Evaluation of Neuropathological Effects of a High-Fat Diet in a Presymptomatic Alzheimer’s Disease Stage in APP/PS1 Mice

Abstract

Alzheimer’s disease (AD) is currently an incurable aging-related neurodegenerative disorder. Recent studies give support to the hypotheses that AD should be considered as a metabolic disease. The present study aimed to explore the relationship between hippocampal neuropathological amyloid-β (Aβ) plaque formation and obesity at an early presymptomatic disease stage (3 months of age). For this purpose, we used APPswe/PS1dE9 (APP/PS1) transgenic mice, fed with a high-fat diet (HFD) in order to investigate the potential molecular mechanisms involved in both disorders. The results showed that the hippocampus from APP/PS1 mice fed with a HFD had an early significant decrease in Aβ signaling pathway specifically in the insulin degrading enzyme protein levels, an enzyme involved in (Aβ) metabolism, and α-secretase. These changes were accompanied by a significant increase in the occurrence of plaques in the hippocampus of these mice. Furthermore, APP/PS1 mice showed a significant hippocampal decrease in PGC-1α levels, a cofactor involved in mitochondrial biogenesis. However, HFD does not provoke changes in neither insulin receptors gene expression nor enzymes involved in the signaling pathway. Moreover, there are no changes in any enzymes (kinases) involved in tau phosphorylation, such as CDK5, and neither in brain oxidative stress production. These results suggest that early changes in brains of APP/PS1 mice fed with a HFD are mediated by an increase in Aβ_1 ‒ 42, which induces a decrease in PKA levels and alterations in the p-CREB/ NMDA2B /PGC1-α pathway, favoring early AD neuropathology in mice.

Keywords

Alzheimer’s disease APPSwe/PS1dE9 hippocampus insulin receptor mitochondria tau

INTRODUCTION

After over a hundred years of intense research into Alzheimer’s disease (AD), the causes of this pathology’s development are yet to be elucidated [1 –5]. What is obvious is that the pathogenesis of AD is a complex and long-lived process [3 –5]. In addition, the current drugs for AD therapy only provide a temporary improvement on cognition [5]. Due to the lack of an effective treatment to halt the progression of AD, it is necessary to investigate the potential pathways involved in this disease onset. Over the last 25 years, different hypotheses have been formulated to explain the causes of the onset of this disease. What seems to be clear is that aging is probably the main key factor in the development of late sporadic AD. In addition to aging, in the early 1990 s John Hardy proposed the “amyloid cascade” hypothesis [6 –8]. This hypothesis states that depositions of the amyloid-β peptide (Aβ) produce senile plaques, that lead to the formation of neurofibrillary tangles, which in turn cause neuronal death that lead to a cognitive decline [6 –10]. In the same time period, the “oxidative stress” hypothesis emerged and several research groups demonstrated the prominent role of oxidative stress in the brain of AD in the brain of the affected patients [11 –13]. Likewise, Swerdlow and Khan, proposed the so-called “mitochondrial cascade” hypothesis, suggesting that mitochondria damage occurs prior to brain Aβ accumulation [14 –17].

More recently, research in AD has established a clear relationship between obesity, insulin resistance, diabetes, and dementia [18 –24]. In addition, Balakrishnan and colleagues demonstrated that obesity increases the plasma levels of Aβ_1 ‒ 42 [20]. Therefore, the control of body weight could be an additional factor for the development of the late sporadic form of AD. Interestingly, the term “Brain Insulin Resistance”, which is defined as reduced tissue responsiveness to the action of insulin, could probably fit better as a potential cause of sporadic AD [25 –41]. An anomaly in the insulin response pathway could affect multiple brain cellular mechanisms such as mitochondria, Aβ metabolism, Aβ production, neuroinflammation, tau phosphorylation, and also learning and memory capacities [33 –44]. Thus, some researchers denominate AD as type III diabetes [22]. Although many preclinical and clinical studies point out to this close relationship between AD and insulin resistance, few preclinical studies have evaluated it in an early stage of the disease known as presymptomatic stage.

The majority of AD preclinical research is carried out using transgenic animal models that have increased Aβ levels compared to wild-type (WT) mice. While the Aβ pathology is mimicked in these models, many other factors associated with the AD pathology are not. The APP/PS1 double transgenic mouse is a genetically modified model that has been generated to try to mimic human familial AD pathology. In the APP/PS1 line, two strategies are combined to reach elevated Aβ levels: overexpression of the mutant human amyloid precursor protein (APP) encoding gene, together with the mutant presenilin-1 (PS1) gene, which additionally impairs amyloid protein processing, leading to elevated Aβ₄₂ levels [45 –47]. The APP/PS1 mouse is a good model to study the early onset of pathological changes, while the Tg2576, APP23, and 3xtg strains are approximate better models for the study of a late onset form of the disease (reviewed by Bilkei-Gorzo [47]). Although none of these models show, in general, brain neuronal loss from cortex and hippocampus, the APP/PS1 strain shows amyloid plaque formation along with tau protein hyperphosphorylation. Furthermore, it has been demonstrated that APP/PS1 mice show early memory loss that becomes evident at the age of 6 months [39 –42].

