Hydroxysafflor Yellow A Ameliorates Homocysteine-Induced Alzheimer-Like Pathologic Dysfunction and Memory/Synaptic Disorder

Abstract

Homocysteine (Hcy) could induce amyloid β (Aβ) accumulation, synaptic dysfunction, and memory impairment as seen in Alzheimer disease (AD), the most prevalent neurodegenerative disorder, which affects more than 25 million people worldwide. Here we investigated the protective effect of hydroxysafflor yellow A (HSYA) on Hcy-induced Aβ accumulation, synaptic dysfunction, and learning and memory deficits. Rats were randomly divided into four groups: Control group, which received normal saline (NS); Hcy group, which received a daily vena caudalis injection of Hcy (400 μg/kg per day); Hcy+HSYA group, which received the same amount of Hcy plus 6 mg/kg per day HSYA intraperitoneally; and HSYA group, which received 6 mg/kg per day HSYA intraperitoneally for 2 weeks. Results showed that simultaneous supplementation of HSYA significantly attenuated Aβ accumulation, improved synaptic function, and reversed Hcy-induced cognitive impairment. Our data suggest that HSYA might be a promising therapeutic candidate for attenuating Hcy-induced AD-like pathological and behavioral deficits.

Introduction

Alzheimer disease (AD) is the most prevalent age-associated dementia, affecting more than 25 million people worldwide with deterioration of memory capacities; it mainly affects people over 65 years old.¹ AD is thought to be caused by the abnormal accumulation of amyloid β (Aβ) peptide derived from sequential cleavage of amyloid precursor protein (APP), and by intracellular neurofibrillary tangles (NFTs) consisting of hyper-phosphorylated microtubule-associated protein Tau.^2,3 There is still no effective therapy for this devastating disease. Aggregated and accumulated Aβ is considered to be a triggering event in the progress of AD.⁴ Thus, arresting Aβ accumulation provides logical strategies for AD therapy.⁵

Hydroxysafflor yellow A (HSYA), a major active chemical compound extracted from the traditional Chinese drug Carthamus tinctorius L., was approved clinically in 2005 by the Chinese State Food and Drug Administration. HSYA has been used for cardiocerebrovascular diseases.⁶ HSYA exhibits numerous pharmacological properties, including anti-platelet aggregation, scavenging oxygen free radicals, lowering blood pressure and heart rate, and anti-inflammation and anti-tumor effects.^7,8 Moreover, HSYA showed neuroprotective effects on Aβ-induced neurotoxicity both in vitro and in vivo.^9,10

Homocysteine (Hcy) is an amino acid in the blood derived from the metabolism of methionine; it plays essential roles in cellular methylation for proteins, lipids, and DNA. Elevation of plasma Hcy is an independent risk factor for AD.^11,12 Mice with hyper-homocysteinemia exhibited a variety of the AD-like neuropathological changes, such as disruption of blood–brain barrier integrity, Aβ accumulation, and cognitive dysfunction.^13
–15 Previous studies reported that vena caudalis injection of Hcy-induced Aβ accumulation and tau hyper-phosphorylation.¹⁶ These data suggest that hyper-homocysteinemia induced by Hcy could serve as a promising animal model for AD studies.

In the present study, we found that HSYA could rescue the impairment of memory retention ability, attenuate Aβ accumulation, and restore the synaptic dysfunction induced by Hcy.

Materials and Methods

Antibodies and chemicals

The specific primary antibodies in this study are listed in Table 1. dl-Hcy was purchased from Sigma Chemical Co. (St Louis, MO). HSYA was obtained from the Chinese National Institute (Beijing, China). The peroxidase-conjugated secondary antibodies were from Pierce Chemical Co. (Pierce, IL).

Table 1.

Summary of Commercial Antibodies Used

Name	Catalog no.	Source	mAb/pAb	Dilution
PS1	ab15456	Abcam	mAb	1:1000
ADAM10	ab84595	Abcam	pAb	1:1000
BACE1	ab2077	Abcam	pAb	1:1000
GAPDH	ab9483	Abcam	pAb	1:2000
Bax	ab69643	Abcam	pAb	1:1000
Bcl-2	#2876	Cell Signaling	pAb	1:1000
Cleaved-PARP	ab2317	Abcam	pAb	1:500
PARP	ab6079	Abcam	pAb	1:1000

mAb, Monoclonal; pAb, polyclonal;

Animals and treatments

Male Sprague–Dawley rats (3 months old, 280±20 grams) were purchased from the Experimental Animal Center of Lanzhou University (Lanzhou, China). All rats were kept in cages under standard laboratory conditions—a 12-hr light–dark cycle and temperature at 22±2°C. All animal procedures were performed according to the Guide for Laboratory Animals Care and Use of Lanzhou University. Rats in the four groups were injected with Hcy (400 μg/kg per day) or saline in the same volume by vena caudalis for 2 weeks with or without an addition of HSYA (6 mg/kg per day) intraperitoneally 1 hr prior to the Hcy treatment. Animals were sacrificed 12 hr after the final injection.

