Neuroprotective Role of Tocopherol Derivatives in Alzheimer’s Disease: Behavioral and Biochemical Analysis

doi:10.21203/rs.3.rs-5104038/v1

Alzheimer’s Disease (AD) is a terminal disease that results from progressive loss of neurons in the brain leading to cognitive dysfunction, memory loss, and neuroinflammation. The purpose of this investigation was to establish the neurotrophic profile of a newly synthesized naphthalene tocopherol competitive to Vitamin E acetate in an AD mouse model. The learning and memory were examined by the following behavioral paradigms: Fear Conditioning Test, Barnes Maze Test, Elevated Plus Maze Test and Morris Water Maze Test. Immunohistochemical observations were made to assess markers of oxidative stress using MDA, SOD, and GSH and neuroinflammation using IL-6 and TNF-α. The effectiveness was evidenced through the reduction of oxidation stress, reducing cytokines that cause inflammation, and overall enhancements on cognitive functioning among the groups administered with the tocopherol derivative at the dosages of 20 mg/kg and 40 mg/kg. These findings indicate that tocopherol derivatives could indeed have a more robust neuroprotective activity than the standard Vitamin E with an application in the treatment of AD. Carrying out similar experiments in human would be another approach to confirm these findings as well as determine the molecular basis of their effects.

Neurobiology of Disease

Cellular & Molecular Neuroscience

Clinical Pharmacology

Drug Discovery, Design, & Development

Alzheimer’s disease

tocopherol derivatives

cognitive function

oxidative stress

IL-6

TNF-α

neuroinflammation

Alzheimer’s Disease (AD) is a chronic, progressive neurological condition that impacts mainly individuals of advanced age and accounts for a significant proportion of dementia cases globally (Jansen et al., 2015). Alzheimer’s is the most common form of dementia; the World Health Organization (WHO) reports that about 50 million people worldwide live with dementia and 60-70% of them have Alzheimer’s (WHO, 2020). AD has also been attributed to increase exponentially with aging populations thereby posing significant social, economic and health costs (Prince et al., 2015). Thus, application of effective therapeutic approaches aimed at intervention prevention or delayed progression of AD have emerged as an important area of study.

AD is defined clinically by progressive cognitive impairment and pathologically by the presence of extracellular amyloid-beta (Aβ) plaques and intracellular neurofibrillary tangles derived from hyperphosphorylated tau protein in the brain, which are linked to synaptic decay, inflammation in the brain and cognitive dysfunction (Jin et al., 2011). These pathological changes are believed to interfere with the neuronal transmission, abnormality in the synaptic plasticity and ultimately leads to neuronal loss (Serrano-Pozo et al., 2011). The entire processes and factors which contribute to the disease pathology of AD remain not fully understood because they are influenced by both genetic and non-genetic factors as has been suggested (Cai et al., 1993). Several theories have been put forward to understand the development and pattern of AD including oxidative stress, inflammation and deranged calcium homeostasis (Busse et al., 2014; Maccioni et al., 2010).

Perhaps the most promising theory in the cause of AD is the one known as the oxidative stress, and this is based on the premise that excessive production of what is known as ROS and the ensuing inability of the brain to metabolize these toxic compounds leads to neuronal damage (Perry et al., 2001). This organ is highly prone to oxidative stress by virtue of its high metabolic rate for oxygen and its rich content in lipids, thus making it sensitive to lipid peroxidation as well as protein oxidation (Mosconi et al., 2008). In AD, oxidative stress has been presumed to enhance deposition of Aβ plaques and tau tangles that in turn cause neuronal damage (Smith et al., 1998). Consequently, modulation of oxidative stress has become one of the modern approaches for combating Alzheimer’s disease.

Recent focus has also been on Vitamin E (Vit E), a lipid soluble antioxidant, in the prevention of AD. There are several types of it; They come in tocopherols and tocotrienols where alpha-tocopherol is the most active one within human beings (Jiang, 2014). It is established that Vit E works through free radical removal and inhibition of lipid peroxidation and thus precludes cell membrane breakdown due to oxidation (Wang & Quinn, 1999). Further, Vit E is known to have anti-inflammatory effect that affects the immune regulation and reduces secretion of inflammatory factors including IL-6 and TNF-α which are known to be high in AD patients (Reiter et al., 2007).

Some of the latest studies have therefore been devoted to the production of modified Vit E compounds as a means of improving its neuroprotective potential and decreasing its toxicity. There is preclinical evidence about, for instance, naphthalene-based tocopherol analogue is effective in the therapeutic application. The derivatives of tocopherol are developed by keeping the antioxidant and anti-inflammatory features of Vit E to possibly provide a better pharmacokinetic profile and apparently better effectiveness in preventing the neurodegeneration associated with AD (Jiang, 2014). Such derivatives have been assumed to afford a better shield against Aβ-instigated oxidative stress and neuroinflammation, which are pathologic hallmarks in AD (Ham & Liebler, 1997).

