Ubisol-Q 10 (a Nanomicellar Water-Soluble Formulation of CoQ 10 ) Treatment Inhibits Alzheimer-Type Behavioral and Pathological Symptoms in a Double Transgenic Mouse (TgAPEswe,PSEN1dE9) Model of Alzheimer’s Disease

Abstract

Alzheimer’s disease (AD) is one of the most common neurodegenerative pathologies for which there are no effective therapies to halt disease progression. Given the increase in the incidence of this disorder, there is an urgent need for pharmacological intervention. Unfortunately, recent clinical trials produced disappointing results. Molecular mechanisms of AD are converging on the notion that mitochondrial dysfunction, oxidative stress, and accumulation of dysfunctional proteins are involved in AD pathology. Previously, we have shown that a water-soluble formulation of Coenzyme Q₁₀ (Ubisol-Q₁₀), an integral part of the electron transport chain, stabilizes mitochondria and prevents neuronal cell death caused by neurotoxins or oxidative stress both in vitro and in vivo. In this study, we evaluated the neuroprotective effects of Ubisol-Q₁₀ treatment in double transgenic AD mice. In the present study, we report that providing Ubisol-Q₁₀ in drinking water (at a dose of ∼6 mg/kg/day) reduced circulating amyloid-β (Aβ) peptide, improved long term memory, preserved working spatial memory, and drastically inhibited Aβ plaque formation in 18-month-old transgenic mice compared to an untreated transgenic group. Thus Ubisol-Q₁₀ supplementation has the potential to inhibit the progression of neurodegeneration, leading to a better quality of life for humans suffering with AD.

Keywords

Alzheimer’s disease amyloid-β plaques long term memory working memory

INTRODUCTION

Alzheimer’s disease (AD), the most common form of dementia, is characterized by progressive decline in cognitive function and is a major cause of morbidity and mortality [1]. The key pathological features associated with AD are loss of neurons in the hippocampal region leading to learning and memory impairments, aggregation and deposition of amyloid fibrils forming neuritic plaques outside neurons, and accumulation of hyperphosphorylated tau that form neurofibrillary tangles (NFT) inside the nerve cell bodies [2, 3]. Although the majority of the AD cases are sporadic, mutations in genes linked to early onset and familial cases of AD have also been identified and contribute to 10% of reported AD cases. Mutations in genes involved in the encoding of amyloid-β (Aβ), presenilin 1 (PS1), and presenilin 2 (PS2) have been linked to early onset autosomal dominant AD. Both PS1 and PS2 are part of γ-secretase enzyme that cleaves amyloid-β protein precursor (AβPP) via the β-secretase pathway to give rise to Aβ [4 –6]. FDA approved treatments currently available are acetylcholinesterase inhibitors and NMDA receptor antagonists which only stabilize symptoms for a short period in early to moderate stages of the disease.

Impaired ubiquitin proteasome system, impaired autophagy, and mitochondrial dysfunction are implicated in a number of aging-related diseases such as AD [7]. The ubiquitin proteasome system is involved in clearing the unwanted damaged proteins that could become toxic to the cell. Mitochondria that generate energy via oxidative phosphorylation are the largest source of reactive oxygen species (ROS) that, if over-produced, damages proteins, lipids, and DNA. Any such increases in damaged protein have to be removed by the proteasomes. Furthermore, ROS can potentially damage those proteasome proteins hence decreasing their activity.

Therefore, targeting one of the deficiencies, mitochondrial dysfunction, could potentially save the ubiquitin proteasome system.

Generally, the focus of AD pathophysiology has been on the accumulation of Aβ plaques and NFT. However, recent evidence indicates that mitochondrial dysfunction and increase in oxidative stress have a larger role in AD and are responsible for synaptic abnormalities and neurodegeneration. Proteomics studies have also shown that the mitochondrial defects are predominantly in complex I, IV, and V of the electron transport chain [8]. This leads to the generation of free radicals. Hence, one of the ideal methods to approach AD treatment would be to restore mitochondrial function, reduce the levels of oxidative stress, and quench the free radicals by using an antioxidant. One such naturally occurring antioxidant is CoQ₁₀, which is part of the electron transport chain and has been extensively studied and tested on animal models of neurodegenerativedisorders [9 –11].

Despite an oil soluble formulation of CoQ₁₀ that showed effective neuroprotection in animal models of neurodegenerative diseases, it failed in clinical trials of Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis [12 –14]. This may have been due to its poor bioavailability. In a rodent model, the effective dose of the oil soluble formulation required to provide significant neuroprotection is 200–1600 mg/Kg/day. This translates to extremely high doses in humans, well above those approved by FDA for human clinical trials. As a result, clinical trials may have failed due to insufficient dosage being given to participants.

Attempts have been made to improve the bioavailability of CoQ₁₀ by making it water-soluble. One such formulation of CoQ₁₀ is Ubisol-Q_10, developed by the National Research Council of Canada in Ottawa. The carrier used for solubilization of CoQ₁₀ consists of polyethylene glycol linked to α-tocopherol via an alkanedioyl linker, sebacic acid, giving rise to the stable non-toxicUbisol-Q₁₀ [15]. Previous studies in our laboratory have demonstrated that Ubisol-Q₁₀ is able to provide significant neuroprotection by halting neurodegeneration in both an environmental toxin model and a genetic susceptibility model of Parkinson’s disease. The bioavailability is so much improved with this formulation that the effective dose required in rodent models is only 6 mg/Kg/day [9, 10]. In addition, Ubisol-Q₁₀ is able to decrease the levels of ROS generated by fibroblasts of AD patients with mutations in the PS-1 gene, stabilizes the mitochondria, and delay the onset of premature senescence in vitro [16, 17]. In the current report, we tested the effects of prophylactic treatment with Ubisol-Q₁₀ in a mouse model expressing chimeric human/mouse AβPP and mutant human PS1. These transgenic mice experienced elevated levels of human Aβ peptide due to the presence of Swedish mutations that favor processing of AβPP via the β-secretase pathway. The efficacy of providing transgenic mice Ubisol-Q₁₀ was evaluated by measuring the prevention of typical changes in pathological features and behavioral symptoms that occurred in non-treated transgenic animals.