Morris water maze

The spatial learning and memory ability was assessed using the Morris water maze.¹⁷ The water maze was performed in a black cylindrical tank. The tank was equally divided into four quadrants. A transparent platform was submerged 1.5 cm below the water surface in the target quadrant. During the 6 days of spatial training, the platform was fixed. The rats were trained to find the platform using cues available in the testing room. A trial began by placing the rat into the water facing the wall of the pool at one of the starting points in the four quadrants. The animals were placed into the water facing the wall and allowed to find the hidden platform within 60 sec and stay on it for 20 sec. If the rat failed, then the rat was guided to the platform and allowed to remain there for 20 sec. On day 7, the swimming pathway and escape latency were examined. On day 9, the platform was removed and the percentage of time spent in the target quadrant and crossing times were tested. The swimming path was analyzed by using a computer-connected video tracking system (Bogazici Biyomedikal, Istanbul, Turkey).

Enzyme-linked immunosorbent assay

For quantitative analysis of Aβ levels in hippocampus, we performed a sandwich enzyme-linked immunosorbent assay (ELISA). Briefly, rat hippocampus was extracted and homogenized, and 200 μg of sample protein was used for ELISA. Aβ40 and Aβ42 in samples were captured with G2-10 and G2-11 respectively, and then were probed specifically with Biotin-WO2 (Abeta GmBH, Heidelberg, Germany). Samples were further developed with horseradish peroxidase (HRP)-NeutrAvidin (Pierce, IL), and the HRP activity was determined by using TMB (3,3′,5,5′-Tetramethylbenzidine) as substrate (Kirkegaard, MD). A standard curve in a linear range of 0.0–50.0 pmol/gram of Aβ (R ²=0.9912) was used for quantitative analysis of Aβ.

Measurement of long-term potentiation

The rats were anesthetized with urethane (1.2 grams/kg) and fixed on the stereotaxic instrument (Narishige, Tokyo, Japan). The temperature of the animal body was maintained at 37°C in a bath circulator. A small hole was drilled at the appropriate coordinates for vertical penetration of the stimulating and recording electrodes immediately after the skull was exposed. The stimulating electrode was placed in a perforant path with coordinates of anteroposterior (AP) −6.9 to −7.1 mm, dorsoventral (DV) −3.4 to −3.6 mm, and mediolateral (ML)±4.4 to±4.6 mm according to the rat brain atlas, and the recording electrode was fixed in CA3 at coordinates of AP −3.4 to −3.6 mm, DV −3.2 to −3.4 mm, and ML±3.4 to±3.6 mm. For each measurement, a stable baseline (±10% change, at least 20 min) was required before treatment of high-frequency stimulation (HFS). Long-term potentiation (LTP) was elicited by HFS consisting of four trains of 50 pulses delivered at 200 Hz with a 2-sec inter-train interval. LTP was assessed by the measurement of excitatory postsynaptic potential and population spike.¹⁸

Western blot

Rat hippocampus were dissected and homogenized on ice in extraction buffer containing 50 mM Tris–HCl (pH 7.5), 1% (vol/vol) Triton X-100, 1% (wt/vol) deoxycholate, 150 mM NaCl, 0.1% (wt/vol) sodium dodecyl sulfate (SDS), 1 mM sodium orthovanadate (Na₃VO₄), 10 mM sodium fluoride (NaF), and 2 μg/mL each of leupeptin, aprotinin, and pepstatin A. The hippocampal extracts were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) gels and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking in 3% non-fat milk for 30 min at room temperature, the membranes were incubated with specific primary antibodies at 4°C overnight and further incubated with horseradish peroxidase (HRP)-linked secondary antibodies. The blots were visualized by electrochemiluminescence (ECL; Pierce, IL). The optical density was analyzed by Kodak Digital software (Eastman Kodak, CT).

