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Neurochemical Research

, Volume 40, Issue 8, pp 1709–1718 | Cite as

Differences Between Tg2576 and Wild Type Mice in the NMDA Receptor–Nitric Oxide Pathway After Prolonged Application of a Diet High in Advanced Glycation End Products

  • Zdena Kristofikova
  • Jan Ricny
  • Jana Sirova
  • Daniela Ripova
  • Irit Lubitz
  • Michal Schnaider-Beeri
Original Paper

Abstract

It has been suggested that advanced glycation end (AGE) products, via cognate receptor activation, are implicated in several diseases, including Alzheimer’s disease. The NMDA receptor–nitric oxide pathway appears to be influenced by AGE products and involved in the pathogenesis of this type of dementia. In this study, C57BL/6J (WT) and transgenic (Tg2576) mice expressing human mutant amyloid precursor protein were kept on prolonged (8 months) diets containing regular or high amounts of AGE products. After the decapitation of 11-months old mice, brain tissue analyses were performed [expressions of the NR1, NR2A and NR2B subunits of NMDA receptors, activities of neuronal, endothelial and inducible nitric oxide synthase (nNOS, eNOS and iNOS)]. Moreover, levels of malondialdehyde and of human amyloid β 1–42 were estimated. We found increased activity of nNOS in WT mice maintained on a high compared to regular AGE diet; however, no similar differences were found in Tg2576 mice. In addition, we observed an increase in NR1 expression in Tg2576 compared to WT mice, both kept on a diet high in AGE products. Correlation analyses performed on mice kept on the regular AGE diet supported close links between particular subunits (NR2A–NR2B, in WT as well as in Tg2576 mice), between subunits and synthase (NR2A/NR2B–nNOS, only in WT mice) or between particular synthases (nNOS–iNOS, only in WT). Correlation analysis also revealed differences between WT mice kept on both diets (changed correlations between NR2A/NR2B–nNOS, between nNOS–eNOS and between eNOS–iNOS). Malondialdehyde levels were increased in both Tg2576 groups when compared to the corresponding WT mice, but no effects of the diets were observed. Analogously, no significant effects of diets were found in the levels of soluble or insoluble amyloid β 1–42 in Tg2576 mice. Our results demonstrate that prolonged ingestion of AGE products can influence the NMDA receptor–nitric oxide pathway in the brain and that only WT mice, not Tg2576 mice, are able to maintain homeostasis among subunits and synthases or among particular synthases. The prolonged application of AGE products enhanced differences between 11-months old Tg2576 and WT mice regarding this pathway. Observed differences in the pathway between WT mice kept on regular or high AGE diets suggest that the prolonged application of a diet low in AGE products could have beneficial effects in older or diabetic people and perhaps also in people with Alzheimer’s disease.

Keywords

NMDA receptor subunits Nitric oxide synthases Advanced glycation end products Mouse model of Alzheimer’s disease 

Introduction

Advanced glycation end (AGE) products are a heterogenous group of molecules whose formation is initiated by a non-enzymatic reaction between reducing sugars such as glucose, and amino groups in proteins, lipids and nucleic acids. Formation of AGE products is irreversible and leads to protease-resistant crosslinking of peptides and proteins. This formation is increased under conditions of chronic hyperglycemia (such as diabetes) in combination with oxidative stress [e.g., 1, 2, 3, 4].

It is suggested that AGE products, especially via the activation of their receptor (RAGE), are implicated in several diseases including Alzheimer’s disease (AD) [1, 2, 3]. Activation of RAGE by AGE products or by amyloid β (Aβ) peptide, probably a key player in AD pathogenesis, results in the release of various proinflammatory mediators [5]. In addition to the above-mentioned crosslinking of peptides and proteins leading to their deposition and amyloidosis (e.g., AGE-modified Aβ promotes accelerated self-aggregation [6]), there are direct links between diabetes as a known risk factor of AD [4, 7] and the mechanisms of oxidative stress/Aβ/inflammation, all involved in the pathogenesis of AD [e.g., 4]. The important role of AGE products in this type of dementia can be supported by their colocalization within Aβ-based plaques or τ-based neurofibrillar tangles [6, 8, 9] and by RAGE overexpression [1] in the brains of AD patients.

N-methyl-d-aspartate (NMDA) receptors are associated with three isoforms of nitric oxide (NO) synthase (i.e., neuronal—nNOS, endothelial—eNOS and inducible—iNOS) through a postsynaptic density protein. It is well-known that the stimulation of NMDA receptors activates the synthesis of NO via this pathway [10, 11]. Functional interactions have been reported, especially between the NR1 subunit of NMDA receptor and nNOS, but also between the NR2A or NR2B subunits and the eNOS/iNOS isoforms [e.g., 12].

