflammation and inhibits BACE-1 in a transgenic model of Alzheimer’s disease-like amyloid pathology. J Neuroinflammation 9:62. doi:10. 1186/1742-2094-9-62
123
