Effect of Memantine Treatment and Combination with Vitamin D Supplementation on Body Composition in the APP/PS1 Mouse Model of Alzheimer’s Disease Following Chronic Vitamin D Deficiency

Abstract

Background:

Vitamin D deficiency and altered body composition are common in Alzheimer’s disease (AD). Memantine with vitamin D supplementation can protect cortical axons against amyloid-β exposure and glutamate toxicity.

Objective:

To study the effects of vitamin D deprivation and subsequent treatment with memantine and vitamin D enrichment on whole-body composition using a mouse model of AD.

Methods:

Male APPswe/PS1dE9 mice were divided into four groups at 2.5 months of age: the control group (n = 14) was fed a standard diet throughout; the remaining mice were started on a vitamin D-deficient diet at month 6. The vitamin D-deficient group (n = 14) remained on the vitamin D-deficient diet for the rest of the study. Of the remaining two groups, one had memantine (n = 14), while the other had both memantine and 10 IU/g vitamin D (n = 14), added to their diet at month 9. Serum 25(OH)D levels measured at months 6, 9, 12, and 15 confirmed vitamin D levels were lower in mice on vitamin D-deficient diets and higher in the vitamin D-supplemented mice. Micro-computed tomography was performed at month 15 to determine whole-body composition.

Results:

In mice deprived of vitamin D, memantine increased bone mineral content (8.7% increase, p < 0.01) and absolute skeletal tissue mass (9.3% increase, p < 0.05) and volume (9.2% increase, p < 0.05) relative to controls. This was not observed when memantine treatment was combined with vitamin D enrichment.

Conclusion:

Combination treatment of vitamin D and memantine had no negative effects on body composition. Future studies should clarify whether vitamin D status impacts the effects of memantine treatment on bone physiology in people with AD.

Keywords

Adipose tissue Alzheimer’s disease animal models body composition bone and bones 3-D imaging memantine muscle vitamin D x-ray micro-CT

INTRODUCTION

Alzheimer’s disease (AD) is a neurodegenerative condition that is characterized pathologically by the development of amyloid-β plaques and neurofibrillary tangles with progressive cognitive decline. AD is the most common cause of dementia in the elderly affecting one-third of individuals over the age of 85 [1]. Weight loss [2 –5], increased body fat [6], decreased muscle mass [6 –8], and decreased bone mineral density (BMD) [6 , 9– 11] are frequently reported in people with AD and these changes have all been associated with cognitive decline [5 , 9]. In addition to changes in body composition, it is estimated that as many as 70– 90% of AD patients have deficient levels of vitamin D [12]. In healthy populations, serum 25(OH)D levels have been correlated with decreased adiposity [11 , 13– 15], increased lean mass [16], and increased BMD [17]. However, it is currently unknown whether vitamin D deficiency exacerbates changes in whole body composition observed in people with AD.

The direct effects of vitamin D supplementation on body composition are also largely unknown. Although low levels of 25(OH)D have been shown to increase body mass index (BMI), there is no evidence that higher levels of 25(OH)D decreases BMI [18]. Conversely, there are many studies that suggest vitamin D has neuroprotective effects in AD. These effects include neurotransmitter regulation [12, 19], prevention of calcium toxicity [12, 19], immune system regulation [12 , 20], induction of amyloid-β clearance [12 , 19– 21], prevention of oxidative stress [19], and neurotrophic factor regulation [12 , 19– 21]. Accordingly, vitamin D deficiency in AD is associated with increased risk of cognitive decline [9 , 22].

Recently, the combination of memantine, an N-methyl-D-aspartic acid (NMDA) receptor antagonist approved for the symptomatic treatment of moderate to severe AD, with vitamin D supplementation has been investigated in the treatment of AD. As a low affinity NMDA receptor (NMDAR) antagonist, memantine primarily targets extra-synaptic NMDARs, which are involved in the regulation of amyloid-β production, while still allowing synaptic NMDARs to maintain physiological synaptic activity [23]. When taken together, memantine and vitamin D have synergistic effects that protect cortical axons against exposure to amyloid-β and glutamate toxicity [24]. The combined effects of memantine and vitamin D have also shown the potential to slow cognitive decline in AD patients compared to memantine or vitamin D alone [25]. Memantine has previously shown the potential to reverse or reduce weight loss in AD patients [26, 27], although other studies have shown that memantine has no such effect [28, 29]. It is currently unknown whether memantine alters body tissue composition in AD; however, recent studies have confirmed that memantine has both central and peripheral effects in alleviating amyloid-β and metabolic pathologies of AD [23, 30]. Based on the associations of memantine and vitamin D with body weight and composition, this combination treatment may also prove effective at mitigating the changes in body composition regularly observed in AD patients.