The aims of the present study were to evaluate the molecular pathways involved in the AD pathology process in APP/PS1 mice fed with a high-fat diet (HFD) [32–37 , 44]. We focus on the evaluation of HFD, together with Aβ_1-42 peptide, are cofactors that trigger the AD pathology in a presymptomatic stage of the disease, at three months of age. Moreover, we tried to detect very early biochemical and molecular neuropathology clues that may allow a better understanding of a link between AD and obesity.

MATERIALS AND METHODS

Animals

APPswe/PS1dE9 (APP/PS1) and C57BL/6 (WT) male mice were used in this study. APP/PS1 animals co-express a Swedish (K594M/N595L) mutation of a chimeric mouse/human APP (Mo/HuAPP695swe), together with the human exon-9-deleted variant of PS1 (PS1-dE9), allowing these mice to secrete elevated amounts of human Aβ peptide. The generation of mice expressing the human mutated forms APPswe and PS1dE9 has already been described [40 –43]. Identification of transgenic mice was carried out from tail clips, using the polymerase chain reaction (PCR) technique with the PCR conditions proposed by the Jackson Laboratory.

Sixty animals were used, divided into four groups: a) WT mice fed with a standard (CT) diet; b) WT mice fed with a high fat diet (HFD), consisting of 25% fat (45 kcal %), mainly from hydrogenated coconut oil, 21% protein (16 kcal %), and 49% carbohydrate (39 kcal %); Cat# D08061110 (Research Diets Inc., New Brunswick, USA); c) APP/PS1 mice fed with a CT diet and d) APP/PS1 mice fed with a HFD (Fig. 1; Table 1). Following in vivo testing, 3-month-old animals were sacrificed and at least 6 mice of each group were used for RNA and protein extract isolation, with an additional 4 mice used for immunofluorescence. The animals were kept under controlled temperature, humidity and light conditions with food and water provided ad libitum. Mice were treated in accordance with the European Community Council Directive 86/609/EEC and the procedures established by the Department d’Agricultura, Ramaderia i Pesca of the Generalitat de Catalunya. Every effort was made to minimize animal suffering and to reduce the number of animals used.

Novel Object Recognition test

The Novel Object Recognition test was used for testing the hippocampal-dependent recognition memory of mice. The task procedure consisted of three phases: habituation, familiarization and test phase. In habituation phase, mice explored individually a circular open-field arena of 40 cm of diameter without object for three consecutive days, 10 min for each session. On the fourth day (familiarization phase), each mouse was placed in the arena containing two identical objects (A+A) in the middle of the field for 10 min. To perform the test phase, mice were returned 24 h later to open-field arena with two objects, one was identical to the day before and the other was a novel object (A+B) for 10 min. The light intensity in the middle of the field was 30 Ix in all phases and the arena and objects were cleaned with 96% ethanol between animals, so as to eliminate olfactory cues. Exploration was defined as the orientation of snout of the animals toward the object, sniffing or touching [49]. The data were measured by discrimination index (DI), which indicates the difference in exploration time between familiar and novel object. Therefore, the exploration time of each object was divided by the total time of exploration, measured in seconds and indicated in percentage [43, 44].

Serum insulin ELISA

Heart puncture was used to collect whole blood samples from 3-month-old WT and APP/PS1 mice at the moment of death after a 5-h morning fast. Blood samples were transferred to Serum-Gel Z microcentrifuge tubes (Sarstedt, Numbrecht, Germany), for serum separation. The samples were collected and kept at room temperature, and the serum was separated by centrifugation for 10 min at 5000× g. Serum insulin levels were measured with Rat/Mouse Insulin ELISA kits (Cat #: EZRMI-13K; EMD Millipore; St. Charles, MO, USA), according to the manufacturer’s instructions, utilizing 10μl of mouse serum.

Total blood cholesterol and triglycerides measurements

Total cholesterol and triglyceride blood levels were measured following 4-h-long fast at the point of sacrifice with Accutrend Plus meter (Roche Diagnostics, Switzerland).

Glucose and insulin tolerance tests

Intraperitoneal glucose tolerance tests (IP-GTT) were performed in accordance with the previously published guidelines [50]. For IP-GTT, mice were fasted overnight for 16 h. The test was performed in a quiet room, preheated to +30°C. The tip of the tail was cut with the heparin-soaked (Heparina Rovi, 5000 IU/ml; Rovi S.A.; Madrid, Spain) scissors, 30 min prior to intraperitoneal glucose injection. Blood glucose levels in the tail vein were measured at –30, 0, 5, 15, 30, 60, and 120 min after the glucose injection with the Ascensia ELITE blood glucose meter (Bayer Diagnostics Europe Ltd.; Dublin, Ireland).