Analysis of plasma Hcy

Rats were anesthetized with 6% chloral hydrate (400 mg/kg) intraperitoneally. Plasma Hcy level was detected by using fluorescence high-performance liquid chromatograph (HPLC), as described previously.¹⁹ Briefly, 60 μL plasma sample was mixed with 30 μL of 10% trichloroethyl phosphate (Sigma, NY) in 0.25 M borate buffer (pH 10.5). A 200-μL amount of 0.5 M perchloric acid containing 0.5 mM EDTA-Na₂ was added. The homogenized samples were centrifuged at 13,000×g for 15 min. To 30 μL of the supernatant, a mixture consisting of 30 μ of 7-fluorbenzo-2-oxa-1,3-diazle-4-sulfonic acid (4.25 mM in 0.1 M borate buffer [pH 10.5] containing 2 mM of EDTA) and 20 μL of 0.5 M NaOH were added. A 10μL sample of the supernatant was analyzed by fluorescence HPLC at an excitation of 385 nm and emission 515 nm. Samples were separated using a reversed-phase column (Thermo, FL). Analysis was performed under isocratic conditions at a flow rate of 1.2 mL/min for 15 min. A standard curve in a linear range of 0.0–40.0 μM of Hcy (R ²=0.9942) was used for quantitative analysis of Hcy.

Statistical analysis

All results were expressed as means±standard deviation (SD) and analyzed using SPSS 16.0 statistical software (SPSS, IL) by one-way analysis of variance (ANOVA) procedure followed by a least significant difference (LSD) post hoc test. p<0.05 was set as statistically significant.

Results

HSYA arrested Hcy induced learning and memory deficits

The Morris water maze test was used to investigate the protective effect of HSYA. The rats were injected with 400 μg/kg per day of Hcy or normal saline (NS) for 2 weeks with/without an addition of HSYA (6 mg/kg per day) intraperitoneally 1 hr prior to Hcy treatment. Rats were trained in the Morris water maze followed Hcy treatment. The latency to search for the hidden platform was much longer in rats injected with Hcy compared with the NS group for both the training and memory tests (Fig. 1A, B). On day 9, decreased crossing times and less time spent in the target quadrant were found in the Hcy-treated rats (Fig. 1C, D). A supplement of HSYA efficiently rescued the Hcy-induced learning and memory deficits.

FIG. 1.

Hydroxysafflor yellow A (HSYA) arrested homocysteine (Hcy)-induced learning and memory deficits. (A) The escape latency to search the hidden platform in the target quadrant from day 1 to day 6. (B) The escape latency to search the hidden platform on day 7. (C) The number to cross the hidden platform on day 9. (D) Time spent in the target quadrant on day 9. (**) p<0.01 compared with the normal saline (NS) group; (##) p<0.01, compared with the Hcy group. Color images available online at www.liebertpub.com/rej

HSYA attenuated Hcy induced Aβ accumulation

As reported previously, vena caudalis injection of Hcy for 2 weeks could induce Aβ accumulation.¹⁶ We explored the effect of HSYA on Aβ accumulation in the brain hippocampus. Hcy increased both Aβ40 and Aβ42 levels in hippocampus, whereas HSYA treatment reversed the effect of Hcy (Fig. 2A, B). To further investigate the possible underlying mechanisms, we detected the expression of α-secretase, ADAM10, β-secretase, BACE1, and PS1 by western blotting. Supplementation of HSYA attenuated the elevation of PS1 induced by Hcy, whereas expression of ADAM10 and BACE1 were not obviously changed (Fig. 2C). To confirm the role of Hcy, we detected the plasma level of Hcy by fluorescence HPLC. A remarkable elevation of Hcy was observed after the injection (Fig. 2D).

FIG. 2.

Hydroxysafflor yellow A (HSYA)-attenuated homocysteine (Hcy)-induced Aβ accumulation. Hcy (400 μg/kg per day) was injected with or without addition of HSYA (6 mg/kg per day) for 2 weeks. Rats were sacrificed, and Aβ40 and Aβ42 levels in hippocampus were measured by enzyme-linked immunosorbent assay (ELISA) (A, B). Amyloid precursor protein (APP)-processing enzymes, α-secretase ADAM10, β-secretase BACE1, and PS1 protein levels were detected by western blotting (C). The plasma level of Hcy was measured by fluorescence high-performance liquid chromatography (HPLC) as described in the Materials and Methods section (D). The data were drawn from three separate experiments. (*) p<0.05; (**) p<0.01 compared with the normal saline (NS) group; (#) p<0.05; (##) p<0.01, compared with the Hcy group. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

HSYA rescued Hcy induced apoptosis

To examine whether HSYA could prevent the apoptosis induced by Hcy, we measured the expression of important molecules involved in the apoptosis pathway. Hcy increased expression of the pro-apoptosis molecule Bax, while inhibiting expression of the anti-apoptosis molecule Bcl-2. HSYA supplementation significantly reversed apoptosis induced by Hcy. The inhibitory effect of HSYA on apoptosis was supported by decreased level of the poly(ADP ribose) polymerase (PARP) cleavage product as well (Fig. 3A, B). These data suggest that HSYA could rescue Hcy-induced apoptosis.

