A prebiotic diet modulates microglial states and motor deficits in α-synuclein overexpressing mice
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The complex mechanisms through which diet impact Parkinson's Disease are unclear, limiting the ability to guide patients to an optimal diet. Here, researchers use a mouse model to test the impact of dietary fiber, revealing changes in gut microbes and immune cells in the brain. This study raises intriguing hypotheses about how diet-induced changes in the microbiome could lead to changes in brain function.
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Abstract
Parkinson’s disease (PD) is a movement disorder characterized by neuroinflammation, α-synuclein pathology, and neurodegeneration. Most cases of PD are non-hereditary, suggesting a strong role for environmental factors, and it has been speculated that disease may originate in peripheral tissues such as the gastrointestinal (GI) tract before affecting the brain. The gut microbiome is altered in PD and may impact motor and GI symptoms as indicated by animal studies, although mechanisms of gut-brain interactions remain incompletely defined. Intestinal bacteria ferment dietary fibers into short-chain fatty acids, with fecal levels of these molecules differing between PD and healthy controls and in mouse models. Among other effects, dietary microbial metabolites can modulate activation of microglia, brain-resident immune cells implicated in PD. We therefore investigated whether a fiber-rich diet influences microglial function in α-synuclein overexpressing (ASO) mice, a preclinical model with PD-like symptoms and pathology. Feeding a prebiotic high-fiber diet attenuates motor deficits and reduces α-synuclein aggregation in the substantia nigra of mice. Concomitantly, the gut microbiome of ASO mice adopts a profile correlated with health upon prebiotic treatment, which also reduces microglial activation. Single-cell RNA-seq analysis of microglia from the substantia nigra and striatum uncovers increased pro-inflammatory signaling and reduced homeostatic responses in ASO mice compared to wild-type counterparts on standard diets. However, prebiotic feeding reverses pathogenic microglial states in ASO mice and promotes expansion of protective disease-associated macrophage (DAM) subsets of microglia. Notably, depletion of microglia using a CSF1R inhibitor eliminates the beneficial effects of prebiotics by restoring motor deficits to ASO mice despite feeding a prebiotic diet. These studies uncover a novel microglia-dependent interaction between diet and motor symptoms in mice, findings that may have implications for neuroinflammation and PD.
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Author Response
Reviewer #1 (Public Review):
Abdel-Hag, Reem et al. investigated the beneficial effects of a fiber-rich diet in the pathology of α-synuclein overexpressing (ASO) mice, a preclinical model of Parkinson's disease. They found that a prebiotic intervention attenuates motor deficits and reduces microglial reactivity in the substantia nigra and striatum. They extended these findings by doing scRNA sequencing, and they identified the expansion of a protective disease-associated microglia (DAM), a microglial subset previously described during the early stages of disease in several mouse models. Interestingly, the data indicate that microglia do not influence the behavior of ASO mice in the early stages of disease progression. However, microglia are the key mediators of the protective effects of prebiotic treatment in ASO …
Author Response
Reviewer #1 (Public Review):
Abdel-Hag, Reem et al. investigated the beneficial effects of a fiber-rich diet in the pathology of α-synuclein overexpressing (ASO) mice, a preclinical model of Parkinson's disease. They found that a prebiotic intervention attenuates motor deficits and reduces microglial reactivity in the substantia nigra and striatum. They extended these findings by doing scRNA sequencing, and they identified the expansion of a protective disease-associated microglia (DAM), a microglial subset previously described during the early stages of disease in several mouse models. Interestingly, the data indicate that microglia do not influence the behavior of ASO mice in the early stages of disease progression. However, microglia are the key mediators of the protective effects of prebiotic treatment in ASO mice. Overall, the conclusions of this paper are well supported by data, but some aspects should be considered to improve the manuscript.
- Colony-stimulating factor 1 receptor (CSF1R) inhibition has been widely used as a method for microglia depletion, however, the impact of this approach on peripheral immune cells is controversial. The authors elegantly showed that most gut-associated immune cell populations were unaffected by PLX5622. However, CSF1R signaling has been implicated in the maintenance of gut homeostasis. Could it be possible that PLX5622 treatment affects directly the gut microbiome composition? Are the beneficial changes in the gut microbiome composition of a prebiotic diet still maintained in combination with PLX5622? CSF1R inhibitors with low brain penetration such as PLX73086 and therefore unable to deplete resident microglia (Bellver- Landete, Victor et al., Nat Commun, 2019) would be helpful to rule out peripheral off-target effects.
We agree that loss of benefits by the prebiotic diet following PLX5622 treatment is possibly due to changes to the microbiome, and cannot exclude this possibility. The mechanism of action of PLX5662 in reshaping the microbiome would most likely involve effects through changes in immune (or other) cell types in the gut, as the drug is not known to have direct effects on the microbiome. As described by the referee, we carefully profiled the mucosal immune system of mice treated with PLX5622 and control chow, and show minor changes associated with the drug. These are control experiments that very few previous studies using PLX5622 have performed, and suggest immune-mediated microbiome changes may be subtle. Further, we do not suggest in the manuscript that microbiome changes, in the first place, mediated the benefits of the prebiotic diet but rather focus the current study on the well-known effect of microglia depletion by PLX5622.
