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Synergistic Deleterious Effect of Chronic Stress and Sodium Azide in the Mouse Hippocampus María José Delgado-Cortés, Ana M. Espinosa-Oliva, Manuel Sarmiento,† Sandro Argüelles, Antonio J. Herrera, Raquel Mauriño, Ruth F. Villarán, José L. Venero, Alberto Machado, and Rocío M. de Pablos* Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Sevilla, 41012-Sevilla, Spain Instituto de Biomedicina de Sevilla (IBiS)-Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, 41013-Sevilla, Spain ABSTRACT: Alzheimer’s disease is the most common cause of dementia in the elderly. Although the primary cause of the disease is presently unknown, to date several risk factors have been described. Evidence suggests that one of these risk factors could be chronic stress. The aim of this work is to demonstrate that chronic stress is able to induce Alzheimer’s disease features after the administration of nontoxic doses of sodium azide. We found that chronic stress increases the levels of several proteins involved in Alzheimer’s disease pathogenesis, such as presenilin 1, presenilin 2, and S100β, besides inducing the aggregation of Tau, ubiquitin, and β-amyloid proteins in the hippocampus. More important, our work shows a synergistic effect of stress and sodium azide treatment leading to significant neuronal death in the mouse hippocampus. Our results point out that chronic stress is a risk factor contributing to amplify and accelerate Alzheimer’s disease features in the hippocampus.

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INTRODUCTION Many neurodegenerative diseases such as Alzheimer’s disease (AD) are characterized by abnormal accumulation of certain proteins in the brain, such as amyloid beta-protein (Aβ), α-synuclein, and Tau. Different neurological disorders, known as taupathies, have been recently described. In these disorders, it has been suggested that modifications in the microtubuleassociated protein Tau could cause neural degeneration in specific regions.1 A common taupathy is AD, the most widespread cause of dementia among the elderly, affecting one in two individuals over the age of 85.2 These processes involve an increased deposition of (Aβ)-40 and -42 proteins into extracellular plaques (β-amyloidosis), synaptic dysfunction, and neuronal death.3 In AD, Tau pathology has been correlated with the level of dementia.4 Although the primary cause of the disease is presently unknown, many hypotheses have been proposed to explain it. The implication of mitochondria in the aging process as well as in neurodegeneration is widely accepted.5−9 Partial inhibition of the mitochondrial respiratory chain produces free radicals, diminishes aerobic energy metabolism, and causes excitotoxic damage creating a deleterious spiral causing neurodegeneration, © XXXX American Chemical Society

a pathological process considered to underlie AD. Selective reduction of complex IV activity is present in post-mortem AD brains.10 Inhibition of this complex could be evoked by chronic sodium azide (NaN3) administration in animals. NaN3 is a chemical of rapidly growing commercial importance with a high acute toxicity. Azide is widely used as a preservative in aqueous laboratory reagents and as the propellant in automobile air bags and aircraft escape chutes, which has increased the potential for direct human exposure through operations such as repair, transportation, manufacturing, assembly, scraping, and dismantling. Handling azide and azide-containing mixtures is of some concern due to both toxicity and combustibility issues.11,12 The treatment in animals with NaN3 causes chemical hypoxia and low energy production in the mitochondria11,13 and impairs learning and memory in rats.14 The brain regions most vulnerable to chronic NaN3 delivery are the cortical and hippocampal areas, the mesencephalic reticular formation, and the central amygdala.15 Taking into account the heterogeneity of the initial factors, and the poor correlation between β-amyloidosis and the degree Received: October 31, 2014

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DOI: 10.1021/tx5004408 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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Chemical Research in Toxicology of cognitive impairment in early stages of AD,16 it is difficult to determine the most important factors in the onset and progression of the disease. Evidence suggests that environmental factors, such as chronic stress, might also accelerate AD pathogenesis.17,18 Stress is a condition of the human experience and an important risk factor in the onset of several diseases. When an organism is stressed, the hypothalamic neurons release corticotropin-releasing hormone, which in turn stimulates the release of adrenocorticotropin hormone from the anterior pituitary. Finally, the adrenocorticotropin hormone induces the secretion of the stress steroid corticosterone (CORT) from the adrenals glands into general circulation.19 Although stress can be beneficial in its acute phase, repeated and severe stressful stimuli produce adverse effects on neuronal functions, especially in those structures involved in stress response, such as the hippocampus. The possible relationship between stress and AD has been pointed out by many authors.17,20 For the foregoing discussion, we studied the effect of chronic stress and CORT on some AD markers in animals treated with nontoxic doses of NaN3.

