Microbiota-derived short chain fatty acids promote Aβ plaque deposition
This article has been Reviewed by the following groups
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
- Evaluated articles (eLife)
Abstract
Previous studies have identified a crucial role of the gut microbiome in modifying Alzheimer’s disease (AD) progression. However, the mechanisms of microbiome-brain interaction in AD, including the microbial mediators and their cellular targets in the brain, were so far unknown. Here, we identify microbiota-derived short chain fatty acids (SCFA) as key metabolites along the gut-brain axis in AD. Germ-free (GF) AD mice exhibit a substantially reduced Aβ plaque load and markedly reduced SCFA plasma concentrations; conversely, SCFA supplementation to GF AD mice was sufficient to increase the Aβ plaque load to levels of conventionally colonized animals. While Aβ generation was only mildly affected, we observed strong microglial activation and upregulation of ApoE upon the SCFA supplementation. Taken together, our results demonstrate that microbiota-derived SCFA are the key mediators along the gut-brain axis resulting in increased microglial activation, ApoE upregulation and Aβ deposition.
Article activity feed
-
###Reviewer #3:
In this study, the authors present data aimed at supporting their conclusion that microbiota-derived SCFA resulting in increased AD pathology, including microglial activation, ApoE upregulation, and A-beta deposition.
First and foremost, the biggest issue with this study is the lack of male versus female comparisons and the very small sample sizes of the mice. Especially given the past literature of microbiome effects on AD pathology, e.g. with antibiotic cocktails, it is essential to look at sufficient numbers of both female and male mice, individually, and not just group them. Moreover, the average number of mice used in each experiment (N=5) are relatively small for making any firm conclusions.
Specific concerns:
Figure 1D: Based on the observation of more smaller plaques in SPF mice vs GF mice, the authors conclude, …
###Reviewer #3:
In this study, the authors present data aimed at supporting their conclusion that microbiota-derived SCFA resulting in increased AD pathology, including microglial activation, ApoE upregulation, and A-beta deposition.
First and foremost, the biggest issue with this study is the lack of male versus female comparisons and the very small sample sizes of the mice. Especially given the past literature of microbiome effects on AD pathology, e.g. with antibiotic cocktails, it is essential to look at sufficient numbers of both female and male mice, individually, and not just group them. Moreover, the average number of mice used in each experiment (N=5) are relatively small for making any firm conclusions.
Specific concerns:
Figure 1D: Based on the observation of more smaller plaques in SPF mice vs GF mice, the authors conclude, "This result highlights the impact of bacterial colonization on early amyloid plaque deposition rather than plaque growth," The problem here is that these mice are 5 months old. It is well-known that SPF APPPS1 mice start depositing at only 6 weeks old. So, they would need much earlier (and later) time points to support this conclusion. In addition, N=5 animals/group is very small and not appropriate for making conclusions.
The authors also need to show total plaque burden distribution in each group and level of variability?
Figure 2: Again, N=5/group is very small for high impact paper. They also need to show plaque burden distribution, especially since there is much more variability in 3 month old animals.
Figure 2D: The authors claim SCFA brings up plaque load to a "significant increase", i.e., 2X GF levels. But what are these values compared to SPF animals? They would need to have data on the 3 month SPF group for comparison sake to make the claim that SCFAs are driving pathology. Otherwise, this is just not convincing.
Figure 3: Westerns should also include 3 month SPF animals. The small differences in CTFalpha and CTFbeta are not convincing. Even if there were a change, how does it account for elevated Abeta?
Figure 4: SCFA trigger microglial activation: the data in this figure fail to support this conclusion:
Fig 4B: Why did the authors perform in situ for CX3CR1 instead of Iba 1 ICC for microglia? The quantification is unconvincing. There should be other CX3CR1 microglia that are not plaque associated, but we don't see these in the field. This brings into question the sensitivity of the in situ analyses? They need to also do Iba1ICC.
Fig 4 C/D: Regarding the statement, "we directly investigated the influence of bacterial colonization on microglial reactivity in the WT background. To this end, we injected brain homogenates from 8 months old APPPS1 mice containing abundant Ab into the hippocampus of GF or SPF WT mice (Fig. 4C) and subsequently analyzed microglial abundance and activation by smFISH. We observed a significant increase in overall microglial cell counts at the peri-injection site of SPF compared to GF WT mice (Fig. 4D)."
This experiment does not support the conclusion since one would expect microglial reactivity to increase in this experimental paradigm. The authors claim more activation in SPF mice, thus "gut microbiome triggers microglial activation and reactivity towards an exogenous insult containing Ab". But, this is unfortunately not supported by the experiments, as performed.
Figure 4F: The ex vivo amyloid clearance assay is not useful or convincing since cultured microglia lose their transcriptional phenotype after 6 hrs in culture (Gosselin et al, Science 2017).
-
###Reviewer #2:
This is an interesting and well-written paper on the relationship between gut microbiota metabolites and AB production. Although previous studies have documented a link between the gut microbiome and Ab pathology, the underlying mechanisms and molecular mediators remain elusive. Here the authors use a germ-free Alzheimer's Disease mouse model to examine the role of short chain fatty acids on amyloidogenesis and neuroinflammation.
The studies thus add another welcome piece to the puzzle of how the microbiota affects the brain.
My comments are relatively minor:
How do the behaviour of GF animals compare with non-GF animals given that cognitive deficits have been reported in them (Gareau et al., 2011)?
