Cholinergic signalling in the forebrain controls microglial phenotype and responses to systemic inflammation
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Evaluation Summary:
Loss or decrease of cholinergic tone occurs during brain aging or in pathologies such as Alzheimer's disease. This elegant study has used in vitro and in vivo approaches to explore the impact of decreased cholinergic signaling on hippocampal/cortical microglial state. The study demonstrates that the neurotransmitter Acetylcholine (ACh) maintain microglial cells in a homeostatic state, preventing their priming towards an activated state that leads to an exacerbated response to an inflammatory stimulus. The study thus provides important insights on the feed-forward contribution of microglial cells to neurodegenerative conditions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Loss of basal forebrain cholinergic projections occurs in Alzheimer’s disease, frontotemporal dementia and in aging. Moreover, nicotinic stimulation is anti-inflammatory in macrophages and microglia but how loss of basal forebrain acetylcholine impacts on microglial phenotype is poorly understood. Here we hypothesized that endogenous ACh maintains homeostatic microglial phenotype and that neurodegeneration-evoked loss of ACh tone, triggers microglial activation. Using the specific immunotoxin, mu-p75 NTR -saporin, we performed partial lesions of the basal forebrain cholinergic nuclei, medial septum and ventral diagonal band. We examined microglial phenotype in the hippocampus, the major projection area for these nuclei, using bulk RNA preparations, Flow cytometry-sorted microglial cells, immunohistochemistry and ELISA to examine responses to cholinergic withdrawal and acute responses to subsequent systemic inflammation with LPS. Basal forebrain cholinergic degeneration elicited lasting activation of microglia in the hippocampus, showing suppression of Sall1 and persistent elevation of Trem2, Clec7a, Itgax and complement genes proportionate to Chat loss. These primed microglia showed exaggerated IL-1β responses to systemic LPS challenge. In normal animals LPS evoked acute increases in extracellular choline, a proxy for ACh release, and this response was lost in lesioned animals. Restoration of basal cholinergic signalling via serial treatments with the nicotinic agonist PNU282,987 resulted in reversion to the homeostatic microglial phenotype and prevented exaggerated responses to acute systemic inflammation. The data indicate that neurodegeneration-evoked loss of cholinergic tone, triggers microglial activation via impaired microglial nicotinic signalling and leaves these microglia more vulnerable to secondary inflammatory insults. The data have implications for neuroinflammation during aging and neurodegeneration and for responses to sepsis and systemic inflammation.
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Reviewer #2 (Public Review):
The present study addresses the hypothesis that a decline in the cholinergic tone in ageing or neurodegenerative diseases such as Alzheimer's disease may lead to an increased microglial reactivity. This hypothesis is supported by several in vitro evidence, indicating that the alpha 7 nicotinic receptor is responsible for the anti-inflammatory activities of ACh on microglia and macrophages; however, the hypothesis has not been fully explored for example by conducting in "in vivo" studies. In particular, the Authors use the mu-p75-saporin immunotoxin injected into the lateral ventricles, to obtain a partial lesion of the basal forebrain cholinergic nucleus, from where cholinergic neurons project to specific regions of the hippocampus. The microglial phenotype is then studied in isolated microglia from the …
Reviewer #2 (Public Review):
The present study addresses the hypothesis that a decline in the cholinergic tone in ageing or neurodegenerative diseases such as Alzheimer's disease may lead to an increased microglial reactivity. This hypothesis is supported by several in vitro evidence, indicating that the alpha 7 nicotinic receptor is responsible for the anti-inflammatory activities of ACh on microglia and macrophages; however, the hypothesis has not been fully explored for example by conducting in "in vivo" studies. In particular, the Authors use the mu-p75-saporin immunotoxin injected into the lateral ventricles, to obtain a partial lesion of the basal forebrain cholinergic nucleus, from where cholinergic neurons project to specific regions of the hippocampus. The microglial phenotype is then studied in isolated microglia from the hippocampus and in homogenates from the same brain area. Intraperitoneal LPS injection is used as secondary inflammatory insult. Changes in hippocampal ACh levels are also measured in freely-moving mice through a microelectrochemical biosensor.
The study is technically sound, well performed and presented. The results are solid and confirm the hypothesis, by showing that the loss of cholinergic tone is associated to a higher microglial reactivity and leaves microglial cells more vulnerable to secondary inflammatory insults, causing an exaggerate response to as compared to control mice.
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Reviewer #1 (Public Review):
This manuscript explores the effect of loss of cholinergic innervation on microglia homeostatic phenotype. The authors show that loss of cholinergic tone "primes" microglial towards an activated phenotype, which makes them more prone to an exacerbated response to an inflammatory stimulus administered when microglial cells have already reverted to a "basal state". They further show that this overresponse is due to impaired nicotinic signalling mediated by the α7-nAChR expressed in microglial cells. The authors conclude that cholinergic signalling normally contributes to maintain microglia cells in a resting state. In its absence or reduction, microglial cells are primed and enter an acute proinflammatory state upon a second stimulus.
This is an interesting and well conducted study that provides a molecular …
Reviewer #1 (Public Review):
This manuscript explores the effect of loss of cholinergic innervation on microglia homeostatic phenotype. The authors show that loss of cholinergic tone "primes" microglial towards an activated phenotype, which makes them more prone to an exacerbated response to an inflammatory stimulus administered when microglial cells have already reverted to a "basal state". They further show that this overresponse is due to impaired nicotinic signalling mediated by the α7-nAChR expressed in microglial cells. The authors conclude that cholinergic signalling normally contributes to maintain microglia cells in a resting state. In its absence or reduction, microglial cells are primed and enter an acute proinflammatory state upon a second stimulus.
This is an interesting and well conducted study that provides a molecular mechanism underlying the feed-forward contribution of microglial cells to conditions such as brain aging or neurodegenerative pathologies such as Alzheimer's disease. A few aspects of the manuscript could however be improved:
Figure 2D. In all graphs there are two samples that do not show any increase in cytokine content with respect to saline treated animals? Are these the same animals? Do the authors have an explanation for their lack of response?
Can the authors provide double immunostaining analysis to show that Iba1 positive cells express α7-nAChR? This will nicely support FACS results given that FACS represent after all a stress for the cells.
Figure 5. The data are somewhat confusing and in part they seem to repeat those reported in Fig 2 and 3. They are not easy to follow for non-specialists in the field and will benefit from re-writing. Furthermore, why looking at circulating levels of the cytokines? It would be more appropriate to determine cytokine content in the brain
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Evaluation Summary:
Loss or decrease of cholinergic tone occurs during brain aging or in pathologies such as Alzheimer's disease. This elegant study has used in vitro and in vivo approaches to explore the impact of decreased cholinergic signaling on hippocampal/cortical microglial state. The study demonstrates that the neurotransmitter Acetylcholine (ACh) maintain microglial cells in a homeostatic state, preventing their priming towards an activated state that leads to an exacerbated response to an inflammatory stimulus. The study thus provides important insights on the feed-forward contribution of microglial cells to neurodegenerative conditions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer …
Evaluation Summary:
Loss or decrease of cholinergic tone occurs during brain aging or in pathologies such as Alzheimer's disease. This elegant study has used in vitro and in vivo approaches to explore the impact of decreased cholinergic signaling on hippocampal/cortical microglial state. The study demonstrates that the neurotransmitter Acetylcholine (ACh) maintain microglial cells in a homeostatic state, preventing their priming towards an activated state that leads to an exacerbated response to an inflammatory stimulus. The study thus provides important insights on the feed-forward contribution of microglial cells to neurodegenerative conditions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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