Immunofluorescence staining

Mice used for immunofluorescence studies were anesthetized by intraperitoneal injection of sodium pentobarbital (80 mg/kg) and perfused with 4% paraformaldehyde (PFA) diluted in 0.1M phosphate buffer (PB). Brains were removed and stored in the same solution overnight (O/N) at 4°C, and then they were cryoprotected in 30% sucrose-PFA-PB solution. Samples were frozen at –80°C and coronal sections of 20μm of thickness were obtained by a cryostat (Leica Microsystems, Wetzlar, Germany).

Free-floating sections were first washed three times with 0.1 mol/l PBS pH 7.35 and after five times with PBST (PBS 0.1 M, 0.2% Triton X-100). Then, they were incubated in a blocking solution containing 10% fetal bovine serum (FBS), 1% Triton X-100, and PBS 0.1 M- 0.2% gelatin for 2 h at room temperature. After that, slices were washed with PBST (PBS 0.1 M, 0.5% Triton X-100) five times for 5 min each and incubated with polyclonal rabbit anti-GFAP (1:1000; Dako, Glostrup, Denmark), rabbit anti-IBA1 (1:1000; Wako Chemical USA) and monoclonal anti-βA 1-42 (12F4, which detects the c-terminus of βA) (1:1000; Covance, USA) primary antibodies at 4°C O/N. Sequentially, sections were washed with PBST (PBS 0.1 M, 0.5% Triton X-100) 5 times for 5 min and incubated with Alexa Fluor 594 goat anti-rabbit and Alexa Fluor 488 donkey anti-rabbit antibodies (1:500; Invitrogen, Eugene, OR, USA) for 2 h at room temperature. The staining for Aβ plaques was performed using S-Thioflavin (ThS 0.002%, Sigma-Aldrich). Slices were incubated for 8 min in the dark at room temperature, washed with 50% ethanol twice for 1 min, and rinsed with PBS 0.1 M. Later, sections were co-stained with 0.1μg/ml Hoechst 33258 (Sigma-Aldrich, St Louis, MO, USA) for 15 min in the dark at room temperature and washed with PBS 0.1M. Finally, the slides were mounted using Fluoromount G (EMS), and image acquisition was performed with an epifluorescence microscope fluorescence filter (BX41 Laboratory Microscope, Melville, NY-Olympus America Inc.). For plaque quantification, similar and comparable histological areas were selected, particularly with the hippocampus and the whole cortical area positioned adjacently.

Immunoblot analysis

To perform western blot analysis, first hippocampi were dissected, frozen, and stored at –80°C until use. After, samples were homogenized in lysis buffer (50 mM Tris-HCl 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100) and protease inhibitor mixture (Complete, Roche Diagnostics, Barcelona, Spain). The homogenates were centrifuged at 12,000 g for 5 min at 4°C, and the protein quantity of the supernatant was determined using a Pierce BCA Protein Assay Kit (Pierce Company, Rockford, MI, USA). Sequentially, 10μg of protein per sample were analyzed using the western blot method. For that, they were denatured at 95°C for 5 min in sample buffer (0.5M Tris–HCl, pH 6.8, 10% glycerol, 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 0.05% bromophenol blue). Samples were separated by electrophoresis on 10% acrylamide gels (100 V cte) and they were transferred to polyvinylidene difluoride (PVDF) sheets (Immobilon-P; Millipore Corp., Bedford, MA, USA) (200 mA cte). Then, membranes were blocked for 1 h with 5% non-fat milk dissolved in TBS-T buffer (mM Tris; 1.5% NaCl, 0.05% Tween 20, pH 7.5), washed with TBS-T without containing milk 3 times for 5 min and incubated with primary antibodies O/N at 4°C, as detailed in Table 2. Subsequently, blots were washed thoroughly in TBS-T buffer and incubated at room temperature for 1 h with a horseradish peroxidase-conjugated IgG secondary antibody (Table 2), followed by enhanced chemiluminescence detection (Immobilon Western, Chemiluminescent HRP Substrate, Millipore) according to the supplier’s instructions. Protein levels were determined using Chemidoc XRS+ Molecular Imager detection system (Bio-Rad), with ImageLab image analysis software. Measurements were expressed as arbitrary units and all results were normalized to the corresponding GAPDH.

RNA extraction and quantification

The hippocampi of mice were homogenized in Trizol reagent (Life Technologies Corporation) for RNA isolation, as described previously [48]. Sequentially, chloroform was added and RNA was precipitated from the aqueous phase with isopropanol at 4°C. The RNA pellet was reconstituted in RNAse-free water, with the RNA integrity determined by Agilent 2100 Bioanalyzer.

