Decrease in age-related tau hyperphosphorylation and cognitive improvement following vitamin D supplementation are associated with modulation of brain energy metabolism and redox state

Abstract

In the present study we examined whether vitamin D supplementation can reduce age-related tau hyperphosphorylation and cognitive impairment by enhancing brain energy homeostasis and protein phosphatase-2A (PP2A) activity, and modulating the redox state. Male F344 rats age 20 months (aged) and 6 months (young) were randomly assigned to either vitamin D supplementation or no supplementation (control). Rats were housed in pairs and the supplementation group (n=10 young and n=10 aged) received subcutaneous injections of vitamin D (1, α25-dihydroxyvitamin D3) for 21 days. Control animals (N=10 young and n=10 aged) received equal volume of normal saline and behavioral testing in water maze started on day 14 after initiation of vitamin D supplementation. Tau phosphorylation, markers of brain energy metabolism (ADP/ATP ratio and adenosine monophosphate-activated protein kinase) and redox state (levels of reactive oxygen species, activity of superoxide dismutase, and glutathione levels) as well as PP2A activity were measured in hippocampal tissues. Our results extended previous findings that: 1) tau phosphorylation significantly increased during aging; 2) markers of brain energy metabolism and redox state are significantly decreased in aging; and 3) aged rats demonstrated significant learning and memory impairment. More importantly, we found that age-related changes in brain energy metabolism, redox state, and cognitive function were attenuated by vitamin D supplementation. No significant differences were seen in tau hyperphosphorylation, markers of energy metabolism and redox state in the young animal groups. Our data suggests that vitamin D ameliorated the age-related tau hyperphosphorylation and cognitive decline by enhancing brain energy metabolism, redox state, and PP2A activity making it a potentially useful therapeutic option to alleviate the effects of aging.

The brain, like most organs, undergoes gradual decline in energy metabolism during aging (Navarro and Boveris, 2007, Boveris and Navarro, 2008). Since neurons require large amounts of energy to carry out their function such as firing of action potentials and neurotransmission, they are vulnerable to the effects of age-related decrease in energy metabolism. The brainy's source of energy primarily comes from glucose delivered via oxygen supply and both of them are diminished during aging as illustrated in clinical imaging studies that show age-dependent reduction of glucose utilization in most human brain regions [reviewed in (Shulman et al., 2004)]. Similarly, experimental studies in rat brain show an age-dependent decrease in oxygen uptake especially in the hippocampus and cortex (LaFrance et al., 2005, Navarro et al., 2008), brain regions involved in memory processing; thus, the decline in energy metabolism during aging most likely contributes to age-related cognitive impairment. When neurons fail to adapt to the age-related decline in basal metabolic rate, they become susceptible to neurodegenerative disorders (Atamna and Frey, 2007, Gibson et al., 2010).

The energy-transducing capacity of mitochondria meets the neuronal energy demands through its most prominent metabolic process, which is oxidative phosphorylation (OXPHOS) generating the universal energy currency, adenosine triphosphate (Yin et al., 2012a). Free radicals are generated during normal mitochondrial function of OXPHOS as a result of electron transfer (Balaban et al., 2005). Steady state levels of free radicals produced are determined by both rate of energy metabolism and the integrity of the redox (oxidation-reduction) systems (Yap et al., 2009, Yin et al., 2012a). Consequently, the decrease in the mitochondrial energy-transducing capacity commonly seen in aging is then associated with a progressive increase in free radical production. The maintenance of redox homeostasis becomes crucial for neuronal function because disruption in energy homeostasis can shift the cells from a reduced state to an oxidized state that can predispose neurons to damage, dysfunction, and death (Yin et al., 2012b). For example, lower glucose availability in both in vivo and in vitro models induces hyperphosphorylation of the tau protein [reviewed in (Kapogiannis and Mattson, 2011)]. Age-related decreased in levels and activity of protein phosphatase 2A (PP2A) can alsoresult in increased tau phosphorylation (Gong et al., 2001). Hyperphosphorylation of the tau protein is linked to the formation of neurofibrillary tangles (Yin et al., 2012a). Because the presence of neurofibrillary tangles is one of the pathological hallmarks of Alzheimer's disease and associated with cognitive impairment (Kapogiannis and Mattson, 2011), it stands to reason that decreased brain energy metabolism and PP2A activity can contribute to the development of neurodegenerative disorders.

Dietary and/or pharmacologic antioxidants and hormones are some of the proposed therapies to combat age-related changes in redox state and cognitive function. One of the hormones gaining increasing support as a therapeutic agent to provide neuroprotection in the aging brain is vitamin D. The prevalence of chronic low serum vitamin D concentration in the elderly is estimated to be 50–80% (Nesby-O'Dell et al., 2002, Coudray et al., 2005). More recently, a meta-analysis report shows that vitamin D concentration is even lower in patients with Alzheimer's disease when compared to age-matched controls (Annweiler et al., 2013). Vitamin D has multiple biological targets mediated by vitamin D receptors (VDR) present in most cells including neurons and glial cells (Eyles et al., 2005) and that the enzyme responsible for the synthesis of the active form of vitamin D is ubiquitous in the brain (Eyles et al., 2005, Kalueff and Tuohimaa, 2007, Buell and Dawson-Hugues, 2008). In the central nervous system, VDR is shown to be located in the human cortex and hippocampus (Eyles et al., 2005), which are key brain areas involved in cognitive functioning. Studies show that in the central nervous system, vitamin D is involved in the regulation of neurotransmission, neuroprotection, and immunomodulation (Brown et al., 2003, Spach and Hayes, 2005, Dursun et al., 2010). Hence, in the present study we examined whether vitamin D supplementation can reduce age-related tau phosphorylation and cognitive impairment by enhancing brain energy metabolism and phosphatase activity, and modulating the redox state.