As the focus is slowly being shifted from neurons being the fundamental unit of the brain toward the neuron-microglia-astrocyte triad, the contribution of astrocytes and microglia in the pathophysiology of AD is becoming increasingly recognized [18, 19]. Studies suggest the presence of reactive astrocytes and primed microglia in the hippocampal region especially at the vicinity of the Aβ plaques [19, 20]. Therefore, in the present study we examined changes in astrocytes and microglia morphology in the transgenic mice treated with Ubisol-Q₁₀ compared with those in the untreated group.

One of the most prominent pathological features of early-stage AD is neurodegeneration in the hippocampus [21]. Hippocampal degeneration significantly impairs cognitive function in humans with AD. Similarly, the ability for rats and mice to form long-term memories and perform spatial memory tasks depend on having a functional hippocampus [22 –24]. Previous research has shown that hippocampal damage, either by directly lesioning the hippocampus [25 –29] or by indirectly affecting it through the development of AD-related transgenic models [20, 30], primarily affects long-term memory and spatial working memory. One possible example of declined spatial working memory in transgenic mice has been observed in their reduced spontaneous alternation in an enclosed opaque Y maze [30, 50]. However later studies failed to replicate such an effect in these animals [51, 52]. If such spontaneous alternation, however, involves exploration among seemingly different arms, preventing mice from viewing extra-maze cues might simply generally reduce such response, which could obscure any cognitive working memory differences between transgenic and wild-type mice. Consequently, animals might display greater exploratory activity over arms that allow them to examine extra-maze cues. Under such conditions, transgenic mice with compromised hippocampi might be less able to remember which arm they have just entered and so be more likely to reenter after fewer entries to other arms to reduce the amount of spontaneous alternation. Based on these findings and our reasoning to account for unreliable spontaneous alternation effects, we conducted the following behavioral tests of working and long-term memory to assess the potential therapeutic effects of Ubisol-Q₁₀.

To examine spatial working and long-term memory, we determined the degree spontaneous alternation over several distributed trials in a translucent (opaque) and in a transparent enclosed Y-maze located in a larger square chamber. We were particularly interested how different groups of mice, wild-type (WT) mice, protected transgenic (T + Ub-Q₁₀) mice, and unprotected transgenic (Tg) mice spontaneously alternate their exploratory activity among arms and reduce (habituated) such or any other exploratory activity in these Y-mazes over trials spaced 24 h apart. To measure these mice’s long-term memory, we examined the extent to which they spontaneously recovered their previously habituated exploratory behavior following a long rest from exposure to the Y-maze. The assumption being that the amount of spontaneous recovery measures some degree of forgetting about prior experience in the maze. This hypothesis is based on the notion that habituation of an animal’s reaction to any initial novel event and its spontaneous recovery following a longer period (rest) away from that event reflects basic learning and memory processes [23, 31]. Untreated transgenic mice were expected to show greater spontaneous recovery compared to treated transgenic mice and wild type mice. Of course there is the possibility that spontaneous alternation will continue to be an unreliable measure of spatial working memory despite our methods. Therefore, we decided to modify an alternative promising novel location/novel object recognition (NL/NOR) task recently introduced by Benice et al. for our Y-maze [32]. In their study, mice were placed in an open field with three distinct objects each located in a different corner. The three objects remained in those locations over the first three trials, followed by a fourth “novel location” trial where one of the objects was moved to the fourth corner, and finally by a fifth “novel object” trial where one of the previously unmoved objects was replaced by a novel object. According to Benice and Raber, increased exploration of the moved object suggests the mouse had remembered the objects’ spatial features, providing insight into their spatial working memory [33]. Additionally, increased exploration of the novel object suggests the mouse had remembered the previous objects’ non-spatial features allowing it to recognize which of the objects had been replaced by a new one.

Taken together, these measures provide a robust indication of spatial and non-spatial working memory processes. We predicted that untreated transgenic mice would explore the moved object but not the novel object less than the treated transgenic mice and wild type mice due to unprotected neurodegeneration in the former group’s hippocampi.

MATERIALS AND METHODS

Animal care

Experiments on the animals were conducted following approval by the University of Windsor’s Animal Care Committee that insured our procedures were in accordance with guidelines of the Canadian Council of Animal Care. One month old double transgenic male mice containing human/mouse chimeric APP and a mutant PS1 gene were purchased from MMRRC, Jackson Laboratory (Maine USA). Fourteen male transgenic mice and nine male C57BL/6J wildtype mice were purchased for a total of 23 mice. Mice were housed in groups of three or four with the exception of three mice that were isolated. Transgenic and wild type mice were separately housed to avoid any social hierarchies due to functional differences in neurological change. Environmental enrichment was provided in their home cages in the form of baby-food jars, overturned cardboard cup holders, and toilet paper tubes. Mice were allowed continuous access to food and drink in their group or single home cages. The colony room housing the mice was maintained at 20°C and placed on a reversed 12 h:12 h dark light cycle. The mice were weighed once a week to ensure they were healthy and did not exhibit any unprecedented weight loss. Following the experimental period the mice were euthanized and perfused using Tyrode’s buffer containing heparin and the tissues were fixed with 10% formalin.