Transgenic mouse models are frequently used to study the effects of AD pathology, allowing important insight into the mechanisms that underlie disease progression. The severity of pathology varies across mouse models of AD based on the degree of genetic manipulation, with more aggressive models containing more alterations to AD-related genes. The double transgenic APPswe/PS1dE9 model is considered a mild model reflecting changes in the early stages of the disease. Specifically, this model presents with amyloid plaques after the age of six months [31], neuronal loss at nine months [32], and evidence of cognitive impairment by 12 months [33], while neu-rofibrillary tangles are absent. This leads to its designation as a model of preclinical AD, representing the preventative window of approximately 20 years during the time that amyloidosis occurs prior to tauopathy and neurodegeneration [34].

The goal of the current study was to evaluate the effect of combined memantine and vitamin D treatment on body composition measured by micro-computed tomography (micro-CT) in the APPswe/PS1dE9 AD mouse model. Our objectives were to assess the effects of vitamin D deprivation on weight and whole-body composition (specifically, characterization of lean, adipose, and skeletal tissue contributions) in the APPswe/PS1dE9 AD model, and to determine whether treatment with memantine, or memantine combined with vitamin D reversed changes in weight and body composition. We hypothesized that vitamin D deficiency would increase body mass and decrease BMD, while the combination of memantine plus vitamin D supplementation in vitamin D deficient mice would reverse these effects.

MATERIALS AND METHODS

Animal subjects

Sixty male APPswe/PS1dE9 mice were obtained from Jackson Laboratories (JAX MMRC Stock #034829; Bar Harbor, ME, USA) at 2.5 months of age. These mice carried the Swedish mutation of the human amyloid precursor protein (APPswe) and the exon-9 deleted mutation of the human presenilin-1 protein (PS1dE9) on a (C57BL/6 x C3H) F2 genetic background. Mice arrived in cohorts of 5 until a total of 60 mice were obtained. The mice were housed individually in standard tub cages with ad libitum access to food and water, and with a covered shelter as the only form of environmental enrichment. A 12 : 12 h light-dark cycle was maintained, with lights on at 7 am. All experimental procedures were completed between the hours of 9 am and 6 pm. Mice were euthanized at 15 months of age. Ethics approval for this study was obtained from the University of Western Ontario Animal Use Subcommittee (AUP#: 2012-040).

Diet and serum vitamin D levels

A summary of the study design is shown in Fig. 1. From 2.5 to 6 months of age, all 60 mice were fed a standard AIN-76A rodent diet from Research Diets, Inc. (D10001; New Brunswick, NJ, USA). This control diet contained 1000 IU vitamin D₃ per kg. At 6 months of age, the surviving 56 mice were randomized to one of four groups. The control group continued to receive the control diet for the remainder of the study. There were 14 mice in the control group at 6 and 9 months of age, and 13 mice at 12 and 15 months of age (Control, n = 14, 14, 13, 13). The other three groups (n = 42) received a vitamin D-deficient diet from Research Diets, Inc. (D08090903; New Brunswick, NJ, USA) with < 1 IU vitamin D₃ per kg from 6 to 9 months of age. Other than the omission of vitamin D from the diet’s vitamin mix, the vitamin D-deficient diet was identical to the standard AIN-76A diet. The vitamin D-deficient group (VitD-, n = 14, 14, 13, 13) continued to receive the vitamin D-deficient diet for the remainder of the study. Starting at month 9, the remaining two groups were fed 20– 30 mg/kg/day of memantine in addition to either the vitamin D-deficient diet (Mem & VitD-, n = 14, 13, 13, 13), or a vitamin D-enriched diet containing 10,000 IU vitamin D₃ per kg (Mem & VitD+, n = 14, 14, 14, 14). These diets were also provided by Research Diets, Inc. (D16030202 and D16030203, respectively; New Brunswick, NJ, USA). The high dose of vitamin D was based on previous studies that have used 7,500 IU/kg [35] and 12,000 IU/kg [36] in mouse models of AD. At 10×the murine recommended dietary dose, 10,000 IU/kg of vitamin D would result in mice receiving approximately 1100 IU vitamin D/kg of body weight each day, assuming food consumption at 11% of body weight per day [37]. This would be comparable to a daily intake of 6,700 IU/day in humans based on dose conversion information provided in Nair et al. [38]. This vitamin D dose is not only safe but has been postulated as a high dose to achieve neuroprotective effects [39, 40].