Experimental results support the involvement of the NMDA receptor–NO pathway in the pathogenesis of AD [e.g., 10, 11] and demonstrate marked changes in this system dependent on the stage of dementia [13]. Moreover, the pathway seems to be influenced by chronic hyperglycaemia or diabetes [for a review, see 14]. For example, adult Wistar rats consuming a high-sucrose diet for 9 weeks are a prediabetic rodent model and display poorer performance of hippocampal-dependent short- and long-term spatial memory and exhibit increased expression of NR1 and NR2A in the hippocampus [15]. In diabetic rats, nitrergic neurons innervating the gastrointestinal organs undergo a selective degenerative process and the animals show a significant reduction in nNOS expression [16, 17]. An analysis of human penile tissue revealed that erectile dysfunction in diabetes may be dependent on AGE-mediated upregulation of iNOS and downregulation of eNOS [18]. In human kidney tissue, it appears that diabetes triggers mechanisms that in parallel enhance and supress NO bioavailability, involving both eNOS and nNOS [19]. Very pronounced effects of AGE products, especially on the NO mediator system, may be mediated either indirectly via RAGE mechanisms or directly through l-arginine-derived AGE products that are structurally analogous to endogenous inhibitors of NOS isoforms. Various in vitro experiments have demonstrated that AGE products inhibit the activities of nNOS/eNOS/iNOS and the expression of nNOS/eNOS [20, 21, 22], and that AGE-modified proteins reduce the release of NO and quench it [21]. In sinoaortic denervated rats, treatment with soluble RAGE daily for 3 weeks restored aortic NO formation through the upregulation of eNOS [23]. In the brain tissue of AD patients, colocalization of AGE products and nNOS [24] or AGE products and iNOS [25] have been found.

The main aim of the present study was to compare the effects of prolonged application of a diet containing either regular or high amounts of AGE products on the NMDA receptor–NO pathway in the brain and to analyze diet-mediated differences between aged wild-type C57BL/6J (WT) mice and aged transgenic mice overexpressing the double Swedish mutated human amyloid precursor protein (Tg2576). Tg2576 mice, one of the accepted genetic models of AD, express high concentrations of human Aβ and display memory deficits by 9–10 months of age [26]. Since Aβ peptides can reduce the activity of many neurotransmitter systems, including the glutamatergic pathway, differences between old Tg2576 and age-related WT mice in the NMDA receptor–NO pathway are not very surprising. Experiments on cortical or hippocampal neurons have revealed that Aβ 1–42 promotes endocytosis of NMDA receptors, leading to a rapid depression of NMDA-evoked currents and that a reduction in cell-surface expression of NR1, but not that of total NR1 levels, protects against glutamate-induced extracellular calcium influx and cell death [27]. Although quantitative receptor autoradiography did not reveal significant changes in the cortical NMDA receptor density in 17-months old Tg2576 mice [28], increased expression of striatal-enriched protein tyrosine phosphatase 61 in the cortex of 12-months old Tg2576 mice supports enhanced internalization of the NMDA receptor via dephosphorylation of NR2B [29]. Old Tg2576 mice can have also decreased nNOS expression/increased nNOS activity and increased iNOS expression/activity in some brain regions [30, 31]. However, some studies have not observed marked changes in iNOS expression, and alterations in nNOS seem to be based on the distance of NOS-positive neurons from plaques [32]. With respect to NO levels in the cortex of Tg2576 mice, these are not changed among 3- to 7-months old mice, are significantly increased at about 9 months and decrease again at the age of 11 months with no further alterations up to 21 months. This developmental profile does not markedly differ between Tg2576 and WT mice [33]. On the other hand, endothelial dysfunction in Tg2576 mice occurs very early and seems to precede further neuropathological features. E.g., impaired endothelium-dependent relaxation and a significant decrease in phosphorylated eNOS expression, but not in total protein, were found in the aorta of 4- to 5-months old Tg2576 mice when compared to age-related WT mice [34]. Cerebrovascular autoregulation appears to be essentially lost even in 2- to 3-months old Tg2576 mice [35]. Thus, we anticipated that the NMDA receptor–NO pathway in the brains of Tg2576 mice should be more vulnerable to the prolonged effects of high AGE products than that in WT mice. It is known that phospholipid-linked AGE products can initiate lipid oxidation in vivo [36, 37]. Therefore, the second aim of this study was to compare alterations in the NMDA receptor–NO pathway with levels of malondialdehyde (MDA), i.e., with a biomarker of oxidative lipid injury.