Control and treatment regimen

Transgenic mice were divided into two groups with eight mice in the treated group and six mice in the untreated group. The untreated group received regular drinking water whereas the treatment group received Ubisol-Q₁₀ supplemented drinking water at a concentration of 50 μg/ml of the CoQ₁₀. Ubisol-Q₁₀ was supplied by Zymes LLC (Hasbrouck, NJ). Supplemented and un-supplemented fresh drinking water was provided once a week. Treatment started one month after their arrival (at 90 days old) and continued for a period of 18 months. A separate group of nine wild-type mice received un-supplemented water under identical conditions.

Enzyme linked immunosorbent assay

In order to measure the levels of Aβ_1-40 in the blood, ELISA was performed on the serum samples using the KHB3482 kit. Standards and sample dilutions were prepared, mixed with detection antibodies and loaded in triplicates to an antibody pre-coated ELISA plate. After incubation, the plates were washed to remove any excess unbound antigen or detection antibody and then the HRP-conjugated antibody was added, followed by a substrate that gave a colorimetric product which could be read after stopping the reaction with sulphuric acid. Standard curve was plotted and the sample concentration was calculated from the line equation obtained from the standard curve.

Antibodies

The following antibodies were used at the mentioned dilutions to perform immunohistochemistry and assess the morphological changes and levels of various proteins in the brain: Mouse monoclonal anti-human beta–amyloid antibody (1:500; Novus Biologicals Cat NBP2-13075), mouse monoclonal anti-neuronal nuclei antibody (1:600; EMD Millipore Cat MAB 377X), rabbit polyclonal anti-glial fibrillary acidic protein antibody (1:500; Novus Biologicals Cat NB300-141), rabbit polyclonal anti-Iba-1 antibody (1:300; Cat NB100-1028), and rabbit polyclonal to 4-hydroxynonenal (4-HNE) (1:200, Abcam California USA, Cat ab46545).

Immunohistochemistry

Following perfusion of five animals from each group, their brains were extracted and stored in 10% formalin at 4°C. Three days prior to sectioning using a microtome the brains were transferred to 30% (w/v) sucrose. The several (approximately 20) sections from each brain included the entire hippocampal region and were each 30μm. On the first day of immunohistochemistry procedure, the slides were washed twice in Tris buffered saline (TBS) for 5 min each following which they were incubated in 1% H₂O₂ for 5 min in order to block any endogenous peroxidase activity. The slides were then rinsed twice for 5 min in TBS to wash off any excess H₂O₂ and then blocked for 30 min each in DAKO universal blocking solution (purchased from Diagnostics Canada Inc., Mississauga) and normal serum (followed instructions on Vectastain ABC Kit, Vector laboratories) in order to block the binding of non-specific IgG. Following blocking the sections were incubated overnight in the primary antibody solution at 4°C. Three sections from each of the five animals in each group were stained with each of the four primary antibodies mentioned above. On the second day, the slides were washed twice in TBS for 5 min and this time to remove any excess antibody before incubating them for 1.5 h in the corresponding biotinylated secondary antibody present in the VectastainABC kit. The TBS washes were repeated and the sections were incubated in avidin biotin complex for a period of 45 min. The TBS washes were then carried out once again and a peroxidase substrate 3, 3‘diaminobenzidine was prepared and added in order to stain the specific antigenic sites on the section to which the antibody was bound to. The slides were dehydrated with first 95% ethanol and xylene and cover-slipped using permount for microscopic visualinspection.

Congo red staining

Three other sections from each animal were subjected to Congo red staining. The day prior to conducting this procedure, we prepared the 0.2% Congo red solution in saturated NaCl solution by allowing it to stir overnight. Before use, the Congo red solution was vacuum filtered and 1% v/v of 1M NaOH was added to make the solution alkaline. The slides subjected to Congo red staining were first rehydrated by immersing them in distilled water for 30 s, incubated in alkaline saturated NaCl for 20 min, following which they were incubated in the Congo red solution for 30 min. The slides were then rinsed by dipping them eight times, first in 95% ethanol, followed by 100% ethanol (repeated twice), and then incubated thrice in xylene for 5 min, after which they were cover-slipped with permount.

The Leica microscope and 4.5AF software were used to visualize and capture images of the region of interest, which was kept consistent across thegroups.

Behavioral testing

Apparatus and materials

Two Y-mazes, constructed from Plexiglas material, consisting of symmetrically radiating arms, each measuring 22 cm long by 6 cm wide with 15 cm high walls were used. One Y maze was transparent and the other was translucent (see Fig. 1A). Either maze could be placed in a 50-cm square grey chamber with 40-cm grey walls. The interior of the chamber was illuminated by an incandescent 60-W lamp positioned 1.5 m above the apparatus. A Sony Digital Video camera was also positioned near this lamp to record a mouse’s movements in the Y-maze. Noldus Ethovision XT10 software was used to track a mouse’s movement in the Y-maze and to determine the amount of time it explored objects during the second phase of the experiment.

Fig.1

Picture of the transparent (A) and translucent (B) Y-maze that used in Phase 1 for behavioral tests of long-term memory and spontaneous alteration. Picture of the three objects positioned at the end of each arm the NL/NOR task in Phase 2 of the behavioral test of spatial and non-spatial working memory, the NL/NOR task (C). A schematic representation of the 3 trial NL/NOR test. D) Familiarization trial: Two stainless steel wire mesh floors and a smooth floor were placed in the arms to provide salient directional cues. Two distinct objects (paper clip and round headed bolt shown here) were randomly placed at the ends of two arms. E) Novel location trial: One of the objects (the paper clip in this example) was moved to the previously empty arm while the other object (the round headed bolt) remained in its previous trial’s arm. F) Novel object trial: The previously unmoved object was replaced with a novel object (butterfly bolt) while the moved object remained in the arm it had been moved to in the previous trial.