Fig. 1

Summary of the study design. VitD-, vitamin D-deficient; Mem, memantine; VitD+, vitamin D-enriched; RIA, radioimmunoassay; Micro-CT, micro-computed tomography. Mice receiving a control diet and memantine supplementation were not included in the current study.

To ensure that the vitamin D levels in the mice were modified by the diet, mice were anaesthetized via isofluorane gas (4% induction, 2% maintenance) and a tail nick procedure was performed for blood draw at months 6, 9, 12, and 15. Blood samples were then sent to Heartland Assays (Ames, IA, USA) for measurement of serum 25(OH)D levels by radioimmunoassay.

Micro-CT imaging

At 15 months of age, the anaesthetized mice were first weighed using a digital balance to obtain their gravimetric weights. Then, they were scanned on a GEHC eXplore Locus Ultra micro-CT imaging system at a peak spectral energy of 80 kVp and tube current of 55 mA. 1000 projections (1024×680 pixels), each 16 ms in duration, were acquired over a 360-degree rotation of the gantry taking approximately 16 s for the acquisition. A Feldkamp, filtered back-projection algorithm was employed to create a 3D data set (512×512×680 voxels) with isotropic 154μm voxel-spacing from the x-ray projections. Internal calibration standards of air, water, and a cortical bone standard (SB3) were included with each scan to ensure that greyscale values (Analog-to-Digital Units, ADU) were calibrated in Hounsfield Units (HU).

Whole body composition of the mice was derived from the reconstructed micro-CT scan data as described by Beaucage et al. [41] and Granton et al. [42]. Suitable grey-scale, signal-intensity values were selected to classify voxels as adipose tissue (AT: from – 380 to – 31 HU), lean tissue (LT: from – 30 to 189 HU), or skeletal tissue (ST: above 190 HU).

Custom software, written in-house, was used to determine the total volume of each tissue type by the summation of the number of voxels in each category. The mass for each of these three tissue types were calculated by multiplying the total volume for each tissue by their respective tissue densities of 0.90 g/cc (AT), 1.05 g/cc (LT), and 1.92 g/cc (ST) [43] and the total CT-derived mouse mass was the sum of these three calculated masses.

For each voxel classified as skeletal tissue, bone mineral content (BMC) was calculated as the fractional content (ratio of skeletal voxel HU to SB3 cortical bone calibrator HU) multiplied by the density equivalent of the SB3 calibrator (1.073 g/cc) [44]. Total BMC is the sum of these BMC values for all skeletal voxels. BMD is the total BMC value divided by the volume of all the skeletal tissue voxels.

Statistical analysis

All statistical analyses were performed using GraphPad Prism Version 8.0 for Mac OS X (GraphPad Software, San Diego, CA). Outliers were identified using the ROUT method [45]. Mice identified as outliers in serum 25(OH)D levels at a specific time point were removed from 25(OH)D analyses at that time point. Mice identified as outliers in whole-body composition measures were also removed from all body composition analyses.

All body composition parameters were assessed for normality via the D’Agostino-Pearson omnibus normality test [46] as well as QQ plots. Body composition measures that were normally distributed were analyzed using one-way ANOVA with treatment (control, VitD-, Mem & VitD-, or Mem & VitD+) as the fixed effect. Body composition measures not normally distributed were instead analyzed using the Kruskal-Wallis test. When the effect of treatment was significant, post-hoc tests were performed to investigate individual differences between treatment groups.

Serum vitamin D levels were analyzed using a mixed-effects model, as implemented in GraphPad Prism 8.0, which fits a compound symmetry covariance matrix using Restricted Maximum Likelihood (REML). In the presence of unbalanced data, as was the case for these measurements, results can be interpreted like repeated measures two-way ANOVA (which is incapable of handling missing values). Age, treatment, and age-by-treatment interaction were considered fixed effects. Homoscedasticity and normality were confirmed using residual plots and QQ plots, respectively. Sphericity was not assumed, so the Geisser-Greenhouse correction was applied [47]. When any fixed effect was found to be significant, post-hoc tests were performed to identify statistically significant differences between time points and treatment groups. In all post-hoc tests, multiplicity adjusted p-values were computed. Significance for all statistical tests was set at p < 0.05.