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Table 1. Stressors Used during the Chronic Variate Stress Treatmenta stressor

duration

forced swimming restraint restraint at 4 °C isolation food deprivation water deprivation

10 min 3h 1.5 h 24 h 24 h 24 h

a Animals subjected to stress were exposed daily to a different stressor from those listed above. These stressors were randomly repeated during the period of treatment. The right column indicates the length of each stimulus applied.

drilled holes at the far end for breathing. Forced swimming was carried out by placing the animal in a glass tank measuring 44 cm × 33 cm × 30 cm, with 22 cm of water depth at 23 °C ± 2 °C. Body weight was measured at the beginning and at the end of the each treatment and was evaluated as an indirect parameter of hypothalamic−pituitary−adrenal axis activation. Immunohistological Evaluation: Neuronal Nuclei (NeuN) and Aβ. Mice were perfused through the heart under deep anesthesia (isofluorane) with 60 mL of 4% paraformaldehyde in phosphate buffer at pH 7.4. The brains were removed and then cryoprotected serially in sucrose in PBS (1×, pH 7.4), first in 10% sucrose (24 h) and then in 20% (24 h) and 30% sucrose until they sank (2−5 days). Brains were then frozen in isopentane at −15 °C, and 25 μm sections were cut on a cryostat and mounted in gelatin-coated slides. The primary antibodies used are listed in Table 2 and were mouse-derived antineuronal nuclei

MATERIALS AND METHODS

Animals and Surgery. Experiments were carried out in accordance with the Guidelines of the European Union Directive 2010/63/EU) and Spanish regulations (BOE 34/11370-421, 2013) for the use of laboratory animals; the study was approved by the Scientific Committee of the University of Seville. Male C57BL/6 mice (25−30 g) were used for these studies. Mice were kept at constant room temperature of 22 °C ± 1 °C and relative humidity (60%) on a 12-h light−dark cycle with free access to food and water for the duration of the study. Mice were anaesthetized with isofluorane, and a mini-osmotic pump was surgically implanted subcutaneously (sc) in the intrascapular area (Alzet Osmotic pumps, U.S. and Canada). The pump was filled, under sterile conditions in accordance with manufacturer’s instructions, with either 200 μL of NaN3 (2.5 mg/kg/day) or 200 μL of isotonic saline solution (0.9%). Pumps had a mean flow rate of 0.25 μL/h. The contents of the pumps take 1 month to move out. The pumps were removed 1 month after implantation. In the case of experiments for 2 months, the pumps were removed after 1 month of implantation and a new pump was implanted under the same procedure/conditions. Nine groups of animals were established according to the different treatments. All animals were implanted with a mini-osmotic pump filled with either saline solution or NaN3 and were subjected or not to chronic stress. Two different time points were tested for each of these conditions (1 or 2 months). These groups of animals were stressed during the last 2 weeks of the cycle (when stressed for 1 month) or during the third, fourth, sixth, and eighth week of the cycle (when stressed for 2 months). We also included two groups of animals that were implanted with a mini-osmotic pump filled with either saline solution or NaN3 and received a sc dose of corticosterone (20 mg/kg) 5 days a week for 3 weeks. After a week off, the treatment was repeated for 3 weeks. One week after each treatment cycle, animals were sacrificed by decapitation (for Western blot experiments) or by perfusion (for immunohistochemistry and immunofluorescence experiments). At least four animals were used for each group. Stress Model. Chronic heterogeneous sequential stress was adapted from different models of variate stress21,22 with modifications. Animals were divided into stressed and nonstressed groups. Nonstressed animals were kept undisturbed in their home cages for 1 or 2 months. A variatestressor paradigm was used for the animals in the stressed groups. The schedule of stressor and the length of each stimulus applied each day are listed in Table 1. Application of stress started at different times from day to day (between 8:00 and 20:00), and stressors were used randomly, in order to minimize its predictability. Restraint was carried out by placing the animal in a 10 cm × 2.5 cm plastic tube and adjusting it with plaster tape on the outside, so the animal was unable to move. There were small