Spontaneous alternation and long-term reference memory task

Mice began this phase at 12 months of age. As already described, we used the Noldus software program to track each mouse’s movements in the Y-maze to determine how often it re-entered an arm after first entering either one other arms or the other two arms (spontaneous alternation) as well as how often it entered each arm regardless of its previous pattern of arm entries and whether it traveled to the end of an arm before turning around and exiting it. We note here that, as already described, we measured mice’s habituation and spontaneous recovery of their exploration in Y-mazes (see Fig. 1A). In this study, we define habituation as a decrease in overall activity or exploratory behavior when exposed to the same, initially unfamiliar environment over repeated exposures. Of the various types of activities of the mouse in the Y-maze that the Noldus program could monitor, we report only two types of exploration in any 5-min session: the number of times a mouse entered arms and consistent effects as a function of treatment. We note that groups did not differ in their proportions of spontaneous alternations within either within either the transparent or translucent maze and therefore exclude these findings from our report. A mouse was considered to have entered an arm when its body’s center-point was 3 cm inside it. We note that mice seldom if ever backed out of an arm at this point but ran at least half way down it and often to its end before turning around and exiting the arm.

This phase of the experiment consisted of two blocks of five sessions each. A daily session consisted of placing a mouse in the center of the Y-maze and allowing it to explore it for 5 min. Inter-trial intervals within each block were kept at 24 h and the interval between blocks (the ‘rest’ period) was 16 days. Mice were randomly assigned to begin the test in either the transparent or a translucent Y-maze which alternated over all 10 sessions. We varied exposure to these two types of mazes to determine if exploration would be affected by an animal’s ability to perceive the larger chamber and if this effect was a function of group treatment. As our subsequent analysis failed to show any effects from this factor, we report animals’ exploration collapsed over it. We expected that mice would habituate to the maze (i.e., reduce their exploration) over the first five trials. Their long-term memory of the maze over the 16-day rest period would be evident in the amount of spontaneous recovery of exploration when reintroduced to it. If such long-term memories were better preserved in wild-type and Ubisol-Q₁₀ treated transgenic mice than in the untreated transgenic mice, the former two groups should show less spontaneous recovery than the latter group. Within any daily session, the maze was sprayed with Accel Prevention Cleaner and Disinfectant (concentration 1:40) and wiped dry between mice.

Working memory

Prior to running this phase of the experiment, one mouse from each group had been sacrificed for brain tissue analysis immediately after having completed the previous phase. We note that another wild type mouse died of natural causes before this phase. Thus we were left with seven wild-type mice, seven treated transgenic mice, and five untreated transgenic mice for this phase. These remaining mice began this task at 18 months of age. We adapted the novel location / novel object recognition (NL/NOR) test of Benice and Raber (2008) for the Y-maze [33]. In this phase mice only ran in the transparent maze. The maze was modified to contain removable arm floors, two of which were made of stainless steel wire mesh and one of smooth polystyrene material. These floor cues provided additional spatial, location cues in the maze. A different object on a covered metal base, was placed at the end of each of these two arms. These two objects were randomly selected from among three objects, a black large paper clasp, a round screw, and a butterfly screw, for the first familiarization trial for each mouse. The third object was reserved as the novel item used to replace one of the initial objects in the third trial of this task. Which of the two arms contained an object during the first, familiarization trial in this task was also randomly determined for each mouse. Each animal received three 5-min trials in the Y-maze: the first being the location familiarization trial with two objects, the second being the novel location test trial where one of these two objects had been moved to the previously ‘empty’ arm, and the third being the novel object test trial where the novel object had replaced the remaining unmoved object of the previous two trials(see Fig. 1B).

Each trial in the working memory test was separated by a 2-min interval during which time the experimenter prepared the maze for the next trial by replacing the two objects from the first trial with identical replicates and moving one of them to the empty arm for the second trial and then for the third trial, replacing the unmoved object from the second trial with the novel object and replacing the previously moved object with an identical replicate. The maze and objects were also cleaned between trials and mice as in the first phase. These procedures were used to control for any possible differential experimenter- or subject-induced odors left on objects or in maze arms from a previous trial.

In tracking a mouse’s movement in the maze, the Noldus software was programmed to determine the amount of time the animal remained within 3 cm from each object during the trial as a measure of its inspection (exploration) of it. From these data, we calculated the proportion of time a mouse spent near each object from its proportion of time it spent exploring both objects rather than from its time (5-min) in the maze. We used this conditional proportion measure as a more precise indicant of a mouse’s object preferences as a function of its spatial and non-spatial changes because mice from each treatment condition, spent a similar overall small proportion of time inspecting both objects on any trial(0.12±0.07).

Statistical analyses

For the long-term memory task (phase 1), proportion of time spent in the arms and number of arm entries was initially analyzed by a 3 (groups: treated transgenic, untreated transgenic, untreated wild type) by 2 (blocks: initial series of habituation trials; following rest, 2nd series of habituation trials) by 5 (trials in each series) ANOVA with repeated measures on the last two factors. As already mentioned, we collapsed these data over the within-subjects factor (maze type). To determine spontaneous recovery on the first session of block 2 from the last session of block 1 for these measures, we conducted another 3 (treatment groups) by 2 (sessions) ANOVA. We conducted subsequent analyses for simple effects based on outcomes from these statistical tests and observations as will be described in the following section of this report.