FIG. 3.

Hydroxysafflor yellow A (HSYA) rescued homocysteine (Hcy)-induced apoptosis. Apoptosis was assessed by measuring the expression of Bax, Bcl-2, and cleaved poly(ADP ribose) polymerase (PARP) (A). Relative intensity was analyzed (B). Data are presented as mean±standard deviation (SD) (n=4). (*) p<0.05 compared with the normal saline (NS) group; (#) p<0.05 compared with Hcy group. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

HSYA reversed Hcy induced synaptic deficits

Synaptic plasticity is essential for learning and memory, and we determined the synaptic transmission by recording LTP. We found that in Hcy-treated rats, the slope and amplitude of field excitatory postsynaptic potential (fEPSP) increased to ∼1.3-fold and 1.4-fold, much lower than the NS group. Adding HSYA restored the amplitude and slope of fEPSP (Fig. 4A–C). These data show that HSYA could efficiently rescue the synaptic deficits induced by Hcy.

FIG. 4.

Hydroxysafflor yellow A (HSYA) reversed homocysteine (Hcy)-induced synaptic deficits. Hcy (400 μg/kg per day) was injected with or without addition of HSYA (6 mg/kg per day) for 2 weeks. Long-term potentiation (LTP) was detected in rats. (A) The representative analog traces of evoked potential before (pre-, broken line) and after (post-, solid line) high-frequency stimulation (HFS). (B) Normalized slope of excitatory postsynaptic potential (EPSP). (C) Normalized population spike (PS) amplitude. The data are expressed as mean±standard deviation (SD) (n=5). Color images available online at www.liebertpub.com/rej

Discussion

AD, which is characterized pathologically by numerous intracellular neurofibrillary tangles and intercellular senile plaques in the affected brain regions, is a specific and the most prevalent form of dementia in the elderly. Until now, the upstream factors triggering the formation of plaques were poorly understood. More than 40% of AD patients showed high Hcy levels in the plasma, displaying more rapid neural atrophy than those with normal Hcy levels.²⁰ Recently Ray et al reported that both AD and vascular dementia (VAD) subjects showed markedly elevated serum Hcy compared to control subjects.²¹ Moreover, Chacón et al. found that elevated total Hcy (tHcy) was significantly associated with dementia, and increased tHcy was a significant risk factor in older (>66 years), but not in younger (55–66 years), subjects in a Venezuelan population that had high prevalence of both increased tHcy and dementia.²² In AD, Aβ accumulation plays an essential role and co-exists with varying degrees of cerebrovascular dysfunctions. The elevated serum Hcy in AD subjects was thought to contribute to the genesis of the vascular pathology of AD.²³ Numerous studies had been performed to explore the direct neurotoxic effect of Hcy using cultured SHSY5Y cells, primary hippocampal neurons, rat brain cortical slices, and rat in vivo.^24
–26 Hyper-homocysteinemia was correlated with brain amyloidosis, Tau phosphorylation, electrophysiological properties, and cognitive impairment.^16,25,27 As a result, high Hcy has been proposed to be an independent risk factor of AD. Attenuating the elevated plasma Hcy level might be a potential strategy for AD therapy.²⁸ In the present study, we found that supplementation of HSYA could effectively rescue Hcy-induced learning and memory deficits, Aβ accumulation, and synaptic dysfunction in rats. Our data suggest that HSYA might serve as a potential candidate for AD therapy.

It is well known that Aβ is produced when APP is sequentially cleaved by β- and γ-secretases.²⁹ PS1 is the catalytic subunit of the β-secretase, and exogenous over-expression of PS1 stimulated Aβ production.^30,31 Previous studies demonstrated that Hcy could increase PS1 expression and promote Aβ production.³² In this study, we found that HSYA significantly decreased both Aβ40 and Aβ42 levels partially through suppressing PS1 expression, which was elevated by Hcy. Unlike previously reported agents, such as VitB12/folate and betaine, HSYA did not affect the plasma Hcy level, suggesting that HSYA might work to counteract the downstream molecules affected by Hcy, or it might function competitively with Hcy and restore the effect of Hcy. Elevated plasma Hcy could induce Aβ accumulation, and HSYA might play a protective role in Hcy-induced Aβ accumulation partially by suppressing PS1 expression.