Table 2. Primary Antibodies Used in the Immunohistochemistry (IH), the Immunofluorescence (IF), and the Western Blot (WB) Analysis antibody NeuN (IH) Aβ (IH) Tau P (IF) ubiquitin (IF) GSK3β (WB) GSK3β P (Tyr 216) (WB) Cdk5 (WB) Tau T (C-17) (WB) Tau P (Ser 404) (WB) ubiquitin (WB) HSP 70 (WB) S-100β (N-15) (WB) PS 1 (N-19) (WB) PS 2 (N-20) (WB) β-actin (WB)

supplier

dilution

incubation

source

Chemicon International Inc. Chemicon International Inc. Santa Cruz Biotech Santa Cruz Biotech Santa Cruz Biotech Santa Cruz Biotech

1:1000

overnight

mouse

1:50

overnight

rabbit

1:100 1:100 1:500 1:500

overnight overnight overnight overnight

rabbit mouse rabbit goat

Santa Cruz Biotech Santa Cruz Biotech Santa Cruz Biotech

1:500 1:500 1:500

overnight overnight overnight

rabbit goat goat

Millipore Santa Cruz Biotech Santa Cruz Biotech Santa Cruz Biotech Santa Cruz Biotech Sigma-Aldrich

1:500 1:500 1:500 1:500 1:500 1:5000

overnight overnight overnight overnight overnight overnight

mouse goat goat goat goat mouse

(anti-NeuN, Chemicon International Inc. 1:1,000) and rabbit-derived anti-Aβ (anti-Aβ, Chemicon, 1:50). Incubations and washes for each antibody were carried out in Tris-buffered saline (TBS; 1×, pH 7.4). All work was done at room temperature. Sections were washed and then treated with 0.3% hydrogen peroxide in methanol for 15 min, washed again, and incubated in a TBS, solution containing 1% horse serum (for anti-NeuN, Vector Laboratories, Burlingame, CA, USA) or 1% goat serum (for anti-Aβ, Vector Laboratories, Burlingame, CA, USA) for 60 min in a humid chamber. Slides were drained and further incubated with the primary antibody in TBS, containing 1% horse serum/goat serum and 0.25% Triton-X-100 for 24 h at 4 °C. B


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Chemical Research in Toxicology Sections were then incubated for 2 h with biotinylated horse antimouse IgG (for anti-NeuN, Vector, 1:200) or goat antirabbit (for anti-Aβ, Vector, 1:200). The secondary antibodies were diluted in TBS, containing 0.25% Triton-X-100, and its addition was preceded by three 10 min rinses in TBS. Sections were then incubated with ExtrAvidin-Peroxidase buffered aqueous solution (Sigma, St Louis, MO, USA; 1:100) for 1 h. Peroxidase was visualized by performing a standard diaminobenzidine−hydrogen peroxide reaction for 5 min. For Aβ immunohistochemistry, cresyl violet counterstaining was used. Immunohistochemistry Data Analysis. Quantification of NeuN cells in the CA1 and CA3 hippocampal areas was estimated according to a modified stereological approach. Brains were cryosectioned at 25 μm thickness. Four of them were systematically and equidistantly (100 μm) sampled from a random starting point along the anterior− posterior axis of the CA1 and CA3 regions. The counted region had a total length of 2,180 μm. For each section, counting boxes (120 μm × 90 μm) were placed at three sites equally spaced along the mediolateral extent of CA3. The area of the CA1 and CA3 regions were estimated using the principle of Cavallieri. NeuN-positive cells were counted only in the CA1 and CA3 region. All data were collected blind to experimental treatment and expressed as the cell per mm3. Immunofluorescence. Animals were perfused, and sections were prepared as described above. Incubations and washes for all the antibodies were carried out in PBS. All work was done at room temperature. For immunofluorescence of phosphorylated Tau (antiTau P) and ubiquitin (antiubiquitin), sections were rehydrated in PBS, for 10 min, and then blocked with PBS, containing 1% goat serum (for anti-Tau P, Vector Laboratories) or horse serum (for antiubiquitin, Vector Laboratories) for 1 h. The slides were washed three times in PBS, then incubated overnight at 4 °C with rabbit-derived anti-Tau P (1:100; Santa Cruz Biotechnology) and mouse-derived antiubiquitin (1:100; Santa Cruz Biotechnology) diluted in PBS, containing 1% goat/horse serum and 0.25% Triton X-100. Sections were incubated with goat antirabbit secondary antibody conjugated to Cy3 (1:300; Vector Laboratories) for anti-Tau P and horse antimouse secondary antibody conjugated to fluorescein (1:300; Vector Laboratories) for antiubiquitin for 1 h at 22 °C ± 1 °C in the dark, and their addition was preceded by three 10 min rinses in PBS. The secondary antibody was diluted in PBS, containing 0.25% Triton X-100. Immunofluorescence was visualized using an Olympus BX61 microscope (Tokyo, Japan). Images were acquired using a digital camera (Olympus DP70) and processed using the software package associated with the camera (Olympus DPCONTROLLER and Olympus DPMANAGER). Western Blot Analysis. The hippocampus was lysed in 15 mM Tris−HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid, and 1 mM phenylmethylsulfonyl fluoride (all from Sigma-Aldrich). The homogenate was centrifuged at 12,000g for 20 min at 4 °C. Protein content of the samples was estimated by the method of micro-Lowry using bovine serum albumin as a standard;23 25 μg of protein was loaded in each lane. Protein samples were separated by SDS−PAGE (10% or 16% depending of the molecular weight of the protein) and transferred onto a nitrocellulose membrane (Novex; Life Technologies, Grand Island, NY, USA). Membranes were blocked with blocking buffer (5% milk in TBST: 20 mM TrisHCl, pH 7.5, 500 mM NaCl, and 0.05% Tween 20) for 1 h at room temperature. Membranes were then incubated using the antibodies listed in Table 2: anti- glycogen synthase kinase 3β (GSK3β; 1:500; Santa Cruz Biotechnology), antiphosphorylated GSK3β (GSK3β P; Tyr 216; 1:500; Santa Cruz Biotechnology), anti-cyclin-dependent kinase 5 (Cdk5; 1:500; Santa Cruz Biotechnology), anti-total Tau (Tau T; C-17; 1:500; Santa Cruz Biotechnology), anti-phosphorylated Tau (Tau P; Ser 404; 1:500; Santa Cruz Biotechnology), anti-ubiquitin (1:500; Millipore), anti-heat shock protein (HSP)-70 (1:500; Santa Cruz Biotechnology), anti-S100β (N-15; 1:500; Santa Cruz Biotechnology), and anti-presenilins (PSs) 1 (N-19) and 2 (N-20; 1:500; Santa Cruz Biotechnology) overnight at 4 °C in blocking buffer: 5% milk in TBST. β-Actin antibody (Sigma-Aldrich; 1:5,000) was used as a loading control. After incubation with the primary antibodies, all membranes were washed in TBST, incubated with peroxidase-conjugated antiimmunoglobulins secondary antibodies (DAKO, Produktionsvej, DK)

