Effect of Vitamin D Supplementation on the Progression of Alzheimer’s Disease in Rats: A Mechanistic Approach

Purpose: A multifaceted treatment approach can be effective for Alzheimer's disease (AD). However, currently, it involves only symptomatic treatment with cholinergic drugs. Benecial effects of high vitamin D levels or its intake in the prevention and treatment of cognitive disorders have been reported. Thus, the present study examined the preventive effect of vitamin D supplementation on AD progression and evaluated its impact on the accumulation or degradation of Aβ plaques. Methods: A single intraperitoneal injection of scopolamine was used to induce AD in rats. Treatment of vitamin D was provided for 21 days after the injection. Various behavioral parameters like learning, spatial memory and exploratory behavior, biochemical alterations in the brain homogenate and histology of the hippocampus were investigated. Results: Our results indicated that scopolamine-induced rats depicted cognitive decits with high Aβ levels and hyperphosphorylated tau proteins in the brain tissue, while vitamin D supplementation could signicantly improve the cognitive status and lower these protein levels. These results were supported by the histopathological and immunohistochemical staining of the hippocampal brain region. Furthermore, mechanistic analysis depicted that vitamin D supplementation improved the Aβ protein clearance by increasing the neprilysin levels. It also reduced the accumulation of Aβ plaques by lowering neuroinammation as well as oxidative stress. Conclusion: The present ndings indicate that vitamin D supplementation can delay AD progression by an increase in Aβ plaques degradation or reducing inammation and oxidative stress.


Introduction
The reach of concern for Alzheimer's disease (AD) spans the globe as the most prevalent form of senile dementia. Currently, 50 million people live with dementia, of which 60-70% are suffering from AD. In 2050, it is predicted to increase to 152 million (WHO 2020). The pathological hallmarks of AD comprise amyloid (Aβ) plaque deposition, hyperphosphorylated Tau (p-Tau), and an abundance of in ammatory markers in the cerebral neurons. All these lead to neuronal injury. Besides, cholinergic de cits, synaptic loss, and oxidative stress are also seen in the neuronal tissue of AD patients leading to neurodegeneration (Grimm et al. 2017;Chai et al. 2019). The key player, Aβ plaques, are formed initially from the degradation of amyloid precursor protein (APP). The enzymes responsible for it includes β-secretase 1 (BACE1) and gsecretase (Durk et al. 2014; Grimm et al. 2017). The degradation products include Aβ  or Aβ 1-40 , of which Aβ 1-42 is more prone to plaques' formation (Durk et al. 2014). These plaques are further cleared via neprilysin (NEP) and insulin-degrading enzyme (IDE) (Grimm et al. 2014; Grimm et al. 2017).
A multimodal treatment approach for AD is effective; however, it currently includes only drugs affecting the pathways of cholinergic systems. Also, none of the current therapies can delay or prevent the progress of AD. Interestingly, preventive strategies (dietary and lifestyle habits) can effectively curb the growth rate and hinder disease progression ( such as decreased BACE1 protein levels leading to reduced Aβ formation or upregulating the gene expression of degrading enzymes (NEP or IDE) leading to Aβ degradation (Grimm et al. 2017). Further, it also reduces senile p-Tau deposition and thus intercepts neuronal death (Grimm et al. 2014). Vitamin D also promotes hippocampal neurodevelopment by upregulating the gene expression of proteins involved in new synaptic genesis (Latimer et al. 2014). Thus, this study examined the effect of vitamin D supplementation on the accumulation or degradation of Aβ plaques in Alzheimer's disease using a scopolamine-induced AD rat model. Further, the impact of long-term administration of vitamin D on the cognitive function of the AD rats was also evaluated.