Methods

Subjects

Male F344 rats age 20 months (aged) and 6 months (young) obtained from Harlan Laboratories (Madison, Wisconsin) were used. Rats were housed individually in a pathogen-free vivarium under controlled condition (temperature 22 ± 1°C and humidity 70 ± 5%) and a 14:10 hour light:dark cycle was maintained. All animals were housed in the same room so that temperature, humidity, and lighting conditions are similar for all groups. Animals had free access to food (Harlan Tekland 8604 rodent chow containing 24.3% crude protein, 2.4 I.U./g vitamin D3, 1.4% calcium, and 1.1% phosphorus, and 14% calories from fat) and water. Animals were also handled daily throughout the study so that they could get acclimated to the research personnel thereby decreasing stress. Experiments started two weeks after arrival of the animals and all experimental protocols in this study were approved by the Institutional Animal Care and Use Committee and in accordance with the National Institutes of Health guidelines.

Vitamin D Supplementation

Two weeks after arrival from the breeder, young and aged rats were randomly assigned to either control (CON) or vitamin D supplementation (vit D) group (Fig. 1). A total of n = 40 rats were used in the study (n=10 animals in each young and aged CON groups; n=10 animals in each young and aged vitamin D groups). The supplementation group received the active metabolite of vitamin D (1, α25-dihydroxyvitamin D3; Calbiochem, LaJolla, CA) 42 I.U./Kg based on our previous work (Briones and Darwish, 2012) where this amount of vitamin D and duration of supplementation produced an elevation of serum levels without any other side effects (e.g. lethargy, weight loss, diarrhea). Vitamin D was prepared daily and dissolved in 1% ETOH (diluted with sterile saline). Injections instead of dietary or water supplementation were chosen to be able to control the amount of vitamin D given because each animal's dietary and water intake is variable. Rats in the CON group received 99% normal saline and 1% ETOH of equal volume to control for the effects of stress induced by the injection. Both vitamin D and normal saline injections were given subcutaneously daily for a total of 21 days and rats were weighed once a week during the supplementation regimen and no group differences were seen. Rats were also monitored daily for possible side effects (n=0) such as apathy, lethargy, and diarrhea.

Figure 1.

Timeline Study timeline. Legend: D – day.

Behavioral Testing

Two weeks after vitamin D or saline injections started, rats were tested in the water maze to evaluate cognitive impairment. All testing were done approximately 2 hours prior to the onset of the dark cycle to ensure that it is close to the rats’ active period. Behavioral testing was performed in the water maze for both spatial (cued-learning and memory) and non-spatial (discrimination learning) tasks. Rats were given a habituation swim and consistent water temperature was maintained throughout the test days to minimize animal stress during behavioral testing. The habituation training was given the day before actual testing for the purpose of teaching the rats to swim and locate the platform using the visible cue and also to rule out visual impairments and mobility problems. The habituation training consisted of 4 trials where a white wooden block (different cue from the one used in the actual testing) was attached to a visible platform and this cue remained in a fixed location and the starting location was also fixed (start position is east quadrant and the platform located in the west quadrant).

Water maze cued learning

During testing, the water maze tub was filled with tepid water (22 ± 2 ° C) and made opaque by the addition of powdered milk. The pool was divided into four quadrants of equal surface area and the starting locations for testing were assigned north, south, east, and west (not actual compass positions but rather relative to the behavioral testing room). The behavioral testing walls were already painted white so just the other distal (to the water maze) visual cues in the rooms were removed during the actual testing. The cued spatial learning and memory (acquisition and recall) task is sensitive to hippocampal dysfunction (Morris, 2003, Winocur et al., 2006). During the actual testing, a 10 cm diameter flower pot positioned in one quadrant halfway between the center and the side of the pool and submerged 2 cm below the surface with a small brass rod mounted on it vertically protruding 10 cm above the water with a red plastic ball mounted at the top served as a visual cue. Testing was conducted the day after completion of the habituation training, wherein the rats received 3 trials/day for 4 consecutive days and the starting location and visual goal positions were changed randomly for every trial. During the trials, swim latency (time to reach the platform) to reach the platform was recorded by a video camera connected to an image analyzer (Water Maze System Version 4.20, Columbus, OH). In addition, swimming speed (path length/swim latency) was used to assess the motoric activity in performing the task.

Water maze discrimination learning task

The rats were tested in the discrimination-learning test after two days of rest following the cued learning and memory task. The discrimination-learning task is sensitive to non-hippocampal dysfunction (Winocur and Hasher, 2004, Macleod et al., 2007). Rats were given 2 trials per day × 4 days and had to discriminate between black and white visible goals to find the hidden platform and all extra-maze cues in the room were again covered. The goal painted black was placed on top of the hidden platform to provide escape (P⁺) from the water (located in the southeast quadrant); whereas the other one painted white was floating (P⁻) and not able to offer sufficient buoyancy to support the rat (located in the southwest quadrant). Both visible goals were placed 10 cm above the water level. For this task, path taken to reach the correct goal was recorded as well as search errors based on the choices of P⁺ compared to P⁻ since the aim was to train the rats to avoid P⁻. Search errors represent the number of P⁻ choices made during the daily trials.

Tissue Preparation

All rats were euthanized using CO₂ asphyxiation and cervical dislocation after completion of the behavioral testing, the brains were quickly removed, and both hippocampi manually dissected then processed. Cervical dislocation was used to control for the effects of anesthesia-mediated tau phosphorylation (Planel et al., 2007). Western blot was used for determination of tau phosphorylation, and adenosine monophosphate-activated protein kinase level. Biochemical assays were used to determine levels of oxidized glutathione (GSH) and glutathione disulfide (GSSG, reduced glutathione), activity of superoxide dismutase, overall levels of reactive oxygen species (ROS), ADP/ATP ratio, and PP2A activity.

