Dietary Lactoferrin Supplementation Prevents Memory Impairment and Reduces Amyloid-β Generation in J20 Mice

Abstract

Lactoferrin (LF) is present in senile plaques and neurofibrillary tangles in the brains of Alzheimer’s disease (AD) patients and amyloid-β protein precursor transgenic (AβPP-Tg) mice. LF has anti-inflammatory and antioxidant functions, which exert neuroprotective effects against AD. However, its effects on memory impairment and AD pathogenesis have not been fully examined. In this study, we examined the effects of LF on memory impairment and AD pathogenesis in AβPP-Tg mice (J20 mice). Nine-month-old J20 mice were fed with control, 2% lactoferrin-containing (LF), and 0.5% pepsin-hydrolyzed lactoferrin-containing (LF-hyd) diets for 3 months. We found that both the LF and LF-hyd diets attenuated memory impairment in J20 mice and decreased brain Aβ₄₀ and Aβ₄₂ levels through the inhibition of amyloidogenic processing of AβPP, as it decreased β-site amyloid protein precursor cleaving enzyme 1 (BACE1) levels. Furthermore, we found for the first time that LF and LF-hyd treatments increased both ApoE secretion and ATP-binding cassette A1 (ABCA1) protein levels in the brains of J20 mice and in primary astrocyte cultures. Moreover, LF and LF-hyd promoted extracellular degradation of Aβ in primary astrocyte cultures. These findings indicate that the reduction in Aβ levels in the brains of mice fed with both the LF and LF-hyd diets may also be mediated by increased ApoE secretion and ABCA1 protein levels, which in turn leads to the enhanced degradation of Aβ in the brains of J20 mice. Our findings suggest that LF and LF-hyd can be used for the treatment and/or prevention of the development of AD.

Keywords

ABCA1 Alzheimer’s disease amyloid-β apolipoprotein E BACE1 ERK1/2 MAPK lactoferrin

INTRODUCTION

Alzheimer’s disease (AD), which is characterized by the loss of memory and other cognitive functions, is a progressive neurodegenerative disease and is the most common cause of dementia in the elderly [1]. The two major histopathological hallmarks of AD are extracellular senile plaques consisting of amyloid-β (Aβ) peptides [2] and intracellular neurofibrillary tangles composed of an abnormal hyperphosphorylated tau protein [3]. Aβ peptides are the products of the proteolytic cleavage of amyloid-β protein precursor (AβPP) by β-secretase and γ-secretase [4]. According to the amyloid hypothesis, an increase in Aβ levels is the result of an imbalance between Aβ production and clearance or a decrease in Aβ proteolytic degradation [5]. Aβ can undergo proteolytic degradation intracellularly in microglia and astrocytes via neprilysin and extracellulary via an insulin-degrading enzyme (IDE) and lipidated apolipoprotein E (ApoE-HDL).

ApoE is the major apolipoprotein in the central nervous system (CNS) and is synthesized and secreted mainly by astrocytes, and also by microglia [6, 7]. AβPP transgenic mice lacking the murine ApoE gene have markedly elevated levels of Aβ peptides in their brains [8]. Conversely, AβPP transgenic mice overexpressing the human ApoE gene exhibit a delay in the onset of plaque deposition and a significant reduction in plaque burden, suggesting that ApoE inhibits Aβ deposition and promotes Aβ clearance [9]. Regarding Aβ clearance, ApoE-HDL binds to Aβ and removes it [10, 11] through either extracellular degradation by IDE or astroglial uptake followed by lysosomal degradation. Furthermore, the lipidation status of ApoE has been shown to regulate its Aβ-binding properties and its degree of lipidation affects the capability of ApoE to promote extra- and intracellular Aβ proteolysis [10].

Lipidation of ApoE is mediated primarily by ATP-binding cassette A1 (ABCA1) in the brain, and ABCA1 transfers cholesterol and phospholipid to ApoE to form HDL particles [12], and this function is ApoE-isoform specific [13, 14]. Direct evidence that ABCA1-mediated lipidation of ApoE affects Aβ degradation has been demonstrated in AβPP transgenic models of AD. ABCA1 knockout mice show decreased levels of lipidated ApoE, and primary astrocyte cultures prepared from ABCA1 knockout mice secrete low levels of ApoE-HDL [15], and the deletion of ABCA1 results in an enhanced Aβ deposition in the PDAPP transgenic mouse model of AD [16]. It has also been reported that the overexpression of ABCA1 results in higher levels of lipidated ApoE leading to the reduction of Aβ deposition [17]. Taken together, these findings indicate that ApoE levels and the ABCA1-mediated lipidation status of ApoE may be crucial to Aβ degradation and clearance in AD pathogenesis.