Animals
Healthy male albino Wistar rats weighing 250-300 gm were used for the experimental study. They were housed in the Institute vivarium (22ºC ± 1ºC and relative humidity 55%-75%) under 12-h:12-h dark/light cycle and were provided with rodent chow and water ad libitum. The behavioral studies were carried out in quiet lab and the animals were transported everyday 30 min prior to the commencement of the evaluation to provide time for acclimatization. All the animals were handled with gentle care to avoid any stressful impact on their behavior. The experimental protocol was approved by the Institutional Ethics Pharmaceuticals Pvt. Ltd., India and was freshly prepared in physiological saline before administration.
Calcitriol (vitamin D 3 ) was a kind gift from Sun Pharmaceuticals Industries Ltd., India.

Experimental protocol and treatment groups
The rats were randomized into 5 groups (6 animals/group) as follows: Group I (NC) received normal saline solution and served as normal control, while Group II (DC) received scopolamine and served as disease control. Both the groups received normal saline orally for 21 days (8 th -28 th day). Groups III (CAL 2.5) and IV (CAL 5) received vitamin D (2.5 and 5 µg/kg/day, respectively) while Group V (DPZ) received donepezil (5 mg/kg/day) for 21 days (8 th -28 th days), orally. Groups II-V received scopolamine (2 mg/kg) as a single dose, intraperitoneally (ip), on the 8 th day.

Neurobehavioral evaluation
The animals were tested for neurobehavioral tests such as Morris water maze (MWM), Modi ed Y-maze, and fear conditioned avoidance tests. The animals were subjected to these behavioral paradigms during the rst week for learning and memory. The tests were repeated post 30 min of the scopolamine injection on the 15 th day and 28 th day (end of the study period) (Fig 1).

Morris water maze test:
The MWM tests assessed spatial memory and learning. It mainly assesses the ability of the animals to navigate a submerged, hidden platform from a start location in an open circular tank. The tailor-made circular tank (110 x 60 cm) was designed for the rats and divided into 4 equal-sized quadrants (N, E, W, and S). The water was lled up to a depth of 35 cm and made opaque using apple green food grade color. A hidden platform (30 cm in height) was submerged 2 cm below the water level in the SW quadrant. Brie y, animals were initially trained for four days (3 trials each day) to navigate a hidden submerged platform in a circular tank using a random set of start locations. The experiment was performed in a dark and silent room. The time taken to locate the platform was termed escape latency time (ELT). The mean ELT was recorded for each animal in all the groups. They were allocated 60 sec to locate the platform in the target quadrant. If the animal failed, it was returned onto the start platform and made to repeat the test after 20 sec. Finally, without any escape platform, the probe trial was conducted on the 5 th day, and provided 120 sec to locate the platform. The time spent in the target quadrant was measured and was

Modi ed Y-maze test
This test evaluated short-term spatial memory and exploratory behavior. The test mainly depends on the natural ability of the rats (exploring the novel environment). The modi ed Y-maze consisted of 3 arms (A, B and C) 40 cm long, 3 cm wide, and 18 cm high. The surface of all the arms was covered with corn husk Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js to prevent any anxious stimuli to rats. During the training session, rats explored the two arms (A and B) for 15 min while the C arm (novel arm) was blocked. After an interval of 1-2 hr, another session was conducted. All the arms were kept open during this session, and the rats were given 5 min to explore them. The number of entries in the novel arm C and the time spent in the novel arm C were recorded (Conrad et al. 1997).

Fear Conditioned Avoidance Test:
This test evaluated associative learning behavior in rats. It was conducted using a transparent box (30 x 30 x 30 cm) with an electric oor framework made of stainless steel. A sound stimulus was provided to deliver foot shocks. Brie y, the experiment was conducted in three phases: familiarization, learning, and retention. The animals were initially placed, individually, on the electric grid and allowed to explore for 4 min. Later, a sound stimulus (conditioned stimulus, 2.9 kHz, 20 sec, 80 dB) followed by a foot shock (0.8 mA, 2 sec) was given. This stimulus was repeated in 5 trials conducted at a time interval of 1 min. The retention test was conducted after 24 hours. The animals were placed on the grid again, and the freezing behavior was recorded for 3 min in the absence of sound and shock (contextual recording). After 3 min, the conditioned stimulus (sound stimulus) was provided, and freezing behavior was again recorded for 2 min (Curzon et al. 2009). The absence of any movements during the observation period except respiration was de ned as freezing behavior. The total freezing time of each animal during the contextual recording and under the fear conditioning was recorded in sec.