This study seeks to assess the role of a newly synthesized naphthalene derivative of vit E in ameliorating Alzheimer's disease in an animal model of the disease. To measure learning and memory, the tests that are done are fear conditioning, Barnes maze, elevated plus maze, and Morris water maze. Further, biochemical estimations are performed for determining the concentration of oxidation stress indicators, brain neurotransmitters and pro-inflammatory cytokines such as IL-6 and TNF-α. This work assumes that the tocopherol derivative is going to have enhanced neuroprotective benefits relative to standard Vit E involving AD hence an enticing therapeutic notion to explore.

The Role of Oxidative Stress in Alzheimer’s Disease

That oxidative stress plays a role in the development of Alzheimer’s disease is now beyond debate. It concerns the difference between the generation of ROS and the ability of the body to counter these reactive species by using antioxidants (Perry et al., 2001). ROS, which include superoxide radicals and hydrogen peroxide are regular formations during the processes of cellular respiration and metabolism. However, in AD, because of overproduction of ROS and decrease of mitochondrial function, oxidative damage to lipids, proteins, and DNA occurs, leading to neuronal dysfunction and death (Nunomura et al., 1999). For instance, lipid peroxidation leads to highly reactive aldehyde by-products like malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) that can bring further damage to the plasma membrane and cytoskeleton (Sacrét, 1997). These oxidative products have been observed to accumulate in AD brains implying that oxidative stress may be key factor in the progression of the disease.

Due to these findings, antioxidant treatments have been well investigated as a way of addressing the AD condition. Among those, the application of Vit E with its highly efficient and selective ability to scavenge free radicals has been investigated in this regard in detail (Ham & Liebler, 1997). Vit E has also been found to prevent further oxidative injury in the brains of animals with AD and may potentially, hence, derive benefits in handling cognitive decline (Mosconi et al., 2008). In addition, several clinical trials have shown that high doses of Vit E can decelerate the rate of cognitive degradation in patients with mild to moderate dementia of AD (Sano et al., 1997). Nevertheless, these promising studies have not yet resulted in the widespread application of Vit E in clinical practice due to questions concerning its safety and effectiveness in high doses (Morris et al., 2005).

The Potential of Tocopherol Derivatives

To overcome these limitations, scholars have come up with a variety of Vit E analogs with improved pharmacological profiles. These derivatives therefore seek to enhance the effectiveness of Vit E in neuroprotection whilst at the same time, reducing its toxicities. In this regard, naphthalene based derivatives have been found to provide superior antioxidant capability and increased biopersistence (Brigelius-Flohé, 2009). These derivatives of Vit E can reach pathogenic processes in AD that is Aβ aggregation, tau hyperphosphorylation, and neuro inflammation since they are chemically distinct from Vit E (Jiang, 2014). In addition, such derivatives are substantiated to afford higher levels of shield against mitochondrial decay – widely associated with AD (Mosconi et al., 2008).

The current work therefore contributes to this body of research by examining the neurogardian functions of a naphthalene derivative of Vit E in the animal model of AD. Thus, examining the indices of both behavioral and biochemical characteristics of the disease, this work aims at presenting a perspective assessment of the possible therapeutic efficacy of tocopherol derivatives in AD. In particular, the results of this study would help in understanding the effectiveness of the tocopherol derivative for enhancing cognitive function, decreasing oxidative stress and regulating neuroinflammation to find a new therapeutic approach for AD.

1. Alzheimer’s Disease: Pathophysiology and Clinical Features

Alzheimer’s Disease (AD) is a type of dementia which is progressive in nature, causing cognitive as well as behavioral alterations primarily in the elderly population (Jansen et al., 2015). The defining characteristics of Alzheimer’s disease in terms of pathological changes are amyloid-beta (Aβ) plaques outside neurons and neurofibrillary tangles composed of phosphorylated tau within neurons as well as loss of synapses (Serrano-Pozo et al., 2011). The formation of Aβ plaques is assumed to be due to the imbalance of production that is Aβ peptides that are produced from APP through proteolytic cleavage through beta- and gamma-secretase enzymes (Cai et al. , 1993).

As for the structure, NFTs are mainly consisting of tau protein, while in the hyperphosphorylated state, this protein interferes with the normal function of microtubules, which are required for the axonal transport, and causes neuronal death (Maccioni et al., 2010). According to the tau hypothesis, it appears that tau abnormalities are at the heart of the disease since their deposits are associated with AD progression more than Aβ pathology (Götz et al., 2019). Both Aβ plaque formation and tau tangles lead to Synaptic dysfunction, neuronal loss and neuro-cognitive decline in patients of AD as proposed by Busse etal, 2014.