RESULTS

Serum vitamin D levels

The number of mice included in the 25(OH)D analysis in each treatment group at months 6, 9, 12, and 15, respectively, was as follows: control, n = 14, 14, 13, 13; VitD-, n = 14, 13, 10, 7; Mem & VitD-, n = 14, 12, 13, 13; Mem & VitD+, n = 14, 14, 14, 8 (Supplementary Table 1). Serum 25(OH)D levels from three mice in the Mem & VitD + group were considered outliers using the ROUT method. Statistical analyses of the 25(OH)D data were performed with these three values both included and excluded. There were no differences in the results of the group comparisons using either approach, although p-values did change slightly as expected. It should be noted that serum 25(OH)D measurements were not available from 18 mice randomly distributed between groups and time points because the samples were lost during transit to Heartland Assays. Since this sample loss can be considered “missing completely at random,” these mice were included in all other assessments for which data were available.

The linear mixed model for serum 25(OH)D levels (Fig. 2) revealed a significant effect of age (F(1.87,81.54) = 51.64, p < 0.0001), treatment (F(3,52) = 139.7, p < 0.0001) and age-by-treatment interaction (F(9,131) = 52.96, p < 0.0001). It is important to note that in the analysis of serum 25(OH)D levels, age incorporates changes to the diet that the mice were being fed at that time point. Post-hoc univariate analysis showed that no significant changes in serum 25(OH)D occurred in the control group, which was fed a vitamin D-sufficient control diet throughout the study (F(2.22,25.93) = 2.43, p = 0.10). In contrast, post-hoc univariate analyses revealed that age (and thus diet) did have an effect on the VitD- (F(1.06,9.92) = 170.2, p < 0.0001), Mem & VitD- (F(1.08,17.29) = 309.3, p < 0.0001), and Mem & VitD + (F(1.40,15.43) = 48.59, p < 0.0001) treatment groups. Post-hoc comparison tests revealed that in all three of these groups, the switch from the control diet to the vitamin D-deficient diet at month 6 resulted in significant declines in 25(OH)D levels by month 9 (p < 0.0001 for all three), with an average decrease of 83%. Moreover, 25(OH)D levels continued to decline in those treatment groups that remained on a vitamin D-deficient diet for the remainder of the study (VitD- and Mem & VitD-), as evidenced by the significant differences between month 9 and 12, and between month 9 and 15 in these groups (VitD-: p < 0.01 and p < 0.05, respectively; Mem & VitD-: p < 0.01 for both). On the other hand, the switch from a vitamin D-deficient diet to a vitamin D-enriched diet at month 9 in the Mem & VitD + group resulted in a large increase in serum 25(OH)D, as expected. At months 12 and 15, not only were 25(OH)D levels in this group higher compared to month 9 (p < 0.0001 and p < 0.01, respectively), they were also more than doubled compared to month 6 (p < 0.0001 and p < 0.05, respectively). Therefore, the enriched diet not only recovered the mice from vitamin D deficiency but increased their serum vitamin D levels above the level of the control diet. Overall, these results indicate that serum vitamin D levels of the mice were modified by the diets as intended. Mice on the vitamin D-enriched diet had more than 10×higher serum 25(OH)D levels compared to the mice in the vitamin D-deficient diet.

Fig. 2

Serum 25(OH)D levels: Radioimmunoassay was used to determine the serum 25(OH)D levels present in blood samples of APPswe/PS1dE9 mice at 6, 9, 12 and 15 months of age. These results verify the manipulation of vitamin D levels in the mice through diet. Data are expressed as mean±SEM. Mem, memantine; VitD+, vitamin D-enriched; VitD-, vitamin D-deficient. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001, ^****p < 0.0001.

Whole-body composition

The number of mice used in the analysis of body composition in each group was as follows after removal of outliers: control, n = 13; VitD-, n = 9; Mem & VitD-, n = 10; Mem & VitD+, n = 14. An example micro-CT image that was segmented into lean, adipose, and skeletal tissue is shown in Fig. 3. There were no differences in total mass and volume between mice of different treatment groups (Fig. 4), using the Kruskal-Wallis test (CT-derived mass: H(3) = 4.32, p = 0.23; gravimetric mass: H(3) = 4.56, p = 0.21; total volume: H(3) = 4.17, p = 0.24). The total body mass measured gravimetrically was not significantly different from that measured by micro-CT using one-way ANOVA (F(3,42) = 0.36, p = 0.79), validating the use of micro-CT to make measurements of tissue mass.