Biochemical parameters
The animals were euthanized by cervical dislocation after the completion of the study period. Brain samples were collected followed by decapitation and washed with ice-cold saline. The samples were divided into two halves for biochemical analysis and histopathological as well as immunohistochemical analysis. For biochemical analysis, the weight of isolated brain tissues was measured, followed by homogenization with ice-cold phosphate buffer saline (pH 7.4, 0.1 M) and centrifugation at 10,000 rpm for 15 min. The supernatant was used for biochemical estimations. The samples for histopathological and immunohistochemical analysis were preserved in 4% formaldehyde at 4℃.

Protein Estimation
The protein content of the brain tissue was measured by Lowry's method using bovine serum albumin as standard (1 mg/mL) (Lowry et al. 1951). The results were stated as tissue concentration/mg protein.

Estimation of enzyme activity
The Acetylcholine-esterase (AChE) inhibitory activity was analyzed in the supernatant using the Ellman method (Ellman et al. 1961) and was expressed as enzyme unit/mg protein. The BACE-1 activity from brain homogenate was analyzed using a β-secretase uorometric assay kit (Biovision, California, USA) according to the instructions provided by the manufacturer. It was expressed as relative uorescence units (RFU).
Estimation of oxidative stress parameters The brain lipid peroxidation was determined by measuring malondialdehyde (MDA) levels (expressed as nmol/mg protein) using thiobarbituric acid reacting substances (Ohkawa et al. 1979). Reduced glutathione (GSH, expressed as nmol/mg protein) (Jollow et al. 1974) and superoxide dismutase (SOD, expressed as IU/mg protein) activity (Sun and Zigman 1978) were measured in the brain homogenate.

Histopathological and immunohistochemical analysis
The standard procedure was followed for H&E staining of the tissue. The brain tissue was xed, dehydrated, impregnated, and embedded in para n for sectioning. Further, the sections were stained with hematoxylin and eosin. For immunohistochemical detection, the hippocampus was observed after xation in 4% formaldehyde. Later, the para n-embedded tissue was sectioned (5 µM) and immunohistochemical staining was performed as per manufacturer's instructions, using antibodies against synaptophysin (Krishgen Biosystems, India). The stained sections of both the analysis were studied and captured with a camera that was connected to an inverted light microscope (Olympus CKX41, Japan, 400x).

Statistical analysis
Results are expressed as mean ± standard error of mean (SEM). Statistical difference between the groups was analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni's posthoc test using GraphPad Prism® software (version 5.01, California, USA). The data were considered statistically signi cant at a p-value of <0.05.

Results
Effect of vitamin D supplementation on the neurobehavioral performance of rats in MWM The mean ELT was signi cantly increased (p<0.001) in the DC rats post 21 days of scopolamine injection compared to normal controls. The same was signi cantly reduced (p<0.01) by the treatment with vitamin D (5 µg/kg/day) as compared to DC rats. Insigni cant differences were found for vitamin D (2.5 µg/kg/day) and donepezil (5 mg/kg/day) (Fig 2a). A similar bene cial effect was observed for the mean time spent in the target quadrant by the scopolamine-induced AD rats treated with vitamin D (5 µg/kg/day, p<0.01) in the MWM test as compared to DC rats, while insigni cant effect was observed with the low dose (2.5 Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js µg/kg/day) (Fig 2b). This result showed an improved retrieval index in the rats supplemented with vitamin D (Fig 2).