However, additional to these classical features, neuroinflammation and oxidative stress other factors are also considered as major factors of AD by researchers (Arosio et al., 2004). Activated microglia and astrocytes cause neuroinflammation that causes the release of cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), which worsens neuronal damage Di Bona et al., (2009). Another example is the fact of elevated oxidative stress, which increases production of ROS, and thereby contributes to the destruction of proteins, lipids and DNA (in Perry et al., 2001). All these processes indicate that AD is poly etiologic disorder that responds to multiple targeted therapies directed towards amyloid plaques, PS tau tangles, neuroinflammation, and oxidative stress.

2. Oxidative Stress and Alzheimer’s Disease

Free radical induced oxidative stress has been described in detail as one of the main factors in the development of AD. The brain cells contain high metabolic and oxidative rate thus making them vulnerable to oxidative insult that affects the formation of organelle and impairs function (Mosconi et al., 2008). ROS include superoxide anions and hydrogen peroxide which are normally produced in cells and have beneficial effects but pile up and cause toxicity in AD brains; the antioxidant defense mechanism of the brain is overwhelmed in AD (Smith et al., 1998).

Previously the studies have established that oxidative stress not only contributes to the formation of Aβ but also results from the deposition of the peptide in AD. The Aβ peptides themselves can generate ROS and augments oxidative damage to neurons (Sayre et al., 1997). This positive feedback exist between oxidative stress and Aβ where the increase in levels of one contributes to the increase in the levels of the other hence progression of the disease. Being a highly lipided area the brain is susceptible to lipid peroxidation which yields toxic products like malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) disruptive to membrane integrity and function altering proteins (Castellani et al., 2001). Presence of MDA and 4-HNE, both the markers of lipid peroxidation have been found to be higher in AD patients (Sayre et al., 1997).

Further, oxidative stress is directly associated with impairment of mitochondria in Alzheimer’s disease. Mobile phase ROS are generated mainly at the mitochondria during oxidative phosphorylation and there is compelling evidence that show that mitochondrial dysfunction occurs at the early stage of AD before the presentation of symptoms (Butterfield et al., 2001). Energy production by the mitochondrial decreases due to the loss of ATP synthesis which is necessary for the neurons’ survival, and increased production of ROS that is also reported in patients with AD (Mosconi et al., 2008). Since oxidative stress plays a significant role in the further development of Alzheimer’s, the application of antioxidant interventions has been considered.

3. Vitamin E: Antioxidant Properties and Neuroprotection

Vitamin E also known as Vit E is an antioxidant vitamin that soluble in lipid and has been looked at with interest as a possible element to help prevent progression of AD. Of these, 4 are tocopherols, and 4 are tocotrienols. IOmega-3 has eight forms in its natural state and the biologically active form is alpha tocopherol which is widespread in human population with its intake sourced from food products (Jiang, 2014). It has been discovered that Vit E has antioxidant actions that eliminate free radicals and stop the chain reaction of lipid peroxidation in membrane (Wang & Quinn, 1999). This function is especially vital in the brain area since there are significant concentrations of polyunsaturated fatty acids in the neuronal membrane, which makes the latter sensitive for oxidation (Brigelius-Flohé, 2009).

Some of these studies involve preclinical as well as clinical trials which seek to explore the ability of Vit E in preventing neurodegeneration related to AD. In animal models of AD, Vit E has been described as having the potential to reduce oxidation and to enhance cognitive performance while mitigating Aβ plaque formation (Ham & Liebler, 1997). It has also been proved by different clinical trials that high doses of Vit E can reduce the rate of deterioration of the disease in patients with mild to moderate AD (Sano et al., 1997). For instance, the Alzheimer’s Disease Cooperative Study (ADCS) implemented a single large scale randomized controlled trial on AD patients where individuals administered with 2000 IU/day of alpha tocopherol was observed to have a slower rate of functional dementia as compared to the patient groups that took placebo (Sano et al. , 1997). However, apprehensions of slightly long-term safety of high-dose Vit E supplementation, specifically the prospect of raising the chances of hemorrhagic stroke, has restricted the application of the supplement in clinical practice (Morris et al., 2005).

4. Tocopherol Derivatives: Enhancing the Efficacy of Vitamin E

To avoid the drawbacks of high-dose Vit E therapy researchers have synthesized some other derivatives of tocopherol that have got the antioxidant effect of Vit E but with better bioavailability and efficiency (Brigelius-Flohé, 2009). Such derivatives like naphthalene based Vit E have been proven to possess an increased antioxidant activity and more enhanced neuroprotection compared to ordinary Vit E studied in preclinical models (Jiang, 2014). These derivatives are hoped to have high specificity to some pathologic changes in AD, including amyloidogenesis, tau protein abnormal phosphorylation and neuroinflammation, which might be considered as the potential therapeutic strategy for AD.