For the NL/NOR task, we conducted a 3 (groups) by 2 (object locations) ANOVA with repeated measures on the second factor to determine the effects of moving an object for the second trial. To determine the effects of replacing the remaining unmoved object with a novel object for the third trial, we conducted a univariate groups ANOVA. Effects from all statistical analyses were considered significant at p≤0.05 (one-tailed).

RESULTS

Ubisol-Q₁₀ maintains reference (long-term) memory in double transgenic mice

Long-term (reference) memory was evaluated in wild-type and in the treated and untreated transgenic mice after 12 months of age as described in the methods section. Analysis of the proportion of time mice spent exploring the arms of the Y-maze only revealed a main effect for groups, F_2,20 =6.31, p < 0.01, due to the untreated transgenic mice spending a significantly lower proportion of time (0.60±0.04) than in either the treated transgenic (0.71±0.03) or the wild-type (0.77±0.03) mice. Other than this effect no evidence of any other effects, including that of spontaneous recovery among the three groups was found. Therefore, we focus our description on arm entry data as shown in Fig. 2A and 2B. As seen in Fig. 2A, and supported by a main effect for sessions within each block, Fs4, 80 =11.12; 3.51, ps≤0.01, animals decreased their number of arm entries primarily from the first session to that on subsequent sessions. A main effect for blocks, F_1,20 =11.42, p < 0.01, resulted from fewer averaged arm entries per session on the second block (23.2±1.5) than on the first block (26.5±1.4). However, there was a group by blocks interaction, F_2,20 =5.59, p < 0.05) resulting from only the wild-type mice making significantly fewer arm entries on the second than first block of sessions (21.0±2.4 versus 28.8±2.2). An examination of spontaneous recovery of exploration between the last session of block 1 and the first session of block 2 following the 16-day rest period clearly shown in Fig. 2B revealed a greater effect in the untreated transgenic mice than in either of the other two groups. Despite the lack of a significant group by session interaction, separate statistical analyses within each group, revealed that only the untreated transgenic mice significantly increased their exploration following the rest period, F_1,5 =8.33, p = 0.035. This striking recovery effect in the untreated transgenic group was likely responsible for their significant main sessions effect, F_1,20 =5.12, p = 0.035. These results suggest that long-term memory, as measured by the degree of spontaneous recovery or dishabituation, was better preserved in the treated transgenic and wild-type mice than in the untreated transgenic mice.

Fig.2

The number of arm entries made per session in the Y-maze for each treatment group (A). Wild type, treated transgenic, and untreated transgenic groups show habituation over sessions, primarily from the first session and onwards. Data are represented as the Mean±SEM. The number of arm entries made on the last session of the first block and the first session of the second block (B). Only the untreated transgenic group show a significant increase in exploration between sessions, demonstrating a large degree of spontaneous recovery or dishabituation that was not observed in the wild type or treated transgenic groups. This suggests long-term memory was preserved in the treated transgenic group compared to the untreated transgenic group. Data are represented as the Mean±SEM. *p < 0.05

Ubisol-Q₁₀ only preserves working spatial memory in double transgenic mice

As seen in Fig. 3A and supported by a significant trial by group interaction F_2,16 =3.67, p = 0.049, moving a familiar object from trial 1 to a new location in trial 2 promoted greater exploration of it in the wild-type and treated transgenic mice but not in the untreated transgenic mice. Further analysis of changes of exploration within each group (one-tailed t-tests), revealed that the treated transgenic group significantly increased its exploration, t_1,6 =2.46, p = 0.025, the wild type showed a non-significant trend in that direction, t_1,6 =1.73, p = 0.065, but the untreated transgenic mice showed a slight, non-significant change in the opposite direction, t_1,6 = –1.58, p = 0.095.

Fig.3

The proportion of time spent exploring a familiar object before and after it moved to a new location (A). The wild type and treated transgenic groups spend more time exploring the object in a novel location whereas the untreated transgenic group shows no difference. This suggests that spatial working memory was preserved in the treated transgenic group compared to the untreated transgenic group. Data are represented as the Mean±SEM. *p < 0.05. Proportion of time spent exploring a novel object (B). The wild type, treated transgenic, and untreated transgenic groups do not significantly differ from each other. Data are represented as the Mean±SEM.

As seen in Fig. 3B, mice in both the wild type and untreated transgenic group similarly explored a novel object slightly more than mice in the treated group. These differences, however did not produce a significant main group effect, F_2,16 =0.58, p = 0.57. Failure to replicate the group effect for non-spatial working memory as measured by novel object preference suggests that unprotected neurodegeneration in transgenic mice disrupted their spatial but not their non-spatial working memory.

Ubisol-Q₁₀ treatment caused a decrease in the circulating Aβ in APP/PS1 transgenic mice

Following the behavior analysis, we conducted experiments to determine if treatment with Ubisol-Q₁₀ reduced changes in the pathological features of AD associated with the APP/PS1 transgenic mouse model. First, the serum levels of human Aβ_1-40 were measured using ELISA to determine any observable differences between the wild type and transgenic groups. Wild type mice that were devoid of the human APP gene had no Aβ_1-40 as expected. There were, however, higher levels of Aβ_1-40 in the serum obtained from transgenic untreated group in comparison to the transgenic treatment group (Fig. 4). The difference between circulating amyloid β between the untreated transgenic mice and Ubisol-Q₁₀ treated transgenic mice was approaching significance (p = 0.084).

Fig.4

Serum levels of human Aβ_1-40. A sandwich ELISA was conducted to analyze serum levels of Aβ_1-40 across the animals. The absorbance was measured at 450 nm and the results were averaged over two independent experiments. The untreated control group show high levels of Aβ in the serum whereas there is a reduction in Aβ for the treated group. (p = 0.084) WT group shows no Aβ expression.

