Journal of Alzheimer's Disease 2018; 64 (3): 835857
Hutton, Craig P., Lemon, Jennifer A, Sakic, Boris, Rollo, C. David, Boreham, Douglas R, Fahnestock, Margaret, Wojtowicz, J. Martin, Becker, Suzannaa
Department of Psychology,
Neuroscience & Behavior,
Hamilton, ON, Canada.
The increasing global burden of Alzheimer's disease (AD) and failure of conventional treatments to stop neurodegeneration necessitates an alternative approach. Evidence of inflammation, mitochondrial dysfunction, and oxidative stress prior to the accumulation of amyloid-? in the prodromal stage of AD (mild cognitive impairment; MCI) suggests that early interventions which counteract these features, such as dietary supplements, may ameliorate the onset of MCI-like behavioral symptoms. We administered a polyphenol-containing multiple ingredient dietary supplement (MDS), or vehicle, to both sexes of triple transgenic (3xTg-AD) mice and wildtype mice for 2 months from 24 months of age. We hypothesized that the MDS would preserve spatial learning, which is known to be impaired in untreated 3xTg-AD mice by 4 months of age.
Behavioral phenotyping of animals was done at 12 and 34 months of age using a comprehensive battery of tests. As previously reported in males, both sexes of 3xTg-AD mice exhibited increased anxiety-like behavior at 12 months of age, prior to deficits in learning and memory, which did not appear until 34 months of age. The MDS did not reduce this anxiety or prevent impairments in novel object recognition (both sexes) or on the water maze probe trial (females only). Strikingly, the MDS specifically prevented 3xTg-AD mice (both sexes) from developing impairments (exhibited by untreated 3xTg-AD controls) in working memory and spatial learning. The MDS also increased sucrose preference, an indicator of hedonic tone. These data show that the MDS can prevent some, but not all, psychopathology in an AD model.
Keywords Alzheimers disease; anhedonia; anxiety; dietary supplements; learning; memory; mice; mild cognitive impairment; reversal learning; transgenic; working memory
From the FULL TEXT Article:
Alzheimers disease (AD) afflicts over 33 million
people worldwide (http://www.who.int) at a cost of
US$ 604 billion and is projected to reach 135 million cases by 2050. Conventional treatments, such as
cholinergic drugs, fail to stop progression, highlighting the need for new treatments . In AD, the rapid decline in cognitive ability reflects damage to brain regions important in learning, memory and mood regulation, notably the hippocampus and prefrontal cortex . Thus, AD is also associated with high
39 levels of depression (43.6%) and anxiety (25.4%) far exceeding those in age-matched controls .
Most research into AD pathogenesis has focused on counteracting the accumulation of misfolded proteins amyloid-β (Aβ) and hyperphosphorylated tau that contribute to plaques and tangles throughout the brains ofADpatients. Thiswork has led to the prominent amyloid cascade hypothesis of AD [6, 7]. Briefly, the cascade hypothesis proposes that excessive production or impaired clearance of Aβ leads to its subsequent accumulation, which triggers a neurotoxic cascade resulting in tau hyperphosphorylation and aggregation, synaptic atrophy, inflammation and oxidative damage, Ca2+ imbalance and ultimately neuronal death.
The amyloid cascade hypothesis is well supported 55 by evidence from in vitro and animal studies . However, it appears to have some serious limitations, most notably the fact that all anti-amyloidogenic
drugs have thus far failed in human clinical trials [10, 11]. Furthermore, downstream effectors such as synapse loss  and reduced levels of brain derived neurotrophic factor (BDNF) [13, 14], rather than plaque or tangle burden, are better predictors of cognitive symptoms in AD patients. Moreover, an emerging literature implicates exacerbation of age-related physiological changes in cognitive decline and AD. Specifically, the preclinical stage of AD, mild cognitive impairment (MCI) , has been linked to elevated levels of inflammation , insulin resistance [17, 18] and oxidative stress , and reduced levels of BDNF  and brain glucose metabolism . Therefore, in contrast to most other studies on AD model animals [21, 22], we chose to focus on prevention rather than treatment.