Lactoferrin (LF), a glycoprotein and member of the transferrin family, is abundant in exocrine fluids such as breast milk, saliva, tears, and in mucosal secretions [18, 19]. Because of its wide distribution in various tissues, LF is a multifunctional protein and is considered to be involved in many physiological functions, including anti-inflammatory activity, cellular proliferation and differentiation, and cancer protection [18, 19]. LF is the main iron-binding protein [20], and it chelates free iron to prevent the formation of reactive oxygen species (ROS) and increases antioxidant capacity [21 –23]. The anti-inflammatory function of LF depends on its binding to free iron and the prevention of oxidative stress formation or the inhibition of the binding of bacterial lipopolysaccharide and inflammatory cells, inducing the inhibition of inflammatory cytokine production. LF has been shown to be involved in neurodegenerative diseases such as Parkinson’s disease and prion disease. LF levels are upregulated in dopaminergic neurons resistant to degeneration in Parkinson’s disease [24], and LF protects against MPP-induced neuronal cell death in the ventral mesencephalon via the reduction of ROS generation [25]. LF treatment prevents neuronal cell death caused by prion proteins through the prevention of ROS formation [26]. Since LF receptors (LFRs) are present on the membrane of vascular endothelial cells in the blood-brain barrier (BBB), exogenous LF can easily cross the BBB; thus, LF has been widely used as a carrier for drug targeting in the brain [27, 28]. It has also been reported that LF is present in senile plaques and neurofibrillary tangles in the brains of AD patients [29] and AβPP transgenic mice, which are used as a mouse model of AD [30], and it may have neuroprotective effects against AD through a combination of its anti-inflammatory and antioxidant functions. Furthermore, Carro et al. demonstrated that salivary lactoferrin concentration was significantly reduced in AD patients when compared to healthy controls, which suggest that lactoferrin is impressive new candidate to be one of the first salivary biomarkers for AD early detection and diagnosis [31]. Although LF has been shown to exhibit anti-inflammatory and antioxidant functions in AD, its effects on memory impairment and AD pathogenesis have not been fully examined.

Thus, in this study, we determined the effects of LF on memory impairment and AD pathogenesis including its effects on Aβ production and degradation, ApoE secretion, ABCA1 expression, and AβPP metabolism in the J20-AβPP transgenic mouse model of AD and in primary rat astrocytes. Here, we report that both LF-containing (LF) and pepsin-hydrolyzed LF-containing (LF-hyd) diets attenuated memory impairment and decreased Aβ levels through the reduction in BACE1 levels in the brains of J20 mice. Furthermore, we demonstrated for the first time that both LF and LF-hyd increased ApoE secretion and ABCA1 protein levels in primary astrocytes and J20 mouse brains, which in turn may decrease Aβ levels via Aβ degradation in the brains of J20 mice. These findings suggest that LF and LF-hyd can be used for the treatment and/or prevention of the development of AD.

MATERIALS AND METHODS

Preparation of LF and pepsin-hydrolyzed LF (LF-hyd)

The bovine LF used in this study was from Morinaga Milk Industry Co., Ltd. (Zama, Japan), which was prepared from fresh skimmed milk by cation-exchange chromatography [32]. LF-hyd was produced by LF digestion with porcine pepsin as described previously with some modifications [33]. Briefly, LF was dissolved in distilled water at 5% (w/v). HCl was added to adjust the pH to 3.0. Porcine pepsin (EC 3.4.23.1; 10 units/mg) was added to a final concentration of 3% (wt/wt of substrate). The hydrolysis reaction was performed at 37°C for 4 h and terminated by heating at 80°C for 10 min. NaOH was added to readjust the pH to 6.0. The hydrolysates were lyophilized and broken down to particles, and homogenized powder was used in theexperiments.

AβPP transgenic mice and lactoferrin treatment

J20 mice, overexpressing human AβPP695 with the Swedish and Indiana mutations under PDGF promoter, were obtained from Jackson Laboratories. These mice exhibit behavioral deficits starting at about 4 months of age and develop robust amyloid plaques by 5 to 7 months of age, and Aβ deposition levels in the brains of these mice increase with age [34]. To investigate the effects of the LF and LF-hyd diets on cognition and Aβ levels in the brains of mice, 9-month-old mice were fed with the control diet, 2% LF diet, and 0.5% LF-hyd diet for 3 months (control diet group, n = 7; LF diet group, n = 6; LF-hyd diet group, n = 7 mice). All mice were housed under a 12-h light/dark cycle and had access to food and water ad libitum. All the experiments were performed in accordance with the Guidelines for Animal Experiments of the Animal Experimentation Committee of Nagoya City University.

Novel object recognition test

The novel objective recognition test is based on the innate tendency of rodents to explore novel objects over familiar ones and is believed to measure episodic memory. The experimental procedure was as described previously with some modifications [35]. Briefly, the novel object recognition test consisted of three different sessions: a habituation session, a training session, and a retention session. A mouse was habituated to a box (40×40 cm² and 40 cm high) by allowing it to explore the box without objects for 3 min for 3 days (habituation session). Twenty-four hours after the last habituation session, the mouse underwent a 5 min training session of exposure to two identical objects in an open field box. The time spent exploring each object was recorded by a video camera. After the training session, the mouse was returned to its home cage. After an interval of 24 h, the mouse was returned to the same box containing two objects, one identical to the familiar object but previously unused and one novel object. The mouse was allowed to explore for 5 min retention session, during which the amount of time exploring each object was recorded. Throughout the experiments, the objects used were matched in terms of their physical complexity and emotional neutrality. A preference index, that is, the ratio of the amount of time spent exploring any one of the two objects (training session) or the novel object (retention session) to the total amount of time spent exploring both objects, was used to measure cognitive function.