I am somewhat surprised that more metabolite differences were not observed between GF & SPF mice as all microbial …
###Reviewer #2:
This is an interesting and well-written paper on the relationship between gut microbiota metabolites and AB production. Although previous studies have documented a link between the gut microbiome and Ab pathology, the underlying mechanisms and molecular mediators remain elusive. Here the authors use a germ-free Alzheimer's Disease mouse model to examine the role of short chain fatty acids on amyloidogenesis and neuroinflammation.
The studies thus add another welcome piece to the puzzle of how the microbiota affects the brain.
My comments are relatively minor:
How do the behaviour of GF animals compare with non-GF animals given that cognitive deficits have been reported in them (Gareau et al., 2011)?
I am somewhat surprised that more metabolite differences were not observed between GF & SPF mice as all microbial metabolites should be only in the latter.
Fig 2B should include all metabolites tested individually
Were the concentrations of the metabolites increased in the plasma following administration in drinking water? The physiological relevance of the doses used in the rescue experiments could be better supported with experimental data
If acetate is most important then it is not clear why they used a pooled cocktail in rescue experiments.
The analysis of transcriptome of brain samples from control- and SCFA-supplemented GF APPPS1 mice is a nice addition but the molecular targets for SCFAs on microglia remains unresolved.
The comments about modulating dietary fibre to reduce central SCFA concentrations are provocative and although beyond the scope of the current study are clearly studies that would be very welcome for the field to test.
The potential effects of SCFAs on HDACs is completely left as a cliff-hanger...
-
###Reviewer #1:
The authors do a good job of citing the prior literature; however, Harach et al., 2017 did diminish my enthusiasm as it covers much of the same ground as this study, limiting the novelty of the current findings.
Essential Revisions:
Experimental perturbation of the proposed pathway. The manuscript leads to a nice model; however, the data is descriptive in nature with any experiments using either genetic or pharmacological approaches to test the proposed mechanisms. The impact of this study would be increased substantially if at least one link between SCFAs and AB, microglia, or ApoE were experimental validated. While most of the text avoids making causal claims based on correlative evidence, the one sentence summary states that SCFAs impact disease "via activation of microglial cells and upregulation of ApoE."
Identify …
###Reviewer #1:
The authors do a good job of citing the prior literature; however, Harach et al., 2017 did diminish my enthusiasm as it covers much of the same ground as this study, limiting the novelty of the current findings.
Essential Revisions:
Experimental perturbation of the proposed pathway. The manuscript leads to a nice model; however, the data is descriptive in nature with any experiments using either genetic or pharmacological approaches to test the proposed mechanisms. The impact of this study would be increased substantially if at least one link between SCFAs and AB, microglia, or ApoE were experimental validated. While most of the text avoids making causal claims based on correlative evidence, the one sentence summary states that SCFAs impact disease "via activation of microglial cells and upregulation of ApoE."
Identify which SCFA matters. The experiments all rely on a mixture of 3 SCFAs making it impossible to determine which compound is responsible. There is also high salt in this mixture which confounds the interpretation further. At a minimum, each individual compound needs to be tested using an equimolar amount of salt as a negative control. The authors should also note issues with oral delivery of SCFAs, which does not necessarily mimic production in the colon. Ideally, tributyrin, or a similar ester for acetate or propionate should be used. Another key missing control is the administration of SCFAs to SPF mice. It is also important to be clear that while SCFAs are sufficient to impact AB, there is no evidence in the paper to suggest that they are necessary, the full scope of "key microbial metabolites" remain to be determined. If the authors want to claim necessity, they would need to deplete specific SCFAs in the presence of a complex gut microbiome.
Be more cautious in discussing the role of the microbiome in Alzheimer's disease. The background discussion includes studies that show correlations in humans and phenotypic differences in germ-free mouse models, which in my opinion are insufficient to claim a causal role in human disease. The authors should discuss the level of evidence in humans for a causal role of the microbiome and its relative impact relative to other risk factors, including any prospective or intervention studies that have been conducted. They should also take care not to extrapolate differences in intermediate phenotypes in mice (plaque levels, microglial activation, and ApoE expression) to human disease. For example, the one sentence summary says, "contributing to AD disease progression". The authors should also discuss whether or not cognitive performance was evaluated in response to SCFAs.
-
##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
Colombo et al. present an intriguing set of findings from the amyloidosis mouse model (APPS1). Rederivation of this model under germ-free conditions led to both decreased plaque load and impaired cognitive performance. Administration of a cocktail of SCFAs and salt (sodium propionate, butyrate, and acetate) significantly increased plaque levels, microglial activation, and ApoE expression. Together, these findings suggest a potential pathway through which the microbiome could impact cognitive performance. The …
##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
Colombo et al. present an intriguing set of findings from the amyloidosis mouse model (APPS1). Rederivation of this model under germ-free conditions led to both decreased plaque load and impaired cognitive performance. Administration of a cocktail of SCFAs and salt (sodium propionate, butyrate, and acetate) significantly increased plaque levels, microglial activation, and ApoE expression. Together, these findings suggest a potential pathway through which the microbiome could impact cognitive performance. The paper is well-written, with a clear description of the current results and a logical flow to the text and figures. These data are a good starting point for further mechanistic dissection and add another welcome piece to the puzzle of how the microbiota affect the brain.
-