Lifestyle based approaches to MCI and AD prevention, such as nutritional supplements, are emerging as alternatives to pharmaceutical compounds [1, 11]. For example, 6 months of supplementation with the omega-3 fatty acid docosahexaenoic acid (1.16 g/d DHA) in healthy adults was found to improve episodic memory, working memory, and attention . Further, in a 68 year longitudinal study, the risk of AD was 60% lower in thosewhodrank polyphenol-containing fruit or vegetable juices at least 3x per week .
Such findings suggest that a combination of polyphenols and unsaturated fatty acids may protect
against MCI and consequently AD. We propose that a particularly promising approach is to use a broad- based multiple ingredient dietary supplement (MDS; Table 1) developed to target age-related alterations in inflammation, oxidative stress, mitochondrial dysfunction, insulin resistance and membrane integrity , all of which are also implicated in AD disease progression as discussed above. This MDS has been shown to reduce age-related declines in spatial learning, brain volume, neuronal atrophy, neuronal death and DNA damage in aged mice [25, 26].
The current study administered the MDS to both sexes of 3xTg-AD mice  and wildtype (WT) mice and compared their behavior across a wide range of measures (Table 2) to vehicle-treated 3xTg-AD and
WT controls. This battery of tests was designed to clarify which behavioral alterations appear in this mouse model at the earliest stages; most previous studies examined behavioral outcomes at 6 months of age or later (e.g., ), reported conflicting sex differences (e.g., [34, 35]), used only a single behavioral measure starting at 24 months of age [34, 36], or evaluated only one sex (e.g., [37, 38]).
The importance of including both sexes is becomng increasingly appreciated, as male and female 3xTg-AD mice exhibit different trajectories of Aβ accumulation  and behavioral abnormalities [33, 35]. Behavioral alterations, progressing from anxiety to learning and memory impairments, emerge in males at 24 months of age [30, 36, 38, 40, 41]), while females develop similar symptoms at 46 months of age [33, 42, 43]. After initial presentation, it is unclear whether memory deteriorates more rapidly in males or females, as different behavioral tests have yielded conflicting results (e.g., [34, 35]). Behavioral deficits are followed by the emergence of amyloid and tau accumulation starting at 36 months of age, with more rapid accumulation in females . Plaques appear later, at 814 months of age in females and 1618 months of age in males [38, 39, 44, 45, 48]. Still later, tangles emerge at 1618 months of age in females [44, 48] and 2126 months of age in males [46, 49].
Given the rate of disease progression described above,we anticipated that by 34 months of age 3xTg-AD mice would exhibit anxiety-like behavior (males only; ) and selective deficits on the Morris water maze (MWM; both sexes ). Moreover, we predicted especially pronounced deficits in the MWM for the high interference reversal trials, during which the mouse is required to neglect the original location of the submerged platform and learn a new location. High interference learning is markedly impaired in MCI patients  and has been shown previously to be dependent upon hippocampal neurogenesis in animal models [51, 52]. Moreover, adult neurogenesis and BDNF are among the earliest biomarkers depleted in rodent models of AD (by 24 months of age ). Given that the MDS (and similar supplements) upregulate neurogenesis [57, 58] and BDNF [57, 5961], we hypothesized that the MDS would ameliorate deficits in the 3xTg-AD mice on water maze reversal trials.
While we have previously observed benefits of the MDS on the age-related decline in spatial learning and memory among wild-type mice following an extended period of supplementation (?1 year ), the present study demonstrates that similar benefits can be obtained in an AD mouse model with only 2 months of supplementation. That strong benefits can be observed after such a short intervention period is remarkable considering that most similar studies (e.g., [21, 22, 103]) report positive results after longer treatment periods (at least 36 months). Specifically, we show here that the MDS prevents the emergence of deficits in working memory and reversal learning that occur in 3xTg-AD by 4 months of age. The reversal learning deficit is expected as part of a human-like MCI phenotype (see Methods for a description), whereas the working memory impairment seems to emerge earlier in 3xTg-AD mice  than in humans . Thus, the MDS may impart a degree of resilience against the deterioration of learning and memory in AD.