Semi-quantitative Real-time-PCR

First-strand cDNA was reverse transcribed from 2μg of total RNA using the High Capacity cDNA Reverse Transcription kit, according to manufacturer’s protocol (Applied Biosystems). Equivalent amounts of cDNA were used for qRT-PCR and each sample was analyzed in triplicate for each gene. TaqMan gene expression essays (Applied Biosystems) were used, as detailed in Table 3, and they were performed on StepOnePlus Real Time PCR system (Applied Biosystems). The values were normalized to gapdh and tbp using the delta-delta Ct method.

Antioxidant assays

One fraction of the cortex samples was homogenized manually by means of a Teflon bar after the addition of Radio Immune Precipitation Assay buffer (RIPA) containing (in mM) 50 TrisHCl, 150 NaCl, 5 NaF and 0.1% of sodium dodecyl sulphate (SDS), 1% Triton X-100, 1% Sodium deoxycholate (final pH 7.4). A cocktail of protein-inhibitors was mixed with RIPA buffer before homogenation (Leupeptin 2 ug/mL, Pepstatine, 2 ug/mL, PMSF 1 mM, NaVOn 1 mM, Aprotinin, 1.7 mg/mL). The homogenates were kept on ice for 15 min and then centrifuged at 2,000 g for 10 min and the precipitated fraction was discarded. Supernatants of cortical extracts were aliquoted and stored at –80°C for TBARS determination. Malondialdehyde (MDA) (which is an end-product of lipid peroxidation), was measured by the thiobarbituric reactive substances (TBARs) assay following the Uchiyama and Mihara method [51]. The formation of MDA-TBA adducts was fluorometrically quantified at an excitation wavelength of 515 nm and an emission wavelength of 550 nm. The calibration curve was determined using tetraethoxypropane. Values were expressed in nmol/mg protein.

The total brain proteins were determined using the Bradford protein assay [52].

Other fractions of the cortex samples were homogenized in cold phosphate buffer (50 mM potassium phosphate, 1 mM EDTA, pH 7.5) and centrifuged at 10,000 g for 10 min (4°C). The resulting supernatant was aliquoted and stored at –80°C for posterior determination of advanced oxidation protein products (AOPP) and antioxidant enzymatic activities.

AOPP content in cortex homogenates was assayed by a modification of Witko–Sarsat’s method [53, 54]. The formation of AOPP was spectrophotometrically measured at 340 nm and results were obtained through a standard calibration curve using 100μL of chloramine-T solution. AOPP concentration was expressed as μmol of chloramine-T equivalents per mg of protein.

Cellular levels of oxidant molecules are controlled by enzymatic and non-enzymatic antioxidants. The major antioxidant enzyme is superoxide dismutase (SOD), which plays a critical role in scavenging superoxide radical. In the same line, another antioxidant enzyme, glutathione reductase (GR) has the role of maintaining the reduced state of the main non-enzymatic antioxidant molecule, glutathione (GSH), by catalyzing the NADPH-dependent reduction from its oxidized form GSSG.

SOD (EC 1.15.1.1) activity was determined by using Arbor Assay Superoxide dismutase colorimetric activity kit (Arbor Assay, Ann Arbor, MI, USA) by measuring the decrease in superoxide products (generated by xanthine oxidase system). Bovine SOD was provided by manufacturers, and, after adding increasing amounts of SOD, a sigmoideal standard curve was obtained. Enzyme’s activity in samples was calculated by carrying out a four-parameter logistic curve (4PLC), fitting and values were expressed as U/mg protein.

GR (EC 1.6.4.2) activity was measured with Cayman Chemical Glutathione Reductase Assay Kit (Cayman Chemical Co., Ann Arbor, MI, USA) by measuring the rate of NADPH oxidation. The oxidation of NADPH to NADP⁺ is accompanied by a decrease in absorbance at 340 nm. Results of GR activity were expressed as mU/mg protein.

Measurement of Aβ peptides in brain tissues by ELISA

Soluble and insoluble Aβ_1 ‒ 40 and Aβ_1 ‒ 42 were measured in cortical extracts, according to a previously published procedure [44]. In brief, the samples were homogenized in an 8× volume of PBS with an AEBSF protease inhibitor cocktail set (Cat # 539131; Calbiochem; La Jolla, CA, USA). The soluble fraction was separated by centrifuging the samples for 10 min at 4000× g. In order to obtain the insoluble fraction, pellets containing insoluble Aβ peptides were solubilized in a 5 M guanidine HCl/50 mM Tris solution by incubating them for 3.5 h in an orbital shaker at room temperature. The levels of soluble and insoluble Aβ_1 ‒ 40 and Aβ_1 ‒ 42 were determined using the commercially available human ELISA kits (Cat # KHB3481 and KHB3441; Invitrogen, Camarillo, CA, USA). The data obtained from the cortical homogenates are expressed as picograms of Aβ content per milligrams of total protein (pg/mg).