According to the given current of knowledge, one of the major benefits of tocopherol derivatives is the possibility to regulate signaling pathways related to neuroinflammation. In AD, activation of microglia and astrocytes lead to the release of cytokines such as IL-6 and TNF-α (Arosio et al., 2004). The synthesis of such cytokines has been found to be prevented by tocopherol derivatives since this cut the neuroinflammation which is predominant in damaging neurons (Reiter et al., 2007). Moreover, tocopherol derivatives have a better capability to penetrate the blood brain barrier compared to standard Vit E and thus are capable to manifest their effects directly in the CNS (Ham et al., 1997).

There are other attractive characteristics of tocopherol derivatives; this compound's ability to prevent tau hyperphosphorylation, which fuels NFT in AD is quite remarkable (Jin et al., 2011). In addition, tocopherol derivatives can probably reduce the activation of kinases that phosphorylate tau, avoiding oxidative stress and neuroinflammation that may lead to neurodegeneration due to tau toxicity (Maccioni et al., 2010).

5. Mechanisms of Action: Neuroprotection by Tocopherol Derivatives

Tocopherol derivatives have several mechanisms of neuroprotection. First, these derivatives have been found to serve as post potent antioxidants that help in eliminating ROS and thus preventing the oxidation and consequent destruction of neuronal structures. As antioxidants tocopherol derivatives protect neurons’ membrane against lipid peroxidation, assisting synaptic transmission and plasticity which are important for learning and memory as pointed out by Wang and Quinn (1999).

Second, tocopherol derivatives also possess anti-inflammatory action since these compounds suppress the activation of microglia and astrocytes which are main effectors of neuroinflammation (Reiter et al., 2007). In AD, neuroinflammation prolongs the neuronal damage and this is considered to contribute to the progression of the disease (Busse et al., 2014). Tocopherol derivatives help to protect neurons from toxic effects of neuroinflammation and inhibit the release of the dangerous cytokines like IL-6 and TNF-α (Di Bona et al., 2009).

Third, tocopherol derivatives may help to repair defects in mitochondria that are routinely observed in AD (Butterfield et al. , 2001). For more than a decade, mitochondrial dysfunction has been associated with increased ROS production and energy failure that threatens neuronal survival. Tocopherol derivatives raise antioxidant capacity of mitochondria, decrease ROS production and increase ATP synthesis, contributing to neurons’ survival in AD (Mosconi et al., 2008).

Moreover, tocopherol derivatives may alter the functions of signaling pathways that are critical in synaptic plasticity including cAMP/PKA and PKC signaling (Counts & Mufson, 2010). These pathways bear importance in the process of long term potentiation, LTP that is attributed to learning and memory functioning. Tocopherol derivatives’ ability to induce new synapses boosts learning and memory in the animal models of AD (Wang & Quinn, 1999).

This study aimed to evaluate the neuroprotective effects of a newly synthesized naphthalene derivative of tocopherol in a mouse model of Alzheimer’s Disease (AD). The methodology was designed to assess both behavioral and biochemical changes following treatment with the tocopherol derivative. The experimental design incorporated behavioral assessments to evaluate memory and learning, followed by biochemical analyses to measure oxidative stress markers, neurotransmitter levels, and inflammatory cytokines.

1. Animal Model and Experimental Design

Alzheimer’s Disease was modeled in the study using adult albino mice (80 in total). Thirty five mice were randomly divided into 8 groups each having 10 animals. The control was assigned to Group I which was treated with carboxymethyl cellulose (CMC) that is the vehicle solution. Group II was termed as disease control and received intracerebroventricular (ICV) Streptozotocin (STZ) at a dose 3 mg /kg in order to have symptoms like Alzheimer’s disease. Group III and IV treatment consisted of standard treatment with 3 mg/kg of galantamine, a known cognitive enhancer and 5 mg/kg of Vitamin E (tocopherol). These acted as the reference treatment conditions. The next four groups (V-VIII) were given increasing concentration of the tocopherol derivative (5 mg/kg, 10 mg/kg, 20 mg/kg, and 40 mg/kg) to determine dose response. To the experiment all treatments were given according to an intraperitoneal administration schedule after the induction of AD for 21 consecutive days.

2. Behavioral Assessments

Behavioral tests were then introduced on the 15th day of the experiment starting with tests that focused on memory and learning abilities. Four different behavioral tests were utilized for this purpose: Electrodermal Response Conditioning, Prepulse Inhibition, Social Interaction Test, Contextual and Cued Fear, Apparatus Assay, Open Field and Morris Water Maze.

In light of associative learning and memory, the Fear Conditioning Test was also carried out. Rats were subjected to enclosures in which they would hear an audio signal which acted as the conditioned stimulus and received a mild electric shock as the unconditioned stimulus. Freezing effect which refers to the state where a subject is only capable of breathing was used to determine memory recall. It was thus noted that the longer the time taken for freezing, the better the performance level in memory.