Detection of Plasma Vitamin D and Calcium Levels

Plasma levels of activated vitamin D (1, α25-dihydroxyvitamin D3) were determined using the commercially available ELISA assay (Immunodiagnostic Systems, Fountain Hills, AZ) according to manufacturer's instructions. After CO₂ asphyxiation, the thoracic cavity was opened and blood collected from cardiac puncture was placed in EDTA coated tubes. Samples were centrifuged (6000 x g for 15 min at 4°C) and stored at 80°C until assaying. Briefly, controls and samples were delipidated and transferred to supplied immunocapsules containing monoclonal antibody to 1, α25-dihydroxyvitamin D3 in suspension with vitamin D-binding protein inhibitor for immunoextraction. After eluting calcidiol from the immunocapsule gel, samples were evaporated in borosilicate glass tubes in a heating block for 30 minutes under nitrogen gas flow. Evaporated samples were reconstituted in assay buffer and incubated overnight with 100 μl of primary antibody solution. Standards, controls, and samples were then pipetted into a 96-well plate precoated with antibodies specific for 1, α25-dihydroxyvitamin D3 then incubated at room temperature for 90 minutes on an orbital plate shaker. After several washes, the chromogen was added to each well and incubated for an additional 30 min. Color reaction was stopped by an equal volume of stop solution and reaction was read in a microplate reader (Bio-Tek, Winooski, VT) at a wavelength of 450 nm (650-nm reference wavelength). The color change for each sample was plotted and measured within the range of the standard curve. Assays were sensitive to 2.5 pg/ml and coefficient of variation was <10%.

For detection of calcium level, blood was also collected from cardiac puncture during euthanasia using syringes containing Ca²⁺-balanced heparin (Radiometer America, Westlake, OH) for ionized calcium and immediately analyzed. Ionized calcium level and pH were measured using the I-Stat Clinical Analyzer (Abbott Laboratories, Princeton, NJ). We measured plasma levels of ionized calcium because this is the active form of Ca²⁺. To determine ionized Ca²⁺ levels, we adjusted the actual values obtained to a pH of 7.4 since acidosis can affect calcium binding.

Western Blot

Levels of adenosine monophosphate-activated protein kinase (AMPK) were assessed using 0.4 g of hippocampal tissue from each rat that was homogenized and centrifuged at 25,000 × g for 20 minutes as previously described (Briones et al., 2006, Briones et al., 2009). Aliquots from the supernatant were removed for protein determination. Protein concentration in samples was determined using the BCA-Protein assay (Pierce, Rockford, IL).

For assessment of insoluble tau phosphorylation, 0.3 g of hippocampal tissue from each rat was homogenized as previously described (Briones et al., 2006, Briones et al., 2009) then centrifuged at 14,000 × g for 10 minutes at 4°C. The supernatant was collected from this low speed spin then incubated in 1% RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 5 mM EDTA, 0.5% Sodium deoxycholate, and 0.1% SDS, pH 8.0) for 10 minutes followed by ultracentrifugation at 200,000 × g at room temperature. The pellets were resuspended in 450 μl of homogenizing buffer then ultracentrifuged at 200,000 × g for 30 minutes at room temperature. The supernatant was removed and the pellet was resuspended again in 200 μl 1x Laemmli sample buffer (Sigma Aldrich, St. Louis, MO) then centrifuged at 14,000 × g for 10 minutes at 4°C to obtain the final insoluble sample.

For Western blot analyses, equal amounts of protein (40 μg) from each rat were loaded and separated by SDS-PAGE gel electrophoresis as previously described (Briones et al., 2009). The protein bands were electrophoretically transferred to nitrocellulose membranes (Amersham, Piscataway, NJ) stained with 0.5% Ponceau Red to visualize total proteins, then destained. Non-specific binding sites were blocked then nitrocellulose membranes were incubated overnight at 4°C with gentle agitation in the primary antibody. The primary antibodies used were: 1) anti-tau (mouse monoclonal, 1:1500; clone S.125.0); 2) anti-phosphorylated tau, Ser⁴⁰⁴ (rabbit polyclonal, 1:2000); 3) anti-phosphorylated tau, Ser²¹⁴ (mouse monoclonal, 1:2000; clone AT100); 4) anti-phosphorylated tau Thr²³¹ (mouse monoclonal, 1:1000; clone AT180); 5) anti-AMPK (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA); 6) anti-phosphorylated AMPK (polyclonal rabbit, 1:1000; Santa Cruz Biotechnology); and 5) anti-β-actin (rabbit polyclonal, 1:2000; Santa Cruz Biotechnology). All tau antibodies were obtained from Thermo Scientific (Rockford, IL). The secondary antibodies used were horseradish peroxidaseconjugated immunoglobulin (Sigma) and the Super Signal chemiluminescense substrate kit (Pierce, Rockford, IL) was used to visualize immunoreactive bands. After visualization, the membranes were then stained with Amido-Black to qualitatively verify protein loading. A series of dilutions were performed and immunoblotted for each antibody to establish that the relationship between protein band and intensity was linear over the range of band intensities observed in the samples. Band visualization was obtained by exposure of membranes to autoradiographic film (Kodak Biomax™). Samples were analyzed in quadruplicates and measurements were averaged and used as one individual data point for statistical analysis. Quantification of differences in protein bands between samples was done using densitometric analysis (Scion Image Beta 4.0.2; Frederick MD). The internal control β-actin was used to standardize experimental values in densitometric analysis.

Biochemical Assays

Levels of ROS, activity of superoxide dismutase (SOD), glutathione level (both reduced and oxidized forms), ADP/ATP ratio, and PP2A activity were evaluated using different biochemical assays and these data provided information on brain energy metabolism, redox state, and phosphatase activity. For determination of SOD activity and levels of ROS, 0.3 g of hippocampal tissue was homogenized in ice-cold phosphate buffer (0.1 M, pH 7.4) containing KCl (140 mM), EDTA (1 mM) and the protease inhibitor phenylmethylsulfonyl fluoride (1 mM) using a Teflon-glass homogenizer. The homogenate was centrifuged at 960 × g for 10 minutes and the supernatant was collected.