Aβ ELISA

The cortex and hippocampus were homogenized in 19 volumes of ice-cold Tris-buffered saline (TBS; 10 mM Tris and 150 mM NaCl, pH 7.6) containing a protease inhibitor cocktail (Roche, Mannheim, Germany). The resulting homogenates were centrifuged at 100,000 rpm for 20 min at 4°C. The supernatants (TBS extracts) were transferred to a new tube and stored at – 80°C until use for soluble Aβ determination and extracellular ApoE levels. The pellets were washed with ice-cold TBS, and then 10 volumes of 6 M guanidine hydrochloride were added to the pellets. The samples were sonicated and incubated at room temperature (RT) for 1 h. Homogenates were centrifuged at 100,000 rpm for 20 min at 4°C. The resulting supernatants were transferred to a new tube and stored at – 80°C until analysis and use for insoluble Aβ determination. The amounts of soluble and insoluble Aβ₄₀ and Aβ₄₂ were assayed using ELISA kits (Wako Pure Chemical Industries, Osaka, Japan). The Aβ levels were normalized to brain tissueweight.

Primary rat astrocyte cultures

Primary cultures of mixed glial cells were prepared from the brains of postnatal day 1 Wister rat pups. Briefly, brain tissue was dissected, stripped of meninges, and minced with forceps. The minced tissue was incubated in 0.125% trypsin and 0.1 mg/ml DNase I in PBS at 37°C for 15 min. The fragments were then dissociated into single cells, and then the cells were cultured for 7 days in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), at which point, microglia were removed by shaking.

Western blotting analysis

Cells were washed with ice-cold PBS and lysed with modified RIPA buffer (50 mM Tris-HCl (pH7.6), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing a protease inhibitor cocktail. The obtained homogenates were incubated on ice for 30 min and centrifuged at 12,000 rpm at 4°C for 10 min to remove cell debris. The protein concentrations in the supernatants were determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). These membranes were then blocked with 5% skim milk in TBS-T buffer for 1 h at room temperature. These membranes were then incubated with primary antibodies, namely, anti-ApoE (1:2000, AB947, Millipore), anti-ABCA1 (1:1000, AB18180, Abcam), anti-Erk1/2 (1:1000, #9102, Cell Signaling), anti-phospho-Erk1/2 (1:1000, #9101, Cell Signaling), anti-APP (1:1000, MAB348, Millipore), anti-PS1 (1:1000, MAB5232, Millipore), anti-ADAM10 (1:1000, MAB19026, Millipore), anti-BACE1 (1:1000, MAB931, R&D), anti-6E10 (1:1000, SIG-39300, Vovanc), anti-C-terminal AβPP (1:1000, A8717, Sigma), and anti-sAβPPβ (1:1000, #10321, IBL) antibodies at 4°C overnight. The membranes were washed and then incubated with an appropriate secondary antibody conjugated to horseradish peroxidase. Immunoreactive bands were visualized with ImmunoStar Zeta or ImmunoStar LD (Wako) and analyzed using an Amersham Imager 680 (GE Healthcare Life Science). Signal intensity was quantitified using ImageJ (NIH, Bethesda, MD, USA).

RNA extraction and real-time PCR

Total RNA was isolated from cultured primary astrocytes using Trizol (Invitrogen) following the manufacturer’s instructions. Reverse transcription was performed using an iScript Select cDNA Synthesis Kit (Bio-Rad). Quantitative real-time PCR was carried out using the GeneAce SYBR qPCR Mix (Nippon Gene, Japan) and 7500 Fast Real-Time PCR System (Applied Biosystems). The expression levels of mRNA were normalized with the corresponding amount of GAPDH mRNA using the comparative threshold cycle method following the manufacturer’s protocols. Amplification was performed using the following primers (sense and antisense): ABCA1 (5’-GGTTTGGGGAGGAAAT-TGAT-3’ and 5’-AACCATCCACA-GCAACCTTC-3’), and GAPDH (5’-GCATCTTCTTGTGCAGTG-CC-3’ and 5’-GAGAAGGCAGCCCTG-GTAAC-3’).

Aβ uptake

A synthetic Aβ₄₂ peptide was purchased from Peptide Institute (Osaka, Japan), dissolved in 0.1% NH₃ to a final concentration of 1 mM, and stored at – 80°C until use. Primary rat astrocytes were pretreated with 100 μg LF or 10 μg LF-hyd for 17 h in serum-free DMEM. Cells were treated with soluble 2 μg/ml Aβ₄₂ peptide for 3 h, and then the medium was collected for the determination of extracellular Aβ₄₂. After collecting the medium, the cells were washed twice with PBS, and then incubated with 0.05% trypsin/EDTA for 20 min at 37°C to remove cell surface-bound Aβ₄₂. Then the cells were lysed with RIPA buffer containing a protease inhibitor cocktail (Roche, Mannheim, Germany). The Aβ₄₂ levels in the medium and cellular lysates were quantified by ELISA and western blotting. For western blotting, the conditioned medium or equal amounts of proteins were subjected to 16% Tris-Tricine gel (Invitrogen) electrophoresis and transferred to PVDF membranes. These membranes were incubated with a mouse monoclonal primary antibody against human Aβ (6E10; Covance,Emeyryville, CA).

