Transcriptional responses to chronic oxidative stress require cholinergic activation of G-protein-coupled receptor signaling

Reviewer #1 (Public review):

Summary:

The researchers aimed to identify which neurotransmitter pathways are required for animals to withstand chronic oxidative stress. This work thus has important implications for disease processes that are caused/linked to oxidative stress. This work identified specific neurotransmitters and receptors that coordinate stress resilience, both prior to and during stress exposure. Further, the authors identified specific transcriptional programs coordinated by neurotransmission that may provide stress resistance.

Strengths:

The manuscript is very clearly written with a well-formulated rationale. Standard C. elegans genetic analysis and rescue experiments were performed to identify key regulators of the chronic oxidative stress response. These findings were enhanced by transcriptional profiling that identified differentially expressed genes that likely affect survival when animals are exposed to stress.

Weaknesses:

Where the gar-3 promoter drives expression was not discussed in the context of the rescue experiments in Figure 7.

Reviewer #2 (Public review):

In this paper, Biswas et al. describe the role of acetylcholine (ACh) signaling in protection against chronic oxidative stress in C. elegans. They showed that disruption of ACh signaling in either unc-17 mutants or gar-3 mutants led to sensitivity to toxicity caused by chronic paraquat (PQ) treatment. Using RNA seq, they found that approximately 70% of the genes induced by chronic PQ exposure in wild type failed to upregulate in these mutants. The overexpression of gar-3 selectively in cholinergic neurons was sufficient to promote protection against chronic PQ exposure in an ACh-dependent manner. The study points to a previously undescribed role for ACh signaling in providing organism-wide protection from chronic oxidative stress, likely through the transcriptional regulation of numerous oxidative stress-response genes. The paper is well-written, and the data are robust, though some conclusions seem preliminary and do not fully support the current data. While the study identifies the muscarinic ACh receptor gar-3 as an important regulator of the response to PQ, the specific neurons in which gar-3 functions were not unambiguously identified, and the sources of ACh that regulate GAR-3 signaling and the identities of the tissues targeted by gar-3 were not addressed, limiting the scope of the study.

Major Comments:

(1) The site of action of cholinergic signaling for protection from PQ was not adequately explored. The authors' conclusion that cholinergic motor neurons are protective is based on studies using overexpression of gar-3 and an unc-17 allele that may selectively disrupt ACh in cholinergic motor neurons (Figure 9F), but these approaches are indirect. To more directly address the site of action, the authors should conduct rescue experiments using well-defined heterologous promoters. Figure 7G shows that gar-3 expressed under a 7.5 kb promoter fragment fully rescues the defect of gar-3 mutants, but the authors did not report where this promoter fragment is expressed, nor did they conduct rescue experiments of the specific tissues where gar-3 is known to be expressed (cholinergic neurons, GABAergic neurons, pharynx, or muscles). UNC-17 rescue experiments could also be useful to address the site of action. Does expression of unc-17 selectively in cholinergic motor neurons rescue the stress sensitivity of unc-17 mutants (or restore resistance to gar-3(OE); unc-17 mutants)? These experiments may also address whether ACh acts in an autocrine or paracrine manner to activate gar-3, which would be an important mechanistic insight to this study that is currently lacking.

(2) The genetic pan-neuronal silencing experiments presented in Figure 1 motivated the subsequent experiments, but the authors did not relate these observations to ACh/gar-3 signaling. For example, the authors did not address whether silencing just the cholinergic motor neurons at the different times tested has the same effects on survival as pan-neuronal silencing.

(3) It is assumed that protection occurs through inter-tissue signaling of ACh to target tissues, where it impacts gene expression. While this is a reasonable assumption, it has not been directly shown here. It is recommended that the authors examine GFP reporter expression of a sampling of the genes identified in this study (including proteasomal genes that the authors highlight) that are regulated by unc-17 and gar-3. This would serve to independently confirm the RNAseq data and to identify target tissues that are subject to gene expression regulation by ACh, which would significantly strengthen the study.

Author response:

Reviewer #1 (Recommendations for the authors):

“The gar-3 promoter expression pattern was not discussed in the context of rescue experiments.”

We agree that the expression pattern of the gar-3 promoter used in our rescue experiments should be clarified. We will include a description of the tissues where the 7.5 kb gar-3 promoter fragment is expressed, based on both prior studies and our own expression data. We will also discuss how the gar-3 cell and tissue expression pattern relates to both our analysis of gar-3 expression in the genome edited strain we generated as well as the observed rescue effects.

Reviewer #2 (Recommendations for the authors):

(1) The site of action of cholinergic signaling was not adequately explored.

We plan to perform additional rescue experiments using heterologous promoters to drive gar-3 expression in specific tissues (e.g. cholinergic neurons, muscle). These experiments will help clarify the sufficiency of unc-17 expression in specific cell types for rescue. However, we point out that cell-specific unc-17 knockdown by RNAi using the unc-17b promoter (expression largely restricted to ventral cord ACh motor neurons) increases sensitivity to PQ in our long-term survival assays. Combined with our analysis of unc-17(e113) mutants, we believe our data offer robust support of a requirement for unc-17 expression in cholinergic motor neurons.

(2) Pan-neuronal silencing experiments were not connected to ACh/GAR-3 signaling.

We will expand our discussion to relate the pan-neuronal silencing results to our analysis of ACh signaling. We used the pan-neuronal silencing to motivate further analysis of various neurotransmitter systems. We note that our studies implicate both glutamatergic and cholinergic systems in protective responses to oxidative stress. The effects of silencing on survival during long-term PQ exposure may therefore be derived solely from cholinergic neurons, glutamatergic neurons, or a combination of both neuronal populations. We hope the reviewer will agree that distinguishing between these possibilities may be quite complicated and is not central to the main message of our paper. We therefore suggest this additional analysis lies outside the scope of this revision.

(3) Inter-tissue signaling and transcriptional regulation by ACh were assumed but not directly shown.

We will generate GFP reporters for a subset of genes (including proteasomal genes) identified in our RNA-seq analysis or assess their expression by quantitative RT-PCR to validate cholinergic regulation. These experiments will help to identify target tissues and confirm transcriptional regulation by cholinergic signaling.

We appreciate the opportunity to revise our manuscript and believe that these additions will significantly strengthen the mechanistic insights and overall impact of our study. Please let us know if further clarification is needed.