at a dilution of 1:3,000 for 1 h at room temperature in TBST. Proteins were visualized using Western blotting chemiluminescence luminal reagent (Santa Cruz Biotech., Santa Cruz, CA, USA). All experiments were repeated in triplicate. The products were analyzed by densitometry using the Quantity One 1-D Analysis software (Bio-Rad, Hercules, CA, USA). Statistical Analysis. Results are expressed as the mean ± SD. Means were compared by the Student’s t-test or by one-way ANOVA followed by the LSD test for posthoc multiple range comparisons. The α value was set at 0.05. STATGRAPHICS Plus 3.0 software was used for the calculations (Statpoint Technologies, Warranton, VA, USA). Hazardous Materials. The following chemicals are hazardous and should be handle carefully. Sodium Azide. The title compound should be kept locked up and kept away from heat and sources of ignition. Empty containers pose a fire risk. Therefore, the residue should be evaporated under a fume hood. Dust should not be ingested or breathed. Precautionary measures should be taken against electrostatic discharges. In case of insufficient ventilation, suitable respiratory equipment should be worn. If ingested, medical advice should be sought immediately, and the container or the label should be shown. Contact with skin and eyes should be avoided, and the compound should be kept away from incompatibles such as metals. Diaminobenzidine. The title compound should be kept away from heat and sources of ignition. Empty containers pose a fire risk. Therefore, the residue should be evaporated under a fume hood. Dust should not be ingested or breathed. Suitable protective clothing should be worn. In case of insufficient ventilation, suitable respiratory equipment should be worn. If ingested, medical advice should be sought immediately, and the container or the label should be shown. Contact with skin and eyes should be avoided, and the compound should be kept away from incompatibles such as reducing agents and moisture. Acrylamide. The title compound should be kept locked up and away from heat and sources of ignition. Empty containers pose a fire risk. Therefore, the residue should be evaporated under a fume hood. Dust should not be ingested or breathed. Suitable protective clothing should be worn, and in case of insufficient ventilation, suitable respiratory equipment should be worn. If ingested, medical advice should be sought immediately and the container or the label should be shown. Contact with skin and eyes should be avoided, and the compound should be kept away from incompatibles such as oxidizing agents, acids, alkalis, moisture. Paraformaldehyde. Keep locked up. Keep container dry and keep away from heat and sources of ignition. Do not ingest and breathe dust. Never add water to this product. In case of insufficient ventilation,