Statistical analysis

Statistical analysis was performed using a statistical package, GraphPad prism software (GraphPad Software, San Diego, CA). Data are presented as the mean±SEM from at least three independent experiments. Statistical significance was analyzed by one-way ANOVA or Student’s t-test. Data were considered significant when p < 0.05.

RESULTS

LF diet attenuates memory impairment in AβPP transgenic J20 mice

To determine whether an LF diet could rescue cognitive impairment in J20 mice, 9-month-old J20 mice were divided into three groups and fed with control, LF, and LF-hyd diets for 3 months, after which we conducted novel object recognition tests, which evaluate short-term memory and recognition ability. The time spent exploring a novel object versus a familiar object is a measure of attention and nonspatial declarative memory. The retention interval is the amount of time the animals must retain the memory of the identical objects presented during the training session prior to the retention session when one of the familiar objects is replaced with a novel one. During the training session, there were no significant differences in exploratory time (or preference) between the two familiar objects (Fig. 1a) in all three groups, suggesting that all groups of mice have similar levels of interest in exploring familiar objects. During the retention session, however, the LF- or LF-hyd-diet-fed J20 mice spent more time exploring the novel object than the familiar object (Fig. 1b). We also calculated the preference index (PI) and observed that the control group showed a preference for the familiar object (PI = 45.77%), whereas the LF- and LF-hyd-diet-fed groups showed a preference for the novel object (PI = 55.98% and 56.85%, respectively) (Fig. 1c). These findings indicate that the LF and LF-hyd diets attenuated short-term memory impairment in J20 mice.

Fig. 1

LF and LF-hyd diets attenuate memory impairment in J20 mice. Nine-month-old J20 mice were fed with control diet, 2% lactoferrin-containing (LF), and 0.5% pepsin-hydrolyzed lactoferrin-containing (LF-hyd) diets for 3 months, after which the novel objective recognition test was carried out. a) The time spent exploring two familiar objects (A and B) during the training session. Data are expressed as the mean±SEM, n = 6-7, *p < 0.05, Student’s t-test. b) The time spent exploring familiar object (A) and novel object (C) during the retention session. Data are expressed as the mean±SEM, n = 6-7, Student’s t-test. c) Preference index (time spent exploring the novel object/time spent exploring the novel and familiar objects)×100% in the retention session. Data are expressed as the mean±SEM, n = 6-7, *p < 0.05 versus control diet, n.s., no significant difference, as determine by one-way ANOVA.

LF diet decreases Aβ in the mouse brain

As AD is pathologically characterized by the production of Aβ, we further examined whether the improved cognitive performance of J20 mice fed with the LF or LF-hyd diet resulted from the altered production of Aβ. We measured the levels of soluble and insoluble Aβ₄₀ and Aβ₄₂ in the cortex and hippocampus by ELISA. The levels of soluble (Fig. 2a, b) and insoluble (Fig. 2c, d) Aβ₄₀ and Aβ₄₂ were significantly lower in the cortex and hippocampus of mice fed with the LF and LF-hyd diets than in those of mice fed with the control diet.

Fig. 2

Effects of LF and LF-hyd diets on the levels of Aβ₄₀ and Aβ₄₂ in the cortex and hippocampus of J20 mice. Nine-month-old J20 mice were fed with control diet, 2% lactoferrin-containing (LF), and 0.5% pepsin-hydrolyzed lactoferrin-containing (LF-hyd) diets for 3 months. Soluble Aβ₄₀ (a) and Aβ₄₂ (b), and insoluble Aβ₄₀ (c) and Aβ₄₂ (d) levels in the cortex and hippocampus of J20 mice were measured by sandwich ELISA. Aβ levels were normalized to brain tissue weight. The data are expressed as the mean±SEM, n = 6-7, *p < 0.05, **p < 0.01, ***p < 0.001 versus control diet, n.s., no significant difference, as determine by one-way ANOVA.

To study the mechanisms underlying the LF-diet-mediated reduction in Aβ levels, we first assessed the levels of AβPP and AβPP processing enzymes, namely ADAM 10 (α-secretase), BACE1 (β-secretase), and PS1 (γ-secretase component) in the cortex of J20 mice by western blot analysis. We found that compared with the control diet, the BACE1 levels were significantly decreased by both LF supplementations, whereas there were no significant differences in the levels of AβPP, ADAM10, and PS1 in the cortex between the control- and LF-diet-fed mice (Fig. 3a-e). Thus, we further measured the levels of AβPP cleavage fragments, including sAβPPα, sAβPPβ, and CTFβ, which are important to explain the production of Aβ in the cortex of J20 mice. As expected, the supplemention of both LF and LF-hyd increased the expression level of sAβPPα, and decreased sAβPPβ and CTFβ levels (Fig. 3a, f-h). Taken together, these findings suggest that LF and LF-hyd diets inhibit the amyloidogenic processing of AβPP through the reduction in BACE1 levels.