On the spontaneous alternation test, we also did not observe a decrease in performance when the starting viewpoint was shifted (i.e., alternation rate on days 15 versus day 6). This suggests the use of an allocentric rather than egocentric navigation strategy . The particular strategy used was of interest, since (in humans) allocentric navigation preferentially engages the hippocampus, while egocentric navigation depends more on parietal association and striatal areas. Furthermore, there is a shift toward egocentric navigation with age  that may reflect changes in hippocampal volume that also occur with age and in AD . Thus, the decreased alternation rate among 34-month-old control 3xTg-AD mice is not due to a change in navigation strategy (i.e., from allocentric to egocentric).
Evidence from the literature suggests that performance on the reversal trials of the water maze depends upon adult hippocampal neurogenesis  (although see ). More generally, adult neurogenesis seems to be important for hippocampal-dependent learning under conditions of high interference [51, 52, 111]. Similarly, in AD patents, deficits on a high-interference, picture-matching memory test are also correlated with Aβ levels . Promoting neurogenesis may therefore impart resilience against Aβ toxicity. In addition, the benefit of the MDS on reversal learning shown here may be due to preserved levels of neurogenesis in MDS-supplemented 3xTg-AD mice, which merits further investigation.
Our wide-ranging battery of tests across different ages has also yielded a more detailed and comprehensive picture of the early behavioral trajectory of the 3xTg-AD mouse than has been available thus far. To our knowledge, this is the first study aimed at preventing the development of symptoms in an AD mouse model using a complex supplement that was administered starting at 2 months of age. The fact that not all our results were positive suggests that the use of supplements alone may not be sufficient to prevent AD onset. Similarly, at least two other recent studies used complex supplements (at least 5 ingredients) in AD model mice. In one , a combination of 11 ingredients, 10 of which are also in the MDS used here, was administered to Tg2576 mice for 6 months starting at 6 months of age (behavioral deficits begin at 3 months of age in this strain). Benefits of varying magnitude were observed for all 3 measures studied: the novel object test and water maze, and levels of Aβ oligomers in whole brain homogenates. However, without the use of a more comprehensive behavioral battery of tests, it is unknown if the treatment could impact other aspects of an AD-like phenotype such as sensory/motor function or emotionality (see below for more information). Another study  using 3xTg-ADmice reported sex-dependent benefits of 4 months of treatment starting at 710 months of age on mitochondrial function (cytochrome C oxidase activity; males only) and a delayed-match-to-position, short-term memory task (only a 30 s delay; males only), using a 29component supplement containing 5 similar ingredients to the MDS(turmeric/curcumin, green tea, ginger, fish oil, and vitamin D). However, the treatment did not improve memory deficits exhibited by the 3xTg-AD mice over longer periods (30 min or 24 h). Additionally, mitochondrial function was reduced in supplemented females, highlighting the importance of studying both sexes during pre-clinical experiments.
Our results suggest that, prior to 4 months of age, the 3xTg-AD mouse models the human behavioral phenotype of an MCI-like syndrome reasonably well, although not perfectly. For instance, the 3xTg-AD mouse did exhibit anxiety-like behavior (both sexes) Ad deficits in olfactory acuity (females only), but not motor co-ordination impairments, prior to 4 months of age. These changes in olfaction and anxiety level were the earliest behavioral symptoms to appear in these animals, developing prior to the spatial learning, working memory, or object recognition memory impairments at 34 months of age. Although symptoms of depression and anxiety are both common in patients with dementia , our results suggest the possibility that, among behavioral symptoms, anxiety or olfactory deficits may appear earlier than anhedonia or memory impairments in some older adults who later develop MCI. These findings are in agreement with those of Marchese et al.  who reported simi1lar findings in male 3xTg-AD mice only. In that study, compared to WT males, 3xTg-AD males exhibited elevated anxiety-like behavior on the step-down test at 1.5 months of age and increased olfactory acuity starting at 6 months of age. Here, we confirm that the anxiety-like behavior is also present in 3xTg-AD females at 12 months of age, and we show that olfactory alterations (increased sensitivity in males, decreased sensitivity in females) are present in 3xTg-AD mice at 12, but not 34, months of age. Our finding of a beneficial effect of the MDS on sucrose preference also suggests that supplementation with the MDS may help protect older adults at risk for MCI from developing anhedonia or other depressive symptoms.