Materials and Methods

Diets

In the study, two diets were used (both from Harlan laboratories): 2018 (regular AGE product content) and 2918 (high AGE product content). The diet containing a regular AGE product content was prepared without exposure to heat, while the diet with a high AGE product content consisted of the same chow exposed to gamma irradiation from a cobalt-60 source as per standard procedure. Both diets were therefore nutritionally equivalent and differed only in the content of AGE products (for a more detail, see [38]). Diets were pelleted by the manufacturer and kept at room temperature.

Animals

All experiments were performed on C57BL/6J (WT) or transgenic (Tg2576) mice obtained from Taconic. Animals were housed one per cage in a temperature-controlled room with a 12-h light–dark cycle and provided with food (low or high AGE diet) and water ad libitum. Animals were weighed weekly and maintained on the diets from 3 to 11 months of age. After evaluation of their cognitive functions at 11 months of age, animals were anesthetized with isoflurane (Baxter Healthcare Corp., Deerfield, IL, USA) and sacrificed by decapitation. The right brain hemisphere was fixed and then used for histological/morphological analyses (the results will be published separately along with those of behavioral testing using the Morris water maze [38]). The left hemisphere was rapidly frozen in liquid nitrogen, stored at −80 °C and then used for neurochemical analyses. All procedures were approved by the Institutional Animal Care and Use Committee of the Sheba Medical Center.

Brain Tissue Homogenization

The frozen whole left hemisphere was homogenized (1:5) in redestilled water (Teflon-glass Potter B. Braun homogenizer: 20 strokes, 1000 rpm) and particular aliquots were either used immediately (for the activities of nNOS, eNOS and iNOS) or rapidly frozen and stored at −80 °C until assayed (for the expression of NR1, NR2A and NR2B or for the concentration of MDA and of soluble/insoluble Aβ 1–42).

Expression of the NMDA Receptor Subunits NR1, NR2A and NR2B by Western Blotting

The experiments were performed in accordance with our previous study [11]. Briefly, 200 µl of frozen homogenates were homogenized in 200 µl of lysis buffer (320 mM sucrose, 10 mM Tris, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 2 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM 2-mercaptoethanol and 0.05 % protease inhibitor cocktail; pH 7.4). The resulting homogenate was centrifuged at 1000g and then the supernatant at 13,400g, both for 20 min at 4 °C. The pellet was resuspended in redistilled water and a part of this sample was resuspended in loading buffer (63 mM Tris, 10 % glycerol, 2 % sodium dodecyl sulfate, 5 % 2-mercaptoethanol and 0.01 % bromophenol blue). The protein concentration was determined by the Bradford method using bovine serum albumin as the standard (Bio-Rad, USA). The resuspended material was subjected to electrophoresis on a 7.5 % polyacrylamide gel (Criterion Cell, Bio-Rad, USA), followed by electroblotting using a Criterion blotter (Bio-Rad, USA). Non-specific binding was blocked with 3 % non-fat dried milk dissolved in 0.1 % Tween in phosphate buffer. Blots were incubated overnight with anti-NMDAR1 (1:100; Millipore, USA) or for 2 h with anti-NMDAR2A/2B (1:500; Millipore, USA) primary antibody. For the loading control, blots were treated with an anti-α-tubulin antibody (1:1500; Exbio, CZ) for 1 h. Then, the blots were washed in phosphate buffer and incubated for 1 h with a horseradish peroxidase-conjugated secondary antibody (1:3000, Dako, Denmark). Detection was performed with a chemiluminescent substrate (Pierce, USA) and evaluated by the Gel Doc Analysis system (Bio-Rad, USA).

Activities of nNOS, eNOS and iNOS

The method was performed in accordance with our previous studies [10, 11]. Briefly, 200 μl of frozen homogenates were mixed with 200 μl of centrifugation buffer [1 mM ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 mM dithiothreitol, 20 mM HEPES, 0.32 M sucrose, 14.6 µM pepstatin and 21 µM leupeptin; pH 7.4] and centrifuged at 1200g for 10 min at 4 °C. Supernatants were added to reaction buffer [centrifugation buffer containing also 200 µM β-nicotinamide adenine dinucleotide phosphate, 50 µM tetrahydrobiopterin and 4.6 µM [14C] arginine (American Radiolabeled Chemicals, Inc.,)] and incubated for 30 min at 37 °C. Some samples also contained 1 µM CaCl2 (nNOS and eNOS) and specific inhibitors (1 mM spermidine for nNOS, 190 µM Nω-nitro-l-arginine methyl ester for nNOS/eNOS and 1 mM aminoguanidine for iNOS, all from Sigma). Final protein concentrations determined by the Bradford method and were equivalent to 0.24 mg/ml in all incubation mixtures. The reaction was terminated by adding stop buffer (30 mM HEPES, 3 mM EDTA, pH 5.5) and by rapid cooling. DOWEX 50WX8-200 (Sigma) was used to separate citrulline from arginine.