Fig. 3

Micro-CT images used for whole-body tissue composition measurement: Whole-body tissue composition of live male APPswe/PS1dE9 mice aged 15 months was obtained from micro-CT images. Tissue was classified as adipose tissue (yellow), lean tissue (red), or skeletal tissue (white) based on previously described image thresholds. The result is shown in a 2D sagittal slice (A), and a rendering of that tissue slice overlaid with a 3D representation of skeletal tissue (B).

Fig. 4

Total mass and volume: The total body mass of APPswe/PS1dE9 mice aged 15 months as measured by weight (A) and micro-CT calculation (B), as well as total body volume calculated by micro-CT (C). Data are expressed as mean±SEM. Mem, memantine; VitD+, vitamin D-enriched; VitD-, vitamin D-deficient.

Lean tissue (LT) mass, LT volume, adipose tissue (AT) mass, AT volume, skeletal tissue (ST) mass, and ST volume determined using micro-CT are shown Fig. 5. Treatment did not have a significant effect on LT mass (F(3,42) = 0.25, p = 0.86), LT volume (H(3) = 4.82, p = 0.19), AT mass (F(3,42) = 0.19, p = 0.90), or AT volume (F(3,42) = 0.18, p = 0.91). However, treatment did have significant effects on ST mass (F(3,42) = 2.90, p < 0.05) and ST volume (F(3,42) = 2.88, p < 0.05). Post-hoc comparison tests revealed that the Mem & VitD- group had significantly higher ST mass (p < 0.05) and ST volume (p < 0.05) compared to the control group. This change was the only significant difference in ST mass and ST volume found between groups.

To account for the size of the mouse, AT, LT, and ST masses and volumes were expressed as percentages of total body mass and volume, respectively (Fig. 6). One-way ANOVA (or Kruskal-Wallis test where appropriate) revealed there were no significant effects of treatment on LT mass (F(3,42) = 0.25, p =0.86), LT volume (F(3,42) = 0.23, p = 0.88), AT mass (F(3,42) = 0.19, p = 0.90), AT volume (F(3,42) =0.20, p = 0.90), ST mass (H(3) = 0.60, p = 0.90) or ST volume (H(3) = 0.54, p = 0.91) when the size of each mouse was considered. This indicates that the proportion of LT, AT, and ST is the same across groups and that the difference in absolute ST mass and ST volume between control and Mem & VitD- mice (Fig. 5) could be due to a small difference in size.

Fig. 5

Whole-body tissue masses and volumes: Individual micro-CT measurements of whole-body lean tissue (LT), adipose tissue (AT) and skeletal tissue (ST) masses (A-C) and volumes (D-F) in APPswe/PS1dE9 mice aged 15 months. Data are expressed as mean±SEM. LT, lean tissue; AT, adipose tissue; ST, skeletal tissue; Mem, memantine; VitD+, vitamin D-enriched; VitD-, vitamin D-deficient. ^*p < 0.05.

Fig. 6

Tissue mass and volume percentages: Individual micro-CT measurements of whole-body lean tissue, adipose tissue and skeletal tissue masses (A-C) and volumes (D-F), expressed as percentages of calculated total body mass and volume, in APPswe/PS1dE9 mice aged 15 months. Data are expressed as mean±SEM. LT, lean tissue; AT, adipose tissue; ST, skeletal tissue; Mem, memantine; VitD+, vitamin D-enriched; VitD-, vitamin D-deficient.

Lastly, the bone mineral content (BMC) and bone mineral density (BMD) of the mice were calculated using the CT-derived ST volume (Fig. 7). One-way ANOVA showed that treatment did not have a statistically significant effect on BMD (F(3,42) = 0.19, p = 0.90), while a Kruskal-Wallis test revealed a significant effect of treatment on BMC (H(3) = 10.86, p < 0.05). Much like the ST results, these results can be explained by slight disparities in size between the control and Mem & VitD- groups, as BMD takes the size of the mouse into account while BMC does not. Post-hoc tests showed a significantly higher BMC in the Mem & VitD- group versus control (p < 0.01). This reflects the fact that the Mem & VitD- group had significantly higher ST volume than controls. Overall, there were no significant differences between treatment groups in any whole-body composition measure when the size of each mouse was taken into consideration. This indicates that neither memantine nor vitamin D had an impact on relative whole-body composition.