Determination of ROS Level

The ROS and nitrogen reactive species (RNS) production was assessed through the 2’,7’-dichlorofluorescein (2’,7’-DCF) oxidation method (Lebel et al., 1992). Briefly, 60 μL of the tissue sample was incubated at 37°C in the dark with 240 μL of 2’,7’-DCF diacetate (H₂DCFDA) reagent (Invitrogen, Carlsbad, CA) in a 96-well plate. H₂DCFDA is cleaved by cellular esterases and form H₂DCF that is oxidized by ROS and RNS that are present in the sample, producing the fluorescent compound DCF. DCF oxidation was measured using a microplate reader at 488 nm excitation and 525 nm emission wavelength. A standard curve, using standard DCF (0.25–10 mM) was performed in parallel with the samples. The fluorescent intensity unit was normalized with the protein content, plotted and measured against the standard curve, and expressed in relative unit production per minute.

Determination of SOD Activity

Total SOD activity was evaluated at room temperature according to previously described method (Misra and Fridovich, 1972). Approximately 200 μl of hippocampal tissue homogenate was added to 3ml of EDTA – sodium carbonate buffer (0.5M) at pH 10.2. The reaction was started by adding 100 μl of epinephrine (30mM in 0.1M HCl) to the homogenate and SOD activity was evaluated by quantifying the inhibition of superoxide-dependent autoxidation of epinephrine and the absorbance of the sample was verified using a microplate reader at a wavelength of 480 nm. One unit of SOD is defined as the amount of enzyme that inhibits the speed of oxidation of epinephrine by 50%. Results were expressed as units/mg protein.

Determination of GSH and GSSG

To quantify levels of glutathione, 0.3 g of hippocampal tissue was homogenized in a cold MES [0.4 M 2-(N-morpholino)ethanesulfonic acid and 2mM EDTA, pH 6.0] buffer (Sigma Aldrich, St. Louis, MO) then centrifuged at 10,000 × g for 15 minutes at 4°C. Aliquots from the supernatant were removed for protein determination. Protein concentration in samples was determined using the BCA-Protein assay (Pierce). The remaining supernatant was deproteinated and stored at -20°C until use. GSH level was measured using the glutathione assay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions. Briefly, samples and standards (provided by the manufacturer) were loaded in a 96-well plate and the Assay cocktail (provided by the manufacturer) was added. GSSG was also measured using the same procedure above but the deproteinated samples were first treated with 1 M 4-vinylpyridine (Sigma, St. Louis, MO). Color absorbance was measured every 5 minutes for a total of 25 minutes using a microplate reader at a wavelength of 405 nm (650-nm reference wavelength). Values of GSH and GSSG for each sample were normalized with the protein content and calculated by plotting them against the standard curve. The inter-assay and intra-assay coefficient of variation are 3.6% and 1.6%, respectively; and the dynamic range of the assay kit for GSH and GSSG are 0-16 μM and 0-8 μM, respectively.

Determination of ADP/ATP Ratio

To evaluate brain metabolism, the ApoSENSOR ADP/ATP Bioluminescence Assay kit was used and the procedure was carried out according to the manufacturer's specifications (BioVision Inc, Milpitas, CA). Approximately, 0.3 g of hippocampal tissue from each rat was homogenized in cold PBS buffer then 100 μl of Nucleotide Releasing buffer was added to the sample. ATP level was measured by adding 1 μl of ATP Monitoring enzyme to the prepared sample then the color reaction was read after 1 minute using the microplate reader at a wavelength of 450 nm (650-nm reference wavelength). The color change was proportional to the total level of ATP. To the same sample, 1 μl of ADP Converting Enzyme was added then the color reaction was read again after 10 minutes to yield ADP data then another reading was done after 1 minute. Data were normalized with the protein content and ADP/ATP ratio was then calculated as: ADP values (data from second reading – data from first reading) / ATP values.

Measurement of Protein Phosphatase 2A

The PP2A Immunoprecipitation Phosphatase BioAssay Kit (Millipore, Temecula, CA) was used according to the manufacturer instructions to determine PP2A activity. Briefly, 0.3 g of hippocampal tissue was homogenized in 40x volume/weight of 20 mM imidazole-HCl pH 7.0, 2 mM EDTA, 2 mM EGTA, 1 mM PMSF, 10 μl/ml of Protease Inhibitor Cocktail P8340 (Sigma-Aldrich), and centrifuged at 2000 g for 5 min at 4°C. After determination of protein concentration, the PP2A catalytic subunit was immunoprecipitated with a monoclonal antibody and protein A agarose then the activity of the immunoprecipitated PP2A was assessed by the release of phosphate from threonine phosphopeptide over a period of 10 min at 30°C. The amount of phosphate released was measured by the absorbance of the molybdate-malachite green phosphate solution at 630 nm using a microplate reader and fluorescent intensity was normalized with the protein content.

Statistical Analysis

The SAS general linear model (SAS Institute, North Carolina) procedures for two-way analysis of variance (ANOVA) were used to examine effects of experimental conditions (vitamin D vs. saline groups) and age (young vs. aged) on the dependent variables. Two-way repeated measures ANOVA was used to examine vitamin D supplementation effects on behavioral performance on the water maze to determine differences in swim latency, path length, search errors, and swimming speed. When appropriate, the SAS CONTRAST statement was used for planned comparisons of the effects of supplementation (vitamin D vs. saline groups) and age (young and aged), and the combination of age and supplementation. All error bars represent ± standard error of the mean (SEM) of the sample size used in the study. In addition, regression analysis was performed to examine the relationship between brain energy metabolism and behavioral performance.

Results

Plasma levels of vitamin D and calcium

To determine age-related vitamin D changes we measured circulating levels of its active form, 1, α25-dihydroxyvitamin D3, and found significantly lower levels in the aged rats (F(3,36) = 9.33, p<0.05) when compared to the young animals (Fig. 2A); but the deficiency seen in the aged rats that received the supplementation was significantly less in comparison to the aged CON animals (F(3,36) = 10.02, p<0.05). Post hoc comparisons showed a significant 1, α25-dihydroxyvitamin D3 deficiency in the aged CON rats to less than 26% of the young CON group suggesting an age-related vitamin D deficiency. Further analysis revealed circulating levels of 1, α25-dihydroxyvitamin D3 is 22% less in the aged CON rats compared to the aged animals that received the supplementation. No significant differences were evident in circulating levels of 1, α25-dihydroxyvitamin D3 in the young animal groups.