Effect of vitamin D supplementation on the spontaneous alternation behavior of rats in modi ed Y-maze test
In the modi ed Y-maze test, scopolamine injection in the DC rats signi cantly decreased the number of entries (p<0.001) as well as the time spent in the novel arm C (p<0.01) as compared to the NC rats.
Treatment with vitamin D (5 µg/kg/day) was able to signi cantly increase the number of entries (p<0.001) (Fig 3a); while the time spent in the novel arm was improved (p<0.05) by vitamin D (5 µg/kg/day) supplementation as well as donepezil (5 mg/kg/day) (Fig 3b) compared to the DC rats. However, vitamin D supplementation at the dose of 2.5 µg/kg/day could not show the bene cial effects in the Y-maze test (Fig 3).
Effect of vitamin D supplementation on the associative learning behavior of rats in fear-conditioned avoidance test The present ndings indicated a signi cant reduction (p<0.001) of the freezing time in the contextual and conditioned avoidance test after scopolamine administration on the 21 st day in the DC rats compared to the NC rats. The calcitriol supplementation at both doses (2.5 and 5 µg/kg/day, p<0.001) as well as donepezil (5 mg/kg/day, p<0.001) led to signi cant improvement of the freezing time in the conditioned avoidance tests (Fig 4a). Additionally, a signi cant improvement of the freezing time in the contextual test was observed with only 5 µg/kg/day calcitriol (p<0.001) and donepezil (p<0.001) (Fig 4b).

Effect of vitamin D on biochemical parameters
After scopolamine injection, the AChE activity in the brain homogenate was signi cantly elevated (p<0.001) due to AD induction in DC rats compared to NC rats. Vitamin D (2.5 µg/kg/day, p<0.01), vitamin D (5 µg/kg/day, p<0.001) and donepezil (5 mg/kg/day) supplementation were able to signi cantly reduce the elevated AChE activity as compared to DC rats ( Table 1).

Effect of vitamin D on AD markers
Since Aβ proteins are majorly responsible for the formation of plaques in AD, we evaluated both the Aβ proteins formed by the activity of BACE1 and g-secretase, i.e., Aβ 1-42 and Aβ 1-40 , respectively. The results in Fig 5a and 5b depicted that Aβ 1-42 and Aβ 1-40 were signi cantly elevated (p<0.001) due to AD induction by scopolamine compared to the normal rats. Vitamin D (5 µg/kg/day) and donepezil (5 mg/kg/day) treatment were able to reduce signi cantly (p<0.001) the elevated Aβ 1-42 (Fig 5a) and Aβ   (Fig 5b) in AD rats. In contrast, supplementation of vitamin D in low dose (2.5 µg/kg/day) was able to signi cantly lower only Aβ 1-42 (Fig 5a; p<0.001) and not Aβ 1-40 (Fig 5b) .
In the same context, the present study indicated that the mean values of p-Tau proteins in the brain homogenate signi cantly increased (p<0.001) in the DC rats compared to the NC rats. Administration of calcitriol (2.5 and 5 µg/kg/day) and donepezil (5 mg/kg/day) in the AD-induced rats signi cantly reduced (p<0.001) the elevated levels of p-Tau proteins compared to the DC rats (Fig 5c).
In line with the above parameters, the enzymes such as BACE1, NEP, and IDE, were also evaluated. A surge in BACE1 activity (p<0.05) in the brain homogenate was found in the DC rats compared to NC rats. Signi cant improvement was observed in BACE1 activity via treatment with vitamin D at doses 2.5 µg/kg/day (p<0.05) and 5 µg/kg/day (p<0.001) as well as donepezil (5 mg/kg/day, p<0.01) (Fig 5d). The IDE levels were signi cantly reduced (p<0.001) in DC rats compared to the normal rats. However, insigni cant differences were found between vitamin D (both doses), or donepezil treated rats and DC rats (Fig 5e). In contrast, the NEP levels were signi cantly improved via treatment with vitamin D (2.5 µg/kg/day, p<0.01), vitamin D (5 µg/kg/day, p<0.001), and donepezil (5 mg/kg/day, p<0.001) compared to the DC rats (Fig 5f). Thus, a shift in the proteolytic processing of the APP in the amyloidogenic pathway was observed by vitamin D supplementation at a high dose after collating the results of all AD markers.