Fig. 7

Bone mineral density and content: Micro-CT measurement of whole-body bone mineral density (A) and bone mineral content (B) in APPswe/PS1dE9 mice aged 15 months. Data are expressed as mean±SEM. BMD, bone mineral density; BMC, bone mineral content; Mem, memantine; VitD+, vitamin D-enriched; VitD-, vitamin D-deficient. ^**p < 0.01.

DISCUSSION

This study examined the effects of vitamin D deprivation on whole-body composition in the APPswe/PS1dE9 mouse model of AD as well as subsequent treatment with memantine and vitamin D enrichment. Treatments were administered through diet, which modified serum 25(OH)D levels as intended (Fig. 3); deficient diets caused a decrease in serum vitamin D while the enriched diet caused an increase in serum vitamin D above the level of the control group, whose serum 25(OH)D level did not change throughout the study. In vitamin D-deficient mice, memantine increased absolute skeletal tissue volume and mass, as well as bone mineral content at 15 months. However, neither memantine or vitamin D deficiency or enrichment had an impact on relative lean tissue, adipose tissue, and skeletal tissue whole-body composition.

In the present study, vitamin D deprivation initiated at six months of age had no measurable effect on weight, body composition, or bone mineral density and content at 15 months of age. This result is contrary to our hypothesis that vitamin D deficiency would increase body mass and decrease BMD. The finding that vitamin D deprivation did not cause changes in weight conflicts with epidemiological studies in humans suggesting increased BMI is associated with low levels of vitamin D [13, 18]. In fact, it has been suggested that each 1.15% decrease in 25(OH)D levels is associated with a unit increase of BMI [18]. Furthermore, low vitamin D levels have been associated with increased adiposity and decreased lean tissue [11 , 13– 15] in healthy human populations, but we did not see any differences in adipose or lean tissue in the current study. The results of the current study do agree with research recently conducted in C57BL/6 J mice fed a vitamin D-deficient diet from 5.5– 11 months of age in which no differences were observed in body fat or lean mass compared to mice on a standard diet [48]. Additionally, other work in C57BL/6 J mice fed a vitamin D-deficient diet from 6– 12 months of age saw a decrease in lean and adipose tissue at month 8, but not by month 12 [49]. However, these investigators also noted trends toward lower muscle fiber size and myofibrillar protein content [49]. Neither of these studies reported significant changes in weight.

Overall, the lack of differences in adipose and lean tissue is surprising, given that the vitamin D receptor (VDR) is expressed in adipose tissues and is central to the regulation of energy and glycolipid metabolism [50]. Specifically, VDR is a nuclear receptor that interacts with the vitamin D response element (VDRE) to modify the expression of genes responsible for regulating metabolism [51]. Polymorphisms of the VDR gene have even been linked to obesity [52]. Due to the large decrease in 25(OH)D levels in our vitamin D-deficient groups, this study would seem sufficiently powered to detect any substantial changes in body composition caused by vitamin D deficiency.

The lack of change in BMD in the vitamin D-deprived group may be due to the calcium content of the mouse diet used, which was 0.5%, corresponding to the estimated murine recommended daily intake for calcium [53]. Studies in vitamin D-deficient rats have shown that calcium homeostasis can be maintained with diets containing 0.4% and even 0.1% calcium [54, 55]. This suggests that there may have been enough calcium present in the diet to compensate for any decrease in calcium absorption due to vitamin D deficiency, eliminating the need for increased bone resorption that would reduce BMD. As a result, bone physiology was maintained in the vitamin D-deficient mice.

The addition of memantine, a common treatment of AD, to the diet of vitamin D-deficient APPswe/PS1dE9 mice had no effect on overall mass or volume, lean tissue mass or volume, or adipose tissue mass or volume. These results complement the findings of a recent study that found no difference in weight when comparing untreated APPswe/PS1dE9 mice to those treated with memantine [30]. This previous study examined the mice at 6 months of age, when mice had only been treated with memantine for two months and were not vitamin D deprived. The current study evaluated body composition at 15 months of age, providing an assessment of the long-term effects of treatment with memantine on body composition under the conditions of vitamin D deficiency.