Fig. 3

Effects of LF and LF-hyd diets on AβPP metabolism in the cortex of J20 mice. Nine-month-old J20 mice were fed with control diet, 2% lactoferrin-containing (LF), and 0.5% pepsin-hydrolyzed lactoferrin-containing (LF-hyd) diets for 3 months. a) Western blot analysis of AβPP, ADAM10, BACE1, PS1, sAβPPα, sAPPβ, CTFβ, and β-actin in the cortex homogenates of 12-month-old J20 mice. b-h) The intensities of bands corresponding to AβPP, ADAM10, BACE1, and PS1, sAPPα, sAβPPβ, and CTFβ were quantified by densitometry, and then normalized to β-actin and expressed as a value relative to the control. All the values are presented as the mean±SEM, n = 6-7. *p < 0.05, ***p < 0.001 versus control diet, n.s., no significant difference, as determine by one-way ANOVA.

LF promotes extracellular Aβ degradation in primary astrocytes

According to the amyloid hypothesis, an increase in Aβ levels is the result of an imbalance between Aβ production and clearance or a decrease in Aβ proteolytic degradation [5]. Therefore, we also determined whether LF can enhance Aβ degradation and clearance. Previous studies have shown that cultured astrocytes can take up and clear soluble Aβ from media [36]. Thus, we investigated the effect of LF on Aβ degradation and clearance using primary astrocyte cultures. Astrocytes were treated with 100 μg/ml LF or 10 μg/ml LF-hyd for 17 h, then soluble Aβ₄₂ was added to the medium at a concentration of 2 μg/ml, and then the cells were incubated for 3 h at 37°C. To remove cell-surface-bound Aβ₄₂, the cells were incubated with 0.05% trypsin/EDTA for 20 min at 37°C. The levels of Aβ₄₂ in the medium and cellular lysates were measured by western blot analysis (Fig. 4a-c) and ELISA (Fig. 4d, e), respectively, and we found that both LF and LF-hyd decreased the Aβ₄₂ levels both in the medium and cellular lysates compared with controls. These results indicate that LF probably promotes extracellular degradation of soluble Aβ₄₂, which leads to the reduction in cell-internalized Aβ₄₂ levels. Taken together, these results indicate that LF and LF-hyd diets also contribute to the degradation of soluble Aβ₄₂, resulting in the reduction in Aβ levels in the brains ofJ20 mice.

Fig. 4

LF and LF-hyd promote the extracellular degradation of soluble Aβ₄₂ in rat primary astrocytes. Primary astrocyte cultures were treated with 100 μg/ml lactoferrin (LF) or 10 μg/ml pepsin-hydrolyzed LF (LF-hyd) for 17 h, and then soluble Aβ₄₂ (2 μg/ml) was added to the media followed by incubation for 3 h at 37°C. The medium was collected for determination of extracellular Aβ₄₂. To remove cell-surface-bound Aβ₄₂, the cells were incubated with 0.05% trypsin/EDTA for 20 min at 37°C. The protein bands of Aβ₄₂ in the conditioned medium (CM, b) and cellular lysates (c) were measured by western blot analysis with an anti-Aβ antibody (representative blot shown in left panel), quantified by densitometry, and then normalized to β-actin and expressed as a value relative to the control (middle and right panels). The Aβ₄₂ levels in the medium (d) and cell lysates (e) also quantified by ELISA. All the values are presented as the mean±SEM of three independent experiments. *p < 0.05, ***p < 0.001 versus control, n.s., no significant difference, as determine by one-way ANOVA.

LF treatment enhances ApoE secretion without changing intracellular ApoE levels

ApoE-HDL plays an important role in Aβ degradation and clearance, and it is lipidated principally through the action of the ATP-binding cassette transporter ABCA1, which is primarily produced by astrocytes in the brain. Thus, we investigated the effect of LF on ApoE secretion and intracellular ApoE levels to clarify the mechanism underlying the enhancement of Aβ degradation by LF. We treated primary astrocyte cultures with different concentrations of LF or LF-hyd: 1, 10, and 100 μg/ml. Twenty-four hours after LF treatment, the culture medium and cells were collected to measure the levels of secreted and intracellular ApoE, respectively, by western blot analysis. We found that 100 μg/ml LF (Fig. 5a) and 100 μg/ml LF-hyd (Fig. 5b) significantly increased secreted ApoE levels in the medium of astrocytes compared with controls, whereas intracellular ApoE levels did not change. These findings indicate that the increase in secreted ApoE levels in the medium of astrocytes treated with LF was not induced by the up-regulation of intracellular ApoE, indicating that LF might promoteApoE efflux.

