Examining Alzheimer’s Disease modifiable risk factors: Impact of physical activity and diet on neuroanatomy and behaviour in mouse models

Reviewer #1 (Public review):

A triple-transgenic (3xTgAD) mouse model of Alzheimer's disease was exposed to a high-fat diet and assigned to one of three interventions: voluntary physical activity, a low-fat diet, and their combination. A high-fat diet significantly increased body weight and induced widespread neuroanatomical changes, with effects modulated by sex and genotype. The combined intervention led to significant weight loss in males of both genotypes. Neuroanatomical analyses revealed that a high-fat diet significantly reduced hippocampal and cerebellar volumes in wild-type mice but had a less pronounced effect on 3xTgAD mice; nevertheless, interventions, particularly the combined approach, increased localized brain volumes in these regions regardless of genotype. Spatial gene enrichment analysis of this pattern identified glucose homeostasis. Overall, these findings suggest that voluntary physical activity and a low-fat diet can modulate brain structure and behaviour, partially counteracting the effects of a high-fat diet, and potentially recruiting biological processes that may support brain health.

The authors describe studies of the 3xTg mouse model of Alzheimer's disease (AD). They set out to study the interactions of diet and exercise on three outcomes: weight gain, MRI, and either the novel object recognition or Morris water maze tasks of memory.

They conclude there are sex and genotype effects on hippocampal volume.

There are several strengths to the study. First, they start out with a great deal of mice. Once they are divided into groups, the sample sizes are not always strong, however. It would be good to know that they were sufficiently powered.

The data are also interesting. Mice were placed on several different diets during the study, which will be of interest to many who question the role of diet in outcomes. They also add exercise as an intervention, and study not only diet but also the combined effect of diet and exercise. This is relevant to those interested in controlling dementia by diet and exercise. Finally, they perform some very interesting analyses to study the data.

That said, the study also has several limitations. For example, it is quite complex. Mice had a standard diet until 2 months of age, then were switched to either a low-fat or a high-fat diet. Some mice had both a different diet and exercise. MRI was performed at 2, 4, and 6 months, when behavior was tested. A drawback of this design is that no assessment of outcomes relevant to this animal model, such as amyloid-beta or tau phosphorylation, was conducted. Also, they used the novel object recognition task, despite stating in the Discussion that this task does not show impairments until well after 6 months of age. They added exercise, but it is not clear whether the animals used the exercise apparatus equally. Also, the animals were housed "communally", so adding an exercise wheel may have made the cage crowded, adding stress to the study. The diets were not simply low- or high-fat because many constituents besides fat content also changed. Regarding fat, the type of fat also changed between diets. Therefore, the gut microbiome was probably affected differently by factors other than fat intake. There was no measurement of food consumption, so some mice may not have eaten as much of the new diet as they did of the old diet they were used to.

Regarding the data, only the outcomes of complex analyses are shown. One would first want to see the changes in body weight and perhaps later how it is analyzed in a more complex way. For behavior, one would first want to see outcomes as typically presented. For example, learning, recall, platform test results from the Morris water maze, and discrimination indices for object recognition. Note that, at one point, I believe the authors note that some groups did not explore thoroughly, which would make novel object recognition hard to interpret. If there was any difficulty with ambulation, both tasks would be hard to interpret.

Regarding MRI, from what can be seen, structures cannot be distinguished clearly. At least some raw data should be shown to demonstrate this and to determine what the data show. The raw data suggest that some of the larger structures can be distinguished, and we should see the data for these areas, even if all areas can't be assessed. Lifestyle interventions can mitigate the effects of diet-induced obesity on body weight, behaviour, and brain anatomy in mouse models. Using a longitudinal design, wild-type and triple-transgenic (3xTgAD) mouse models of Alzheimer's disease were exposed to a high-fat diet and assigned to one of three interventions: voluntary physical activity, a low-fat diet, and their combination. A high-fat diet significantly increased body weight and induced widespread neuroanatomical changes, with effects modulated by sex and genotype. The combined intervention led to significant weight loss in males of both genotypes. Neuroanatomical analyses revealed that a high-fat diet significantly reduced hippocampal and cerebellar volumes in wild-type mice but had a less pronounced effect on 3xTgAD mice; nevertheless, interventions, particularly the combined approach, increased localized brain volumes in these regions regardless of genotype. Multivariate integration of behavioural and neuroanatomical measures identified a brain pattern linking hippocampal and cerebellar volumes to intervention and behavioural performance. Spatial gene-enrichment analysis of this pattern identified biological processes, including glucose homeostasis, as potential biological mechanisms underlying intervention effects. Overall, these findings suggest that voluntary physical activity and a low-fat diet can modulate brain structure and behaviour, partially counteracting the effects of a high-fat diet, and potentially recruiting biological processes that may support brain health. In the end, the authors focus primarily on the hippocampus and discuss the cerebellum, but it seems that changes occur throughout the brain. The choice to focus on the hippocampus and cerebellum needs to be supported.

To gain further insight, the authors analyze genes across different brain regions using the Allen Brain Atlas. Although this seems reasonable in theory, once one realizes how many genes are shared across diverse brain regions, one wonders how such an analysis was conducted. More understanding of this approach, as well as how it was validated, is important. In the end, the authors conclude that the glucose homeostatic pathways were primarily altered, and one would like to understand whether that is indeed true and whether it is the only set of pathways that were changed.