Interestingly, a high co-occurrence of type 2 diabetes and AD has led researchers to hypothesize a type 3 diabetes in which central and peripheral amyloid-β pathologies contribute to both cognitive and metabolic alterations [23]. A recent study investigating the effects of memantine in APPswe/PS1dE9 mice fed with a high-fat diet modelling obesity and type 2 diabetes found that memantine improved peripheral markers of metabolism, including insulin-resistance, while diminishing weight gain [30]. The authors hypothesize that memantine exerts these effects by acting centrally to reduce hypothalamic insulin resistance through decreased amyloid-β levels, and peripherally by increasing pancreatic insulin release through inhibition of pancreatic NMDA receptors. While the present study found no changes in weight, or lean or adipose tissue with the addition of memantine, it would be very intriguing to evaluate the effects of memantine and vitamin D on body composition in APPswe/PS1dE9 fed a high-fat diet similar to the mentioned study. This is of particular interest considering that obesity and adiposity are known to decrease circulating levels of 25(OH)D [56].

In the present study, 15-month-old vitamin D-deficient APPswe/PS1dE9 mice treated with memantine for 6 months showed a significant increase in skeletal (bone) tissue mass and volume. We also observed a significant increase in bone mineral content. The mice in the current study were vitamin D-deficient before memantine administration, to model the 70– 90% of AD patients who have deficient levels of vitamin D [12]. Previous studies in humans found that memantine had either no effect on weight in AD patients [28, 29], or showed increased weight [26, 27].

When the memantine treatment was supplemented with vitamin D, serum 25(OH)D level increased substantially, and this eliminated the increase in skeletal tissue observed in the memantine treated group. Specifically, the combination treatment of memantine with vitamin D enrichment maintained skeletal tissue mass and volume at the same levels as that observed in the mice with the normal diet and the vitamin D deprived diet. Additionally, these mice showed no changes in lean tissue or adipose tissue mass and volume. To our knowledge, this is the first study to investigate the combined effects of vitamin D and memantine on whole-body composition in an AD mouse model.

The high dose of vitamin D₃ used in this study (10,000 IU/kg of diet) was chosen based on previous studies in AD mouse models that used diets containing 7500 IU/kg (5xFAD mouse model) [35] and 12,000 IU/kg (APPswe/PS1dE9 mice) [36] of vitamin D₃. In particular, the study by Yu et al. in APPswe/PS1dE9 mice found that high dose vitamin D enrichment of 12,000 IU/kg correlated with a reduction in amyloid plaques, amyloid-β peptides and inflammation, and an increase in nerve growth factor [36]. Similarly, Morello, et al. found the 7500 IU/kg vitamin D dosage led to improved working memory and neurogenesis in 5xFAD mice when delivered in the early stage of the disease [35]. In the present study, a high dose of 10,000 IU/kg vitamin D in the diet led to 2.3×higher serum 25(OH)D levels in APPswe/PS1dE9 mice relative to those on a standard diet containing 1000 IU/kg. This dose would be comparable to humans receiving 6,700 IU/day [37, 38], which research suggests may be required to achieve neuroprotective attributes through antioxidative mechanisms, neuronal calcium regulation, and immunomodulation [40]. These high doses have been recently tested in a clinical trial in humans to improve cognition and demonstrated to be safe [39].

When normalized to total body mass and volume, no differences were observed in lean tissue, adipose tissue, or skeletal tissue in any group. Similarly, no differences were observed in bone mineral density between groups. However, these results are not surprising as the normalization process utilized total body mass or volume, which is not an independent variable since total body mass and volume include skeletal tissue.

It is particularly interesting that an increase in skeletal tissue was observed with the addition of memantine in vitamin D-deprived mice but not in mice receiving vitamin D-enrichment. This observation suggests a differential effect of memantine on bone based on vitamin D status. Given that NMDARs are present in bone [57], NMDAR antagonists such as memantine have previously been suggested for their potential benefits in peripheral conditions such as osteoporosis [58]. Although conflicting reports exist in the literature, most agree that inhibition of NMDAR will lead to decreased bone resorption, either through direct effects on osteoclast activity or indirect regulation of osteoclast differentiation or apoptosis [57]. Additionally, NMDAR activation is known to have effects on vitamin D synthesis and levels of parathyroid hormone, a key hormone in bone resorption and remodeling [57]. Specifically, prolonged activation of NMDAR can cause decreased blood levels of 1,25(OH)₂D₃— an effect attributed to a decrease in 1α-hydroxylase, the enzyme responsible for conversion of 25(OH)D₃ into 1,25(OH)₂D₃ [59]. This decreases negative feedback to the parathyroid gland and increases levels of parathyroid hormone and bone remodeling markers [59]. However, it is not yet known whether the opposite effect would occur, specifically that NMDAR inhibition would result in increased vitamin D synthesis and decreased parathyroid hormone levels.