Determination of MDA

MDA quantification was performed in accordance with the literature [39, 40]. Derivatization of samples was carried out in 2 ml capacity plastic centrifuge tubes fitted with screw-on caps. 100 µl of 10 % brain homogenate (or standard solution) in buffer (10 mM Na-phosphate, pH 7.4; 100 mM NaCl; 0.75 mM aprotinin; 2.5 mM leupeptin; 1 mM pepstatin; 1 mM PMSF) was mixed with 0.15 ml H2O + 0.01 ml 0.5 % butylated hydrosytoluene (Sigma) + 0.375 ml H3PO4 + 0.125 ml 50 mM 2-thiobarbituric acid (TBA, Sigma) and then heated for 1 h at 100 °C in a dry bath incubator. Following heat derivatization, samples were placed on an ice-water bath for 5 min to cool, with 0.5 ml n-butanol was subsequently added to each vial for extraction of the MDA–TBA complex. Tubes were vortexed for 5 min and then centrifuged for 15 min at 15,000g to separate the phases. The upper organic phase was used for the MDA assay. A standard stock solution of 1,1,3,3-tetraethoxypropane (TEP) was prepared from 23 ml (ca. 23 mg) diluted in 10 ml of 1 % H2SO4, then incubated for 40 min at 40 °C to hydrolyze TEP into MDA (final concentration ca. 10 mM; 2.2 mg/ml). Standards for calibration curve construction were prepared by dilution of the stock standard to obtain concentrations of 5, 4, 2, 1, 0.5, and 0 (blank) mM TEP. The concentration of MDA was checked by its absorbance at 244 nm (e = 13,700, 1 % H2SO4). The standard curve was prepared fresh for each analysis. Chromatographic system consisted of a Dionex pump P580 (Dionex Corporation, Sunnyvale, USA), an UltiMate 3000 autosampler (Dionex, Thermo Fisher Scientific, Waltham, USA), a Shimadzu RF-350 fluorescence detector (Shimadzu Corporation, Kyoto, Japan) with 532 nm excitation and 560 nm emission wavelengths, and a reversed phase silica column (Separon SGX C18, 7 μm, 3.3 × 150 mm; Tesek, Czech Republic). The mobile phase (50 mM K-phosphate pH 6.8/40 % methanol) was pumped isocratically at 0.5 ml/min and 5 μl of the sample (in n-butanol) was injected by the autosampler every 4 min. Data acquisition and processing were performed with Chromeleon (v. 6.8; Dionex, Thermo Fisher Scientific) software and quantification was based on the peak area of standards and samples.

Concentrations of Soluble and Insoluble Aβ 1–42

Frozen homogenates were homogenized again (10 % w/v; individually from each animal) in PBS buffer supplemented with protease inhibitors (Roche Biochemicals, Indianapolis, USA) and 1 % Triton X-100 (PBS-T) on ice. After first centrifugation (100,000g for 30 min at 4 °C), the pellet was washed one more times with the same volume of PBS-T and centrifugated again under the same conditions. Combined supernatants were assayed for “soluble Aβ 1–42”. The pelet was extracted with 50 mM Tris-HCl pH 8.0/5 M guanidin-HCl containing protease inhibitors (in the same volume as the original homogenate) with sonication (3 × 3 s) and left for cca 2 h at a room temperature. After centrifugation (13,000g for 20 min), this extract was used for assay of “insoluble Aβ 1–42”. Levels of human peptide were measured using colorimetric ELISA kit (Invitrogen Corporation, Camarillo, USA) according to manufacturer recommendation. Supernatants for “soluble Aβ 1–42” were used without dilution; extracts of “insoluble Aβ 1–42” were diluted 1:5000 in “Diluent Buffer” included in the kit.

Statistical Analysis

Statistical analysis was performed by means of BMDP statistical software (University of California, Los Angeles). We applied either one-way ANOVA (ANOVA) for global analysis and pooled variance of the Student’s t test for pairwise comparisons (program 7D) or ANOVA with repeated measures (program 2V) and subsequently paired t test (program 3 D). Correlation analyses were performed using the 6D program; the equality of correlation coefficients in the two groups was examined by means of a test based on Fisher’s Z-transformation (Z-test). All data are presented as mean ± SEM.