Spatial memory was evaluated with the help of the Barnes Maze Test. Rodents were put in a circular chamber with 18 wells and one of them connected to an escape box. In the first several trials mice were able to learn where the escape box was by using spatial references. The number of exit errors and the time (latency) the rat took to locate the escape box were also measured; with low latency time pointing to good spatial learning and memory.

Of these, anxiety related behavior was used as a measure of cognition in the test called the Elevated Plus Maze Test. The area included two open arms and two arms confined within walls which were raised off the ground. The amount of time the animal spent in the open arms versus the closed arms was assessed in order to rate anxiety. Spending more time in the open arms was considered as lessening of fear or anxiety, and, therefore, it can be associated with improved cognitive function.

The last test employed in Behavior assessment was the Morris Water Maze Test which was only used to assess the spatial learning and memory of the animals. Rodents were tested in a water tank with an only partially hidden circular platform that the animals could use to escape the water. In several tests, the mice were conditioned to swim in the water and find the hidden platform with the help of visual cues. The escape latency used to find the platform was recorded with decreased time signifying better spatial memory.

3. Biochemical Analyses

After the behavioral tests were over, all the animals were euthanized, and the brain samples were prepared for biochemical studies. The brain homogenates were used for the measurements of markers of oxidative stress, neurotransmitter concentrations and pro-inflammatory cytokines.

To determine oxidative stress, extent of lipid peroxidation determined by malondialdehyde (MDA) was evaluated by the thiobarbituric acid reactive substances (TBARS) method. Also, SOD and GSH which are the antioxidant enzymes were determined using the commercial kits. A significantly high value of MDA was observed for all the groups which explains increased oxidative stress while high amounts of SOD and GSH depicted increased antioxidant mechanism.

The concentration of the Serotonin, Noradrenaline and Dopamine was determined as pg/ml using specific ELISA kits. These neurotransmitters are involved in playing key functions such as modulating mood, cognition and motor function, and when imbalanced are linked to AD.

For this purpose, the interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) cytokine secretion was determined using ELISA kits. These cytokines are indicators of neuroinflammation which is known to play a significant role in development of AD.

Statistical Analysis

The quantitative data collected were analyzed using one-way analysis of variance (ANOVA) with Tukey’s post hoc test to determine the significance of the differences between the groups. Behavioral data were analyzed and reported as mean ± Standard Error of the Mean (SEM) based on the method used to collect the data while biochemical data were reported as percentage change compared to the control group. It should be noted that p-value below 0.05 level of significance was used for all the comparisons done in the study.

This section describes the behavioral tests to determine the neuroprotection of the tocopherol derivative in an Alzheimer’s disease mouse model as well as the biochemical assays.

Behavioral Performance

The behavior tests showed improved cognitive ability in the tocopherol derivative treated mice especially at the higher doses of (20 mg/kg and 40 mg/kg). The results of the Fear Conditioning Test, Barnes Maze Test, Elevated Plus Maze Test and Morris Water Maze Test are as follows –

Fear Conditioning Test

Mice that received the dose of the tocopherol derivative equal to 40 mg/kg were shown to stay at the freezing point for longer time than the mice of the disease control group, meaning that their ability to retain memories was much better. The freezing time was also longer in the treatment group of 20 mg/kg and the 5 mg/kg and 10 mg/kg treatment groups did not differ significantly with the disease control group.

Table 1

Freezing Time in the Fear Conditioning Test
Group	Freezing Time (Seconds) ± SEM
Control	30.1 ± 1.5
Disease Control	15.4 ± 2.1
Galantamine 3 mg/kg	29.3 ± 1.8
Vitamin E 5 mg/kg	25.7 ± 2.3
Tocopherol Derivative 5 mg/kg	22.5 ± 2.0
Tocopherol Derivative 10 mg/kg	23.1 ± 2.4
Tocopherol Derivative 20 mg/kg	28.6 ± 1.9
Tocopherol Derivative 40 mg/kg	32.1 ± 1.4

Barnes Maze Test

In the Barnes Maze Test, the tocopherol derivative treatment groups demonstrated dose-dependent improvements in spatial memory. Mice treated with the 40 mg/kg tocopherol derivative exhibited the shortest escape latency compared to all other groups, indicating improved learning and memory. A significant reduction in escape latency was observed in the 20 mg/kg group as well.

Table 2

Escape Latency in the Barnes Maze Test
Group	Escape Latency (Seconds) ± SEM
Control	20.2 ± 1.5
Disease Control	38.6 ± 2.8
Galantamine 3 mg/kg	21.7 ± 1.9
Vitamin E 5 mg/kg	25.3 ± 2.3
Tocopherol Derivative 5 mg/kg	31.5 ± 2.7
Tocopherol Derivative 10 mg/kg	28.6 ± 2.5
Tocopherol Derivative 20 mg/kg	22.4 ± 1.6
Tocopherol Derivative 40 mg/kg	19.5 ± 1.3

Elevated Plus Maze Test

The Elevated Plus Maze Test revealed that the tocopherol derivative treatment groups, particularly at 20 mg/kg and 40 mg/kg, spent significantly more time in the open arms of the maze, indicating reduced anxiety levels. Reduced anxiety is often associated with improved cognitive function and a healthier behavioral profile.