Overall, it is unclear why memantine caused an increase in skeletal tissue under the condition of vitamin D deprivation but not vitamin D enrichment. One plausible explanation for our findings combines the effects of vitamin D on calcium homeostasis [60, 61] along with the idea that NMDAR inhibition causes decreased bone resorption [57] (Supplementary Figure 1). In control mice, the amount of bone resorption by osteoclasts is in balance with bone mineralization by osteoblasts, resulting in maintenance of skeletal tissue. In the vitamin D-deprived mice, as discussed earlier, the amount of dietary calcium was likely high enough to maintain blood calcium levels despite vitamin D deficiency [54, 55], resulting in normal levels of parathyroid hormone and osteoclast activity. Consequently, skeletal tissue was maintained in the vitamin D-deprived mice. In this physiological state, adding an NMDAR antagonist such as memantine could have caused decreased osteoclast activity and overall bone resorption [57], explaining the gain in skeletal tissue and bone mineral content observed in the vitamin D-deprived mice treated with memantine. On the other hand, in the mice treated with vitamin D supplementation as well as memantine, we propose that the vitamin D induced an increase in bone resorption via maturation of preosteoclasts into osteoclasts [60] that balanced the memantine-induced reduction in bone resorption such that skeletal tissue was maintained at the same level as controls.

This explanation is speculative, and the present study cannot speak to its validity as blood calcium, parathyroid hormone, and phosphorous levels were not measured. Additionally, measurements of plasmatic levels of S-adenosylmethionine, S-adeno-sylhomocysteine, and homocysteine as indicators of the homocysteine-methionine (HM) cycle would improve the interpretability of results. The HM cycle plays a key role in metabolism; impairment in its regulation has been identified as a risk factor for dementia [62]. In a model of experimental autoimmune encephalomyelitis, vitamin D has been shown to modify the HM cycle through upregulation of betaine-homocysteine methyltransferase-1 (BHMT1) [63], an enzyme responsible for recycling toxic homocysteine into methionine [56]. Future studies should be aimed at clarifying the relationship between NMDAR inhibition, vitamin D, and bone physiology— particularly with memantine as the NMDAR antagonist— as it may have important implications for the use of memantine in the treatment of AD patients who are often vitamin D deficient.

Another possible explanation may relate to the improvement in mobility that is known to occur under memantine administration [64]. Movements with contact to the ground are osteogenic, so improved motor abilities may also contribute to increased skeletal tissue in mice treated with memantine. Future evaluation of longitudinal gait data for the mice included in the present study could provide further clarity regarding the contribution of this mechanism to skeletal tissue gain.

There are several limitations to this study that should be considered when interpreting the results, the first being that whole-body composition was only measured at a single time point. It is therefore unknown whether the treatment scheme caused short-term changes in body composition in the APPswe/PS1dE9 mice that reflect those observed in human AD patients. Although there is known to be amyloid accumulation in the APPswe/PS1dE9 model by 15 months, this time point may represent a very early phase of human disease. Future work should take a longitudinal approach to investigate these possible changes and follow mice beyond 15 months. The current study was also limited to a single mouse model of AD. Other mouse models, particularly those exhibiting both amyloid and tau pathology should be considered for future studies. Finally, transgenic mouse models like APPswe/PS1dE9 contain genetic mutations that are associated with familial AD. Therefore, caution must be taken when generalizing these findings to sporadic AD.

CONCLUSIONS

Vitamin D deprivation in APPswe/PS1dE9 mice did not alter lean tissue, adipose tissue, or skeletal tissue body composition. In APPswe/PS1dE9 mice that were deprived of vitamin D, memantine increased absolute skeletal tissue volume and mass, as well as bone mineral content compared to those on a regular diet at 15 months. However, memantine did not cause the same increase in skeletal tissue in mice that were vitamin D-enriched. Neither memantine nor vitamin D treatment had an impact on whole-body composition when the total mass or volume of each mouse was taken into consideration. Therefore, we conclude that memantine treatment in combination with vitamin D enrichment caused no detrimental effects related to body composition in the APPswe/PS1dE9 mouse model of AD.

Footnotes

ACKNOWLEDGMENTS

This study was funded by the Schulich School of Medicine and Dentistry, University of Western Ontario, Canada, the Canadian Institutes of Health Research (MD/PhD Studentship, Foundation Grant FDN 148474), and the Research Centre on Autonomy and Longevity, University Hospital of Angers, France.

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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