A Diet Enriched in Palmitate and Deficient in Linoleate Exacerbates Oxidative Stress and Amyloid-β Burden in the Hippocampus of 3xTg-AD Mouse Model of Alzheimer’s Disease

Abstract

Epidemiological studies have suggested a positive correlation between saturated fat intake and the risk for developing Alzheimer’s disease (AD). While diets-enriched in the saturated free fatty acid (sFFA) palmitate has been shown to induce cognitive dysfunction and AD-like pathology, polyunsaturated fatty acids (PUFA) such as linoleate have been suggested to protect against AD in mouse models. However, the underlying cellular and molecular mechanisms that mediate the deleterious effects of palmitate or the protective effects of linoleate remain to be characterized. We fed 9-month-old cohorts of triple transgenic AD mice (3xTg-AD) and their-matched controls with a palmitate-enriched/linoleate-deficient diet for three months and determined the impact of the diet on oxidative stress, Bace1 promoter transactivation status, and amyloid-β (Aβ) burden. The palmitate-enriched/linoleate-deficient diet causes a profound increase in oxidative stress burden characterized by significant oxidative damage to lipids, proteins, and nucleic acids concomitant with deficits in the endogenous antioxidant defense capacity in the hippocampi of 3xTg-AD mice. These effects were also associated with increased NF-κB transcriptional activity resulting in NF-κB-mediated transactivation of the Bace1 promoter that culminated in higher BACE1 expression and activity, and Aβ production. Our study unveils a novel mechanism by which a diet enriched in the sFFA palmitate and deficient in the PUFA linoleate exacerbates AD-like pathology involving signaling cross-talk between oxidative stress and NF-κB activation as a critical underlying factor in upregulating BACE1 activity and increasing Aβ burden.

Keywords

Alzheimer’s disease amyloid-β BACE1 linoleate oxidative stress palmitate

INTRODUCTION

Diets rich in saturated fat are associated with cognitive dysfunction, deficits in learning, as well as impairments in memory and executive function in the elderly [1 –5]. Palmitic acid (palmitate) is the most abundant saturated free fatty acid (sFFA) in the diet [6, 7], plasma [8], and the brain [9]. Palmitate is found in various foods that we consume every day including meat, milk, butter, and cheese. Furthermore, this sFFA can also be made endogenously from conversion of excess carbohydrates to palmitate via de novo lipogenesis [10]. Linoleic acid is found in vegetable oils, nuts, seeds, and dairy and meat products. Higher palmitate and other sFFA levels in the plasma observed under pathophysiological conditions such as diabetes, obesity, and metabolic syndrome are associated inversely with cognitive function [8]. Brain palmitate and sFFA pool is reflective and constitutive of endogenous palmitate from recycling of brain phospholipids as well as peripheral circulating palmitate in the plasma, derived from de novo lipogenesis in the liver and emanating from dietary sources that crosses the blood-brain-barrier with non-saturable kinetics [11, 12]. Saturated fat-enriched diets also evoke learning and memory impairment, cognitive dysfunction, as well as behavioral deficits in a multitude of rodent models [13 –15]. Conversely, polyunsaturated fatty acids (PUFA) such as linoleate may protect against AD [16, 17]. Despite being clearly implicated in either improving or worsening cognitive impairment and the risk for neurodegenerative disorders, the underlying pathophysiological phenomena and molecular entities that mediate the deleterious effects of palmitate-enriched or linoleate deficient diets are poorly characterized. Oxidative stress has emerged as one of the most multi-faceted and dynamic pathophysiological entities involved in the etio-pathogenesis of neurodegenerative disorders, including AD [18 –20]. Oxidative stress is involved in the physiology of aging [21, 22], the greatest risk factor for AD. Oxidative stress is known to foster amyloid-β (Aβ) genesis [23] and hyperphosphorylation of tau [24], two key molecular entities that embody the pathological hallmarks of AD. Furthermore, oxidative stress has been known to increase the enzymatic activity of β-site APP-cleavage enzyme 1 (BACE1), [25 –27], the enzyme that catalyzes the rate-limiting step in Aβ genesis [28, 29]. Oxidative stress evokes NF-κB (Nuclear factor of kappa-light-polypeptide gene enhancer of activated B cells) activation, a major transcriptional regulator of BACE1 [30, 31]. Brain oxidative stress is a cardinal pathophysiological hallmark exhibited in the triple transgenic mouse model of AD (3xTg-AD mice) [32] and the AD brain [18 , 33]. The AD brain is characterized by an elevation in the entire gamut of spectrum of oxidative stress markers [34, 35] that show lipid peroxidation-induced damage [36], oxidative damage to proteins and DNA [18 , 37], as well as deficits in endogenous anti-oxidant defense systems [20, 38]. In this study, we determined the effects of a palmitate-enriched/linoleate deficient diet on the key molecular entities that collectively constitute the oxidative stress burden, global antioxidant defense capacity, and the expression of key constituent proteins that serve as endogenous antioxidants, and the status of NF-κB activation in the hippocampi of 3xTg-AD mice. Furthermore, we investigated the inter-relationship between oxidative stress and NF-κB activation and functional impact of this oxidative stress-induced NF-κB activation on BACE1 expression and activity, as well as Aβ burden, in the hippocampi of the 3xTg-AD mice and delineated the associated molecular mechanisms.

MATERIALS AND METHODS

Mouse experiments

The 3xTg-AD mice (on a C57BL/6J background) are from a colony maintained for more than 10 generations in our facility. Our mice colony was developed from 3xTg-AD mice obtained from Dr. Frederic Calon, Laval University. Dr. Calon’s colony was derived from a colony generated from homozygous founders obtained from Dr. Frank LaFerla on a C57BL/6J x129SVJ background. The mice were housed in individually ventilated cages at an ambient room temperature (23–25°C) and ambient relative humidity ranging between 50–70%. The mice were maintained on 12:12 h light:dark cycle and allowed access to food and water ad libitum. The 3xTg-AD mice (all males, nine months of age) and their matched controls (non-Tg mice) were fed a palmitate-enriched/linoleate-deficient diet (TD 110616, Harlan Teklad, 2.2% w/w palmitic acid and 0.8% linoleic acid) or a corresponding control diet (TD 85172, Harlan Teklad, 0.8% w/w palmitic acid and 2.2% linoleic acid) for three months (n = 6 per group). The diets were isocaloric in relation to each other and the respective composition of the diets is shown in Table 1. The food-intake was monitored for the span of 24 h, once every two weeks, and body weights were measured every two weeks. No differences in food intake or body weight were found between the groups. By the end of the feeding regimens, mice were anesthetized with sodium pentobarbital and perfused transcardially with Dulbecco’s phosphate-buffered saline. Brains were promptly removed and hippocampi immediately dissected and homogenized. Six hippocampi from each hemisphere per group were randomly chosen for western blotting, RT-PCR, Aβ assay, and immunoprecipitation analysis; the remaining 6 hippocampi from each group were used for the oxidative stress, FRET-based BACE1 activity, ChIP and NF-κB DNA binding activity assays. All animal procedures were carried out in accordance with the U.S. Public Health Service Policy on the Humane Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of North Dakota.