Statistical analysis

All data are presented as means±SEM, and differences are considered significant at p < 0.05. Differences between samples/animals were evaluated using Student’s t-test, and 2-way ANOVA, with Tukey’s post hoc test. Both the statistical analysis and the graphs presented here were created with the GraphPad InStat software V5.0 (GraphPad Software Inc., San Diego, CA, USA).

RESULTS

Evaluation of peripheral metabolic parameters in wild type and APPswe/PS1dE9 mice fed with a HFD

Dietary administration of control and HFD started at the time of weaning (21 days) until the age of 3 months. As expected, the intake of HFD produced a progressive obesity (F_(1,50) = 120, p < 0.0001). At the day of sacrifice, body weight reached a 124% increase in WT HFD versus WT CT (p < 0.0001), and a 139% increase in APP/PS1 fed HFD versus APP/PS1 CT group (p < 0.0001) (Fig. 2A, B).

Likewise, two-way ANOVA statistical test indicated a significant effect of the diet (F_(1,28) = 5.681, p < 0.05) and diet-strain interaction (F_(1,28) = 5.681, p < 0.05). HFD administration resulted in a significant increase in blood glucose levels in APP/PS1 mice fed with HFD versus APP/PS1 CT 104.6 versus 80.43 mg/dl (p < 0.05), but not in WT HFD versus WT CT (85 versus 85 mg/dl) (Fig. 2D). Although two-way ANOVA showed a significant effect of the diet (F_(1,33) = 4.998, p < 0.05) and strain (F_(1,33) = 7.944, p < 0.01), we found no detectable differences in glucose utilization by IGTT assay between 3-month-old APP/PS1 HFD versus WT HFD, however, we detected a significant increase in APP HFD versus WT CT (Fig. 2E). Moreover, diet-induced obesity increased plasmatic insulin levels (F_(1,15) = 28.20 p < 0.0001) with concentrations of 4.46 pM/ml in WT HFD (0.94 pM/ml in WT CT; p < 0.05) and 5.9 pM/ml in APP/PS1 HFD (1.01 in APP/PS1 CT; p < 0.005) (Fig. 2F).

We also analyzed triglyceride levels, and two-way ANOVA showed a significant effect of the diet (F_(1,16) = 29.35, p < 0.0001) but none of the strain (F_(1,15) = 0.9289, p > 0.05). Higher blood triglyceride concentrations were found in HFD fed mice, with 169.8 mg/dl in WT HFD (102.5 in WT CT; p < 0.05) and 199.8 mg/dl in APP/PS1 HFD (106 in APP/PS1 CT group; p < 0.005) (Fig. 2G).

Taken together, our data indicated a possible acceleration of an HFD-induced peripheral metabolic phenotype in APP/PS1 animals compared to control mice in a presymptomatic stage of experimental AD. Based on the evidence, APP/PS1 mice maintained on a standard laboratory diet present normal baseline glucose metabolism. We continued exploring the potential relationship between diet-induced obesity and the AD-type amyloidosis in the brain of APP/PS1 mice. Thus, in the following steps, we proceeded to study the effects of HFD on the brain and attempted to identify molecular pathways related to hippocampal metabolic signaling.

Evaluation of the effects of a high-fat diet on cognitive impairment in APPswe/PS1dE9 mice

We explored the possibility that HFD might accelerate cognitive impairment in 3-month-old WT and APP/PS1 mice which are not behaviorally impaired at this age [40 –44]. Two-way ANOVA indicated a significant effect of the diet (F_(1,17) = 11.15, p < 0.01) and strain (F_(1,17) = 7.849, p < 0.05). Our data showed that APP/PS1 mice fed with HFD did not have an impaired capacity to learn. It was evaluated by a Novel Object Recognition test, comparing APP/PS1 HFD fed mice versus mice fed with CT diet. However, the intake of HFD significantly increased the memory loss compared with WT mice (Fig. 3A, p < 0.05). The total exploration time was analyzed using two-way ANOVA and did not show significant changes (p > 0.05)

Moreover, we studied changes in molecules directly involved in the early stages of memory consolidation processes. To this end, we analyzed the transcriptional activity of genes related to synaptic plasticity and memory, such as Arc, Fos, and Bdnf. They showed no significant alterations in APP/PS1 mice fed with HFD compared with APP/PS1 mice fed with CT diet (Fig. 3A, p > 0.05).

Furthermore, our western blot data demonstrated no differences in the protein levels of protein kinase A (PKA), in contrast to data obtained from the phosphorylation at serine 133 of cyclic AMP response element-binding protein (CREB). CREB protein levels showed a significant decrease in the hippocampus from APP/PS1 HFD fed mice compared with APP/PS1 fed with CT diet (Fig. 3B, p < 0.05).