Figure 2.

Plasma Levels of Vitamin D and Calcium. Two-way ANOVA shows significant deficiency in plasma circulating levels of both vitamin D (A) and calcium (B) in the aged rats when compared to the young animals. But post hoc comparisons show that supplementation of vitamin D mitigated both deficiencies. *p <0.05, **p <0.01. Legend: CON – control; vit D – vitamin D supplementation group; ANOVA – analysis of variance.

Measurement of ionized calcium showed age-related significant decrease (F(3,36) = 10.19, p<0.05) in plasma levels (Fig. 2B). However, supplementation with vitamin D in the aged rats modulated the reduction in plasma levels of ionized calcium (F(3,36) = 9.83, p<0.05). That is, ionized calcium level in the aged CON group is 35% lower that the aged vit D animals. Post hoc comparisons showed no significant difference in ionized calcium in the young animal group. Together, these results suggest an age-related deficiency in vitamin D metabolism accompanied by a decrease in ionized calcium but giving supplementation modulated hypovitaminosis D and minimized the reduction in calcium. These results are hardly surprising since vitamin D is essential in maintaining calcium homeostasis (Dusso, 2013).

Vitamin D mitigates age-related cognitive decline in the water maze tasks

Rats were tested in the water maze for cued learning and memory as well as the discrimination learning tasks. In the cued learning and memory task, within-subjects effect was seen for swim latency across the test days given that all rats learned to perform the task over the four testing days. An overall significant main effect of age was seen in mean swim latency (Fig. 3A) and the aged CON group demonstrated longer mean swim latency overall compared to all groups (F(3,36) = 8.19, p<0.05). Meanwhile, a significant main effect of vitamin D (F(3,36) = 8.42, p<0.05) was also seen in that the aged rats that received the supplementation demonstrated decrease mean swim latency compared to the aged CON group. Although the aged vit D group performed significantly better when compared to the aged CON rats, they still demonstrated impairment in comparison to the young animal groups. No significant effects of age on swimming speed was seen (F(3,36) = 5.67, p=0.07). No significant difference was seen in mean swim latency between the young animal groups. Additionally, no interaction effect was seen between age and vitamin D in mean swim latency.

Figure 3.

Behavioral Testing. Two-ANOVA with repeated measures show that aged rats have significantly longer mean swim latency in the cued learning and memory task (A) in comparison to the young animals. Post hoc comparisons show that the aged CON group has the greatest deficit. Analysis of performance in the discrimination learning task using 2-way ANOVA with repeated measures also show that aged rats took significantly longer paths to reach the goal (B) and made significantly more errors in discriminating between the correct and incorrect goals (C) compared to the young animal groups. Post hoc comparisons show that age-related performance deficits are attenuated in the vit D group. *p <0.05 – significantly different from the young animal groups, **p <0.01 – significantly different from all groups. Legend: CON – control; vit D – vitamin D supplementation group; ANOVA – analysis of variance.

Measurement of path taken to reach the goal (path length) in the discrimination learning task showed within-subjects effect (Fig. 3B) where all rats showed a continuous decrease in the swimming distance covered to reach the platform across the test days. Overall significant main effects of age (F(3,36) = 9.31, p<0.05) and vitamin D (F(3,36) = 8.93, p<0.05) were seen where the aged CON animals often took the path to the incorrect goal compared to all groups while the aged vit D group performed better but still showed impairment in comparison to the young animals. No significant difference was seen when the young animal groups were compared. No significant interaction effect (p = 0.10) was seen between age and vitamin D in mean path length. Analysis of the number of errors made by the rats in locating the correct goal showed an overall significant main effect of age (F(3,36) = 9.01, p<0.05) and vitamin D (F(3,36) = 8.39, p<0.05) where aged CON rats regularly chose P^- (the incorrect goal) in most of the trials. Meanwhile, aged rats that received the vitamin D supplementation committed less error but their performance is still significantly impaired in comparison to the young animals (Fig. 3C). On the contrary, no significant differences were seen in performance of the young CON and young vit D groups in discerning the correct goal. In addition, no significant interaction effect was seen between age and vitamin D (p = 0.08). Together, these data suggest that age can result in the impairment of complex cognitive task but providing vitamin D supplementation may slow down this cognitive decline.

Vitamin D enhances brain energy homeostasis

We measured total AMPK and phosphorylated AMPK (pAMPK) to obtain the pAMPK/AMPK ratio as an indication of cellular energy status (Fryer et al., 2002, Kleman et al., 2008). No significant group differences were seen in total AMPK level (Fig. 4A). However, analysis of pAMPK showed significant main effects of both age (F(3,36) = 11.09, p<0.05) and vitamin D (F(3,36) = 10.88, p<0.05) where aged rats showed significantly decrease pAMPK in comparison to the young animal groups (Fig. 4B). But providing vitamin D supplementation to the aged rats significantly increased pAMPK levels by 27% compared to the aged CON group. Post hoc comparison showed that although the pAMPK levels of aged rats that received vitamin D are higher than the aged CON group, it was still significantly lower by 20% and 23% in relation to the young CON and young vit D rats, respectively. Comparison of the young rat groups did not show any significant difference on pAMPK.

Figure 4.

Indicators of Brain Energy Metabolism. Two-way ANOVA shows no significant group differences in the total AMPK levels (A) but the level of phosphorylated AMPK (B) and ratio of pAMPK/AMPK (C) are significantly decreased in the aged rats compared to the young animals. Post hoc comparisons show that providing vitamin D supplementation is able to mitigate the age-related decreased on pAMPK and pAMPK/AMPK ratio. Representative Western blots of total AMPK and pAMPK are shown on the right upper panel. Two-way ANOVA also shows significant age-related increased in ADP/ATP ratio (D) and post hoc comparisons show that giving vitamin D supplementation diminished the effects of aging on ADP/ATP ratio. Linear regression also shows that in the aged rat groups increased ADP/ATP ratio is significantly related to increased number of errors made on the last day of behavioral testing (E). *p <0.05, **p <0.01. Legends: 1 – aged control; 2 – aged vitamin D; 3 – young control; 4 – young vitamin D; CON – control; vit D – vitamin D supplementation group; ANOVA – analysis of variance.