Table 1

Composition of the control chow diet and palmitate-enriched/linoleate-deficient diet

	Control diet	Palmitate-enriched/linoleate-deficient diet
Protein	23.60 % w/w	23.60 % w/w
Carbohydrates	65.80 % w/w	65.80 % w/w
Total Fat	5.60 % w/w	5.60 % w/w
Total Energy	4.08 Kcal/gram	4.08 Kcal/gram
Myristic acid (14:0)	0.10 % w/w	0.10 % w/w
Palmitic acid (16:0)	0.80 % w/w	2.20 % w/w
Stearic acid (18:0)	0.20 % w/w	0.20 % w/w
Palmitoleic acid (16:1)	trace	trace
Oleic acid (18:1)	1.20 % w/w	1.20 % w/w
Gadoleic acid (20:1)	trace	trace
Linoleic acid (18:2 n6)	2.20 % w/w	0.80 % w/w
Linolenic acid (18:3 n3)	0.20 % w/w	0.20 % w/w
Arachidonic acid (20:4 n6)	trace	trace
EPA (20:5 n3)	0.10 % w/w	0.10 % w/w
DHA (22:6 n3)	0.30 % w/w	0.30 % w/w

Western blot analysis

Whole cell, cytosolic, and nuclear homogenates from the mouse hippocampi were prepared as previously described [39]. Proteins (10–40 μg) were resolved on SDS-PAGE gels followed by transfer to a polyvinylidene difluoride membrane (BioRad, Hercules, CA) and incubation with the monoclonal antibodies listed in Table 2. The origin, source, the dilutions of the respective antibodies used for this study is compiled in Table 2. β-actin was used as a gel loading control for whole cell and cytosolic homogenates, whereas Histone H3 was used as a gel loading control for nuclear homogenates. The blots were developed with enhanced chemiluminescence (Clarity^TM Western ECL blotting substrate, BioRad) and imaged using a LiCOR Odyssey Fc imaging system (LiCOR Biosystems, Lincoln, NE).

Table 2

List of monoclonal and polyclonal antibodies used in the study

Antibody	Dilution	Amount	Host	Application	Manufacturer	Catalog #	Antibody Registry
β-Actin	1:2500	2 μg	mouse	WB	Santa Cruz BioTechnology	sc-47778 (C4)	AB_626632
BACE1	1:1000	5 μg	rabbit	WB	MilliporeSigma	AB5832	AB_2258785
CBP	1:500	10 μg	rabbit	WB	Cell Signaling Technology	7389 (D6C5)	AB_10949313
Histone H3	1:1000	2 μg	rabbit	WB	Santa Cruz BioTechnology	sc-8654-R (C16)	AB_1125026
p-Ser^32/36 IκBα	1:250	20 μg	mouse	WB	Cell Signaling Technology	9246 (5A5)	AB_2267145
IκBα	1:1000	5 μg	rabbit	WB	Cell Signaling Technology	9242	AB_331623
p-Ser^176/180 IKKα/β	1:250	20 μg	rabbit	WB	Cell Signaling Technology	2697 (16A6)	AB_2079382
IKKβ	1:500	10 μg	rabbit	WB	Cell Signaling Technology	2678 (L570)	AB_2122301
Na⁺ K⁺ -ATPase	1:1000	5 μg	rabbit	WB	Cell Signaling Technology	3010	AB_2060983
NCoR1	1:1000	5 μg	rabbit	WB	Cell Signaling Technology	5948	AB_10834809
p50	1:1000	5 μg	rabbit	WB	MilliporeSigma	06-886	AB_310284
p65	1:1000	5 μg	rabbit	WB	Cell Signaling Technology	4764 (C22B4)	AB_823578
p65	1:1000	5 μg	rabbit	ChIP	Cell Signaling Technology	8242 (D14E12)	AB_10859369
p300	1:250	20 μg	rabbit	WB	Abcam	ab59240	AB_944579
RNAPIIA	1:250	5 μg	rabbit	ChIP	Abcam	ab26721	AB_777726
SOD1	1:100	5 μg	rabbit	IP	ThermoFisher Scientific	LF-PA0013	AB_1087612
SOD2	1:500	5 μg	rabbit	IP	ThermoFisher Scientific	PA1-31072	AB_2286296
SMRT (NCoR2)	1:1000	5 μg	rabbit	WB	Abcam	ab5802	AB_305122

Immunoprecipitation

Mouse hippocampal tissues were homogenized using a non-denaturing lysis buffer (20 mM Tris HCl, 137 mM Nacl, 2 mM EDTA, 1% Nonidet P-40, 10% glycerol) supplemented with protease/phosphatase inhibitors as previously described [40]. The homogenate containing the equivalent to 750 μg of total protein was pre-cleared by incubation with protein A/G coated agarose beads for 30 min at 4°C to reduce the non-specific binding of proteins to the beads. The equivalent of 750 μg of the pre-cleared lysate was incubated separately with 5 μg of anti SOD1 mouse monoclonal antibody (ThermoFisher Scientific, Waltham, MA, Catalog # LF-PA0013) or 5 μg of anti SOD2 rabbit monoclonal antibody (ThermoFisher Scientific, Catalog # PA1-31072) overnight at 4°C followed by capturing of the immune-complex by the addition of protein A/G agarose beads and incubation overnight. The beads containing the immune-complex were washed 3x with the lysis buffer followed by centrifugation and discarder of the supernatant. The beads were suspended in the lysis buffer and centrifuged to pellet the beads. The pellet (beads) contain,ing the SOD1 and SOD2 enriched fraction were subjected to SOD1 and SOD2 activity assays respectively.

Quantitation of Aβ₄₀ and Aβ₄₂ levels by enzyme-linked immunosorbent assay (ELISA)

Aβ₄₀ and Aβ₄₂ levels in the mouse hippocampi were quantified using an ELISA immunoassay kit (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol and as described earlier [41, 42]. The mouse hippocampal tissues were subjected to a sequential extraction procedure [43] to generate the following sub-cellular fractions containing the respective Aβ species—TBS (Tris buffered saline)-soluble Aβ (water soluble Aβ), 2% SDS (sodium dodecyl sulfate) soluble Aβ (detergent soluble Aβ), and 70% formic acid soluble Aβ (TBS insoluble Aβ and detergent insoluble Aβ). Aβ levels in the mouse hippocampus were normalized to total protein content in the samples (pg/mg protein).

Quantitative real time RT-PCR analysis

Total RNA was extracted from the mice hippocampal tissue using the 5 prime “PerfectPure RNA tissue kit” (5 Prime, Inc., Gaithersburg, MD) following manufacturer’s instructions and as described previously [44, 45]. cDNA was obtained by reverse transcribing 1 μg of extracted RNA using an iScript cDNA synthesis kit” (BioRad). cDNA was obtained by reverse transcribing 1 μg of extracted RNA using an iScript cDNA synthesis kit” (BioRad). The quantitative Real-time RT-PCR analysis was performed using TaqMan chemistry using “Assays-on-Demand” probes (ABI, Foster City, CA) for mouse Bace1 (Bace1 gene) (Mm00478664_m1). The expression of specific transcripts amplified was normalized to the expression of 18 s rRNA. The data were quantified and expressed as fold-change compared to the non-Tg mice fed a control diet by using the Δ ΔC_T method.