Figure 1. Body weight gain along the treatments. Body weight was monitorized along the treatments by weighing the animals once a week. The first weight was recorded before starting the treatments. Results are the mean ± SD of four animals and are expressed as percentage of body weight gain. Statistical significance, Student’s t-test: *, p < 0.05; **, p < 0.01 compared with the control group. C


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Chemical Research in Toxicology

Figure 2. Effect of different treatments on hippocampal neurons. (A) Coronal section showing NeuN immunoreactivity in control animals. (B) NeuN immunoreactivity after the treatment with NaN3 for 1 month. (C) Coronal section showing NeuN immunoreactivity after the treatment with NaN3 for 2 months. (D) NeuN immunoreactivity after 1 month of chronic stress. (E) NeuN immunoreactivity after 2 months of chronic stress. (F) NeuN immunoreactivity in mice treated with NaN3 for 1 month and stressed for 1 month as described previously. (G) NeuN immunoreactivity in mice treated with NaN3 for 2 months and stressed for 2 months as described previously. The combined action of NaN3 and chronic stress for 2 months produced a loss of NeuN-positive neurons. (H) Coronal section showing NeuN immunoreactivity after the treatment with CORT for 2 months. (I) Coronal section showing NeuN immunoreactivity after the cotreatment with NaN3 and CORT for 2 months. (J) Quantification of the number of NeuN-positive cells after each treatment. Results are the mean ± SD of four independent experiments, and are expressed as cells per mm3. Statistical significance (ANOVA followed by the LSD posthoc test for multiple comparisons): a, compared with the control; *, compared with the rest of treatments; p < 0.01. Scale bar: A−I, 250 μm; a−i, 50 μm. Abbreviations: NaN3, sodium azide-treated animals; SS, stressed animals; CORT, corticosterone-treated animals.

weight increase of mice was 12.6%. The weight gain over the treatment was significant in all cases (p < 0.01; Figure 1), all groups gaining less weight than the control group (p < 0.05 for the stress group, p < 0.01 for the NaN3 and the stress+ NaN3 groups). Effect on Hippocampal Neurons. As we have mentioned before, the hippocampus (a cerebral structure especially affected in AD) is one of the brain areas most affected by the treatment with NaN3. Hence, we performed NeuN immunostaining to detect neurons in the CA1 and CA3 layers of the hippocampus. A stereological dissector was used to estimate the number of neurons in these areas. Neither stress (or corticosterone) nor

wear suitable respiratory equipment. If ingested, seek medical advice immediately, and show the container or the label. Contact with skin and eyes should be avoided, and the compound should be kept away from incompatibles such as oxidizing agents, reducing agents, metals, and acids.

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RESULTS Change in Body Weight. Stress and NaN3 exerted a negative effect on the body weight gain of the animals; whereas the weight of control animals increased 26.7%, it increased 22.6% in stressed animals and 21.0% in animals treated with NaN3. Upon the combination of both stress and NaN3, the body D


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Figure 3. Effect of different treatments on tau phosphorylation and the amount of several kinases in the hippocampus. Proteins from the hippocampus of mice from the different treatments assayed were separated by electrophoresis, transferred to nitrocellulose membranes, and stained using rabbit derived anti-GSK3β, goat derived anti-GSK3β P, rabbit derived anti-Cdk5, goat derived anti-Tau, and goat derived anti-Tau P antibodies. Total optical density of each band was calculated. Results are the mean ± SD of four independent experiments and are expressed as intensity relative to control bands. Statistical significance (ANOVA followed by the LSD posthoc test for multiple comparisons): *, p < 0.05; **, p < 0.01, compared with the rest of treatments. Abbreviations: NaN3, sodium azide-treated animals; SS, stressed animals; CORT, corticosterone-treated animals.

stressed animals decreased the number of neurons in the CA3 to 78.3% of control values after 1 month (p < 0.01), and to 61.5% after 2 months (p < 0.01; Figure 2J). However, no differences were found in the CA1 area after the stress treatment (data not shown). Tau Phosphorylation. Stress (or corticosterone) and NaN3 had no significant effect on the phosphorylation of Tau