Impaired NMDA receptor (NMDA-R) function may contribute to the cognitive deficit observed in AD. Thus, its alteration could be linked to AD pathogenesis. For this reason, we studied changes via western blot analysis in the NMDA-R total protein levels (NMDAR1 and NMDAR2B, and their phosphorylated form). Our results show a significant decrease in the phosphorylation of NMDAR2B in hippocampal homogenates from APP/PS1 mice fed with HFD (Fig. 3B, p < 0.01). However, no significant differences were detected in the protein levels of NMDAR1 between the groups (Fig. 3B).

High-fat diet does not enhance oxidative stress in APP/PS1 mice

Given the crucial role of oxidative stress in the pathogenesis of AD, several biomarkers were assessed in the brain homogenates from APP/PS1 and WT mice (fed HFD and CT diet). As shown in Fig. 4, there were no significant differences in lipid peroxidation, protein oxidation, and antioxidant enzymes (i.e., SOD, GSH-px, MDA, and protein carbonyls) (p > 0.05, respectively).

High-fat diet promotes AD-type brain amyloid deposition in APP/PS1 mice

Previous studies have demonstrated that the increase of APP was correlated with insulin resistance and pro-inflammatory gene expression [40]. In agreement with them, we demonstrated that animals fed with HFD show a significant increase in mRNA expression and protein levels of APP in APP/PS1 mice (Fig. 5A, B, p < 0.05, p < 0.01 respectively). Furthermore, while HFD did not increase β-secretase protein levels, ADAM10 (non-amyloidogenic pathway) was significantly decreased in APP/PS1 fed with HFD (Fig. 5A, p < 0.05). In addition, the protein levels of IDE (insulin degrading enzyme), an enzyme involved in the degradation of βA, showed a significant decrease in this group (Fig. 5C, p < 0.05).

We also evaluated the effect of the HFD on βA deposits in the brain of APP/PS1 mice using ThS to detect the fibrillar plaques, and 12F4 antibody to detect Aβ_1 ‒ 42 diffuse plaques (Fig. 6A, B).

Two-way ANOVA showed a significant effect of the strain (F_(1,8) = 23.33, p < 0.01). Besides, our immunofluorescence data demonstrated a significant early accumulation of cortical βA diffuse and fibrillar plaques in APP/PS1 fed with HFD during 2 months (Fig. 6A, p < 0.05). These data were associated with an increase in the levels of brain Aβ_1 ‒ 40 soluble and insoluble and Aβ_1 ‒ 42 insoluble peptides (Fig. 6C, p < 0.05).

Evaluation of the effects of HFD on insulin signaling and tau in the hippocampus of APPswe/PS1dE9 mice

Previous studies have already demonstrated alterations in insulin signaling pathways in brains of AD patients; however, the exact time of the onset of these alterations in experimental models of AD remains unclear. Thus, in a new series of experiments we investigated the mechanisms through which HFD could promote an AD-like amyloidosis in APP/PS1 animals compared to APP/PS1 fed with a CT diet. The results were obtained measuring mRNA levels of gene expression involved in IR signaling in the brain. For this reason, we evaluated mRNA expression of preproinsulin 1 (Ins1), insulin receptor (Insr), insulin receptor substrates 1 (Irs1) and 2 (Irs2), insulin-like growth factors I (Igf1) and II (Igf2), IGF receptor (Igfr) as well as insulin-like growth factor-binding protein 2 (igfbp2), at 3 month of age (Fig. 7). Our data showed no changes in mRNA levels in early stages of AD (p > 0.05).

Since tau phosphorylation is a hallmark of AD, we continued to study the potential molecular mechanisms leading to the AD neuropathology. We evaluated the phosphorylation of tau, downstream targets of the insulin signaling pathway, the protein kinase AKT/PKB pathway and the regulation of GSK3β downstream signaling in the brain of HFD and CT APP/PS1 mice. Our western blot data analysis of total and phosphorylated forms of AKT indicated no changes in the levels of this protein, and when we analyzed downstream substrates of this pathway, they remained unaltered in the hippocampus of both APP/PS1 mice fed with HFD and APP/PS1 mice fed with CT diet at 3 months of age (Fig. 8A, p > 0.05). Moreover, our data also revealed that HFD did not affect major kinases (GSK3β, ERK1/2, and CDK5) involved in tau phosphorylation in the hippocampus at this age (Fig. 8A, p > 0.05). In addition, no significant changes in several tau phosphorylation sites were detected (Fig. 8B, p > 0.05).

PGC-1α is altered in the hippocampus of APPswe/PS1dE9 mice fed with HFD

Previous reported data suggest that PGC-1α expression is decreased in the brain of AD patients and that this decrease could be accompanied by mitochondrial alterations [55 –58]. Our western blot data analyses demonstrated that HFD significantly decreases PGC1α protein levels, suggesting that PGC-1α could be involved in an initial step leading to alterations in mitochondrial biogenesis and metabolic function (Fig. 9, p < 0.05). However, downstream effectors of PGC-1α, such as nuclear respiratory factor 1 (NRF 1) and nuclear respiratory factor 2 (NRF 2), which control the nuclear genes that encode for mitochondrial proteins, and mitochondrial transcription factor A (TFAM), were not altered in the hippocampus of 3-month-old APP/PS1 mice fed with HFD.