Total reactive oxygen species (ROS) and reactive nitrogen species (RNS) measurement

Total ROS/RNS levels were determined in the hippocampal tissue of mice using the “OxiSelecttrademark In Vitro ROS/RNS Assay Kit (Green Fluorescence)” from Cell Biolabs, Inc. (San Diego, CA, Catalog # STA-347) following manufacturer’s protocol. In brief, hippocampal tissue was denaturated in Triton X-100, resuspended in PBS at 10–50 mg/mL, and sonicated on ice. The insoluble particles were removed by spinning at 10,000 g for 5 min, and the homogenates assayed immediately. Data was normalized to the intensity of fluorescence in the hippocampal homogenates of the non-Tg mice fed the control diet (set as unit value) with other experimental groups expressed as a fold-change and includes determination made in six different animals from each group.

Measurement of 4-hydroxynonenal (4-HNE) protein adducts

The 4-HNE conjugated stable protein adducts levels were determined in the hippocampal tissue of mice using the “OxiSelecttrademark HNE Adduct Competitive ELISA Kit” from Cell Biolabs, Inc. (Catalog # STA-838) following manufacturer’s protocol. Data was normalized to the intensity of the absorbance measured at 450 nm in the hippocampal homogenates of the non-Tg mice fed the control diet (set as unit value) with other experimental groups expressed as a fold-change and includes determination made in six different animals from each group.

Measurement of malondialdehyde (MDA) protein adducts

The MDA conjugated stable protein adducts levels were determined in the hippocampal tissue of mice using the “OxiSelecttrademark MDA Adduct Competitive ELISA Kit” from Cell Biolabs, Inc. (Catalog # STA-832) following manufacturer’s protocol. Data was normalized to the intensity of the absorbance measured at 450 nm in the hippocampal homogenates of the non-Tg mice fed the control diet (set as unit value) with other experimental groups expressed as a fold-change and includes determination made in six different animals from each group.

Measurement of 8-iso-Prostaglandin F2α (8-iso-PGF2α)

The levels of 8-iso-PGF2α in the hippocampal tissue homogenates were determined using the “OxiSelecttrademark 8- iso-Prostaglandin F2α ELISA Kit” from Cell Biolabs, Inc. (Catalog # STA-837) following manufacturer’s protocol. Data was normalized to the intensity of the absorbance measured at 450 nm in the cortical and hippocampal homogenates of the non-Tg mice fed the control diet (set as unit value) with other experimental groups expressed as a fold-change of the and includes determination made in six different animals from each group.

Measurement of protein carbonyl content and oxidative modification of proteins

The levels of protein carbonyl derivatives, serving as surrogate determinant of oxidative protein damage, collectively representing the oxidative modification of the amino-acid residues proline, arginine, lysine, and threonine of the entire proteome was determined using the “OxiSelecttrademark Protein Carbonyl ELISA Kit” from Cell Biolabs, Inc. (Catalog # STA-310) following manufacturer’s protocol. Data was normalized to the intensity of the absorbance measured at 450 nm, in the hippocampal homogenates of the non-Tg mice fed the control diet (set as unit value) with other experimental groups expressed as a fold-change and includes determination made in six different animals from each group.

Measurement of protein tyrosine nitration content

The extent of oxidative nitration of tyrosine residues of the entire proteome was determined in the mouse hippocampal tissue homogenates using the “OxiSelecttrademark Nitrotyrosine ELISA Kit” from Cell Biolabs, Inc. (Catalog # STA-305) following manufacturer’s protocol. Data was normalized to the intensity of the absorbance measured at 450 nm, in the hippocampal homogenates of the non-Tg mice fed the control diet (set as unit value) with other experimental groups expressed as a fold-change and includes determination made in six different animals from each group.

Measurement of 8-hydroxydeoxyguanosine (8-OHdG) and 8-hydroxyguanosine (8-OHG) levels

The levels of 8-OHdG and 8-OHG were measured as surrogate indices of oxidative damage to DNA and RNA respectively, using the OxiSelecttrademark Oxidative DNA Damage ELISA Kit (Catalog # STA-320) and OxiSelecttrademark Oxidative RNA Damage ELISA Kit (Catalog # STA-325) from Cell Biolabs, Inc., following manufacturer’s protocol. Data was normalized to the intensity of the absorbance measured at 450 nm, in the hippocampal homogenates of the non-Tg mice fed the control diet (set as unit value) with other experimental groups expressed as a fold-change and includes determination made in six different animals from each group.

Determination of oxidized glutathione (GSSG), reduced glutathione (GSH), and total glutathione content

The total glutathione, GSSG, and GSH content in the mouse hippocampal tissue homogenates was determined using the “Glutathione (GSSG/GSH) detection kit” from Enzo Life Sciences, Inc. (Farmingdale, NY, Catalog # ADI-900-160) following manufacturer’s protocol. Data was normalized to the intensity of the absorbance measured at 405 nm and 414 nm, in the hippocampal homogenates of the non-Tg mice fed the control diet (set as unit value) with other experimental groups expressed as a fold-change and includes determination made in six different animals from each group.

Superoxide dismutase (SOD) activity assay

The total activity of SOD1 and SOD2 in the mouse hippocampal homogenates was measured using the “Superoxide Dismutase Assay Kit” from Cayman Chemicals (Ann Arbor, MI, Catalog # 706002) following manufacturer’s protocol. The immunoprecipitated SOD1 or SOD2 fractions, as described under the “Immunoprecipitation” section of this manuscript, were used as inputs for the respective activity assays. Data was normalized to the intensity of the absorbance measured at 440 nm and 460 nm, in the hippocampal homogenates of the non-Tg mice fed the control diet (set as unit value) with other experimental groups expressed as a fold-change and includes determination made in six different animals from each group.

Glutathione peroxidase (GPx) activity assay

The total activity GPx in the mouse hippocampal homogenates was measured using the “Glutathione Peroxidase Assay Kit” from Cayman Chemicals (Catalog # 703102) following manufacturer’s protocol. Data was normalized to the intensity of the absorbance measured at 340 nm, in the hippocampal homogenates of the non-Tg mice fed the control diet (set as unit value) with other experimental groups expressed as a fold-change and includes determination made in six different animals from each group.

Glutathione-disulfide reductase (GSR) activity assay

The total activity GPx in the mouse hippocampal homogenates was measured using the “Glutathione Reductase Assay Kit” from Sigma Aldrich (St. Louis, MO, Catalog # GRSA) following manufacturer’s protocol. Data was normalized to the intensity of the absorbance measured at 340 nm and 412 nm, in the hippocampal homogenates of the non-Tg mice fed the control diet (set as unit value) with other experimental groups expressed as a fold-change and includes determination made in six different animals from each group.

FRET-based BACE1 activity assay

BACE1 activity in the membrane fractions from the hippocampi of respective cohorts of mice was determined using a FRET-based kit from Sigma-Aldrich (Catalog # CS0010) following manufacturer’s protocol and as described previously [43]. Data was normalized to the intensity of the absorbance measured in the hippocampal homogenates of the non-Tg mice fed the control diet (set as unit value) with other experimental groups expressed as a fold-change and includes determination made in six different animals from each group.