When the pAMPK/AMPK ratio was quantified, significant main effects of both age (F(3,36) = 11.29, p<0.05) and vitamin D (F(3,36) = 10.84, p<0.05) were also seen in that aged rats showed significantly decrease pAMPK/AMPK ratio in comparison to the young animal groups (Fig. 4C). Post hoc comparisons showed that pAMPK/AMPK ratio in the aged rats given vitamin D supplementation was 24% higher compared to the aged CON. However, the pAMPK/AMPK level of aged rats that received vitamin D was still 19% and 24% lower when compared to the young CON and young vit D rat groups, respectively. Comparison of the young rat groups did not show any significant difference on pAMPK/AMPK ratio.

Analysis of ADP/ATP ratio to determine brain metabolism also showed significant main effects of both age (F(3,36) = 10.22, p<0.05) and vitamin D (F(3,36) = 9.13, p<0.05) in that aged rats had significantly increased ADP/ATP ratio in comparison to the young animal groups (Fig. 4D). Post hoc comparisons showed that aged rats given vitamin D supplementation had 22% lower ADP/ATP ratio compared to the aged CON. However, the ADP/ATP ratio of aged rats that received vitamin D was still 27% and 21% higher compared to the young CON rats and the young vit D group, respectively. Comparison of the young rats did not show any significant group difference on ADP/ATP ratio. A significant positive relationship (r² = .82, p<0.05) was also seen between ADP/ATP ratio and the number of errors committed on the last day of behavioral testing (Fig. 4E). Together these results suggest that the capacity to maintain energy metabolism is compromised in aging but using vitamin D supplementation can diminish the effects of age on energy metabolism. Our results also suggest that decreased in ATP supply possibly contributed to age-related cognitive decline.

Vitamin modulates age-related changes in redox state

We measured ROS level, activity of the enzymatic antioxidant SOD, and level of the antioxidant glutathione to evaluate redox state. Assessment of ROS level showed a significant main effect of age (F(3,36) = 9.20, p<0.05) and vitamin D (F(3,36) = 8.92, p<0.05) in that increased dichlorofluorescein expression was seen in the aged rats when compared to the young animals (Fig. 5A). Post hoc comparison showed that providing vitamin D supplementation significantly decreased the expression of dichlorofluorescein in the hippocampus of aged rats by 21% in relation to the aged CON group. However, providing vitamin D to the young animals did not produce any significant effect in dichlorofluorescein expression. When total SOD activity was measured, our results also showed a significant main effect of age (F(3,36) = 8.06, p<0.05) and vitamin D (F(3,36) = 8.89, p<0.05) where decreased SOD activity was seen in the aged rats compared to the young animals but this age-related decreased in antioxidant enzyme activity was modulated by vitamin D supplementation by 21% (Fig. 5B). Meanwhile, no significant vitamin D effect was seen in SOD activity in the young animals.

Figure 5.

Indicators of Redox State. Two-way ANOVA was used to analyze redox state and post hoc comparisons were done using the SAS Contrast statements. Level of ROS is significantly increased (A) while activity of SOD is significantly decreased (B) in the aged rats in comparison to the young animals. As well, level of GSH is significantly decreased (C) but GSSG level (D) is significantly increased in the aged rats compared to the young animals. Accordingly, an age-related decreased in GSH/GSSG ratio (E) is seen. Nevertheless, giving vitamin D supplement modulated the age-related changes in redox state. *p <0.05, **p <0.01. Legend: CON – control; vit D – vitamin D supplementation group; ANOVA – analysis of variance.

We also measured GSH and GSSG levels and significant main effects of age and vitamin D were seen. Levels of the reduced form of glutathione (GSH) were significantly decreased in the aged rats (F(3,36) = 10.16, p<0.05) but levels of the oxidized form (GSSG) were significantly increased (F(3,36) = 9.43, p<0.05). However, giving vitamin D supplementation mitigated and age-related changes in GSH (F(3,36) = 8.17, p<0.05) and GSSG (F(3,36) = 7.94, p<0.05) levels (Fig. 5C and 5D). When GSH/GSSG ratio was analyzed, we found significantly lower values in the aged rats (F(3,36) = 10.44, p<0.05) compared to the young animals (Fig. 5E). Conversely, providing vitamin D supplementation to the aged rats moderated the reduction in GSH/GSSG ratio by 39%. Comparison of the young animal groups showed no significant difference in GSG and GSSH levels, as well as GSH/GSSG ratio. These results suggest an age-related imbalance in redox state exists but giving vitamin D supplementation modulated this imbalance.

Vitamin D mitigates age-related tau hyperphosphorylation by enhancing phosphatase activity

Next we examined changes in tau phosphorylation in the hippocampus associated with aging and vitamin D supplementation. Using Western blot, we used three antibodies targeting several key tau phosphoepitopes. The phosphorylated tau antibodies detected pre-neurofibrillary tangle phospho-tau protein (pThr²³¹), intraneuronal neurofibrillary tangle phospho-tau protein (pSer²¹⁴), and extracellular neurofibrillary tangle phospho-tau protein (pSer⁴⁰⁴) according to earlier studies (Augustinack et al., 2002, Cui et al., 2012). Our results showed no significant group differences in total tau levels (F(3,36) = 5.05, p=0.11) but a significant increased in hippocampal tau phosphorylation at the pre-tangle (F(3,36) = 9.75, p<0.05), intraneuronal tangle (F(3,36) = 9.59, p<0.05), and extracellular tangle sites (F(3,36) = 10.02, p<0.05) were seen in the aged rats compared to the young animals (Fig. 6A-C). However, vitamin D supplementation significantly modulated age-related tau hyperphosphorylation at the pre-tangle, intraneuronal tangle, and extracellular tangle sites by 33%, 37%, and 31%, respectively. No significant increase in tau phosphorylation was seen in the young animal groups.