Ubisol-Q₁₀ treatment resulted in drastic decrease in the Aβ plaques the brain of APP/PS1 transgenic mice

Following 15 months of treatment with either Ubisol-Q₁₀ supplemented drinking water or regular water, the transgenic and wild type mice were sacrificed and their brains sections were subjected to immune-histochemical analysis with anti-human Aβ antibody or Congo red staining to assess the extent of amyloid plaque burden across the treatment groups. As illustrated in Fig. 5A, all wild type mice as expected showed no immunoreactivity with human Aβ immunostaining. In contrast, all the untreated transgenic mice displayed very high levels of human amyloid plaque deposits in the hippocampal region, a characteristic feature of the APP/PS1 transgenic mice model. However, the Ubisol-Q₁₀ treated transgenic group displayed a drastic difference from the untreated group by showing little if any human amyloid plaque in three mice and the other two showing some but substantially lower levels of Aβ plaques. Similar results were obtained with Congo red staining as well (Fig. 5B), further validating the immunohistochemistry results. This clearly indicates that prophylactic treatment with Ubisol-Q₁₀ is able to decrease the extent of amyloid plaque deposit in this transgenic mouse model (Fig. 5A, B).

Fig.5

Immunohistochemical staining for Aβ shows a decrease in plaque formation in treated groups (A). Each image represents a brain section from an individual animal. The untreated group exhibits abundant plaque formation. The first two images in the treated group demonstrate a near complete reduction of Aβ deposition while the third image demonstrates a mouse with a 70% reduction in plaque deposition. The WT groups had no Aβ deposition. Scale bar = 500μm. Congo Red staining demonstrates a decrease in Aβ plaque formation in treated groups (B). Each image represents an individual animal. Congo red staining was used to confirm anti-human Aβ antibody staining results. The same trend was observed where the untreated groups have high levels of plaque while the treated animals demonstrate a near-complete reduction on observed plaques. Scale bar = 100μm.

Ubisol-Q₁₀ treatment resulted in activation of astrocytes and inhibition of microglial activation in APP/PS1 transgenic mice

The morphological changes of the glial cells, namely astrocytes and microglia, were then analyzed and compared between groups. The images demonstrate increased astrogliosis in the brains of Ubisol-Q₁₀ treated transgenic mice, when brain sections were stained with anti-GFAP antibody (a marker for astrocytes, which exhibit longer and increased branching per cell). However, in comparison, the untreated transgenic and wild type groups appeared to show moderate levels of astrocyte activation with shorter and fewer branches per cell (Fig. 6A).

Fig.6

GFAP staining to investigate astrocyte activation (A). Treated groups demonstrate increased astrocyte activity; this is indicated by a change in morphology where there are more branches, of longer lengths per astrocyte. In comparison, the untreated and WT groups demonstrate moderate activation of astrocytes, with shorter and less branches per astrocyte. Scale bar = 100μm. Iba1 staining to investigate microglial activation (B). The level of microglial activation is indicated by the cells morphology and pattern of gene expression. The untreated group exhibits enhanced microglial clumping, as shown by A and B. The transgenic treated and WT groups both express morphology that resembles resting microglia and lack any visible clumping. and exhibit resting microglia as noted by the small cell body, with evenly located thin. Scale bar = 100μm.

The extent of microglia activation was analyzed with anti-Iba1 antibody, a marker for microglia. The treated transgenic and wild type groups have resting microglia indicated by small cell body with equally distributed, thin, long, highly branched cellular processes, whereas the untreated transgenic group had reactive microglia, with large pleomorphic cell bodies that appear to clump around the amyloid plaque. Hence Ubisol-Q₁₀ treatment is able to bring about changes (activation of astrocytes and inhibition of microglia) in the morphology of glial cells in this transgenic AD mouse model and potentially alter disease pathology (Fig. 6B).

Ubisol-Q₁₀ treatment inhibited the loss of neurons in hippocampus in APP/PS1 transgenic mice

A significant loss of neurons in the hippocampal region has been found in humans with AD), and histological evidence indicates thinning of the stratum pyramidal layer as a result of neurodegeneration [34]. Therefore, we stained hippocampal sections in three animals from each group by an immunohistochemical procedure with anti-NeuN antibody, a marker for differentiated neurons as described previously [35]. There were fewer neurons in the CA1 region of the hippocampus in the untreated transgenic mice than in the Ubisol-Q₁₀-treated transgenic mice, which also had fewer neurons than wild-type mice (Fig. 7A). These findings indicate reduction of cell death in the hippocampus in the Ubisol-Q₁₀ treated mice (Fig. 7A).

Fig.7

Inhibition of loss of neurons in hippocampal region by Ubisol-Q₁₀ treatment in transgenic AD mice (A): Microscope images show NeuN immunohistochemistry staining indicating neuronal cells in the hippocampus of sections from different groups. Qualitatively, there were fewer neuronal cells in the untreated-transgenic mice compared to Ubisol-Q₁₀-treated group and wild type mice. The figure is representative of at least three sections stained for each animal and three animals in each group. Qualitative analysis of oxidative stress levels in the lower half of the brain (B). Microscope images show 4-HNE immunohistochemistry staining at 100x magnification. Untreated transgenic mice exhibit increased 4-HNE staining, reflective of oxidative stress levels, in comparison to both treated and wild type groups. Scale bar = 20μm.