Chromatin immunoprecipitation (ChIP) analysis

ChIP analysis was performed to evaluate the extent of p65 binding to the NF-κB binding elements in the BACE1 promoter region using “SimpleChIP^TM Enzymatic Chromatic IP kit” from Cell Signaling Technology (Boston, MA) following manufacturer’s instructions. The relative abundance of the p65 antibody (Catalog #8242, Cell Signaling Technology) precipitated chromatin containing the NF-κB binding site in the Bace1 promoter region was determined by qPCR using sequence specific primers (Qiagen Inc., Valencia, CA) (Table 3). The amplification was performed using the “StepOnePlus” PCR System (ABI). The fold enrichment of the bound p65 in the Bace1 promoter region was calculated using the Δ ΔC_T method which normalizes ChIP C_T values of each sample to the % input and background. The data was further normalized with the non-Tg mice fed the control diet (set as unit value) with other experimental groups expressed as a fold-change and includes determination made in six different animals from each group.

Table 3

List of primers used for ChIP analysis

Species	Gene	Gene ID	Primer pair	Reference Sequence	Primers
Mouse	Bace1	23821	Forward	NT_039472.8	5’- GAGAGCCCCTCAGCTTCTTT-3’ [121]
Mouse	Bace1	23821	Reverse	NT_039472.8	3’- GGGGCCCTGATAGCTTAAAA-5’ [121]

NF-κB DNA binding activity assay

The DNA binding activity of NF-κB was determined, as a surrogate measure for the transcriptional activity of NF-κB, by the ELISA method using a kit from Abcam (Cambridge, MA, Catalog # ab133112) following manufacturer’s protocol. Briefly, nuclear homogenate equivalent to 20 μg of the protein content was added to each of the wells of the 96-well plate containing the double stranded DNA sequence harboring the consensus NF-κB binding sequence (NF-κB response element) coated onto the wells. The nuclear extract was allowed to hybridize with the coated double stranded DNA sequence harboring the consensus NF-κB response element in the plate overnight at 4°C. The activated NF-κB complex was detected by addition of a specific primary antibody directed against the p65 subunit of the NF-κB dimeric complex bound to the exogenous response element followed by incubation with a secondary antibody conjugated to HRP provide a sensitive colorimetric readout at 450 nm. Data was normalized to the intensity of the absorbance measured at 450 nm, in the hippocampal homogenates of the non-Tg mice fed the control diet (set as unit value) with other experimental groups expressed as a fold-change and includes determination made in six different animals from each group.

Statistical analysis

The significance of differences among the samples was assessed by non-parametric Kruskal-Wallis One Way Analysis of Variance followed by Dunn’s post-hoc test. Statistical analysis was performed with GraphPad Prism 6. Quantitative data for all the assays are presented as mean values±SD (mean values±standard deviation) with unit value assigned to control and the magnitude of differences among the samples being expressed relative to the unit value of control as fold-change. Quantitative data for ELISA analysis are presented as mean values±SD with absolute levels of Aβ₄₀ and Aβ₄₂ reported.

RESULTS

Palmitate-enriched/linoleate-deficient diet causes oxidative stress and leads to increase in lipid peroxidation, oxidative protein damage, and nucleic acid oxidation in the hippocampi of 3xTg-AD mice

Oxidative stress is intricately involved in the etio-pathogenesis of AD [18 , 46]. We first determined the effects of palmitate-enriched/linoleate-deficient diet on the levels of total ROS and RNS in the hippocampal homogenates of nine-month old 3xTg-AD mice and their age-matched controls (non-Tg mice) fed a control diet or a palmitate-enriched/ linoleate-deficient diet for three months. The total ROS/RNS levels were significantly higher in the hippocampi of 3xTg-AD mice fed with the control diet relative to the non-Tg mice fed the same diet, indicative of higher basal oxidative stress in the hippocampi of 3xTg-AD mice (Fig. 1A). More importantly, feeding palmitate-enriched/linoleate-deficient diet for three months significantly exacerbated this oxidative stress burden as the total ROS/RNS levels in the hippocampi of 3xTg-AD mice were significantly higher compared to the total ROS/RNS levels in the hippocampi of the non-Tg mice fed the control diet. The increase in ROS/RNS levels resulted in an increase in peroxidative damage to lipids resulting in the formation of lipid peroxidation products that include 4-HNE [47, 48], (MDA [49], and 8-iso PGF2α [50]. We next determined the abundance of the aforementioned lipid peroxidation products in the hippocampi of mice fed the control diet or the palmitate-enriched/linoleate-deficient diet. We found a significant increase in levels of all the three measured lipid peroxidation products, namely 4-HNE, MDA, and 8-iso PGF2α in the hippocampi of 3xTg-AD mice fed the control diet compared to the non-Tg mice on the same diet (Fig. 1B). More interestingly, feeding palmitate-enriched/linoleate-deficient diet for three months significantly exacerbated this lipid peroxidative damage as the levels of 4-HNE, MDA, and 8-iso PGF2α in the hippocampi of 3xTg-AD mice were significantly higher than the levels of 4-HNE, MDA, and 8-iso PGF2α in the hippocampi of 3xTg-AD mice fed the control diet (Fig. 1B). Oxidative and nitrosative protein damage is a cardinal feature of the AD brain [18]. We determined the protein carbonyl content and the extent of tyrosine nitration as a surrogate measure of oxidative and nitrosative protein damage in the hippocampus. The basal levels of the protein carbonylation and tyrosine nitration were significantly higher in the hippocampi of 3xTg-AD mice fed the control diet relative to the non-Tg mice fed the same diet (Fig. 1C). Palmitate-enriched/linoleate-deficient diet significantly increased the protein carbonyl content and the tyrosine nitration levels of the global proteome in the hippocampi of both the 3xTg-AD mice and non-Tg mice (Fig. 1C). Oxidative damage to DNA plays a seminal role in the pathogenesis of AD and other neurodegenerative disorders [51]. The levels of 8-OHdG and 8-OHG are considered as ubiquitous surrogate markers of oxidative damage to DNA and RNA, respectively [52], and the levels of these nucleic acid adducts are increased in the postmortem AD brain [51]. We found that the basal levels of 8-OHdG and 8-OHG were significantly higher in the hippocampi of 3xTg-AD mice fed the control diet relative to the non-Tg mice fed the same diet (Fig. 1D). Palmitate-enriched/linoleate-deficient diet significantly increased the levels of 8-OHdG and 8-OHG in the hippocampi of non-Tg mice and exacerbated the levels of 8-OHdG and 8-OHG in the hippocampi of 3xTg-AD mice (Fig. 1D). Our results show that palmitate-enriched/linoleate-deficient diet increases oxidative damage to cellular macromolecules that is manifested by a profound increase in peroxidative damage to lipids, oxidative modification of proteins, and oxidative damage to nucleic acids.

Fig.1

Palmitate-enriched/linoleate-deficient diet exacerbates oxidative stress and enhances oxidative damage to lipids, proteins, and nucleic acids in the hippocampi of 3xTg-AD and 3xTg-Control mice. Feeding 9-month-old 3xTg-AD mice and non-Tg mice a palmitate-enriched/linoleate-deficient diet (P⁺/L^–) for three months results in a significant increase in oxidative stress as determined by an increase in total ROS/RNS levels (A), oxidative damage to lipids as determined by higher levels of lipid peroxidation products, 4-HNE, MDA, and 8-iso PGF2α (B), oxidative damage to proteins as determined by higher levels of protein carbonylation and protein nitration (C), and oxidative damage to nucleic acids as determined by higher levels of 8-OHdG and 8-OHG (D) in the hippocampus. Data is expressed as Mean±SD and includes determination made in six different mice of each group. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 versus non-Tg mice fed a control diet; ^Δ p < 0.05, ^ΔΔ p < 0.01 versus 3xTg-AD mice fed a control diet; ^† p < 0.05, ^†† p < 0.01, versus non-Tg mice fed the palmitate-enriched/linoleate-deficient diet (P⁺/L^–).