This raises another point: what occurs in a normal wild-type mouse on the standard diet during the first 6 months of life? Do the glucose homeostatic pathways change simply due to age? Sex? It may be that, with age, the mice become more sedentary, which is why. Once that is resolved, what occurs on the standard diet for the 3xTg mice? Perhaps they are more active or more sedentary, regardless of diet or exercise? Thus, the studies end up raising more questions than answers.

Given so much work has already been done, it seems best to simply reorganize the presentation with raw data first, followed by the analysis. For the second section, the implicit assumptions of the analyses should be very clear so that the analyzed data are understood and believable. Limitations of the assumptions, pooling some groups, etc., need to be clear.

Figures. In Figure 1, the weekly measurements are not shown. The points are connected, so an unbroken line is shown. Around the line are lighter lines indicating errors, but with all the lines and colours, one does not know what standard errors surround the values for any given group. This makes the data hard to interpret. In later figures, significant differences are indicated with asterisks, but this seems to be done inconsistently.

In the text, more caution is needed for some assertions. For example, it is not clear that a 2- to 6-month-old is an adolescent. Opinions about the ages of mice that correspond to human life stages have always been debated. Another example is indicating that male mice might gain weight differently than females, as if it were an outcome of diet or exercise. This is because male rodents continue to gain weight in adulthood, but females stabilize because estrogen limits appetite. Additionally, females may not show group differences because they are more variable. This can relate to their estrous cycle. If stressed or housed without males nearby, they may not have a regular estrous cycle, which can then affect their outcomes. This may be particularly true for behavior when they may have been tested during different estrous cycle phases, if they had estrous cycles.

Reviewer #2 (Public review):

Summary:

This manuscript describes an investigation into the effect of diet and exercise interventions in WT and transgenic (male and female) mice who are exposed to either a high-fat or a low-fat diet. The outcome variables include MRI volume and brain morphology, as well as memory performance. First, this study measured the impact of genotype (WT vs 3xTgAD mice), then examined the impact of a high-fat or low-fat diet in each group, and finally examined the impact of a low-fat diet, exercise, or a combined low-fat diet and exercise intervention. This is an important study as it allows us to better understand how changes to lifestyle can affect neurocognitive function and potentially change a person's AD risk.

Strengths:

(1) The study uses a well-controlled longitudinal design, allowing the authors to track how diet and exercise interventions influence brain and behaviour over time.

(2) The integration of multiple levels of analysis (brain imaging, behaviour, and multivariate modelling) provides a rich and comprehensive assessment of intervention effects.

(3) The inclusion of both genotype and sex as key variables strengthens the relevance and interpretability of the findings, given known differences in risk and response across groups.

Weaknesses:

There are a lot of analyses in this paper, and I had a little bit of trouble distilling the major take-home messages. For example, I was left wondering:

(1) If the effect of genotype and the effect of the high-fat diet were consistent in the current study compared to the authors' previous work (e.g. Rollins et al., 2019). A more direct report on the consistency of these findings (maybe even an overlap map, if possible) would benefit the reader.

(2) How consistent/different are the volumetric and morphometric (DBM) results from each other? Especially in the regions of interest (hippocampus and cerebellum), are increases in volumes always related to "expansion" of a given region using DBM? Some of the similarities are reported in the results, but for transparency, a side-by-side table comparing the results across techniques for each effect of interest might provide more clarity.

(3) I was interested in the Partial Least Squares approach that the authors used to investigate how patterns of brain measures relate to the behavioral variables. Because they are presented mostly in the supplement (except for Figure 6E), it's difficult to map the LVs described onto the univariate contrasts in Figures 2-5. In general, greater clarity is needed regarding how the PLS-derived latent variables relate to the univariate findings, and whether the emphasis on LV3 reflects a principled selection or post hoc interpretation.

(4) If I understand the results correctly, there were only modest differences in behavior reported, and the patterns were somewhat inconsistent across sex and genotype. In fact, the authors report that the high-fat diet alone did not impair memory on the Morris Water maze (line 323). The discrepancy between robust neuroanatomical effects and relatively modest behavioural changes raises important questions about the functional significance of the observed structural alterations.

(5) On line 507, the authors state, "Notably, 3xTgAD mice already show smaller brain volumes at baseline, which may constrain the detectable impact of the diet." Is this true for the entire brain or just the hippocampus and cerebellum? Would a global reduction in brain volume due to the 3xTgAD AD model affect the interpretation of the intervention effects?

Reviewer #3 (Public review):

Summary:

The authors sought to determine the individual and combined effects of exercise and low-fat diet consumption on regional brain volume and cognitive function in triple-transgenic Alzheimer's disease mice and wild-type controls.

Strengths:

(1) A strength of this study is its longitudinal design, which captures regional changes in brain volume across the interventions tested.

(2) Its comprehensive design includes 10 groups and is well-powered to isolate genotype-, sex-, diet- and exercise-related effects (and interactions).

(3) The analyses of volumetric and voxel-based measures are comprehensive.

Weaknesses:

(1) Use of automated tracking for NOR data reduces confidence in the behavioural data.

(2) No measures of Ab or tau pathology appear to be performed.

(3) Mice from the critical 'combined' intervention groups are not included in the PLS regression model that integrates behavioural and brain data.

(4) Analyses of behavioural data include a large number of variables without adequate justification.