Results

Although the results of the global analysis (ANOVA) did not indicate significant changes in the case of NMDA receptor subunit expressions (Table 1) or NOS activity (Table 2) in our experiments performed on WT and Tg2576 mice, the results of pairwise tests supported two significant differences between particular groups. Namely, we observed a significant increase to 104.5 % in NR1 expression of Tg2576 compared to WT mice, both kept on a high AGE diet (Table 1), and to 125.6 % in nNOS activity in WT mice maintained on a high AGE diet when compared to a regular AGE diet (Table 2).
Table 1

Expression of NMDA receptor subunits

Groups

n

NR1

WT—regular AGE

13

0.962 ± 0.013

WT—high AGE

12

0.931 ± 0.010

Tg2576—regular AGE

7

0.954 ± 0.016

Tg2576—high AGE

8

0.973 ± 0.011+

ANOVA

 

F(3, 36) = 2.14, p = 0.1126

  

NR2A

WT—regular AGE

13

0.883 ± 0.034

WT—high AGE

12

0.925 ± 0.037

Tg2576—regular AGE

7

0.921 ± 0.058

Tg2576—high AGE

8

0.827 ± 0.048

ANOVA

 

F(3, 36) = 1.02, p = 0.3972

  

NR2B

WT—regular AGE

13

1.120 ± 0.060

WT—high AGE

12

1.175 ± 0.063

Tg2576—regular AGE

7

1.112 ± 0.079

Tg2576—high AGE

8

0.991 ± 0.073

ANOVA

 

F(3, 36) = 1.21, p = 0.3200

Data are presented as mean ± SEM

Statistical significance (pooled variance of the Student’s t test) was calculated with respect to WT mice kept on a high AGE diet (+ p < 0.05)

Table 2

Activity of NO synthases

Groups

n

nNOS (nmoles/30 min/mg of proteins)

WT—regular AGE

13

1092.5 ± 60.6

WT—high AGE

12

1372.1 ± 123.8*

Tg2576—regular AGE

7

1318.7 ± 92.5

Tg2576—high AGE

8

1201.6 ± 104.3

ANOVA

 

F(3, 36) = 1.84, p = 0.1569

  

eNOS (nmoles/30 min/mg of proteins)

WT—regular AGE

13

503.2 ± 36.9

WT—high AGE

12

542.9 ± 49.6

Tg2576—regular AGE

7

452.4 ± 84.7

Tg2576—high AGE

8

448.6 ± 57.4

ANOVA

 

F(3, 36) = 0.68, p = 0.5677

  

iNOS (nmoles/30 min/mg of proteins)

WT—regular AGE

13

12.3 ± 4.4

WT—high AGE

12

15.7 ± 5.1

Tg2576—regular AGE

7

10.2 ± 3.6

Tg2576—high AGE

8

13.4 ± 7.5

ANOVA

 

F(3, 36) = 0.18, p = 0.9114

Data are presented as mean ± SEM

Statistical significance (pooled variance of the Student’s t test) was calculated with respect to WT mice kept on a regular AGE diet (* p < 0.05)

Figure 1 shows the concentration of MDA in the brain tissue of WT and Tg2576 mice. We found significantly increased MDA levels in both Tg2576 groups compared to the corresponding WT mice (increased to 138.2 % in mice fed on a regular AGE diet and to 150.9 % in those on a high AGE diet), but no marked changes mediated by the diets (only non-significant enhancements to 103.9 % in WT and to 113.5 % in Tg2576 mice).
Fig. 1

Concentration of MDA in the mouse brain. Experiments were performed on brain homogenates from WT mice kept on a regular AGE diet (13 animals), WT mice kept on a high AGE diet (12 animals), Tg2576 mice kept on a regular AGE diet (seven mice) and finally Tg2576 mice kept on high AGE diet (eight mice). Data are expressed as µmol/g and are presented as mean ± SEM. Results of ANOVA: F(3, 36) = 12.78, p < 0.001. Statistical significance (pooled variance of the Student’s t test) was calculated with respect to WT kept on a regular AGE diet (**p < 0.010) or WT kept on a high AGE diet (++ p < 0.010)

Figure 2 demonstrates levels of soluble and insoluble human Aβ 1–42 in Tg2576 mice. Results of ANOVA with repeated measures and of paired t tests supported significant differences between levels of soluble and insoluble peptide (the levels of insoluble compared to soluble peptide were significantly increased to 201.4 % in mice fed on regular and to 203.9 % in mice fed on high AGE diet). However, the results did not indicate significant effects of diets.
Fig. 2

Concentrations of human Aβ 1–42 in the brain tissue of Tg2576 mice. Experiments were performed on homogenates from Tg2576 kept on regular AGE diet (seven mice) and Tg2576 kept on high AGE diet (eight mice). Data were expressed as pg/ml and are presented as mean ± SEM. Results of ANOVA with repeated measures with both diets as a grouping factor and differences between soluble and insoluble Aβ 1–42 (solubility) as a within factor: diets—F(1, 13) = 2.91, p = 0.1119, solubility—F(1, 13) = 129.47, p < 0.001, interaction—F(1, 13) 1.61, p = 0.2261. Statistical significance (paired t test) was calculated with respect to soluble levels of Aβ 1–42 in Tg2576 mice maintained on regular (***p < 0.001) or to those maintained on high AGE diet (+++ p < 0.001)