NaN3 produced a significant decrease in the number of neurons in the CA1 and CA3 layers when applied individually. However, a significant and synergic decrease of neurons of the CA3 layer was detected when they were combined (Figure 2). After two months of treatment with NaN3 and corticosterone, the number of neurons in the CA3 decreased to 83.6% of control values (p < 0.01; Figure 2I,J). The treatment with NaN3 in E


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Figure 4. Effect of different treatments on PS1, PS2, and S100β levels in the hippocampus. Proteins from the hippocampus of mice from the different treatments assayed were separated by electrophoresis, transferred to nitrocellulose membranes, and stained using goat derived anti-PS1, anti-PS2, and anti-S100β antibodies. Total optical density of each band was calculated. Results are the mean ± SD of four independent experiments and are expressed as relative intensity of control bands. Statistical significance (ANOVA followed by the LSD posthoc test for multiple comparisons): *, p < 0.05; **, p < 0.01, compared with the rest of the treatments. Abbreviations: NaN3, sodium azide-treated animals; SS, stressed animals; CORT, corticosterone-treated animals.

in the production of Aβ. As it has been described above for other markers, both PS1 and PS2 remained unchanged for the different treatments assayed, except for the administration of NaN3 for 2 months in stressed animals (173.4% and 193.3% of control values for PS1 and PS2, respectively; p < 0.01 in both cases; Figure 4). The brain levels of the protective protein S100β are usually elevated when the tissue is damaged. Again, no significant changes were found in the different treatments assayed except for the administration of NaN3 for two months in stressed animals (161.6% of control values, p < 0.05; Figure 4). Effect on Ubiquitin and HSP70. The amount of the protective chaperone HSP70 in the hippocampus showed no significant changes after the different treatments assayed (Figure 5). On the contrary, the levels of uibiquitin increased in all of the treatments assayed (p < 0.01) with respect to control values. All of the increases ranged between 135 and 167%, except the increase found in the stressed animals receiving NaN3 for 2 months (252.7% of control values; Figure 5).

when applied alone, but the amount of phosphorylated Tau increased to 178.6% of control values (p < 0.01) in stressed animals receiving NaN3 for 2 months (Figure 3), in a synergic way similar to the effect observed on the number of CA3 neurons. The total amount of Tau protein did not change after the treatments. To further evaluate the effect on Tau phosphorylation, the amounts of two protein kinases (GSK3β and Cdk5) involved in Tau phosphorylation were also measured in the hippocampus after the treatments (Figure 3). Again, there was no significant effect except for the stressed animals receiving NaN3 for 2 months; the amount of Cdk5 increased synergically to 144.5% of control values (p < 0.05) after this combined treatment; the amount of phosphorylated GSK3β remained without changes in all treatments, but the amount of total GSK3β increased synergically to 190.1% of control values (p < 0.01). Effect on Presenilins and the S100β Protein. Mutations in PS1 and PS2 genes are associated with familial forms of AD; as components of the γ-secretase complex, they are involved F


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Figure 5. Effect of different treatments on ubiquitin and HSP70 levels in the hippocampus. Proteins from the hippocampus of mice from the different treatments assayed were separated by electrophoresis, transferred to nitrocellulose membranes, and stained using mouse derived antiubiquitin and goat-derived anti-HSP70 antibodies. Total optical density of each band was calculated. Results are the mean ± SD of four independent experiments and are expressed as the relative intensity of control bands. Statistical significance (ANOVA followed by the LSD posthoc test for multiple comparisons): a, compared with the control; *, compared with the rest of treatments; p < 0.01. Abbreviations: NaN3, sodium azidetreated animals; SS, stressed animals; CORT, corticosterone-treated animals.

Protein Aggregates. Deposits of protein aggregates, especially of Aβ (amiloyd plaques) and hyperphosphorylated Tau (neurofibrillary tangles, NFT), are key features of AD. Ubiquitin is usually associated with these deposits. The administration of NaN3 for 2 months in stressed animals had a clear-cut effect on protein aggregates (Figure 6), inducing the accumulation of phosphorylated Tau protein (Figure 6A−F), ubiquitin (Figure 6G,H) and Aβ (Figure 6I,J). However, this effect was not observed when chronic stress or inhibition of complex IV by NaN3 was used separately.