Effects of HFD on glial cells in the hippocampus of APPswe/PS1dE9 mice

Increasing evidence has demonstrated that the activation of glial cells may play an important role in the development of AD [58 –60]. Furthermore, it may lead to neurotoxic damage through the generation of inflammatory responses. The analysis of our immunofluorescence images of glial cells in 3-month-old APP/PS1 mice fed with CT diet and APP/PS1 mice fed with HFD was done using an antibody that detects the glial acidic fibrillar protein (GFAP), which detects astrocytes, and the Iba1 antibody (ionized calcium binding adaptor molecule 1), which targets microglial cells. The results obtained revealed an accumulation of GFAP and Iba 1-positive activated cells around amyloid deposits/aggregates in both groups (Figs. 10 and 11). Also, the effect of HFD in the astrocytes and microglia reactivity was analyzed in different areas of the hippocampus such as cornu ammonis 1 (CA1), cornu ammonis 3 (CA3) and gyrus dentatus (GD). We did not detect changes in glial activation between dietary groups (Figs. 10 and 11).

DISCUSSION

Currently, one of the most important challenges of the 21st century is to develop drugs that can slow and modify the evolution and progression of AD. It is clear that to achieve this goal, it is necessary to determine which biochemical pathways are modulated by the Aβ neurotoxin. Presently, the underlying mechanisms through which Aβ exerts its neurotoxic effects, responsible for cognitive dysfunction at the early stage of AD, are not well understood. However, recent preclinical studies strongly suggest that obesity and type 2 diabetes are potential risk factors for AD development [61 –64]. The intake of HFD, an experimental model of diet-induced obesity, may lead to type 2 diabetes, and may have an effect on the biochemical regulation of the central nervous system. The HFD model had previously been used to show that diet plays an important role as a regulator of brain function, the content of lipids in the brain and processes affecting neuronal plasticity. Moreover, since myelin membranes have a very high lipid composition in cholesterol and fatty acids, the choice of diet can affect its integrity. In addition, it has been reported that alterations in the insulin/IGF-1-AKT and PGC-1alpha signaling pathways are involved in the process of myelinogenesis [65, 66].

Willete and colleagues demonstrated that insulin resistance in humans is associated with an increase in brain Aβ deposition [67, 68]. Preclinical reports have shown that HFD intake, in AD transgenic mice models, increases insulin resistance, Aβ deposition, tau phosphorylation and favors cognitive impairment [69 –71]. In contrast, some studies argue that a HFD does not increase Aβ deposition and phosphorylation of tau protein [72, 73]. It is important to point out that this controversy could be due to the fact that all these preclinical studies have been carried out with aged mice, in which plaques are already established and memory loss has already occurred.

Therefore, it is necessary to take action at the beginning of the disease’s process, in a presymptomatic stage, and investigate how the HFD modifies those biological parameters responsible to trigger AD. For this reason, we used APP/PS1 mice, a model of familial AD that produces high levels of Aβ_1 ‒ 42, one of the key factors responsible for the onset of this illness, although probably not the only one [39–44 , 63].

Thus, we demonstrated that HFD results in a decrease in PGC-1α and IDE levels, an increase in plaque formation, as well as alterations in the non-amyloidogenic pathway in the hippocampus of 3-month-old mice. Unexpectedly, there was no increase in oxidative stress.

IDE is a protease involved in the degradation of Aβ and insulin [74]. Recent studies suggest that peripheral IDE could be a useful biomarker for the detection of AD [74 –76]. The analysis of western blot data in the hippocampus suggests that the decrease in the protein levels of this enzyme may be associated with a reduced Aβ clearance. It is widely known that both insulin and Aβ compete for the availability of IDE, but the enzyme is much more selective for insulin than Aβ. Taking this into account, it could be possible that its reduction could favor a decrease in Aβ degradation that may lead to an increase in plaque formation [75]. In addition, our results suggest that HFD could increase plaque formation through an increase in the activity of the non-amyloidogenic pathway due to a significant reduction of ADAM10 protein level in HFD-fed APP/PS1 mice. [77 –79]. In contrast, we did not find alterations in the protein level of BACE1 by western blot analysis. Likewise, in agreement with previous studies, the mRNA expression and protein levels of APP were significantly increased by HFD, suggesting that monitoring of APP protein levels may be an additional marker of both obesity and AD [27, 43]. Interestingly, our data are in line with previous studies from Pandini and colleagues, where insulin in neuroblastoma cells reduced Aβ production through the non-amyloidogenic pathway, decreased mRNA levels of APP (the precursor of Aβ peptides) and increased IDE activity and expression [28].