Fig. 5

Effects of LF and LF-hyd on expression levels of extracellular ApoE, cellular ApoE, and ABCA1 in rat primary astrocytes. Primary astrocyte cultures were treated with the indicated concentrations of lactoferrin (LF, a) or pepsin-hydrolyzed LF (LF-hyd, b), and then the conditioned medium was collected 24 h later for the detection of secreted ApoE and cells were lysed for the detection of levels of cellular ApoE and ABCA1. The protein bands of secreted ApoE in the medium, cellular ApoE, ABCA1, and β-actin were detected by western blot analysis (representative blot shown in left panel), quantified by densitometry, then normalized to β-actin and expressed as a value relative to control (right panel). All the values are presented as the mean±SEM of three independent experiments. *p < 0.05, **p < 0.01 versus control, as determine by one-way ANOVA.

LF increases ABCA1 levels by inhibiting its degradation in primary astrocytes

ABCA1 is a cholesterol transporter that transfers excess cellular cholesterol and phospholipid to lipid-poor apolipoproteins, which bind to ApoE to form ApoE-HDL, which leads to improved clearance and degradation of Aβ. Therefore, we investigated the effect of LF on ABCA1 levels using primary astrocyte cultures, and we found that 100 μg/ml LF and 10 μg/ml LF-hyd significantly increased ABCA1 protein levels (Fig. 5a, b) without affecting its mRNA expression levels (Fig. 6a). These findings indicate that the upregulation of ABCA1 levels by LF may be due to the inhibition of ABCA1 degradation by LF. Thus, ABCA1 degradation was examined after LF treatment with cycloheximide, an inhibitor of de novo protein synthesis. To examine ABCA1 protein turnover, primary astrocytes were incubated with 100 μg/ml LF or 10 μg/ml LF-hyd in the presence or absence of cycloheximide for 4 h. ABCA1 levels decreased after 4 h in the control cells. In contrast, ABCA1 degradation was inhibited in cells treated with LF and LF-hyd (Fig. 6b). This finding suggests that LF critically affects ABCA1 stability.

Fig. 6

Effects of LF and LF-hyd on ABCA1 mRNA expression and ABCA1 degradation in rat primary astrocytes. a) Primary astrocyte cultures were treated with the indicated concentrations of lactoferrin (LF) or pepsin-hydrolyzed LF (LF-hyd) for 24 h. ABCA1 mRNA expression levels were determined by real-time PCR analysis. The expression level of ABCA1 mRNA was normalized to the corresponding amount of GAPDH mRNA and expressed as a value relative to that of the control. All the values are presented as the mean±SEM of three independant experiments, as determine by one-way ANOVA. b) LF and LF-hyd decreased ABCA1 degradation. Primary astrocyte cultures were incubated with or without cycloheximide (40 μg/ml) in the presence or absence of lactoferrin (LF) or pepsin-hydrolyzed LF (LF-hyd) for 4 h and then cells were lysed. The cell lysates were analyzed by western blotting with an anti-ABCA1 and β-actin antibodies. Typical bands representative of three independent experiments with similar results are shown.

LF diet increases extracellular ApoE and cellular ABCA1 levels, but not cellular ApoE levels in the mouse brain

Next, we measured ApoE and ABCA1 levels in the brains of J20 mice fed with LF or LF-hyd. ApoE levels in TBS-soluble brain homogenates as extracellular ApoE were measured by western blot analysis. Compared with mice fed with the control diet, extracellular ApoE levels were significantly increased in mice fed with LF and LF-hyd. We also measured intracellular ApoE and ABCA1 levels in RIPA-soluble brain homogenates. We found that both LF diets increased ABCA1 levels but not intracellular ApoE levels compared with the control diet(Fig. 7a, b).

Fig. 7

Effect of the LF and LF-hyd diets on the expression levels of extracellular ApoE, cellular ApoE, and ABCA1 in J20 mice brains. Nine-month-old J20 mice were fed with control diet, 2% lactoferrin-containing (LF), and 0.5% pepsin-hydrolyzed lactoferrin-containing (LF-hyd) diets for 3 months, and then protein extracts were analyzed by western blot analysis. For extracellular ApoE levels, the cortex was homogenized in TBS solution and then the supernatants were used. The supernatants and brain homogenates were analyzed by western blot analysis (representative blot shown in a), quantified by densitometry, then normalized to β-actin and expressed as a value relative to control (b). All the values are presented as the mean±SEM, n = 6-7, *p < 0.05, **p < 0.01 versus control diet, n.s., no significant difference, as determine by one-way ANOVA.

LF inhibits ABCA1 degradation through Erk activation

The above-mentioned findings showed that LF and LF-hyd inhibited ABCA1 degradation. Previous studies have shown that the inhibition of Erk activation enhanced ABCA1 degradation in CHO and HuH7 cells [37], and LF can activate Erk in the human lymphoblastic T Jurkat cell line [38]. Therefore, we investigated the effect of LF on the activation of Erk to clarify the mechanism underlying the LF-mediated inhibition of ABCA1 degradation. Primary astrocyte cultures were treated with 100 μg/ml LF or 10 μg/ml LF-hyd for 30 min, and then cell lysate was collected to determine phosphor-Erk1/2 (pErk1/2) levels by western blot analysis. LF and LF-hyd significantly increased pErk levels compared with untreated controls (Fig. 8a). Furthermore, we observed that the pretreatment of cells with the Erk1/2 inhibitor PD98059 decreased both ABCA1 and secreted ApoE levels in the medium of cells without altering cellular ApoE levels (Fig. 8b). We also measured pErk1/2 levels in the cortex of mice fed with LF or LF-hyd by western blot analysis. As expected, both the LF and LF-hyd diets increased pErk1/2 levels (Fig. 8c). Taken together, these findings suggest that LF increases ABCA1 levels in primary astrocyte cultures and in the mouse brain by the inhibition of ABCA1 degradation through Erk activation.