Ubisol-Q₁₀ treatment reduces levels of oxidative stress in APP/PS1 transgenic mice

Oxidative stress has been shown to be implicated in the development AD pathology. We evaluated the changes in levels of oxidative stress using immunohistochemical staining for 4-HNE, a lipid peroxidation product that accumulates in the cell membrane due to oxidative stress, in the brain tissues of the Ubisol-Q₁₀ treated and untreated transgenic mice as well as the wild type. Results shown in Fig. 7B indicated that indeed there was an increase in oxidative stress level indicated by increased staining of 4-HNE in untreated transgenic mice whereas as it was significantly inhibited in the Ubisol-Q₁₀ treated mice. There was a basal level of 4-HNE staining in the wild-type mice. These results indicate that Ubisol-Q₁₀ treatment inhibited the burden of oxidative stress in transgenic mice.

DISCUSSION

Mitochondrial dysfunction and oxidative stress play a crucial role in the pathophysiology of AD and has been shown to precede the key pathological changes associated with the disease. Hence, development of antioxidant drugs that can help improve mitochondrial health holds promise as effective therapies for AD. We have previously shown that Ubisol-Q₁₀ is able to reduce the levels of ROS, ameliorate mitochondrial dysfunction, and delay premature senescence in PS1 mutated AD familial type 3 fibroblasts [17]. Recently research has been focused on level of CoQ₁₀ and its role in neuroprotection targeting mitochondria [36, 37].

In the current report, we have demonstrated the efficacy of prophylactic administration of Ubisol-Q₁₀ in altering behavioral deficits and pathology associated with the APP/PS1 transgenic AD mouse model. Recently we have reported unprecedented effectiveness of Ubisol-Q₁₀ as an inhibitor of Parkinsonian neurodegeneration in rodent models of disease at a very low dose of 6 mg/kg/day [10, 11]. Oral Ubisol-Q₁₀ feeding blocked almost instantly the neurodegenerative processes activated by MPTP or Paraquat [9 –11]. Although the etiological factors triggering AD and PD are different, they ultimately cause neuronal death by common mechanism(s). Therefore, it is conceivable that similar degree of neuroprotection can be achieved in AD.

Administration of Ubisol-Q₁₀ for a period of 15 months is able to decrease the load of circulating human Aβ₄₀ as well as provide near complete inhibition of human amyloid plaque formation in these transgenic mice genetically predisposed to form human amyloid plaque deposition at 6–8 months of age. The differences in the plaque deposition in the treated transgenic group (as shown in Fig. 5A, B) could be attributed to Ubisol-Q₁₀ being provided in their drinking water and differences in their water intake in their group cages. Although these results are qualitative and very clear, we did count the number of Congo-red stained amyloid plaques in five different vision fields and found that semi-quantitatively its was between 20–25 plaques/field in the untreated transgenic mice compared to only 5–9 plaques/field in the Ubisol-Q₁₀ treated mice.

Previous studies in our lab have shown that Ubisol-Q₁₀ is not toxic and is able to cross the blood-brain barrier in order to provide significant neuroprotection and halt neurodegeneration in rodent models of PD [9 –11]. The current findings suggest that Ubisol-Q₁₀ could be a novel treatment option for AD and could ameliorate the pathological features related to mitochondrial dysfunction, oxidative stress, and the accumulation of protein aggregation and autophagy. This formulation is very different and 50 times more effective than the oil-soluble CoQ₁₀ that failed the clinical trial. The effective doses for Oil-soluble CoQ₁₀ were 400 mg/kg/day in animal models, translating to 28 grams/day for a 70 kg person that could not be given [12, 38]. In contrast, the effective dose of Ubisol-Q₁₀ in animal model is 6 mg/kg/day, and could be easily given to patients (420 mg/day for a 70 kg person).

There could be multiple factors involved in the pathophysiology of AD as there is a wide array of clinical manifestations observed in patients suffering from AD. Currently there is no rodent model available that can fully mimic the decline in cognitive capacities as well as the key pathological features observed in AD. However, we were able to show that Ubisol-Q₁₀ was able to decrease the Aβ deposition in the APP/PS1 transgenic mice model. Studies suggest that an imbalance between accumulation and clearance of Aβ leads to an increase in the levels of Aβ₄₀ and Aβ₄₂, which has the tendency to aggregate and form plaques. There are two different schools of thought, which suggests that this plaque deposition could either precede or follow mitochondrial dysfunction and oxidative stress [39, 40]. Our results suggest that Ubisol-Q₁₀ is able to prevent the Aβ deposition by bringing about mitochondrial stability, improving the efficiency of energy production, and decreasing the levels of ROS (Fig. 7B), which also confirms the role played by mitochondria in ageing and in AD.

Previously we have shown that Ubisol-Q₁₀ treatment in the fibroblasts derived from AD patients could prevent premature senescence and induce autophagy [17]. It could be possible that Ubisol-Q₁₀ treatment of transgenic mice could accelerate depletion of oligomerized Aβ and complete digestion of these proteins via phagolysosomes. Therefore, the decrease in circulating Aβ (Fig. 4) could be indicative of efficient autophagy and clearance of Aβ in the brain.

4-HNE is a lipid peroxidation product that accumulates in the cell membrane following ROS exposure or increase in oxidative stress and has been used as a marker for general oxidative stress in the brain previously [53]. Our results clearly indicated that indeed there is an increase in 4-HNE staining in the transgenic mice that was significantly produced in the Ubisol-Q₁₀ treated mice. This confirming Ubsiol-Q₁₀ treatment inhibits oxidative stress in transgenic mice. Neurodegeneration in the hippocampus, one of the key pathological features of AD is not clearly observed in the APP/PS1 transgenic mice. However, we did find decreased numbers of NeuN positive neuronal cells in CA1 region of hippocampus in untreated transgenic mice compared to Ubisol-Q₁₀ treated mice. These results are similar to that observed previously by Ping et al. who demonstrated that APP23 transgenic mice had decreased number of neuronal cells in CA1 region ofhippocampus [41].