Palmitate-enriched/linoleate-deficient diet causes a significant reduction in the endogenous anti-oxidant defense capacity and increases glutathione depletion thereby augmenting the oxidative stress burden

The superoxide ( $O_{2}^{• - -}$ ) radical is the most abundant and highly reactive ROS in the mamalian cells [53] that is constitutively generated by non-enzymatic processes in the mitochondria and other specific enzymatic processes in the cytosol [54, 55]. The enzymes SOD1 (also called Cu/Zn SOD) and SOD2 (also called MnSOD) are the first line of endogenous defense against the superoxide anion ( $O_{2}^{• - -}$ ) radical and chemically reduce it to the less deleterious pro-oxidant hydrogen peroxide molecule [56]. We immunoprecipitated SOD1 and SOD2 using specific antibodies from the hippocampal homogenates and determined the total SOD1 and SOD2 enzymatic activity in the respective immunoprecipitated lysates. The total enzymatic activities of SOD1 and SOD2 were significantly lower in the hippocampi of the 3xTg-AD mice fed the control diet relative to the non-Tg mice fed the same diet. Importantly, the palmitate-enriched/linoleate-deficient diet significantly decreased the total enzymatic activities of SOD1 and SOD2 in the hippocampi of both non-Tg and 3xTg-AD mice (Fig. 2A). Glutathione is an indispensable component and the most abundant thiol of the endogenous antioxidant defense response of the mammalian cells to peroxide-induced damage and oxidative stress [57]. Glutathione exists in the free thiol-form (GSH) that is active and the effete oxidized disulfide-form (GSSG) apportioned among the cytosol, mitochondria, and the endoplasmic reticulum [57]. The depletion of total glutathione (GSH and GSSG) as well as an increase in ratio of GSSG/GSH is indicative of the prevailing oxidative stress burden [57, 58]. The basal levels of the thiol-form of glutathione (GSH) as well as total glutathione levels (GSH and GSSG) were significantly decreased concomitant with the increase in levels of GSSG (oxidized disulfide-form) in the hippocampi of 3xTg-AD mice fed the control diet compared to non-Tg mice fed the same diet (Fig. 2B). The palmitate-enriched/linoleate-deficient diet also caused a significant depletion of total glutathione (Fig. 2C) and GSH concomitant with the increase in levels of GSSG in the hippocampi of 3xTg-AD mice and non-Tg mice suggesting that the palmitate-enriched/linoleate-deficient diet exacerbates the prevailing total glutathione and GSH depletion in the hippocampi of 3xTg-AD mice. The antioxidant effects of GSH are mediated by glutathione peroxidases (GPx) that catalyze the reduction of hydrogen peroxide or organic hydroperoxides using GSH as the reducing agent and consequently generating oxidized GSSG [59]. We determined total GPx activity in the hippocampal homogenates of mouse cohorts. Palmitate-enriched/linoleate-deficient diet significantly reduced the total GPx activity in the hippocampi of the 3xTg-AD mice and non-Tg mice (Fig. 2D). The basal total GPx activity was not significantly different between the 3xTg-AD mice and non-Tg mice fed the control diet (Fig. 2D). Glutathione-disulfide reductase (GSR) catalyzes the reduction of oxidized GSSG into the reduced active GSH form of glutathione. Palmitate-enriched/linoleate-deficient diet significantly decreased the total GSR activity in the non-Tg mice (Fig. 2D) and exacerbated the prevailing decrease in GSR activity in the hippocampi of 3xTg-AD mice (Fig. 2D). Collectively, our data show that the palmitate-enriched/linoleate-deficient diet decreases the activities of endogenous anti-oxidant enzymes, both in the hippocampi of non-Tg and 3xTg-AD mice that leads to glutathione depletion and reduced threshold to combat oxidative stress insult.

Fig.2

Palmitate-enriched/linoleate-deficient diet increases glutathione depletion and elicits deficits in the endogenous antioxidant defense system in the hippocampi of 3xTg-AD and 3xTg-Control mice. Feeding nine-month old 3xTg-AD mice and non-Tg mice a palmitate-enriched/linoleate-deficient diet for three months results in glutathione depletion and a concomitant significant reduction in the endogenous anti-oxidant defense capacity as determined by a decrease in the enzymatic activities of SOD1 and SOD2 (A), a decrease in free glutathione (GSH) levels concomitant with an increase in oxidized glutathione (GSSG) levels (B), a significant reduction in total glutathione levels (C), and an attenuation in the enzymatic activities of glutathione peroxidase (GPx) and glutathione-disulfide reductase (GSR) (D). Data is expressed as Mean±SD and includes determination made in six different mice of each group. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 versus non-Tg-mice fed a control diet; ^Δ p < 0.05, ^ΔΔ p < 0.01 versus 3xTg-AD mice fed a control diet; ^† p < 0.05, ^†† p < 0.01, versus non-Tg mice fed the palmitate-enriched/linoleate-deficient diet (P⁺/L^–).

Palmitate-enriched/linoleate-deficient diet exacerbates and synergizes the prevalent NF-κB activation in the hippocampi of 3xTg-AD mice