Fig. 8

LF and LF-hyd increase ABCA1 levels through Erk activation. a) Primary astrocyte cultures were treated with 100 μg/ml lactoferrin (LF) or 10 μg/ml pepsin-hydrolyzed LF (LF-hyd) for 30 min, and cells were lysed for the detection of levels of total Erk (t-Erk) and phospho (p)-Erk. The cell lysates were analyzed by western blot analysis (representative blot shown in left panel), and then p-Erk and t-Erk levels were quantified by densitometry, and p-Erk levels normalized to t-Erk levels, and expressed as a value relative to control (right panel). **p < 0.01, ***p < 0.001 versus control. n.s., no significant difference, as determine by one-way ANOVA. b) Primary astrocyte cultures were pretreated (1 h) with or without 20 μM PD98059 (Erk inhibitor), subsequently the cells were treated with 100 μg/ml lactoferrin (LF) or 10 μg/ml pepsin-hydrolyzed LF (LF-hyd) for 24 h. The cell lysates were analyzed by western blot analysis (representative blot shown in left panel), and then p-Erk and t-Erk levels were quantified by densitometry, and p-Erk levels normalized to t-Erk levels, and expressed as a value relative to control (right panel). All the values are presented as the mean±SEM of three independent experiments. *p < 0.05, **p < 0.01 versus control, #p < 0.05 versus PD98059 treatment, as determined by one-way ANOVA. c) Effect of the LF and LF-hyd diets on expression levels of p-Erk and t-Erk in J20 mice brains. Nine-month-old J20 mice were fed with control diet, 2% lactoferrin-containing (LF), and 0.5% pepsin-hydrolyzed lactoferrin-containing (LF-hyd) diets for 3 months, and then protein extracts in the cortex of mice were analyzed by western blot analysis (representative blot shown in left panel), quantified by densitometry, then normalized to β-actin and expressed as a value relative to control (right panel). All the values are presented as the mean±SEM, n = 6-7, *p < 0.05 versus control diet, n.s., no significant difference, as determine by one-way ANOVA.

DISCUSSION

AD is a progressive neurodegenerative disorder, a hallmark of which is the deposition of Aβ, which plays an important role in AD pathogenesis. The soluble and small oligomeric forms of Aβ induce synaptic dysfunction, cognitive deficits, and neuronal degeneration [39]. Therefore, the inhibition of Aβ production and the elimination of toxic Aβ species in the early stages of AD seem to be promising strategies for reducing synaptic damage and relieving cognitive impairment [40].

J20 mice show age-related Aβ deposition and progressive memory impairment [41]. In agreement with this study, J20 mice showed memory impairment at the age of 12 months. However, when these mice were fed with the LF or LF-hyd diet from the age of 9 months for 3 months, memory impairment was attenuated, suggesting that the LF diets prevent hippocampal dysfunction. Regarding the molecular mechanisms by which the LF diets attenuated memory impairment in J20 mice, it has been reported that cognitive dysfunction is caused by the extracellular accumulation of soluble Aβ assemblies [42], and Aβ oligomers may play a crucial role as the earliest effectors of synaptic dysfunction and early memory loss associated with dementia in AD. In this study, we showed that the LF and LF-hyd diets decreased soluble and insoluble Aβ₄₀ and Aβ₄₂ levels in the brains of J20 mice, suggesting that the LF-diet-mediated prevention of memory impairment is a result of the reduction in Aβ levels. Furthermore, the LF and LF-hyd diets significantly decreased BACE1 levels associated with the reduction in CTFβ and sAβPPβ levels, whereas there were no significant changes in AβPP, ADAM10, and PS1 levels, indicating that the LF diets may regulate the metabolism of AβPP to CTFβ and reduce soluble and insoluble Aβ levels in the brains of mice.

Aβ is generated from AβPP by two sequential steps of proteolytic cleavage by β-secretase and γ-secretase. Inflammation clearly occurs in pathologically vulnerable brain regions of AD and J20 mice, and inflammation has been shown to upregulate BACE1 levels. Moreover, the expression and the activity of BACE1 are elevated in the brains of late-onset sporadic AD patients. The anti-inflammatory function of LF depends on its binding to free iron, prevention of oxidative stress formation, and inhibition of inflammatory cytokine production [43, 44]. Indeed, a recent report showed that the intranasal delivery of LF to AβPP/PS1 mice suppressed the transcription of pro-inflammatory cytokines such as TNF-α and IL-6 and reduced the formation of GFAP (a maker of astrocytes) immunoreactivity [45]. Although we did not examine the effect of LF on the expression of pro-inflammatory cytokines and the immunohistochemical studies for glial cell activation because of limited samples, the LF-mediated reduction in BACE1 levels may be due to the anti-inflammatory function of LF. Further studies are required to test this hypothesis.