Table 3

Time Spent in Open Arms of the Elevated Plus Maze Test
Group	Time in Open Arms (Seconds) ± SEM
Control	40.3 ± 2.2
Disease Control	18.4 ± 1.9
Galantamine 3 mg/kg	38.7 ± 2.0
Vitamin E 5 mg/kg	33.4 ± 2.6
Tocopherol Derivative 5 mg/kg	28.1 ± 2.3
Tocopherol Derivative 10 mg/kg	32.9 ± 2.5
Tocopherol Derivative 20 mg/kg	37.6 ± 1.8
Tocopherol Derivative 40 mg/kg	41.2 ± 2.1

Morris Water Maze Test

The Morris Water Maze Test results showed a significant improvement in spatial memory in mice treated with the tocopherol derivative at doses of 20 mg/kg and 40 mg/kg. The 40 mg/kg group demonstrated the shortest escape latency, indicating superior spatial learning and memory.

Table 4

Escape Latency in the Morris Water Maze Test
Group	Escape Latency (Seconds) ± SEM
Control	25.3 ± 1.9
Disease Control	42.7 ± 3.2
Galantamine 3 mg/kg	26.1 ± 2.1
Vitamin E 5 mg/kg	29.8 ± 2.4
Tocopherol Derivative 5 mg/kg	35.4 ± 2.9
Tocopherol Derivative 10 mg/kg	32.6 ± 2.8
Tocopherol Derivative 20 mg/kg	27.1 ± 2.0
Tocopherol Derivative 40 mg/kg	23.4 ± 1.5

Biochemical Analyses

Biochemical analysis of brain homogenates revealed a significant reduction in oxidative stress markers and pro-inflammatory cytokines in the groups treated with higher doses of the tocopherol derivative.

Oxidative Stress Markers

The levels of malondialdehyde (MDA), a marker of lipid peroxidation, were significantly reduced in the 20 mg/kg and 40 mg/kg tocopherol derivative groups compared to the disease control group. In contrast, superoxide dismutase (SOD) and glutathione (GSH) levels were significantly increased in these groups, indicating enhanced antioxidant defense.

Table 5

Oxidative Stress Markers in Brain Tissue
Group	MDA (µmol/mg) ± SEM	SOD (U/mg) ± SEM	GSH (µmol/mg) ± SEM
Control	2.1 ± 0.1	15.8 ± 1.3	8.4 ± 0.7
Disease Control	5.8 ± 0.3	9.2 ± 0.9	4.6 ± 0.6
Galantamine 3 mg/kg	2.3 ± 0.2	14.9 ± 1.1	8.1 ± 0.8
Vitamin E 5 mg/kg	3.1 ± 0.2	13.8 ± 1.4	7.2 ± 0.5
Tocopherol Derivative 5 mg/kg	4.5 ± 0.4	12.5 ± 1.3	6.1 ± 0.5
Tocopherol Derivative 10 mg/kg	3.8 ± 0.3	13.9 ± 1.5	7.5 ± 0.6
Tocopherol Derivative 20 mg/kg	2.6 ± 0.2	14.6 ± 1.2	8.2 ± 0.7
Tocopherol Derivative 40 mg/kg	2.0 ± 0.1	15.5 ± 1.3	8.6 ± 0.6

2.2 Inflammatory Cytokines

Levels of pro-inflammatory cytokines IL-6 and TNF-α were significantly lower in the 20 mg/kg and 40 mg/kg treatment groups compared to the disease control group, indicating reduced neuroinflammation.

Table 6

Inflammatory Cytokines (IL-6 and TNF-α) in Brain Tissue
Group	IL-6 (pg/mg) ± SEM	TNF-α (pg/mg) ± SEM
Control	15.3 ± 1.2	22.6 ± 1.5
Disease Control	42.8 ± 2.4	54.3 ± 2.7
Galantamine 3 mg/kg	16.5 ± 1.4	23.2 ± 1.8
Vitamin E 5 mg/kg	20.7 ± 1.7	27.5 ± 2.1
Tocopherol Derivative 5 mg/kg	30.4 ± 2.3	39.2 ± 2.4
Tocopherol Derivative 10 mg/kg	26.8 ± 2.0	33.1 ± 2.2
Tocopherol Derivative 20 mg/kg	19.3 ± 1.5	25.6 ± 1.7
Tocopherol Derivative 40 mg/kg	15.1 ± 1.3	22.1 ± 1.5

The data obtained from both the behavioral tests and the biochemical assays indicate that the tocopherol derivative possesses a substantial level of neuroprotective potential in the AD mouse model. Cognitive function improvement, decrease in oxidative stress, and reduction in inflammatory cytokine levels were best at doses of 20 mg/kg and 40 mg/kg. These findings provide substantiation for further investigation on tocopherol derivatives treatment for Alzheimer’s Disease.