Previous reported data suggest that oxidative stress is present in several areas of the AD brain [76]. Those studies showed that oxidative stress causes damage to proteins, lipids, DNA, RNA, and carbohydrates in AD brain [11 , 81]. Zhang and colleagues reported that at 3.5 months, APP/PS1 mice showed an increase in reactive oxygen species (ROS), which was associated with memory loss [42]. Studzinski and colleagues demonstrated that a mouse model of AD fed with a western diet allows for the observation of cerebral oxidative stress and the initiation of the development of Aβ brain pathogenesis [81]. In our study, APP/PS1 mice fed with a HFD did not show any significant increase of the ROS production in the brain. Therefore, our data do not verify that ROS participates in the early stages of experimental AD neuropathology promoted by HFD.

Another key question asked in this study was whether 3-month-old APP/PS1 mice fed with a HFD showed defects in insulin receptor substrate (IRS) mRNA expression and insulin signaling pathway as it was previously shown in experimental models of AD [82, 83]. This work demonstrated that HFD-feeding does not induce significant alterations in molecules involved in the IRS pathway in the hippocampus at 3 months of age. Besides, the insulin signaling pathway was not altered, since our western blot data indicated that the levels of hyperphosphorylated tau in the hippocampus were not modified as well as the activation of GSK3β. Therefore, we could conclude that the early brain abnormalities produced by obesity in APP/PS1 mice were not associated with alterations in the insulin receptor signaling pathway. Similarly, other kinases, such as ERK and CDK5, which are involved in tau phosphorylation, were not activated by the HFD.

Qin and colleagues were the first to report a reduction in the expression of PGC-1α in AD brains, suggesting that its decrease may represent a risk factor for onset and progression of dementia [55]. Thus, the preservation of its levels might be a molecular mechanism, which could confer protection against AD dementia [55 –58]. Furthermore, downregulation of PGC1α has been associated with in associated with the development of skeletal muscle insulin resistance and it might be inferred that this protein could be a common link between the two diseases [85 –90]. Recent evidence suggests that PGC1α and other transcription factors have a very important function in the regulation of mitochondrial biogenesis in the brain [57 , 88–90]. In addition, it participates in the formation and maintenance of synapses in hippocampal neurons and in the regulation of NMDA activity [90]. Likewise, PGC1α has a powerful suppressive effect on ROS production [88]. Moreover, it has been demonstrated that experimental inhibition of PGC1α expression correlates with the elevation of Aβ peptide generation through mechanisms involving the regulation of the non-amyloidogenic (α-secretase) and amyloidogenic (β-secretase) processing of APP [88].

Transcriptional regulation of PGC1α expression is known to be regulated by CREB [91 –94]. A direct link between CREB phosphorylation and transcriptional regulation at the PGC-1α promoter has been observed in neuronal cells [92].

Our results indicated that the APP/PS1 mice fed a HFD show significant memory loss compared to WT mice fed a CT diet. This suggests a synergistic effect between diet and levels of Aβ that favors cognitive loss. Likewise, at the molecular level, our data revealed that the hippocampal protein levels of p-CREB were significantly decreased in APP/PS1 mice as a consequence of a HFD intake. In addition, it has been reported that CREB plays an essential role in hippocampal-based memory formation [92 –98]. However, while p-CREB is downregulated, probably due to a decrease in PKA levels, HFD did not induce changes in the transcription of early genes involved in the memory process, such as Arc and Fos, or changes in Bdnf. Similarly, the upregulation of NMDAR2B subunit phosphorylation might be a compensatory mechanism to overcome their defective activity underlying the memory impairment that occurs in APP/PS1 mice at 6 months of age.

We showed that the hippocampal expression levels of proteins downstream of PGC1α signaling such as NRF-1 and TFAM were not modified by HFD. These data could indicate that at 3 months of age, not enough time has elapsed to detect changes in the mitochondrial biogenesis [43]. Thus, an increase in APP, associated with impairment of ADAM-10 jointly with an IDE decrease, probably contributes to the increase of hippocampal Aβ levels in APP/PS1 mice fed with a HFD. In addition, Aβ could alter the levels of PKA, which inhibits p-CREB, and decreases hippocampal PGC-1α levels, which is involved in α- and β-secretase modulation. In conclusion, our findings suggest that early alterations in Aβ/PKA, which decrease p-CREB/ PGC1α mediated by HFD in APP/PS1 mice in a presymptomatic stage, may represent a potential risk factor for the onset and progression of AD dementia (Fig. 12). Therefore, their preservation in the brain might be a molecular drug target mechanism conferring protection against metabolic AD.

Footnotes

ACKNOWLEDGMENTS

This work was funded by the Spanish Government’s “Ministerio de Economia y Competitividad” (CIBERNED, CB06/05/0024) and Generalitat de Catalunya (Suport als Grups de Recerca Consolidats 2014SGR 525).

Authors’ disclosures available online ().

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