Correlation analysis performed on WT mice kept on a regular AGE diet revealed significant correlations among particular components of the NMDA receptor–NO synthase pathway (Table 3). In particular, one positive correlation (between NR2A and NR2B) and three negative correlations (between NR2A and nNOS, between NR2B and nNOS and between nNOS and iNOS) were found. Four significant differences between WT mice kept on regular or high AGE diets were observed as well; namely, these were the loss of marked negative correlations (between NR2A and nNOS or between NR2B and nNOS), a shift from a mild negative to a mild positive correlation (between nNOS and eNOS) and a shift from a mild positive to a marked negative correlation between eNOS and iNOS). No significant positive or negative correlations were observed between MDA levels and particular components of the NMDA receptor–NO synthase pathway (data not shown).
Table 3

Results of correlation analyses among particular components of the NMDA–NO pathway in the brains of WT animals

Parameters

Regular AGE (n = 13)

High AGE (n = 12)

Z-test

CC

p

CC

p

p

NR1 versus NR2A

−0.345

0.248

−0.103

0.750

0.577

NR1 versus NR2B

−0.188

0.538

−0.085

0.794

0.819

NR1 versus nNOS

+0.447

0.126

−0.223

0.487

0.123

NR1 versus eNOS

−0.397

0.179

−0.474

0.120

0.836

NR1 versus iNOS

−0.007

0.981

−0.106

0.744

0.829

NR2A versus NR2B

+0.960

<0.001***

+0.989

<0.001***

0.155

NR2A versus nNOS

−0.810

<0.001***

+0.213

0.506

0.003**

NR2A versus eNOS

+0.211

0.489

−0.043

0.895

0.576

NR2A versus iNOS

+0.499

0.082

+0.032

0.922

0.261

NR2B versus nNOS

−0.717

0.006**

+0.210

0.512

0.015*

NR2B versus eNOS

+0.187

0.541

−0.014

0.965

0.658

NR2B versus iNOS

+0.421

0.152

−0.013

0.968

0.315

nNOS versus eNOS

−0.503

0.080

+0.468

0.125

0.021*

nNOS versus iNOS

−0.656

0.015*

−0.347

0.268

0.356

eNOS versus iNOS

+0.141

0.646

−0.655

0.021*

0.044*

CC correlation coefficient

Statistical significance: * p < 0.05; ** p < 0.01; *** p < 0.001

Correlation analysis performed on Tg2576 mice did not reveal the positive or negative correlations observed in WT mice except for the highly positive correlations between NR2A and NR2B (Tg2576 mice on a regular AGE diet: CC = +0.922, p = 0.003, Tg2576 mice on a high AGE diet: CC = +0.992, p < 0.001). In addition, the results of the correlation analyses did not reveal significant differences in the NMDA receptor–NO pathway in Tg2576 mice in either the regular or high AGE diets (data not shown), again in contrast to WT mice (Table 3). No significant correlations were found between MDA levels and particular components of the NMDA receptor–NO pathway in Tg2576 mice kept on a regular AGE diet; nevertheless, correlations between iNOS activity and MDA were significantly changed in Tg2576 mice in comparison with WT mice, both kept on a high AGE diet (WT mice kept on a regular AGE diet: CC = −0.249, p = 0.412, Tg2576 mice kept on a regular AGE diet: CC = −0.048, p = 0.919, difference between groups p = 0.727; WT mice kept on a high AGE diet: CC = +0.043, p = 0.894, Tg2576 mice kept on a high AGE diet: CC = −0.804, p = 0.016, difference between groups p = 0.039). Although correlation analysis revealed significant correlations between Aβ 1–42 and nNOS activity, either in Tg2576 on regular or in these on high AGE diet, differences between groups were not statistically significant (nNOS vs soluble Aβ 1–42: CC = +0.500, p = 0.253 for mice on regular AGE, CC = +0.734, p = 0.038 for mice on high AGE, difference p = 0.563; nNOS vs insoluble Aβ 1–42: CC = −0.238, p = 0.607 for mice on regular AGE, CC = +0.741, p = 0.036 for mice on high AGE, difference p = 0.0748). Differences between Tg2576 mice kept on diets containing low or high AGE products reached out only on borderline significance in the case of soluble Aβ 1–42 and iNOS acitivity (CC = +0.720, p = 0.068 for mice on regular AGE, CC = −0.359, p = 0.382 for mice on high AGE, difference p = 0.0557). Analogical correlation result was found also between levels of soluble and insoluble Aβ 1–42 (CC = +0.326, p = 0.475 for Tg2576 kept on regular AGE, CC = +0.907, p = 0.002 for Tg2576 kept on high AGE, difference p = 0.0806). No significant correlations or significant differences between particular groups were observed between levels of MDA and those of Aβ 1–42.