Microbiome profiling and additional experiments transferring microbiota from diet-treated animals, with and without PLX5622, to naïve mice would be needed to determine the functional effects of gut bacteria on microglial activation and motor symptoms. The use of PLX73086 is also an excellent way to address this point, as are several additional approaches. Comprehensively investigating the indirect contributions of the microbiome to motor symptoms in ASO mice represent a separate series of studies, in our respectful opinion. Nonetheless, this is an important caveat of our work and we now include the following text in the Discussion section to address this point: “Our study does not rule out indirect effects of PLX5622 that include reshaping the microbiome to promote motor symptoms in prebiotic diet-fed mice”. We thank the referee for this comment.
- The authors claimed that microglial depletion eliminates the protective effects of the prebiotic diet in ASO mice by showing increased levels of aggregated aSyn in the SN (Fig 5G). However, microglial depletion also has the same effect on WT mice. How do authors interpret this result?
The referee raises an astute point. Microglia appear to play a complex role in PD and mouse models, with both positive and negative effects demonstrated in various context (for example, PMIDSs: 29401614, 32086763). A primary and non-exclusive function of microglia is the removal of -synuclein accumulations (PMIDs: 32170061, 34555357). Importantly, there is no change in motor behavior in prebiotic-fed WT mice with or without PLX5622 treatment, as expected (see Figs. 5D-F). We have been careful in the manuscript to not suggest that microglia effects on motor symptoms are via a process that include -synuclein aggregates, as this has not been convincingly shown in this mouse model at the time point we are studying (ie., 22 weeks of age). While it would be straightforward to add a statement suggesting why -synuclein levels increase in WT mice on drug, our preferred remedy here is to point out this observation so it does not go unnoticed, but refrain from speculation in the absence of data since this is not a major point of the study. We have now inserted the statement “However, in prebiotic-fed WT and ASO mice, depletion of microglia significantly increased levels of aggregated αSyn in the SN, while levels in the STR remained unchanged (Figure 5G-H).” We thank the referee for this important comment.
- What is the rationale for doing a long-term (17 weeks) prebiotic intervention? Have the authors considered doing a short-term intervention? The prebiotic diet should change quickly the gut microbiome composition within a few days or weeks.
We have previously shown that long-term microbiome depletion is required to impact motor performance in ASO mice (similar timeline as current prebiotic study) (Sampson et al., Cell, 2016). In unpublished data, short-term antibiotic treatment (4 weeks before motor testing) is unable to improve motor symptoms in ASO mice. Thus, we chose a timeframe for the current prebiotic studies guided by empiric data, but further details on dose intervals remain unknown. We agree that the microbiome should rapidly respond to the prebiotic diet, but it is unknown if this response is durable or would the ‘pre-treated’ microbiome profile re-establish at some time after removal of the experimental diet. We respectfully suggest that these more specialized studies are better suited for future projects.
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eLife assessment
The complex mechanisms through which diet impact Parkinson's Disease are unclear, limiting the ability to guide patients to an optimal diet. Here, researchers use a mouse model to test the impact of dietary fiber, revealing changes in gut microbes and immune cells in the brain. This study raises intriguing hypotheses about how diet-induced changes in the microbiome could lead to changes in brain function.
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Reviewer #1 (Public Review):
Abdel-Hag, Reem et al. investigated the beneficial effects of a fiber-rich diet in the pathology of α-synuclein overexpressing (ASO) mice, a preclinical model of Parkinson's disease. They found that a prebiotic intervention attenuates motor deficits and reduces microglial reactivity in the substantia nigra and striatum. They extended these findings by doing scRNA sequencing, and they identified the expansion of a protective disease-associated microglia (DAM), a microglial subset previously described during the early stages of disease in several mouse models. Interestingly, the data indicate that microglia do not influence the behavior of ASO mice in the early stages of disease progression. However, microglia are the key mediators of the protective effects of prebiotic treatment in ASO mice. Overall, the …
Reviewer #1 (Public Review):
Abdel-Hag, Reem et al. investigated the beneficial effects of a fiber-rich diet in the pathology of α-synuclein overexpressing (ASO) mice, a preclinical model of Parkinson's disease. They found that a prebiotic intervention attenuates motor deficits and reduces microglial reactivity in the substantia nigra and striatum. They extended these findings by doing scRNA sequencing, and they identified the expansion of a protective disease-associated microglia (DAM), a microglial subset previously described during the early stages of disease in several mouse models. Interestingly, the data indicate that microglia do not influence the behavior of ASO mice in the early stages of disease progression. However, microglia are the key mediators of the protective effects of prebiotic treatment in ASO mice. Overall, the conclusions of this paper are well supported by data, but some aspects should be considered to improve the manuscript.