Emerging evidence showed Aβ deposition could increase pro-apoptosis factor expression, while suppressing anti-apoptosis molecule expression. In an Hcy-induced AD-like rat model, increased apoptosis was observed, as shown by increased expression of pro-apoptosis Bax expression and PARP cleavage. Hcy also decreased expression of the anti-apoptosis molecule Bcl-2. Supplementation of HSYA could efficiently rescue Hcy-induced apoptosis. Our in vivo study was consistent with previous in vitro studies.^9,33

Synaptic plasticity, which was disrupted in AD patients and AD animal models, could be assessed by LTP and was a prerequisite of learning and memory.^34,35 By detecting the alterations of EPSP slope and PS amplitude, well-accepted parameters for LTP measurement in vivo, we found that Hcy injection inhibited LTP in rat hippocampus, whereas supplementing with HSYA ameliorated the inhibitory effect of Hcy on LTP.

Taken together, we demonstrated in the present study that supplementation of HSYA significantly rescued Hcy-induced behavior impairment, Aβ accumulation, apoptosis, and synaptic dysfunction in rats. Our data suggest that HSYA might be a promising therapeutic candidate for attenuating Hcy-induced AD-like pathological and behavioral deficits.

Footnotes

Author Disclosure Statement

The authors have no conflict of interest to declare.

References

Qiu

, Kivipelto

, von Strauss

. Epidemiology of Alzheimer's disease: Occurrence, determinants, and strategies toward intervention. Dialogues Clin Neurosci, 2009; 11:111–128.

Bassil

, Mollaei

. Alzheimer's dementia: A brief review. J Med Liban, 2012; 60:192–199.

Bettens

, Sleegers

, Van Broeckhoven

. Genetic insights in Alzheimer's disease. Lancet Neurol, 2013; 12:92–104.

Iwata

, Higuchi

, Saido

. Metabolism of amyloid-beta peptide and Alzheimer's disease. Pharmacol Ther, 2005; 108:129–148.

Honjo

, Black

, Verhoeff

. Alzheimer's disease, cerebrovascular disease, and the beta-amyloid cascade. Can J Neurol Sci, 2012; 39:712–728.

, Zhu

, et al. Hydroxysafflor yellow A enhances survival of vascular endothelial cells under hypoxia via upregulation of the HIF-1 alpha-VEGF pathway and regulation of Bcl-2/Bax. J Cardiovasc Pharmacol, 2008; 52:191–202.

, Wang

, Jin

, Zang

. Hydroxysafflor yellow A alleviates early inflammatory response of bleomycin-induced mice lung injury. Biol Pharm Bull, 2012; 35:515–522.

Nie

, Zhang

, et al. The effects of hydroxysafflor yellow A on blood pressure and cardiac function. J Ethnopharmacol, 2012; 139:746–750.

Kong

, Xian

, Ip

, et al. Protective effects of hydroxysafflor yellow A on beta-amyloid-induced neurotoxicity in PC12 Cells. Neurochem Res, 2013; 38:951–960.

10.

Zhu

, Wang

, Ma

, et al. Neuroprotective effects of hydroxysafflor yellow A: In vivo and in vitro studies. Planta Med, 2003; 69:429–433.

11.

Sachdev

. Homocysteine, cerebrovascular disease and brain atrophy. J Neurol Sci, 2004; 226:25–29.

12.

Van Dam

, Van Gool

. Hyperhomocysteinemia and Alzheimer's disease: A systematic review. Arch Gerontol Geriatr, 2009; 48:425–430.

13.

Zhuo

, Portugal

, Kruger

, et al. Diet-induced hyperhomocysteinemia increases amyloid-beta formation and deposition in a mouse model of Alzheimer's disease. Curr Alzheimer Res, 2010; 7:140–149.

14.

Kamath

, Chauhan

, Kisucka

, et al. Elevated levels of homocysteine compromise blood-brain barrier integrity in mice. Blood, 2006; 107:591–593.

15.