It is well characterized that oxidative stress induces NF-κB activation in a multitude of biological systems [60]. We initially determined the activation status of NF-κB by measuring the levels of phosphorylated IKKα/β (Inhibitor of Nuclear Factor Kappa-B kinase subunit alpha/beta) and phosphorylated IκBα (Nuclear Factor of Kappa B Inhibitor alpha) in the cytosolic extracts as well as the abundance of the p65 and p50 subunits in the nuclear homogenates. The palmitate-enriched/linoleate-deficient diet increased the levels of p-Ser^32/36 IκBα and p-Ser^176/180 IKKα/β in the cytosolic homogenates (Fig. 3A,C) concomitant with a pronounce increase in the levels of p65 and p50 subunits in the nuclear homogenates (Fig. 3B,D) from the hippocampi of 3xTg-AD mice and non-Tg mice. The basal status of NF-κB activation as surrogately determined by the levels of phosphorylated IKKα/β and IκBα in the cytosolic fractions as well as by the abundance of the p65 and p50 subunits in the nuclear fractions, was higher in the hippocampi of 3xTg-AD mice fed the control diet relative to the non-Tg mice cohorts (Fig. 3A-D). Palmitate-enriched/linoleate-deficient diet further exacerbated this increase in NF-κB activation with the levels of p-Ser^32/36 IκBα and p-Ser^176/180 IKKα/β (Fig. 3A,C) as well as the translocation of p65 and p50 subunits into the nucleus (Fig. 3B,D) higher in the hippocampi of 3xTg-AD mice fed the palmitate-enriched/linoleate-deficient diet versus the non-Tg mice fed the control diet (Fig. 3A-D). To determine whether the palmitate-enriched/linoleate-deficient diet-induced increase in p65 and p50 subunits in the nucleus resulted in a commensurate increase in NF-κB transcriptional activity, we determined the NF-κB DNA-binding activity in the nuclear fractions. The binding of the NF-κB to the exogenous oligonucleotide probe constituting a consensus NF-κB response element was significantly increased in the hippocampi of control diet-fed 3xTg-AD mice relative to the control diet-fed non-Tg mice (Fig. 3E). Importantly, the palmitate-enriched/linoleate-deficient diet exacerbated the prevalent increase in NF-κB DNA-binding activity in the hippocampi of 3xTg-AD mice compared to the non-Tg mice fed a control diet (Fig. 3E). To further validate the finding that an increase in NF-κB DNA-binding activity translates into increase NF-κB transcriptional activity, we determined the extent of chromatin-bound NF-κB that is associated with the transcriptional machinery. The transcriptional activity of the chromatin-bound NF-κB is positively regulated by the coactivator complex comprised of p300 and CBP (CREB-binding protein) [61] as well as negatively regulated by a corepressor complex comprising of SMRT (silencing mediator for retinoid or thyroid-hormone receptors, also called NCoR2) and NCoR1 (nuclear receptor co-repressor 1) [62]. We immunoprecipitated the chromatin-bound NF-κB by performing ChIP with p65 antibody and determined several components of the core transcriptional machinery associated. In the reverse cross-linked NF-κB (p65subunit) ChIP fraction, the abundance of the co-activators, p300 and CBP, appeared increased while the abundance of the co-repressors, SMRT (NCoR2) and NCoR1, appeared reduced in the hippocampi of 3xTg-AD mice fed the palmitate-enriched/linoleate-deficient diet compared to the non-Tg mice fed a control diet (Fig. 3F). Collectively, our data shows that the palmitate-enriched/linoleate-deficient diet increases NF-κB transcriptional activity in the hippocampi of 3xTg-AD mice.

Fig.3

Palmitate-enriched/linoleate-deficient diet increases the NF-κB activation in the hippocampi of 3xTg-AD and non-Tg mice. Representative western blots and densitometric analysis show that show that feeding 9-month old 3xTg-AD mice and non-Tg a palmitate-enriched/linoleate-deficient diet for three months results in pronounced NF-κB activation as determined by increase in phosphorylation of IKKα/β at Ser^176/180 and IκBα at Ser^32/36 residues in the cytosolic homogenates (A,C); as well as increase in the levels of the p65 and p50 subunits of NF-κB in the nucleus (B,D). DNA-binding activity assays as a surrogate measure for the relative transcriptional activity of NF-κB demonstrates the increase binding of NF-κB in the nuclear homogenates from the hippocampi to the exogenous generic motif constituting the κB response element (E). Western blotting analysis of reverse cross-linked NF-κB - ChIP samples clearly shows increased association of the known NF-κB coactivators, p300 and CBP; as well as decreased association of NF-κB corepressors, SMRT and NCoR1, with the chromatin-bound NF-κB (F). Data is expressed as Mean±SD and includes determination made in six different mice of each group. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 versus non-Tg mice fed a control diet; ^ΔΔ p < 0.01 versus 3xTg-AD mice fed a control diet; ^† p < 0.05, ^†† p < 0.01, versus non-Tg mice fed the palmitate-enriched/linoleate-deficient diet (P⁺/L^–).

Palmitate-enriched/linoleate-deficient diet synergizes and exacerbates the existing increase in the binding of NF-κB to the Bace1 promoter as well as the subsequent augmentation in Bace1 promoter activity in the hippocampi of 3xTg-AD mice

We have delineated a critical role of the dimeric transcription factor NF-κB in the palmitate-induced increase in BACE1 expression through transcriptional mechanisms [31, 63]. We determined whether the palmitate-enriched/linoleate deficient diet-induced increase in NF-κB DNA-binding activity to the exogenous oligonucleotide sequence constituting the κB response-element (as shown in Fig. 3E) resulted into changes in the extent of NF-κB binding to the endogenous κB response-element in the Bace1 promoter. Our ChIP-qPCR assay showed that the palmitate-enriched/linoleate-deficient diet accentuates the prevailing increase in NF-κB binding to the proximal Bace1 promoter in the hippocampi of 3xTg-AD mice relative to the non-Tg mice fed a control diet (Fig. 4A). To further determine the functional relevance of this enhanced NF-κB binding to the Bace1 promoter, we determined the transcriptional activation status of the Bace1 promoter. We determined the recruitment and occupancy of RNA polymerase II (RNAPII) at the transcription start site (TSS) in the Bace1 promoter by ChIP-qPCR. The recruitment and occupancy of RNAPII was significantly increased at the TSS in the Bace1 promoter in the hippocampi of 3xTg-AD mice fed the palmitate-enriched/linoleate-deficient diet compared to the non-Tg mice fed a control diet (Fig. 4B). We subsequently determined whether the increase in binding of NF-κB to the proximal Bace1 promoter resulted in a commensurate increase in BACE1 expression. The mRNA expression of BACE1 was significantly augmented in the hippocampi of 3xTg-AD mice fed the palmitate-enriched/linoleate-deficient diet compared to the non-Tg mice fed a control diet (Fig. 4C). Furthermore, this increase in BACE1 mRNA expression resulted in an increase in BACE1 protein levels in the whole cell homogenates as well as within the membrane fractions from the hippocampi 3xTg-AD mice fed the palmitate-enriched/linoleate-deficient diet compared to the non-Tg mice fed a control diet (Fig. 4D, E). As BACE1 is a transmembrane protein [28, 29], we determined the BACE1 enzymatic activity in the membrane fractions from the hippocampi of mice. The total BACE1 enzymatic activity was significantly higher in the membrane fractions from the hippocampi 3xTg-AD mice fed the palmitate-enriched/linoleate-deficient diet compared to the control diet-fed non-Tg mice (Fig. 4F).

Fig.4

Palmitate-enriched/linoleate-deficient diet increases the binding of NF-κB to the Bace1 promoter that is associated with enhanced BACE1 expression and enzymatic activity in the hippocampi of 3xTg-AD mice. ChIP-qPCR analysis shows that palmitate-enriched/linoleate-deficient diet increases the p65 NF-κB binding to the proximal Bace1 promoter (A), and the recruitment and occupancy of RNAPII at the NF-κB binding sites in the Bace1 promoter in the hippocampi of 3xTg-AD mice (B). Real-time RT-PCR analysis shows that palmitate-enriched/linoleate-deficient diet increases BACE1 mRNA expression in the hippocampi of 3xTg-AD mice (C). Representative western blots (D) and densitometric analysis (E) show that palmitate-enriched/linoleate-deficient diet increases the protein levels of BACE1 in cytosolic homogenates and membrane fractions from the hippocampi of 3xTg-AD mice. Enzyme activity assays show that palmitate-enriched/linoleate-deficient diet increases the enzymatic activity of BACE1 in the membrane fractions from the hippocampi of 3xTg-AD mice (F). Data is expressed as Mean±SD and includes determination made in six different mice of each group. ^** p < 0.01, ^*** p < 0.001 versus non-Tg mice fed a control diet; ^ΔΔ p < 0.01, ^ΔΔΔ p < 0.001 versus 3xTg-AD mice fed a control diet; ^† p < 0.05, ^†† p < 0.01, versus non-Tg mice fed the palmitate-enriched/linoleate-deficient diet (P⁺/L^–).