Figure 6.

Tau Phosphorylation in the Hippocampus. Upper panel left: Representative Western blots of total tau and tau phosphorylation at different sites. Two-way ANOVA shows significantly increase tau phosphorylation at the pre-neurofibrillary tangle (pThr²³¹), intraneuronal neurofibrillary tangle (pSer²¹⁴), and extracellular neurofibrillary tangle (pSer⁴⁰⁴) sites (A-C) in the aged rats compared to the young animals. Post hoc comparisons shows that providing vitamin D supplementation tempered the age-related hyperphophorylation of the tau protein. *p <0.05, **p <0.01. Legends: 1 – aged control; 2 – aged vitamin D; 3 – young control; 4 – young vitamin D; CON – control; vit D – vitamin D supplementation group; ANOVA – analysis of variance

We also evaluated PP2A activity and found it to be significantly decreased (F(3,36) = 9.43, p<0.05) in the aged group (Fig. 7A). However, administration of vitamin D in the aged rats enhanced PP2A activity by 29%. PP2A subunit B examined in this study is an important serine and threonine phosphatase that plays a critical role in central nervous system function (Liu and Wang, 2009) and our data demonstrating decreased PP2A and increased tau phosphorylation in the aged animals given vitamin D compared to the aged control rats suggest that the supplementation was able to modulate age-related tau hyperphosphorylation through PP2A actions. These results suggest that providing vitamin D supplements in aged rats was able to regulate tau hyperphosphorylation by upregulating PP2A activity.

Figure 7.

Protein Phosphatase 2A (PP2A) Activity. Two-way ANOVA shows that PP2A activity is significantly decreased in the aged rats in comparison to the young animals (A). But post hoc comparisons show that giving vitamin D supplement modulated the age-related decreased in PP2A activity. *p <0.05. Legend: CON – control; vit D – vitamin D supplementation group; ANOVA – analysis of variance.

Discussion

In the present study we demonstrate that age-related vitamin D deficiency is associated with tau hyperphosphorylation, decreased brain energy metabolism and PP2A activity, and reduced redox state in the hippocampus and that these neuropathological processes are accompanied by cognitive decline. To our knowledge this is the first study to link the effects of vitamin D on tau phosphorylation and brain energy metabolism in the normal aging brain. Aging is associated with declines in energy production in the brain as well as parallel changes in redox status with a pro-oxidant shift that may be due in part through the mitochondrial generation of superoxide radical and hydrogen peroxide (Boveris and Navarro, 2008, Yin et al., 2012a).

Here we show that energy homeostasis is altered in the aging hippocampus evidenced by decrease pAMPK/AMPK ratio as well as increased ADP/ATP ratio. Our data are in line with previous reports on decreased AMPK activity and/or decreased responsiveness to AMPK activity in different tissues as a function of age (Greer et al., 2007, Yap et al., 2009). AMPK is regarded as a central sensor of cellular energy status or a metabolic fuel gauge conserved along the evolutionary scale in eukaryotes that senses changes in the intracellular AMP/ATP ratio (Kahn et al., 2005). Activation or phosphorylation of AMPK maintains cellular energy stores, switching on catabolic pathways that produce ATP, mostly by enhancing oxidative metabolism and mitochondrial biogenesis, while switching off anabolic pathways that consume ATP (Kahn et al., 2005). The notion of age-related decline in energy status seen in the present study is in line with previous reports on the gradual decline in brain energy metabolism during aging (Navarro and Boveris, 2007, Boveris and Navarro, 2008).

Our data also show increased ADP/ATP ratio in the aged rats suggesting that the ATP-generating capacity of the aging brain is diminished possibly leading to a lower ability to maintain energy supply. The decline in energy metabolism during aging increases the brain's susceptibility to neurodegenerative disorders (Atamna and Frey, 2007, Gibson et al., 2010), and the decreased ability to maintain ATP supply most likely contributes to age-related cognitive impairment since it will be difficult to adapt to the high demands imposed by energy-consuming brain activity such as learning and memory. Indeed, our data show a significant relationship between ADP/ATP ratio and performance on the last day of behavioral testing in the learning and memory task. Age-related decrease in brain energy homeostasis can also potentially lead to the disruption in redox state.

Our data show age-related decrease in redox state shown as (i) increased ROS level, (ii) lower levels of reduced glutathione, increased levels of oxidized glutathione, and decreased GSH/GSSG ratio, and (iii) decreased activity of the SOD enzymatic antioxidant, when compared to the young animals and these data are consistent with the free radical theory of aging (Harman, 1992). The increased ROS level seen in the present study is consistent with previous reports showing age-related upregulation in free radical production resulting in higher susceptibility for oxidative stress (Calabrese et al., 2010, Nunomura et al., 2012, Flynn and Melov, 2013). The beneficial effects of vitamin D on regulating age-related changes in redox homeostasis are shown in our data on decreased ROS level, and increased SOD activity and glutathione levels in the aged rats given the supplementation. SOD as an enzymatic antioxidant is responsible for converting the ROS superoxide radical to hydrogen peroxide so that it can be eliminated by glutathione peroxidase (Hu et al., 2009). Here me measured both the reduced and oxidized forms of glutathione instead of the glutathione enzymatic antioxidant.

Glutathione, a potent antioxidant, is present in the cell in reduced (GSH) and oxidized (GSSG) forms and act as a redox buffer especially in the brain (Dringen and Hirrlinger, 2003). The reduced GSH serves as a substrate for enzymes that scavenge ROS with the concomitant production of oxidized GSSG; and the oxidized GSSG is recycled back to GSH (Dringen and Hamprecht, 1998). Thus, the GSH/GSSG coupling acts to maintain the redox environment of the cell promoting survival. It stands to reason then that the dysregulation of GSH/GSSG homeostasis can potentially contribute to the vulnerability, initiation, and progression of neurodegeneration (Fukui and Moraes, 2008, Johnson et al., 2012).