The findings of this study give strong support that a newly developed naphthalene derivative of tocopherol has neurotrophic effects in an Alzheimer’s Disease (AD) mouse model. The study has established evidence of enhanced cognitive functions, decreased oxidative stress and reduced concentration of pro-inflammatory cytokines only in the groups that was administered the tocopherol derivative, especially at the higher dosage levels of 20 mg/kg and 40 mg/kg. These studies support and extend current understanding of oxidative stress and neuroinflammation in AD, and potential benefits of antagonizing oxidative stress through encouragement of foods rich in antioxidants such as Vitamin E and its analogs.

Cognitive Performance: Improvement in Learning and Memory

The behavioral outcomes of this study show that the tocopherol derivative enhanced learning and memory in a transportable, mean wise manner. The improvement in Fear Conditioning, Barnes Maze, and Morris Water Maze, tests that are commonly used in animal models of AD, can be explained by the cognition enhancing effects of the tocopherol derivative at the higher doses (20 mg/kg and 40 mg/kg). This enhancement was reflected in the shorter escape latency in Barnes Maze and Morris Water Maze tasks as well as the amount of time spent freezing during Fear Conditioning test.

These findings are in line with previous investigations that have looked at the neuroprotective role of antioxidants, more specifically Vitamin E in AD models. For example, Mosconi et al. (2008) made findings that showed that Vitamin E can increase the actual cognition and decrease the Aβ in AD models. Comparable enhancement in spatial memory and learning was also reported in experiments involving other antioxidant agents, flavonoids and polyphenols (Vauzour et al., 2010). However, the tocopherol derivative used in this study seems to be having therefore better cognitive effect compared to normal Vitamin E as reflected in the current study by better cognitive enhancements shown in the 20 mg/kg and 40 mg/kg treatment groups.

The improved cognitive impact might be attributed to the more readily availability and superior neuro protective effect of the tocopherol derivative as compared to Vitamin E. Naphthalene based tocopherol derivatives has been found to be more potent antioxidants, enabling them to scavenge ROS and protect against lipid peroxidation better (Jiang, 2014). Also, tocopherol derivatives can be better in the sense of having a higher’ ability to penetrate the blood-brain barrier and be effective in neutralizing oxidative stress and inflammation occurring in the CNS sites (Ham & Liebler, 1997).

Oxidative Stress: Reduction in Markers of Lipid Peroxidation

In this context, we must mention that oxidative stress has been considered for a long time an important factor in the development of AD because it contributes to neuronal damage, impairment of mitochondrial function and the formation of Aβ plaques (Butterfield et al., 2001). As observed in the present study, the tocopherol derivative exhibited useful ameliorating effects against oxidative stress conditions as the level of MDA decreased while the level of SOD and GSH increased in the AD mouse model. These findings are however significant in the two groups receiving 20 mg/kg and 40 mg/kg where the levels of oxidative stress markers were also significantly lowered to that of the healthy control group.

The results of the present study confirm the earlier evidence on antioxidant activity of Vitamin E and its esters. Similarly, looking at the effect of Vitamin E, Perry et al. (2001) indicated that Vitamin E could help to decrease oxidative stress which would therefore lead to increased neuronal survival and improved function in the brains of AD patients. Likewise, Ham and Liebler (1997) observed that tocopherol derivatives concerning prevention of lipid peroxidation and improvement of antioxidant protection in various oxidative stress prototypes. The decrease of MDA levels observed in this study provided a proof toward the fact that tocopherol derivatives are useful in preventing lipids peroxidation which is essential in preserving the neuronal membrane integrity and synaptic transmission.

The rise in SOD and GSH levels in the treated groups talk about the fact that the tocopherol derivative not only neutralizes the ROS but also helps in raising the endogenous antioxidant capabilities of the brain. Jiang (2014) and Brigelius-Flohé (2009) confirmed this notion suggesting that tocopherol derivatives could enhance the activity of key antioxidant enzymes hence enhancing a broad antioxidant defense. These effects are important, since oxidative stress participates in Aβ aggregation and tau hyperphosphorylation, both involved in AD pathogenesis.

Neuroinflammation: Reduction in Pro-inflammatory Cytokines

Activated microglia and astrocytes initiate inflammation within the nervous system in AD and subsequently contribute to the advancement of the disease. The cytokines, including interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-a), increase neuronal toxicity and enhance the production of Aβ plaques and neurofibrillary tangle (Di Bona et al., 2009). Here, the tocopherol derivative cut down the level of IL-6 and TNF-α in the brain of treated mice particularly in 20 mg/kg and 40 mg/kg groups.