Colony-stimulating factor 1 receptor (CSF1R) inhibition has been widely used as a method for microglia depletion, however, the impact of this approach on peripheral immune cells is controversial. The authors elegantly showed that most gut-associated immune cell populations were unaffected by PLX5622. However, CSF1R signaling has been implicated in the maintenance of gut homeostasis. Could it be possible that PLX5622 treatment affects directly the gut microbiome composition? Are the beneficial changes in the gut microbiome composition of a prebiotic diet still maintained in combination with PLX5622? CSF1R inhibitors with low brain penetration such as PLX73086 and therefore unable to deplete resident microglia (Bellver- Landete, Victor et al., Nat Commun, 2019) would be helpful to rule out peripheral off-target effects.
The authors claimed that microglial depletion eliminates the protective effects of the prebiotic diet in ASO mice by showing increased levels of aggregated aSyn in the SN (Fig 5G). However, microglial depletion also has the same effect on WT mice. How do authors interpret this result?
What is the rationale for doing a long-term (17 weeks) prebiotic intervention? Have the authors considered doing a short-term intervention? The prebiotic diet should change quickly the gut microbiome composition within a few days or weeks.
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Reviewer #2 (Public Review):
The manuscript by Abdel-Haq and colleagues is a descriptive study providing evidence that mice displaying motor impairment related to Parkinson's disease fed with a prebiotic diet show a decrease in the severity of this impairment (some, but not all, of the motor functions tested). Their data indicate that microglial cells are required to mediate the beneficial effect of the prebiotic treatment. Indeed, in the absence of microglial cells, the prebiotic treatment is no longer able to attenuate the motor deficit. This manuscript is of interest to a wide audience as it provides further evidence that links motor impairment related to PD to events occurring in the gut (gut-brain axis). Furthermore, some of the new findings presented in the manuscript highlight the contribution of immune mechanisms as key …
Reviewer #2 (Public Review):
The manuscript by Abdel-Haq and colleagues is a descriptive study providing evidence that mice displaying motor impairment related to Parkinson's disease fed with a prebiotic diet show a decrease in the severity of this impairment (some, but not all, of the motor functions tested). Their data indicate that microglial cells are required to mediate the beneficial effect of the prebiotic treatment. Indeed, in the absence of microglial cells, the prebiotic treatment is no longer able to attenuate the motor deficit. This manuscript is of interest to a wide audience as it provides further evidence that links motor impairment related to PD to events occurring in the gut (gut-brain axis). Furthermore, some of the new findings presented in the manuscript highlight the contribution of immune mechanisms as key contributors to the pathophysiological process leading to PD.
This is an interesting study showing for the first time that the beneficial effect of a prebiotic treatment in the context of motor impairment related to PD is mediated by microglial cells. Since these cells are of macrophagic origin, their data support the concept that the immune system plays a role along the gut-brain axis during the pathophysiological process leading to PD. The sequencing data may be of additional value to some. Considering that the authors had a model system where clear beneficial motor impairment was observed, it is surprising that they did not investigate further whether the dopaminergic system in the SN and STR was modified in relation to the prebiotic treatment and microglial depletion.
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Reviewer #3 (Public Review):
Abdel-Haq presents a comprehensive analysis of the impact of dietary fiber on the ASO mouse model. They describe diet-induced changes in the gut microbiota, microbial metabolites, host gene expression, microglial activation, and motor deficits. Pharmacological inhibition of microglia highlights the importance of these cells for the impact of prebiotics, raising intriguing hypotheses for future studies.
Strengths include the rigor and reproducibility of these studies, the clarity of the presentation, and the timely focus on microglial interactions with the gut microbiome.
The major weakness is the descriptive nature of these studies and the lack of reduction in the mechanism. Only a single model is used and there is no attempt to test the translational relevance of these findings in humans. The putative …
Reviewer #3 (Public Review):
Abdel-Haq presents a comprehensive analysis of the impact of dietary fiber on the ASO mouse model. They describe diet-induced changes in the gut microbiota, microbial metabolites, host gene expression, microglial activation, and motor deficits. Pharmacological inhibition of microglia highlights the importance of these cells for the impact of prebiotics, raising intriguing hypotheses for future studies.
Strengths include the rigor and reproducibility of these studies, the clarity of the presentation, and the timely focus on microglial interactions with the gut microbiome.
The major weakness is the descriptive nature of these studies and the lack of reduction in the mechanism. Only a single model is used and there is no attempt to test the translational relevance of these findings in humans. The putative pathway (fiber→bacteria→SCFA→microglia) has already been reported, so the data is largely confirmatory in nature.
Despite these concerns, this work adds to the growing literature on the gut-brain axis and will be helpful for motivating continued studies in mice and human cohorts. However, caution should be advised for using these results to motivate specific dietary recommendations to patients.
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