Troen

, Shea-Budgell

, Shukitt-Hale

, et al. B-vitamin deficiency causes hyperhomocysteinemia and vascular cognitive impairment in mice. Proc Natl Acad Sci USA, 2008; 105:12474–12479.

16.

Zhou

, Chen

, Zhao

, et al. Acetyl-L-carnitine attenuates homocysteine-induced Alzheimer-like histopathological and behavioral abnormalities. Rejuvenation Res, 2011; 14:669–679.

17.

Morris

, Garrud

, Rawlins

, O'Keefe

. Place navigation impaired in rats with hippocampal lesions. Nature, 1982; 297:681–683.

18.

Wang

, Wang

, Wu

, et al. Melatonin attenuates scopolamine-induced memory/synaptic disorder by rescuing EPACs/miR-124/Egr1 pathway. Mol Neurobiol, 2013; 47:373–381.

19.

Zhang

, Tian

, Wei

, et al. Homocysteine induces tau phosphorylation by inactivating protein phosphatase 2A in rat hippocampus. Neurobiol Aging, 2008; 29:1654–1665.

20.

Clarke

, Smith

, Jobst

, et al. Folate, vitamin B12, and serum total homocysteine levels in confirmed Alzheimer disease. Arch Neurol. Nov, 1998; 55:1449–1455.

21.

Ray

, Khemka

, Behera

, et al. Serum homocysteine, dehydroepiandrosterone sulphate and lipoprotein (a) in Alzheimer's disease and vascular dementia. Aging Dis, 2013; 4:57–64.

22.

Chacon

, Molero

, Pino-Ramirez

, et al. Risk of dementia associated with elevated plasma homocysteine in a Latin American population. Int J Alzheimers Dis, 2009; 2009.

23.

de la Torre

. Alzheimer disease as a vascular disorder: Nosological evidence. Stroke, 2002; 33:1152–1162.

24.

Park

, Ko

, Jang

, Kwon

. Activation of AMP-activated protein kinase alleviates homocysteine-mediated neurotoxicity in SH-SY5Y cells. Neurochem Res Aug, 2013; 38:1561–1571.

25.

Schaub

, Uebachs

, Beck

, Linnebank

. Chronic homocysteine exposure causes changes in the intrinsic electrophysiological properties of cultured hippocampal neurons. Exp Brain Res, 2013; 225:527–534.

26.

Borowczyk

, Tisonczyk

, Jakubowski

. Metabolism and neurotoxicity of homocysteine thiolactone in mice: Protective role of bleomycin hydrolase. Amino Acids, 2012; 43:1339–1348.

27.

Zhuo

, Pratico

. Normalization of hyperhomocysteinemia improves cognitive deficits and ameliorates brain amyloidosis of a transgenic mouse model of Alzheimer's disease. FASEB J, 2010; 24:3895–3902.

28.

Seshadri

, Beiser

, Selhub

, et al. Plasma homocysteine as a risk factor for dementia and Alzheimer's disease. N Engl J Med, 2002; 346:476–483.

29.

Kapoor

, Wang

, Hsu

, et al. Retinoic acid-elicited RARalpha/RXRalpha signaling attenuates Abeta production by directly inhibiting gamma-secretase-mediated cleavage of amyloid precursor protein. ACS Chem Neurosci Mar 26 2013.

30.

Okochi

, Tagami

, Yanagida

, et al. gamma-secretase modulators and presenilin 1 mutants act differently on presenilin/gamma-secretase function to cleave Abeta42 and Abeta43. Cell Rep, 2013; 3:42–51.

31.

Kimberly

, Wolfe

. Identity and function of gamma-secretase. J Neurosci Res, 2003; 74:353–360.

32.

Lin

, Hsieh

, Chen

, Hu

. S-Adenosylhomocysteine increases beta-amyloid formation in BV-2 microglial cells by increased expressions of beta-amyloid precursor protein and presenilin 1 and by hypomethylation of these gene promoters. Neurotoxicology, 2009; 30:622–627.

33.

Wang

, Bai

, Chen

, et al. Homocysteine induces apoptosis of rat hippocampal neurons by inhibiting 14-3-3epsilon expression and activating calcineurin. PLoS One, 2012; 7:e48247.

34.

Johnstone

, Raymond

. Postsynaptic protein synthesis is required for presynaptic enhancement in persistent forms of long-term potentiation. Front Synaptic Neurosci, 2013; 5:1.

35.

Huang

, Zhu

, Zhang

, et al. mTORC2 controls actin polymerization required for consolidation of long-term memory. Nat Neurosci, 2013; 16:441–448.