Discussion

Effects of Prolonged Application of a Diet with a High Content of AGE Products in WT Mice

Our results performed on 11-months old WT mice revealed only one significant change mediated by prolonged application of a high AGE diet when compared to that of a regular AGE diet. Namely, we found an increase in nNOS activity to 125.6 % (Table 2) which could be interpreted as a compensatory reaction of a relatively healthy organism (old WT mice used as a model of normal aging), to an AGE-mediated drop in nNOS expression, as reported in many studies [20, 21, 22]. Nevertheless, some observed trends to alterations in particular components of the NMDA receptor–NO pathway [e.g., the drop in NR1 expression to 96.8 %, the increase in NR2A expression to 104.8 % and the increase in NR2B expression to 104.9 % (Table 1) and the increases in eNOS and iNOS activities to 107.9 and 127.6 %, respectively (Table 2)] should not be neglected e.g., with respect to the results of the correlation analysis. We assume that mild enhancements in NR2A or NR2B expression could contribute to an increase in nNOS activity because the marked negative interactions between NR2A and nNOS or between NR2B and nNOS were significantly attenuated by the prolonged effects of a high AGE diet (Table 3). In addition, AGE products probably also influenced the homeostasis between nNOS and eNOS or between eNOS and iNOS in the brains of WT mice (Table 3).

Our results obtained in WT mice thus support the sensitivity of the cerebral NMDA receptor–NO pathway to AGE products, in good accordance with previous studies [e.g., 20, 21, 22], and suggest that nNOS activity may be a more sensitive biomarker of alterations mediated by AGE products in vivo than e.g., MDA levels reflecting oxidative injury of lipids (Fig. 1). Since NMDA receptors are important for learning and memory [41] and high AGE products are associated e.g., with faster rate of decline in memory in non-demented young elderly [42], our results of marked differences in cerebral NMDA receptor–NO pathway between WT mice kept on both diets could indicate possible beneficial effects of the prolonged application of a diet containing low AGE product contents on cognitive dysfunction of older healthy or diabetic people [e.g., 7]. Recent research indicates that e.g., AGE products-induced microglial activation can be inhibited through dietary intake [43] and that the inhibition of microglial activation improves cognitive deficits in an animal model of AD [44].

Effects of Prolonged Application of a Diet Containing a High AGE Product Content in Tg2576 Mice and Comparisons with WT Mice

We found no significant differences between old Tg2576 mice (used as a model of AD) kept on diets containing regular or high AGE product contents in terms of NMDA receptor subunits (Table 1), NOS activities (Table 2), MDA levels (Fig. 1) or of soluble/insoluble Aβ 1–42 (Fig. 2). Moreover, correlation analysis did not reveal significant positive or negative correlations in Tg2576 mice (except for homeostasis between NR2A and NR2B) in contrast to WT mice. These results can be interpreted as attenuated regulation of the NMDA receptor–NO pathway (probably due to weakened interactions between subunits and synthases or through altered homeostasis among particular synthases rather than among particular subunits), in accordance with previous studies reporting attenuated NMDA-receptor signaling [27] or impaired cerebrovascular autoregulation and attenuated endothelium-dependent vasodilatation [35] in Tg2576 mice.