of the models, which probably involves the reduction in hippocampal and/or cortical cholinergic neurotransmission. The model based on the perfusion of NaN3 has shown to induce neuronal damage in primary cortical and cerebellar granule neurons cultures27 and in cerebrocortical slice cultures.28 However, to date, neuronal damage after NaN3 administration in vivo is not documented. We therefore wanted to study the effect of non-neuronal-toxic doses of NaN3 in a number of neurons under stress conditions in the most important sensitive areas, the CA1 and the CA3 layers of the hippocampus. Our results show that the treatment with either NaN3 or stress for 1 or 2 months did not induce neuronal death in either the CA1 or the CA3 layer. However, strikingly, the combination of both induced a neuronal loss of about 40% in the CA3 layer, although the CA1 area was not affected. These data suggest that the CA3 layer of the hippocampus is especially sensitive to the combination of chronic stress and NaN3. In fact, the dendrites and spines of CA3 pyramidal neurons belong to one of the most plastic network systems in the brain since they receive major presynaptic inputs from the mossy fiber terminals that are constantly renewed in the process of adult neurogenesis of the dentate granule cells.29,30 Therefore, our work shows a significant death of pyramidal neurons in the hippocampus after the treatment with NaN3 but only under stress conditions. Pathologically, AD is characterized by the formation of two aggregates, namely, NFT and senile plaques. NFT consist of filamentous aggregates of hyperphosphorylated Tau in microtubules.31,32 Post-translational phosphorylation of Tau allows the formation of characteristic paired helical filaments, one of the hallmarks of AD. Our results show an increase in the protein levels of phosphorylated Tau in the CA3 layer of the

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DISCUSSION AD is a devastating form of chronic neurodegenerative disease in adults that causes dementia and death of the affected individuals. The onset and severity of the symptoms in the sporadic form of the disease shows extensive individual variations, leading to the idea that patient-related external factors might play a significant role in the development of the disease. Stress could be one of such factor since the physiological consequences of both acute and chronic stress on several systems have been well documented.24 For example, an association between the development of cognitive dysfunction and exposure to stressful events early in life has been suggested.25 A number of groups have shown that the AD phenotype in both transgenic and nontransgenic models of the disease is worsened by chronic stress or corticosterone administration. Three nontransgenic models try to mimic behavioral and morphological features of dementia and AD: permanent bilateral carotid artery occlusion, intracerebroventricular injection of streptozotocin, and brain mitochondrial cytochrome oxidase inhibition by NaN3.26 Learning and memory are impaired in all G


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Figure 6. Effect of NaN3 and stress on the aggregation of Tau P, ubiquitin, and Aβ in the hippocampus. Immunofluorescence of rabbit-derived antiTau P on control animals (A), a Tau P positive control (B), and after the combined treatment of NaN3 and stress for 2 months (C). (D,E,F) Highmagnification images of an area within the images in panels A, B and C, respectively. Immunofluorescence of ubiquitin on control animals (G) and after the combined treatment of NaN3 and stress for 2 months (H). (a,d,g) DAPI staining from control conditions. Immunohistochemistry showing deposits of Aβ on control animals (I) and after the combined treatment of NaN3 and stress for 2 months (J). Scale bars: A, B, and C, 50 μm; a, d, and g, 25 μm. D, E, and F, 25 μm; G and H, 50 μm; and I and J, 10 μm.

hippocampus of the animals treated with NaN3 and stressed for 2 months. Once we found the hyperphosphorylation of Tau, we analyzed the levels of Cdk5 and GSk3β, whose overactivation/ up-regulation has shown to enhance the formation of NTF.33 Cdk5 is a serine/threonine protein kinase that is involved in neuronal migration, synaptic function, myogenesis, neurite outgrowth, and differentiation of nerve cells.34 Cdk5 is considered as an important protein involved in the hyperphophorylation of Tau in AD.35 GSK3β is a multifunctional serine/threonine protein kinase that also plays an important role in the process of Aβ production and Tau hyperphosphorylation.36−38 Its overexpression decreases the levels of nuclear β-catenin and results in Tau hyperphosphorylation and neurodegeneration in the AD brain.39 GSK3β is inactivated by the phosporylation of serine 9 and activated by phosphorylation in tyrosine 216.40

Therefore, we have performed Western blot analysis of these two proteins in animals treated with NaN3, stress, and the combination of both. Our results have demonstrated a significant upregulation of Cdk5 only in response to NaN3 combined with stress, thus giving a rationale for the hyperphosphorylation of Tau. Analysis of GSK3β showed a significant level of increase in the NaN3/stressed animals compared with that of the control group. However, the amount of P-GSK3β is not affected in this experimental condition. The tyrosine phosphorylation of GSK-3β might facilitate substrate phosphorylation but is not strictly required for kinase activity.41 In our particular model, the results suggest that GSK-3β does not require previous phosphorylation in order to target Tau protein downstream. To further elucidate the mechanisms underlying the neuronal death in the stressed animals we studied the levels of several H