Effect of vitamin D on oxidative stress markers
The levels of MDA depict the extent of lipid peroxidation in the brain, which was signi cantly elevated (p<0.001) in the scopolamine-induced DC rats compared to the normal rats. Additionally, GSH was found to be signi cantly elevated (p<0.001). At the same time, SOD was reduced signi cantly (p<0.001) in the brain homogenates of DC rats compared to the normal rats. Treatment with vitamin D (both doses) signi cantly improved the lipid peroxidation (p<0.001) and reduced glutathione levels (p<0.001) in the brain tissue compared to the DC rats. The levels of SOD were found to be signi cantly reduced by administration of vitamin D only at a higher dose (5 µg/kg/day) as compared to the DC rats ( Table 1).

Effect of vitamin D on histology
Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js The qualitative histopathological observations of the hippocampal sections of DC rats depicted the loss of cell density and pyramidal cell structure compared to the NC rats. The intercellular spaces were found to be increased due to cell loss, which led to the loss of the dense neuronal network in the hippocampus of AD-induced rats following scopolamine injection post 21 days. Further, loss of cognitive function was derived from the collapse of the three primary functional layers of dentate gyrus correlated with progressive memory decline. Supplementation of vitamin D for 21 days prevented the degradation in the cellular density and the functional layers of the dentate gyrus, thus preventing cognitive loss. However, the higher dose was more effective qualitatively than the lower dose of vitamin D (2.5 µg/kg/day). The standard drug, donepezil, restored the hippocampal cellular density, but the bene cial effect on the dentate gyrus function was not prominent (Fig 7).

Effect of vitamin D on immunohistochemical analysis
The immunohistochemical staining of the rat hippocampal sections for synaptophysin indicated that the hippocampal cells in the normal rats appeared with light staining and lean processes. In contrast, the DC rats, treated with scopolamine, showed thick processes and densely stained bodies. This indicated loss of synaptic plasticity in the DC rats. The rats treated with vitamin D (both doses) and donepezil (5 mg/kg/day) for 21 days showed a reduction in the stain density compared to DC rats. This reduction could be inferred as restoring functional neuronal cells indicating neurogenesis in the hippocampus (Fig  8).