Comparisons of Tg2576 mice with corresponding WT groups (as a model of AD compared to non-demented controls) revealed significant differences only in animals kept on a high AGE diet. We concede that our 11-months old animals were still relatively young in contrast to 22-months old Tg2576 mice, which display altered nNOS expression/activity compared to age-related WT mice [31]. We suppose that the existing moderate alterations in the NMDA receptor–NO pathway in 11-months old Tg2576 (see studies on the age-dependent progressive alterations in Tg2576, e.g., [30]) are more robust by the application of high AGE products for a longer period time. In particular, we found the significant increase in NR1 expression to 104.5 % (Table 1), the shift (only with a borderline significance) from mild negative to significant positive correlations between nNOS activity and levels of insoluble Aβ 1–42 (“Results” section) and finally the significant shift from a mild positive to a marked negative correlation between iNOS activity and MDA levels (“Results” section). The enhancement in NR1 expression in Tg2576 was surprising, especially with respect to the observed Aβ-mediated reduction in cell-surface expression of NR1 [27], but this corresponds with the effects of AGE products in a prediabetic rodent model [15]. The trend to the marked positive correlation between nNOS activity and insoluble Aβ 1–42 levels (see also the similar difference in the case of nNOS activity and levels of soluble peptide, “Results” section) could be in accordance with the compensatory reaction to AGE-mediated drop in nNOS expression, observed in WT mice, but weakened in Tg2576 via attenuated regulation. Moreover, the result could well correspond with observed increased nNOS activity in the brain of AD people [10]. Since MDA levels were significantly increased in both groups of Tg2576 mice compared to the corresponding WT mice (to 138.5 % in animals kept on a regular AGE diet and to 150.2 % in these kept on a high AGE diet, Fig. 1), the pronounced negative correlation between iNOS activity and MDA levels exclusively in Tg2576 mice kept on a high AGE diet could be interpreted as a trend toward an AGE-mediated drop in iNOS activity [20, 21, 22] under conditions of overexpressed Aβ (see also the non-significant decrease in iNOS activity to 82.9 % in Tg2576 mice kept on a regular AGE diet and to 85.4 % in Tg2576 mice kept on a high AGE diet when compared to the corresponding WT mice, Table 2). This result could also support an increasing role of oxidative lipid injury in alterations in iNOS activity in Tg2576 mice.

One would expect alterations in eNOS activity with regard to the significant changes reported in AD patients [10], diabetic people [18, 19] and Tg2576 mice [34, 35] and to the high sensitivity of eNOS protein to AGE products in vitro experiments [20, 21, 22]. We suggest that our results of the relatively intact eNOS activity in WT as well as Tg2576 mice (Table 2) could be based on antagonistic trends evoked either by a high AGE product diet in WT mice (eNOS activity could be mildly increased with regard to the significant shift from a negative to a positive correlation with increasing nNOS activity and to the significant negative correlation with moderately decreased iNOS activity, Table 3) either by overexpressed Aβ in Tg2576 (eNOS activity could be decreased, on the contrary [34, 35], see also the non-significant decrease to 82.9 % in Tg2576 mice kept on a regular AGE diet and to 85.4 % in Tg2576 mice kept on a high AGE diet when compared to the corresponding WT mice, Table 2). However, our 11-months old mice were still relatively young and our experimental conditions (measurements were performed on brain supernatants, not on isolated brain capillaries) could play a role, too.

Although the results of this study did not reveal significant differences between Tg2576 mice kept on a high AGE diet when compared to these on a regular AGE diet, the observed changes in the NMDA receptor–NO pathway evoked by the prolonged application of a diet high in AGE products in Tg2576 mice, in comparison with the corresponding WT mice, could indicate possible beneficial effects of a diet low in AGE products also in people with AD; however, this research is in its early stages.

Summary

Our experiments performed on the brains of 11-months old WT mice revealed that prolonged application of a diet containing a high content of AGE products can significantly influence the NMDA receptor–NO pathway (especially via compensatory changes in nNOS activity, perhaps through attenuated interactions between NR2A/NR2B and nNOS or via altered homeostasis among particular synthases). 11-months old Tg2576 mice overexpressing human mutant Aβ peptides were not able to fully regulate this pathway (except for a functional regulation between NR2A and NR2B). Differences in the NMDA receptor–NO pathway in 11-months old Tg2576 and age-related WT mice were better distinguishable when a diet containing high amounts of AGE products was applied and especially changes in NR1, nNOS and iNOS were involved. Since NMDA receptors are important for learning and memory and high AGE products are associated e.g., with faster rate of decline in memory in non-demented young elderly, our results of marked differences in cerebral NMDA receptor–NO pathway between WT mice kept on both diets could indicate possible beneficial effects of the prolonged application of a diet containing low AGE product contents on cognitive dysfunction in older healthy or diabetic people and perhaps also in people with AD.

Notes

Acknowledgments

The research was supported by Ministry of Education, Youth and Sports of the Czech Republic (Project KONTAKT II-LH13254) and by Grant Agency of the Czech Republic (Project P304/12/6069).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standard

All procedures performed in the study were in accordance with the ethical standards and were approved by the Institutional Animal Care and Use Committee of the Sheba Medical Center.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Zdena Kristofikova
    • 1
  • Jan Ricny
    • 1
  • Jana Sirova
    • 1
  • Daniela Ripova
    • 1
  • Irit Lubitz
    • 2
  • Michal Schnaider-Beeri
    • 2
    • 3
  1. 1.Alzheimer’s Disease CenterNational Institute of Mental HealthKlecanyCzech Republic
  2. 2.The Joseph Sagol Neuroscience CenterSheba Medical CenterRamat GanIsrael
  3. 3.Department of PsychiatryIcahn School of Medicine at Mount SinaiNew YorkUSA

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