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proteins involved in AD pathology. S100β is a protein that promotes neurite growth, stimulates the proliferation of astrocytes, and elevates the levels of free calcium in both neurons and astrocytes. However, its overexpression could have deleterious consequences, including the overgrowth of dystrophic neurites that characterize the Aβ neuritic plaques whose presence helps diagnose AD. Marshak et al.42 reported that S100β is overexpressed in the brains of AD patients, particularly in the temporal lobe, where neuritic plaques are concentrated. Our results show that the levels of S100β are increased in the animals treated with NaN3 and stressed for 2 months, whereas there were no changes in the levels of this protein in the rest of experimental conditions. Other important proteins involved in AD are PS1 and 2, key protein members of the γ-secretase complex. Aβ is produced by the cleavage of amyloid precursor protein through β and γ-secretase. It is known that abnormal enhanced activity of β and γ-secretase may underlie Aβ overproduction. Therefore, it is well documented that mutations of PS1 and PS2 are causative for increased Aβ production in familial AD.42−44 To explore the mechanisms producing the stress-induced elevation of Aβ, we studied the expression levels of PS-1 and PS-2, key functional proteins in γ-secretase. A significant increase of PS-1 and PS-2 in NaN3/stress treated animals was observed in the CA3 layer of the hippocampus after 2 months, indicating that stress may promote Aβ42 production by enhancing the activity of γ-secretase. Hence, we performed immunhistochemistry techniques in order to demonstrate increased Aβ deposition. Again, we found Aβ plaques only in the animals treated with NaN3 and stressed for 2 months. Protein degradation was investigated in an effort to understand the neuronal damage induced by the combination of stress and NaN3. The ubiquitin−proteasome system and the autophagy− lysosomal pathway are the main cellular pathways for protein degradation; ubiquitin is common to both pathways. Ubiquitinated proteins have been consistently described in the protein aggregates found in many neurodegenerative diseases.45 Our results show an increase in the protein levels of ubiquitine only in the animals treated with NaN3 and stressed for 2 months. The HSP family constitutes the main control system for protein quality. It controls the synthesis, folding, and protein degradation processes in the nucleus, endoplasmic reticulum, cytoplasm, and mitochondria.46−49 Deleterious protein aggregation is prevented by chaperone binding.50,51 HSP70 seems to be involved in a number of neurodegenerative disorders showing accumulation of protein aggregates such as AD.52−56 In our experimental conditions, chronic stress slightly decreases the levels of HSP70 in the NaN3-treated animals for 2 months. Therefore, the protective role of HSP70 could be diminished in the hippocampus of these animals.

Article

AUTHOR INFORMATION

Corresponding Author

*Faculty of Pharmacy, University of Sevilla, Profesor Garciá González 2, 41012-Sevilla, Spain. Phone: +34 954553808. E-mail: [email protected]. Present Address †

M.S.: Gray Institute for Radiation Oncology and Biology, Department of Oncology, University of Oxford, Oxford OX3 7LJ, U.K. Funding

This work was supported by grants of Junta de Andaluciá (P10-CTS-6494) and Ministerio de Economiá y Competitividad (SAF2012-39029). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS Aβ, amyloid beta-protein; AD, Alzheimer’s disease; Cdk5, cyclin-dependent kinase 5; CORT, corticosterone; GSK3β, glycogen synthase kinase 3β; GSK3β P, phosphorilated GSK3β; HSP, heat shock protein; NaN3, sodium azide; NeuN, neuronal nuclei; NFT, neurofibrillary tangles; PS, presenilin; Tau P, phosphorylated Tau; Tau T, total Tau; TBS, Tris-buffered saline

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REFERENCES

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CONCLUSIONS Our results point out that chronic stress produced a significant appearance of AD features under conditions of mitochondrial dysfunction that were not observed when chronic stress or inhibition of complex IV by NaN3 was used separately. The molecular study of the different experimental groups allow us to suggest that stress could be considered as a potential factor to induce this AD-like scenario due to the abnormal appearance of key proteins involved in the impairment of mitochondrial function. Our results may also help understand the devastating effects of stress on the hippocampus and highlight the need to reduce the effects of stress on the growing population of elderly people. I


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