According to the amyloid hypothesis, an increase in Aβ levels is the result of an imbalance between Aβ production and clearance or a decrease in Aβ proteolytic degradation [5]. The ɛ4 allele of the ApoE gene is currently the strongest genetic risk factor for late-onset AD [46]. Data from human studies and animal models suggest that ApoE primarily affects AD pathogenesis through the alteration of aggregation of Aβ and Aβ clearance from the brain [47]. Altering the amount of ApoE in the brain affects Aβ deposition and clearance. Furthermore, the lipidation of ApoE (ApoE-HDL) is mediated by ABCA1, and ApoE-HDL binds to Aβ. ApoE secretion and the ABCA1-mediated lipidation status of ApoE are highly important to the ApoE capability to promote Aβ extra- and intracellular proteolysis. Thus, we also investigated the effect of LF on Aβ clearance and degradation. Here, we found that LF treatment promoted ApoE secretion and increased ABCA1 levels in primary astrocyte cultures and in the brains of J20 mice, which may reduce Aβ levels in the brains of J20 mice.

In this study, LF treatment promoted ApoE secretion without altering intracellular ApoE levels. Iron accumulation is implicated in neurodegenerative diseases including AD [48, 49], and elevated iron levels in the cortex and hippocampus of an aged brain make it susceptible to the development of an AD-like pathology [50]. Accumulation of iron in the brain may facilitate the aggregation and deposition of Aβ, resulting in the formation of extracellular Aβ plaques where ApoE is colocalized [51, 52]. Moreover, recent studies showed that iron inhibits the secretion of ApoE in cultured human adipocytes and mouse primary astrocytes [53, 54]. LF is the main iron-binding protein [20], and it chelates free iron to prevent the formation of ROS and increases antioxidant capacity [21 –23]. Therefore, it is reasonable to postulate that LF treatment may enhance ApoE secretion via the chelation of free iron. Another possible explanation may be that LF-mediated increases in ABCA1 levels promote ApoE secretion. It has been shown that a lack of ABCA1 expression in ABCA1 KO mice inhibits ApoE secretion from astrocytes [55], and ABCA1 was found to facilitate ApoE secretion from human monocyte-derived macrophages [56]. Additional studies are required to determine the mechanisms by which LF increases ApoE secretion.

In this study, we found that LF treatment increased ABCA1 levels without changing its mRNA expression levels, indicating that LF may modulate ABCA1 degradation. ABCA1 mRNA expression is mainly regulated by the liver X receptor (LXR), retinoid X receptor (RXR), and peroxisome proliferator-activated receptors (PPARs) [57 –59]. ABCA1 levels are also modulated through lysosomal and ubiquitin-dependent degradation [60 –62]. Recently, it has been reported that the inhibition of mitogen-activated protein kinase Erk1/2 increases ABCA1 protein degradation [37, 63] and that LF can activate Erk [64]. Here, we also confirmed that LF treatment increased Erk phosphorylation in primary astrocytes and in the brains of J20 mice fed an LF diet, indicating that LF inhibits ABCA1 degradation through Erk activation.

Hyperlipidemia is one of the factors responsible for severe cardiovascular and cerebrovascular diseases. It is well known that the deletion of ApoE in mice exhibited hyperlipidemia and spontaneous aortic atherosclerosis [65, 66]. In addition, Tangier disease caused by ABCA1 mutation exhibits a severe high-density lipoprotein (HDL) deficiency [67]. These lines of evidence indicate that ApoE and/or ABCA1 deficiency, both of which reduced ApoE-HDL levels resulting in hyperlipidemia. Thus, the increase in ApoE and ABCA1 should enhance the HDL generation but not LDL generation. Although, we did not measure plasma lipid levels in this study, it has been reported that lactoferrin treatment reduced triglycerides and total cholesterol levels in plasma of mice [68]. Therefore, lactoferrin-induced ApoE secretion and the increase in ABCA1 levels may exert some positive effects on lipid metabolism. To support this hypothesis, further investigation for plasmid lipid profiles and body weight change by lactoferrin treatment is necessary.

In summary, we have shown that both the LF and LF-hyd diets attenuated memory impairment in J20 mice after 3 months of administration. This finding can be explained at least in part by two pathways: 1) the reduction in soluble and insoluble Aβ levels through decreased BACE1 levels and 2) the enhancement of Aβ degradation through the enhancement of ApoE secretion and increase in ABCA1 protein levels. Both LF and LF-hyd may therefore be used for the treatment and/or prevention of the development of AD.

Footnotes

ACKNOWLEDGMENTS

This work was supported by a Grant-in-Aid for Scientific Research B (16H05559) and a Grant-in-Aid for challenging Exploratory Research (15K15712) (to M.M.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. This work was also supported by the Project of translational and clinical research seed A from Japan Agency for Medical Research and Development (AMED, A-128) (to M.M.). We acknowledge the assistance of the Research Equipment Sharing Center at the Nagoya City University.

Authors’ disclosures available online ().

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