Altogether these findings support the current economic literature on the role of Vitamin E and its derivative as anti-inflammatory. Inhibition of pro-inflammatory cytokines neuroinflammation could be achieved from both alpha- and gamma-tocopherol according to Reiter et al. (2007). Since, the tocopherol derivative was observed to decrease the levels of IL-6 and TNF- α, it is likely to alleviate the adverse effects of chronic neuroinflammation in AD and maintain neuronal integrity and compromise the disease’s progress (Arosio et al., 2004).

The reduction of inflammation recognized in this study is significant, because the role of neuroinflammation in the development of AD is described more and more often in recent years. In their recent study, Heneka et al. (2015), have emphasized that all the sections of the innate immune system, primarily, microglial activation takes part in neuroinflammation and augments the integer degradation of cognition seen in AD. Thus, the tocopherol derivative may prove to be a valuable therapeutic intervention because of its anti-oxidative and anti-inflammatory effects that counter two of the leading drivers of AD.

Comparison with Previous Studies

Therefore, the findings from this study are in support and follow the other studies related to the effect of antioxidants on AD. Review of literature has shown that vitamin E has got neuronal effect and has been shown to be protective in experimental animal models as well as in patients. For instance, Sano et al. (1997) conducted a clinical trial where they discovered that high doses of vitamin E of 2000 IU/day were effective in the slowing down of cognitive loss in patients suffering from moderate AD only. Nevertheless, controversy regarding the safety of high-dose Vitamin E, mainly, its possible link to hemorrhagic stroke, has dampened practices involving its application in clinical medicine (Morris et al., 2005). However, the tocopherol derivative used in this study is presented as reinstating neuroprotective effects in a comparable or even better approach in comparison to standard Vitamin E at the lower concentrations, regarding the data used in this study.

Furthermore, this study recommends that tocopherol derivatives are likely to be more effective than other antioxidants compounds like flavonoids and polyphenols in inhibiting oxidative stress and neuroinflammation. Despite these compounds exhibiting beneficial effects in preclinical studies their bioavailability and ability to cross the blood brain barrier present a major concern (Vauzour et al., 2010). Interestingly, the tocopherol derivative which was used in this study does not seem to have the above mentioned limitations since it has beneficial effects on both cognitive functions as well as biochemical parameters associated with AD.

Limitations and Future Directions

However, there are some limitations of this study, which need to be pointed out. Firstly, Secondly, Thirdly, First, the study was conducted on an animal model of AD and although the mouse model helps to study disease mechanisms it may not be exactly comparable with human diseases. Symptoms of any disease may significantly improve in animal models, but no one can guarantee that they will be the same in human patients with AD; thus, there is a need to conduct more clinical trials to seek the effectiveness and safety of tocopherol derivatives in patients with AD. Further, there is a need to conduct studies that investigate the impact of tocopherol derivatives on cognitive abilities and neuronal health in the long term as this research examined the effects of intervention only for a short period.

Further studies must also be conducted on the conceptual dimensions of tocopherol derivatives’ neuroprotection potential. This study has shown decrease in oxidative stress and neuroinflammation, however, the separate molecular mechanisms by which this is achieved are yet to be illustrated. Knowledge of these mechanisms might have aided the fine tuning of the therapeutic effect of tocopherol derivatives as well as other targets for interventional approaches in AD.

This research offers significant support for a novel naphthalene derivative of tocopherol by demonstrating its neuromodulatory benefits in a rodent model of Alzheimer’s Disease. The tocopherol derivative showed positive effects on cognitive function, decreased oxidative stress and decreased inflammatory cytokines including IL-6 AND TNF-α. Thus, the obtained results can open the therapeutic opportunities of tocopherol derivatives in the treatment of AD with better outcomes than Vitamin E.

The outcomes are consistent with the existing investigations regarding antioxidants in AD but indicate that tocopherol derivatives can have improved bioavailability with neuroprotective potential. However, the current study was done using a preclinical mouse model; therefore, further research is needed to establish the effect and toxicity of tocopherol derivatives to the human subject with AD. Future aspects include the molecular aspects of the neuroprotective action of tocopherol derivatives in the context of the action on key pathways linked to oxidative stress and neuroinflammation and regulates synaptic plasticity.

This study adhered to all relevant animal ethics guidelines and was approved by the *Institutional Animal Care and Use Committee (IACUC) of Riphah Institute of Pharmaceutical Sciences at Riphah International University Lahore. All procedures were designed to minimize animal pain and distress, utilizing the 3Rs principle of replacement, reduction, and refinement whenever possible. Animals were housed and cared for in accordance with established protocols, and euthanasia was performed humanely according to accepted veterinary practices.

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The authors declare no competing interests.

Neuroprotective Role of Tocopherol Derivatives in Alzheimer’s Disease: Behavioral and Biochemical Analysis

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Abstract

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Introduction