Palmitate-enriched/linoleate-deficient diet exacerbates the Aβ burden in the hippocampi of 3xTg-AD mice

We determined the physiological and functional coupling of this palmitate-induced increase in BACE1 expression and enzymatic activity to the Aβ burden in the hippocampi of the 3xTg-AD mice fed the palmitate-enriched/linoleate-deficient diet. We performed a sequential extraction procedure that allowed determination of the water soluble Aβ (TBS soluble fraction), water insoluble but detergent soluble Aβ (2% SDS soluble fraction), and detergent insoluble (70% formic acid soluble fraction) Aβ fractions in the hippocampi of cohorts of 3xTg-AD and the non-Tg mice. Palmitate-enriched/linoleate-deficient diet exacerbated the Aβ₄₀ and Aβ₄₂ abundance in water soluble fraction (TBS soluble fraction) by 40.51% and 35.37%, respectively (Fig. 5A). The increase in Aβ₄₀ and Aβ₄₂ content in the detergent soluble fraction (2% SDS soluble fraction) was relatively more pronounced with the Aβ₄₀ and Aβ₄₂ levels being 92.17% and 91.04% higher, respectively, in the hippocampi of 3xTg-AD mice fed the palmitate-enriched/linoleate-deficient diet relative to the non-Tg mice fed a control diet (Fig. 5B). Furthermore, palmitate-enriched/linoleate-deficient diet caused a 75.11% and 51.92% increase in the Aβ₄₀ and Aβ₄₂ content, respectively, in the 70% formic acid soluble fraction (TBS insoluble and 2% SDS insoluble fraction) from the hippocampi of 3xTg-AD mice relative to the non-Tg mice fed a control diet (Fig. 5C). We further computed the total Aβ₄₀ and Aβ₄₂ burden by integrating the respective data values from the three aforementioned extraction fractions—TBS soluble fraction, 2% SDS soluble fraction, and the 70% formic acid soluble fraction; by a process of summation. Palmitate-enriched/linoleate-deficient diet increased the total Aβ₄₀ and Aβ₄₂ content by 65.18% and 51.47%, respectively, in the hippocampi of 3xTg-AD mice relative to the non-Tg mice fed a control diet.

Fig.5

Palmitate-enriched/linoleate-deficient diet increases the Aβ burden in the in the hippocampi of 3xTg-AD mice. ELISA immunoassays show that palmitate-enriched/linoleate-deficient diet (P⁺/L^–) increases TBS-soluble (water soluble) Aβ levels (A), 2% SDS-soluble (water insoluble and detergent soluble) Aβ levels (B), 70% formic acid soluble (water insoluble and detergent insoluble) Aβ levels (C), and total Aβ burden (D) in the hippocampi of 3xTg-AD mice. Data is expressed as Mean±SD and includes determination made in six different mice of each group. ^*** p < 0.001 versus non-Tg mice fed a control diet; ^ΔΔ p < 0.01, ^ΔΔΔ p < 0.001 versus 3xTg-AD mice fed a control diet. u.d., undeterminable.

DISCUSSION

A multitude of studies have associated saturated fat intake to cognitive dysfunction and the risk for developing AD [2 , 64–66]. A plethora of studies have also demonstrated that saturated fat-enriched diets evoke learning and memory deficits as well as cognitive deficits in a host of animal models [13–15 , 67]. Although decreased cognitive function is suggested to be associated with diets high in total, trans, and saturated fat, the contribution of each dietary fat in cognitive function is still unclear. At the molecular level, a considerable number of laboratory animal studies have shown that high-fat diets precipitate an increase in Aβ burden by mechanisms that are dependent on commensurate increases in BACE1 expression as well as independent of fluxes in BACE1 activity [68 –70]. The composition of the diets used in the aforementioned studies was diverse resulting in a very wide spectrum of dietary variables, in terms of lipid content, lipid composition, and caloric density. Furthermore, other studies have either high cholesterol or high sucrose added to the prevailing high fat diets, which may significantly exacerbate the cognitive dysfunction and Aβ burden elicited by high fat diets or even induce the effects independent of the high fat content of the diet. The confounding factors emanating due to the heterogeneity of the diets used in the aforementioned studies was further compounded by the lack of caloric parity between the diets. Our palmitate-enriched/linoleate-deficient diet feeding paradigm is devoid of some the confounding variables inherent in other studies. Specifically, the palmitate-enriched/linoleate-deficient diet relative to the control diet is disparate only in the content of palmitate (2.2% w/w versus 0.8% w/w) and linoleate (0.8% w/w versus 2.2% w/w), respectively. Furthermore, the palmitate-enriched/linoleate-deficient diet is isocaloric relative to the control diet and therefore the confounding variable of lack of caloric parity is largely obviated.

The role of oxidative stress in the etio-pathogenesis of AD is well characterized and has been the subject of numerous thematic reviews. We show that the palmitate-enriched/linoleate-deficient diet increases oxidative stress burden by targeting a wide range of facets that contribute to the oxidative stress burden. We demonstrate that the palmitate-enriched/linoleate-deficient diet increases lipid peroxidation and oxidative damage to proteins. Lipid peroxidation is a key biochemical component that contributes to the etio-pathogenic mechanisms that underlie the predisposition and the risk fo development of AD [71, 72]. Increased lipid peroxidation has been reported in 3xTg-AD mice [73] and our study recapitulates that finding and demonstrates that the palmitate-enriched/linoleate-deficient diet exacerbates lipid peroxidation. Furthermore, our study also unveiled that the palmitate-enriched/linoleate-deficient diet increases the oxidative damage to neural proteins, as surrogately determined by the total protein carbonylation load, in the hippocampi of 3xTg-AD mice and non-Tg mice. Increased carbonyl content of neural proteins is a bona-fide marker of oxidative stress in the brain and highly prevalent in the AD brain [74, 75]. Also, augmented protein carbonylation has been reported in the brains of the 3xTg-AD mice at the earliest pathological changes [76, 77]. Our study is novel in that it shows that the palmitate-enriched/linoleate-deficient diets increase the protein carbonylation burden, both in the brains of the non-Tg mice and 3xTg-AD mice. Deficiencies in endogenous antioxidant capacity have been posited as the decisive contributing factor in the increased oxidative stress burden observed in AD [16, 51]. SOD1 and SOD2 are the first line of the endogenous antioxidant defense against superoxide ( $O_{2}^{• - -}$ ) radical [56]. The enzymatic activities of SOD1 and SOD2 have been shown to be reduced in the postmortem AD brain [78]. Our study found that the palmitate-enriched/linoleate-deficient diet causes a profound decrease in the enzymatic activities of SOD1 and SOD2 in the hippocampi of 3xTg-Control and 3xTg-AD mice. GSH is an integral component of the endogenous antioxidant system that combats ROS and peroxide-induced damage to membrane lipids (lipid peroxidation) and carbonylation of proteins [57]. Our study showed a profound decrease in free GSH levels concomitant with an increase in oxidized form of glutathione (GSSG), indicative of reduced endogenous antioxidant capacity or existence of pronounced oxidative stress.