The notion that the aged brain is vulnerable to the development of neurodegenerative disorder because of disruption in redox homeostasis is supported in this study as our data show that age-related increased in ROS levels, decreased SOD activity, and GSH/GSSG ratio are accompanied by a concomitant increased in the phophorylation of the tau protein. Tau is a major microtubule-associated protein that plays a large role in the outgrowth of neuronal processes and the development of neuronal polarity (Gotz et al., 2013). Tau promotes microtubule assembly and stability, and is abundantly present in the neuronal axons of the central nervous system. However, recent studies show that tau is also expressed in glial cells (Fuster-Matanzo et al., 2012). The phosphorylation of tau regulates microtubule binding and assembly but hyperphosphorylation destabilizes microtubules by decreased binding of tau to microtubules (Hashiguchi and Hashiguchi, 2013), resulting in aggregation of the protein leading to the formation of neurofibrillary tangles (Gotz et al., 2013), a pathological feature in most neurodegenerative diseases, especially Alzheimer's disease. In the present study, we detected significantly increased tau phosphorylation at the pre-tangle (phosphorylation at thr²³¹), intraneuronal tangle (phosphorylation at ser²¹⁴), and extracellular tangle (phosphorylation at ser⁴⁰⁴) sites in the hippocampus of aged rats; and these data are consistent with previous reports on age-related increase in the phosphorylation of the tau protein (Schmitt et al., 2012, Ramcharitar et al., 2013). However, our study is the first to show that providing vitamin D supplementation significantly limited the age-related hyperphosphorylation of tau proteins suggesting a usefulness of this neurohormone in decreasing the risk for neurofibrillary tangle formation.

The mechanism whereby vitamin D regulates age-related changes in tau hyperphosphorylation in this study is most likely related to its ability to modulate PP2A activity. Although the brain has several protein phosphatases, PP2A make up more than 70% of the serine/threonine phosphatase activity in mammalian cells (Liu et al., 2005). The phosphorylation of tau that suppresses its microtubule binding and assembly activities in adult mammalian brain is primarily regulated by PP2A and not by PP2B (Liu et al., 2005, Liu and Gotz, 2013); and PP2A also regulates the activities of several tau kinases in the brain (Liu and Gotz, 2013). Our data on increased tau phosphorylation and decreased PP2A activity is hardly surprising because of reports that show: (1) transient decreased in PP2A activity in the presence of anesthesia and hypothermia induced by anesthesia results in reversible abnormal tau hyperphosphorylation in the normal brain (Planel et al., 2007); and (2) non-transient decreased in PP2A activity in Alzheimer's brain correlates with irreversible abnormal tau hyperphosphorylation (Iqbal et al., 2009).

Collectively, the age-related biological changes in tau, energy metabolism, and redox state seen in the current study is associated with cognitive decline, which is not surprising since studies show that oxidative stress resulting from a decline in energy metabolism and tau hyperphosphorylation coincide with the onset of cognitive impairment (Federico et al., 2012). Our data show that impairment in cognitive function is manifested in tasks that involve both hippocampal and non-hippocampal function. But a key finding in this study is that providing vitamin D supplementation mitigates the age-related behavioral changes. Vitamin D is an important hormone with well-characterized effects in the whole body system in early life as well as in later life (DeLuca et al., 2013). It is highly likely that vitamin D is able to regulate age-related cognitive decline because of its role in: (1) regulating cellular function, and (2) binding to the molecules of the endogenous antioxidant systems resulting in the neutralization of oxygen molecules before they can harm cells as free radicals, and thus protects cells against oxidative stress [reviewed in (Chung et al., 2006)]. Studies show that vitamin D is a cellular regulator via its actions on signal transduction and because of this it is possible that this hormone is able to modulate neuronal function thereby minimizing their energy needs (Eyles et al., 2003, Eyles et al., 2007).

In the past decades, our knowledge about vitamin D and its biological activity has significantly improved. Reports on vitamin D deficiency in the elderly (Coudray et al., 2005, Annweiler et al., 2010, Annweiler et al., 2012) is extended in the present study where we show that aged animals are deficient in the active form of vitamin D, 1, α25-dihydroxyvitamin D3. The age-related deficiency in vitamin D demonstrated here may be due to decrease production and/or increase metabolic clearance rate because of the high levels of 24-hydroxylase, enzyme that metabolize vitamin D, seen in the elderly [reviewed in (Tuohimaa et al., 2009)]. The lack of significant difference in circulating levels of 1, α25-dihydroxyvitamin D3 in the young animals is hard to explain but it may be that the amount of supplement used in the present study is not sufficient to cause an increase in plasma levels in conditions where no deficiency exist.

A surprising finding in this study is that vitamin D supplementation was only able to modulate age-related biological and behavior changes and not totally overcome the effects of age on tau phosphorylation, brain energy metabolism, disruption in redox state, and cognitive function. It is possible that due to the complexity of brain function, there is no single “anti-aging therapy” that truly reverses the effects of the aging process. Although this study does not prove a causal relationship between vitamin D and neurological function or vitamin D and tau hyperphophorylation in aging, the data presented suggest an immense potential for this neurohormone to slow down age-related impairment in cognitive and biological functioning. Given that neurodegeneration and cognitive impairment are likely to play a central role in the health care system and society in the near future, use of vitamin D is an easy, inexpensive, and safe way to modify the risk factors associated with aging.

Highlights for Review.

• Vitamin D regulates age-related changes in the rat brain energy metabolism, redox homeostasis, and phosphatase activity.

• Vitamin D supplementation modulates age-related tau hyperphosphorylation in the rat brain.

• Modulation of brain energy metabolism in rat brains by vitamin D is accompanied by decreased age-related cognitive deficit.