Discussion
The present study demonstrates a potential bene cial role of vitamin D supplementation in delaying AD progression in rats due to improvements in the formation or clearance of amyloid plaques, in ammation, and oxidative stress. The scopolamine-induced amnesia model for induction of AD induction in rats was employed in the study. Scopolamine gains access to the brain via blood-brain barrier and causes an increase in AChE levels. It also increases oxidative stress and neuroin ammation in the rat brain, nally leading to cognitive decline and developing AD-like symptoms ( Further, the fear-conditioned avoidance test was conducted to evaluate emotional memory and associative learning. Fear conditioning response is a hippocampus and amygdala dependent memory function. Loss of functional connectivity between the hippocampus and amygdala may be responsible for memory loss in the early stages of AD AD (Hamann et al. 2002;Varinthra et al. 2021). The rats injected with scopolamine indicated a reduction in fear memory retrieval (decrease in freezing time), while treatment with vitamin D or donepezil signi cantly improved the contextual as well as conditioned fear memory retrieval. This improvement may indicate the ability of vitamin D to restore the synaptic plasticity between the hippocampus and amygdala, as observed earlier in an experimental model of AD (Durk et al. 2014). Moreover, the loss of fear conditioning memory is associated with the burden of Aβ plaque deposition (Hanna et al. 2012), and improvement in this memory retrieval may indicate a decrease in Aβ plaque deposition.
Further, the results of the neurobehavioral test corroborate with the loss of synaptophysin staining in the hippocampal cells during immunohistochemistry analysis. Synaptophysin is a presynaptic vesicle protein in the hippocampus, basal ganglia, and cortex, used as a biomarker for synaptic plasticity. A decrease in this protein is associated with the cognitive de cit in AD patients (Hajjar et al. 2013;Zufferey et al. 2013). Vitamin D supplementation depicted a dose-dependent protective effect observed as more viable neuronal cells than DC rats (Fig 8). In a nutshell, these results provide proof for improving cognitive behavior and effective memory consolidation in scopolamine-induced AD rats by supplementing vitamin D.
The pathological hallmarks of AD, i.e., extracellular Aβ plaque formation and intracellular p-Tau resulting in neuro brillary tangles, are held responsible for the cognitive decline, including memory loss (spatial, episodic, emotional, recognition) as well as signi cant in ammatory response (Wang et al. 2015). The balance between the formation (BACE1 and g-secretase) and degradation (IDE and NEP) of Aβ proteins maintains the Aβ levels in the brain (Hafez et al. 2017). In the current study, the bene cial effects of vitamin D supplementation (5 µg/kg/day) and donepezil (5 mg/kg/day) were observed in reducing the disease burden. Previous studies reporting the lowering of Aβ proteins due to vitamin D supplementation Additional anti-in ammatory mechanisms of vitamin D may be responsible for the decrease in Aβ protein accumulation and improvement in cognitive functions in AD rats. Accumulation of Aβ plaques and p-tau proteins can activate a cascade of neuroin ammation including proin ammatory mediators like IL-1β, IL-6, TNF-α, or IFN-g from microglial cells. These proin ammatory cytokines can mediate neurotoxicity leading to AD (Wang et al. 2015). These mediators can also reduce the endocytosis and phagocytosis of Aβ proteins by the microglial cells (Varinthra et al. 2021). Further, NF-κβ is reported to be important in maintaining learning, memory as well as synaptic plasticity. Activation of NF-κβ signaling can stimulate the BACE1 activity and thus increase the Aβ protein accumulation (Jha et al. 2019). In addition, IFN-g is reported to be signi cantly high in AD patients (Belkhelfa et al. 2014) that can result in increased hyperphosphorylation of soluble tau proteins (Zheng et al. 2016). In the current study, supplementation of vitamin D in AD-induced rats demonstrated signi cant anti-in ammatory activity in a dose-dependent manner. Our results are consistent with another study showing a decrease in in ammatory mediators (IL-1β, IL-6, NF-κβ) in the hippocampal regions of the brain due to vitamin D supplementation (Farhangi et al. 2017). The decrease in the in ammatory mediators may also inhibit the over-activation of hippocampal cells and thus can reduce the accumulation of Aβ plaques (Varinthra et al. 2021).
Oxidative stress is a characteristic feature of AD brains along with Aβ plaques and neuro brillary tangles (Zhao and Zhao 2013). Aβ peptide is associated with the generation of reactive oxygen species that can further lead to aggregation and plaque formation (Ahmad et al. 2017). Lipid peroxidation is signi cantly high in several brain regions in AD. Additionally, glutathione and antioxidant enzymes like SOD are also reduced in AD brains. Together, the raised oxidative status may result in neuronal damage and thus, neuronal death as well as synaptic loss (      Alzheimer's disease induced rats treated with vitamin D (5 µg/kg/day, po); DPZ: Alzheimer's disease induced rats treated with donepezil (5 mg/kg/day, po). Data expressed as means ± SEM (n=6 animals per group). *-p<0.05, **-p<0.01, ***-p<0.001 compared to DC rats; $ -p<0.05, − p < 0.01, $ -p<0.001 vs. NC rats.

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