Interaction between neurons and glial cells is important for maintaining normal brain homeostasis and hence it is essential to elucidate their contribution and role in disease pathophysiology. PET studies suggest that astrocytes are highly activated in the initial stages of AD following which there is a decline [42]. Astrocytes could initially aid in slowing down the disease progression following which it is unable to continue this role due to the toxic environment created by Aβ and ROS. Astrocytes activation observed in the transgenic treatment group along with reduction in Aβ plaque shows that the neuroprotective property of astrocytes is resumed with Ubisol-Q₁₀. The mechanism of astrocytes activation is still unclear, but these cells could play a role in supporting the survival of neurons in the treated transgenic group by providing essential growth factors. Indeed, the neuroprotective role of astrocytes has been shown in two recent studies [43, 44].

In contrast to astrocytes there are resting microglia in the transgenic treatment mice in comparison with the untreated transgenic mice that have more reactive microglia clumping around Aβ plaques. The presence of reactive microglia, followed by increase in pro-inflammatory cytokines and neuroinflammation is a viscous cycle [19]. The presence of resting microglia in the treated transgenic group similar to the wild type group suggests that Ubisol-Q₁₀ is able to reduce the toxic effects posed by the Aβ in this transgenic mouse model.

Even though the mechanism of neuroprotection by Ubisol-Q₁₀ needs to be further elucidated, these results suggest the significant role of mitochondria and how reducing ROS to basal levels is effective in preventing mitochondrial dysfunction.

It is especially noteworthy that only non-treated transgenic mice in the present study showed spontaneous recovery of previously greatly habituated exploratory activity in the enclosed Y-maze and impaired spatial but not non-spatial object recognition working memory in that maze. Such greater dishabituation and impaired spatial working memory in our untreated transgenic mice extends and replicates findings from other research demonstrating behavioral disinhibition by transgenic mice to enter covered arms in the elevated plus maze and impaired spatial working memory but not spatial object recognition in the open field [45]. Moreover, such spatial memory deficits along with our observations of changes in the hippocampus in untreated transgenic mice is consistent with earlier extensive research supporting and extending O’Keefe and Nadel’s (1978) original idea about the involvement of this structure in spatial cognition [46 –48]. Perhaps the major importance of the present study is how the transgenic animal model of AD can be applied to preclinical assessment of possible treatments of AD. To accomplish this goal requires recognition that human AD is characterized by gradual progressive cognitive disintegration typically beginning with impaired spatial working memory preceding that of non-spatial working memory [49]. Therefore, future research with this animal model of AD requires investigation of tracking the development of similar declines in reference (long-term) and different types of working (short-term) memory over these animals’ life span. Thus, we need to determine whether dishabituation of exploratory activity increases rather than declines and whether deficits in object recognition working memory extend from its spatial to non-spatial aspects over repeated testing in aging untreated transgenic mice compared with that in aging treated transgenic and normal untreated wild type mice.

Footnotes

ACKNOWLEDGMENTS

Authors are grateful to Mr. Joseph Szecsei, Magdalena Szecsei and their family for their generous donation and establishing of the Szecsei Parkinson and Neurodegenerative Disease Research Fund in support of this study. We acknowledge grants from the University of Windsor’s Faculty of Science and Faculty of Arts, Humanities, and Social Science. Finally, we acknowledge Iva Okaj and Lauren Culmone for their technical assistance.

Authors’ disclosures available online ().

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Ubisol-Q 10 (a Nanomicellar Water-Soluble Formulation of CoQ 10 ) Treatment Inhibits Alzheimer-Type Behavioral and Pathological Symptoms in a Double Transgenic Mouse (TgAPEswe,PSEN1dE9) Model of Alzheimer’s Disease

Abstract

Keywords

INTRODUCTION

MATERIALS AND METHODS

Animal care

Control and treatment regimen

Enzyme linked immunosorbent assay

Antibodies

Immunohistochemistry

Congo red staining

Behavioral testing

Apparatus and materials

Spontaneous alternation and long-term reference memory task

Working memory

Statistical analyses

RESULTS

Ubisol-Q10 maintains reference (long-term) memory in double transgenic mice

Ubisol-Q10 only preserves working spatial memory in double transgenic mice

Ubisol-Q10 treatment caused a decrease in the circulating Aβ in APP/PS1 transgenic mice

Ubisol-Q10 treatment resulted in drastic decrease in the Aβ plaques the brain of APP/PS1 transgenic mice

Ubisol-Q10 treatment resulted in activation of astrocytes and inhibition of microglial activation in APP/PS1 transgenic mice

Ubisol-Q10 treatment inhibited the loss of neurons in hippocampus in APP/PS1 transgenic mice

Ubisol-Q10 treatment reduces levels of oxidative stress in APP/PS1 transgenic mice

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

Ubisol-Q₁₀ maintains reference (long-term) memory in double transgenic mice

Ubisol-Q₁₀ only preserves working spatial memory in double transgenic mice

Ubisol-Q₁₀ treatment caused a decrease in the circulating Aβ in APP/PS1 transgenic mice

Ubisol-Q₁₀ treatment resulted in drastic decrease in the Aβ plaques the brain of APP/PS1 transgenic mice

Ubisol-Q₁₀ treatment resulted in activation of astrocytes and inhibition of microglial activation in APP/PS1 transgenic mice

Ubisol-Q₁₀ treatment inhibited the loss of neurons in hippocampus in APP/PS1 transgenic mice

Ubisol-Q₁₀ treatment reduces levels of oxidative stress in APP/PS1 transgenic mice