Our current study implicates NF-κB as a mediator and downstream effector of oxidative stress induced augmentation in BACE1 activity and Aβ genesis. NF-κB has been widely implicated in the regulation of BACE1 expression and activity in response to a multitude of upstream physiological, biochemical, and signaling stimuli [63 , 80]. While the activation of NF-κB signaling and transcriptional activity by oxidative stress and ROS is well established [60], it is important to consider the findings of reciprocal pro-oxidant effects of NF-κB activation [81]. NF-κB activation is known to induce the expression of components of pro-oxidant enzyme systems that include the NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) subunit NOX2 (gp91 phox) [82], neuronal nitric oxide synthase or NOS1 [83], and inducible nitric oxide synthase or NOS1 [84]. Our current study shows that the palmitate-enriched/linoleate-deficient diet increases NF-κB activation that could be a cause as well as consequence of the associated oxidative stress observed. Furthermore, the NF-κB driven BACE1 expression and the subsequent exacerbation of Aβ burden could be an indirect phenomenon contingent on the NF-κB-induced oxidative stress. Also, the palmitate-enriched/linoleate-deficient diet-induced increase in BACE1 expression and the subsequent exacerbation of Aβ burden could be partially independent of NF-κB-mediated transcriptional upregulation. A multitude of studies have found that high fat diets cause an increase in BACE1 activity and escalation of Aβ burden in transgenic mouse models of AD [68–70 , 85–87]. However, the aforementioned studies utilized a dietary regimen that was not isocaloric. Furthermore, some studies utilized diets that contained high sucrose content [87] or high cholesterol [69 , 86] concomitant with the high lipid content. Diets high in sucrose [88] and enriched in cholesterol [89] are known to increase Aβ plaque in rodents. Our study is therefore unique, as it utilized a dietary regimen enriched in one sFFA, i.e. palmitate, and deficient in one PUFA, i.e., linoleate, without the increase in any other macronutrient density as well as maintaining caloric parity. Studies that have determined the effects of exogenous palmitate in immortalized cell-lines and primary cultures have found the palmitate increases BACE1 expression by transcriptional upregulation by mechanisms that involve STAT3 (Signal Transducer and Activator of Transcription 3) activation in astrocytes [90] and GPR40 (G-Protein Coupled Receptor 40)-mediated activation of Akt/mTOR (mechanistic Target of Rapamycin)/ HIF1α (Hypoxia Inducible Factor 1 alpha) and Akt/NF-κB signaling pathways in human SK-N-MC neuronal cells [91]. These studies have demonstrated that palmitate increases BACE1 expression and Aβ genesis by mechanisms involving an increase in ceramide levels that promotes oxidative stress and neuroinflammation [90 , 92–94].

In the context of the current study, the upstream mechanisms that underlie the palmitate-enriched/linoleate-deficient diet induced increase in oxidative stress remain to be determined. A recent study found that the palmitate-induced activation of the Non-Receptor Tyrosine Kinase (NRTK), Src causes an increase in superoxide ( $O_{2}^{• - -}$ ) formation through NADPH oxidase activation in rat insulinoma INS-1D cell-line and dissociated islet pancreatic islet cultures [95]. Palmitate has also been shown to cause mitochondrial dysfunction in a plethora of cell-culture model systems and primary cultures [96]. Additionally, palmitate increases mitochondrial $O_{2}^{• - -}$ formation in dissociated rat pancreatic islet cultures [97], insulinoma INS-1E cells [98], cardiomyocytes [99], vascular smooth muscle cells [100], endothelial cells [101], and adipose tissue [102]. However, whether the deletrious effects we demonstarte are due mainly to the increase in the content of palmitate or whether these effects are exacerbated with the concomittant reduction in linoleate needs to be determined in our future studies.

In conclusion, our data shows that palmitate-enriched/linoleate-deficient diet increases the oxidative stress burden and exacerbates the oxidative damage to lipids, proteins, and nucleic acids in the hippocampi of 3xTg-AD mice. This enhanced oxidative stress burden is intricately associated with NF-κB activation leading to increased BACE1 expression and enzymatic activity that culminates in the exacerbation of the Aβ burden. Our study delineates the role of increased NF-κB activation and transcriptional activity in the upregulation of BACE1 expression and the exacerbation of Aβ burden in the hippocampi of 3xTg-AD mice, in response to palmitate-enriched/linoleate-deficient diet-induced oxidative stress. Further studies are warranted to determine the molecular basis of this inter-relationship between palmitate-enriched/linoleate-deficient diet, induction of oxidative stress, and NF-κB activation in the context of the etio-pathogenic mechanisms involved in AD.

Footnotes

ACKNOWLEDGMENTS

This work was supported by a Grant from the NIH (R01AG045264) to Dr. Othman Ghribi.

Authors’ disclosures available online ().

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A Diet Enriched in Palmitate and Deficient in Linoleate Exacerbates Oxidative Stress and Amyloid-β Burden in the Hippocampus of 3xTg-AD Mouse Model of Alzheimer’s Disease

Abstract

Keywords

INTRODUCTION

MATERIALS AND METHODS

Mouse experiments

Western blot analysis

Immunoprecipitation

Quantitation of Aβ40 and Aβ42 levels by enzyme-linked immunosorbent assay (ELISA)

Quantitative real time RT-PCR analysis

Total reactive oxygen species (ROS) and reactive nitrogen species (RNS) measurement

Measurement of 4-hydroxynonenal (4-HNE) protein adducts

Measurement of malondialdehyde (MDA) protein adducts

Measurement of 8-iso-Prostaglandin F2α (8-iso-PGF2α)

Measurement of protein carbonyl content and oxidative modification of proteins

Measurement of protein tyrosine nitration content

Measurement of 8-hydroxydeoxyguanosine (8-OHdG) and 8-hydroxyguanosine (8-OHG) levels

Determination of oxidized glutathione (GSSG), reduced glutathione (GSH), and total glutathione content

Superoxide dismutase (SOD) activity assay

Glutathione peroxidase (GPx) activity assay

Glutathione-disulfide reductase (GSR) activity assay

FRET-based BACE1 activity assay

Chromatin immunoprecipitation (ChIP) analysis

NF-κB DNA binding activity assay

Statistical analysis

RESULTS

Palmitate-enriched/linoleate-deficient diet causes oxidative stress and leads to increase in lipid peroxidation, oxidative protein damage, and nucleic acid oxidation in the hippocampi of 3xTg-AD mice

Palmitate-enriched/linoleate-deficient diet causes a significant reduction in the endogenous anti-oxidant defense capacity and increases glutathione depletion thereby augmenting the oxidative stress burden

Palmitate-enriched/linoleate-deficient diet exacerbates and synergizes the prevalent NF-κB activation in the hippocampi of 3xTg-AD mice

Palmitate-enriched/linoleate-deficient diet synergizes and exacerbates the existing increase in the binding of NF-κB to the Bace1 promoter as well as the subsequent augmentation in Bace1 promoter activity in the hippocampi of 3xTg-AD mice

Palmitate-enriched/linoleate-deficient diet exacerbates the Aβ burden in the hippocampi of 3xTg-AD mice

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

Quantitation of Aβ₄₀ and Aβ₄₂ levels by enzyme-linked immunosorbent assay (ELISA)