Regarding the observed anxiety, there is evidence of increased HPA axis activation in 34-month-old 3xTg-AD mice due to upregulation of glucocorticoid and mineralocorticoid receptors, but not corticosteroid levels, in CA3 and the DG of the hippocampus . If this upregulation is also present at 12 months of age in this strain, then it might explain the increased anxiety in transgenic mice evident in our plus maze and open field data andwould merit further research. Additionally, the stress response (increased corticosterone levels) of 3xTg-AD females, but not males, following water maze testing is increased over WT females between 615 months of age , emphasizing the relevance of comparing performance on tests of spatial memory to anxiety measures in both males and females when interpreting treatment effects.
The MDS did not reverse this pre-existing anxiety or prevent the appearance of deficits in novel object recognition (both males and females) or long-term recall on the water maze probe trials (females only) in 3xTg-AD mice by 34 months of age. Previous work with the MDS in 12month-old C57BL6 mice demonstrated superior object recognition memory following 9+ months of supplementation, suggesting that longer treatment periods may have a positive impact on recognition memory in 3xTg-AD mice as well . In fact, given the strength of some results with our relatively short treatment period suggests that some of the AD features that proved refractory to supplementation might still showsome benefit by a longer treatment period. It also appears that the MDS negatively impacts olfactory acuity in 34-month-old females of both genotypes. This was surprising, since previous work on the MDS has found beneficial effects on olfactory function in older animals
(9 months and older [26, 27]), suggesting that this side effect is age-dependent and is unlikely to be an issue if treatment is started at a later age in wild type animals. The sources of these deficits require further investigation.
Our recent work suggests that the MDS in combination with aerobic exercise  may yield greater benefits than either treatment alone, particularly when elevated levels of stress are involved in a disease phenotype, such as is the case for the 3xTg-AD mouse [35, 113]. Again, the benefit of supplementation (and perhaps the interaction with exercise) may have been improved with a longer supplementation period. With respect to the current water maze, if 3xTg-AD females experience a greater stress response to testing than males at 34 months of age, it could explain why they, but not 3xTg-AD males, exhibited a deficit in long term memory based on the water maze probe trials that was not responsive to the MDS alone. Given that impairment in 3xTg-AD females on the probe trials was relatively small (i.e., they could still remember the platform location), we recommend that future studies of young 3xTg-AD mice use converging evidence from additional performance measures, some of which (e.g., platform crossings or average proximity to the platform) are more sensitive to group differences than the time spent in the target quadrant .
Our previous finding that both exercise and the MDS, but neither alone, were sufficient to improve anhedonia, hippocampal neurogenesis, BDNF, and hippocampal size in chronically stressed mice  suggests that theMDSand exercise together may prevent the deficit in probe trial performance observed here in 3xTg-AD females. This is consistent with other literature showing increased effects of combined diet and exercise [59, 116, 117].
Taken together, the results presented here suggest that as little as 2 months of supplementation with the MDS protects cognitive functions such as working spatial memory in juvenile 3xTg-AD mice. These functions are supported by highly plastic structures such as the prefrontal cortex and hippocampus that are affected most severely in AD . However, such a short period of supplementation was insufficient to preserve recognition memory or reverse pre-existing anxiety, functions which depend more on other brain areas such as the perirhinal cortex (supports object recognition ) or amygdala (affects anxiety-like behavior ). Future studies are indicated to determine if longer periods of supplementation or combination with exercise can reverse these other impairments or impact AD-relevant biomarkers (e.g., amyloid-β or hyperphosphorylated tau or BDNF). Regardless, the present data suggest that dietary supplements can obtain remarkable results, at least in mice. Ours and other studies suggest that supplements may serve as a key part of a multi-pronged intervention program to protect against AD-related behavioral changes.