PREreview of Olfaction regulates peripheral mitophagy and mitochondrial function

This review reflects comments and contributions from Hector Fugihara Kroes, Marina Schernthanner, Femi Arogundade, Luciana Gallo & Pablo Ranea-Robles. Review synthesized by Pablo Ranea-Robles

This study revolves around the molecular signaling mechanisms by which olfaction connects the environmental sensing of perturbed conditions with the regulation of peripheral mitophagy and mitochondrial function. The authors employed the model organism C. elegans to explore the role of olfaction in regulating mitochondrial function and the mitochondrial unfolded protein response (mt-UPR). The study investigated how the ablation of specific olfactory neurons (AWC) affects mitochondrial content, function, mt-UPR, and resistance to pathogenic bacteria in worms. The authors found that AWC ablation led to mt-UPR activation, and reduced mitochondrial oxidative phosphorylation and content. These effects were mediated by serotonergic signaling and the mitophagy machinery, specifically involving PDR-1/Parkin. The study also shows that AWC ablation confers resistance to Pseudomonas aeruginosa infection, which was partially dependent on the mt-UPR transcription factor ATFS-1 and fully dependent on the mitophagy machinery protein PDR-1/Parkin. One of the novel findings of this study is that AWC neuron-mediated signaling may impact mitophagy processes upstream of changes in the mt-UPR. Overall, this study provides valuable insights into the regulation of mitophagy and mitochondrial function through olfactory signaling in C. elegans. The introduction covers the topic of study, identifies relevant gaps in the field, and prepares the reader for the results that are shown afterwards. The authors successfully demonstrate the role of specific olfactory neurons in regulating peripheral mitophagy and mitochondrial function. The authors employ a combination of genetic, pharmacological, and functional assays to investigate the role of AWC neuron signaling in regulating mitochondrial biology, adding depth to their findings. However, we think further clarification is needed on several aspects of the study in order to strengthen the conclusions and the implications.

Major comments:

• The study uses C. elegans as a model organism, which is well-established in mitochondrial biology research. However, we think that the title may lead to confusion regarding the implications of the study. The inclusion of the model organism used in the title will provide a more accurate representation of the research findings and help readers understand the context of the study.

• On the same line, while the results in a human cell culture line are promising, caution should be exercised when extrapolating these findings to mammalian/human biology, including the fact that they are assuming OXPHOS will be affected because there is an increase in mitochondrial ROS. Further experiments using additional mammalian cell lines and animal models would be useful to validate the relevance of these findings to higher organisms. At the same time, we acknowledge that studying mitochondrial function in C. elegans provides valuable insights into basic biological processes that can have implications for understanding human health and disease. We just recommend the authors to acknowledge these limitations and emphasize the need for further research in mammalian models in their discussion.

• The authors mention multiple times that AWC ablation leads to changes in mitochondrial dynamics. However, the study does not show mitochondrial shape changes or provide evidence of altered fusion and fission dynamics. To strengthen the conclusions, it would be beneficial for the authors to include additional experiments related to mitochondrial dynamics in response to AWC ablation. Alternatively, the authors could modify the wording used when describing the changes in mitochondrial biology to present a more accurate vision of those changes.

• Most of the data supporting the conclusions of this study comes from the AWC-depleted state. However, the data related to odorant-mediated changes in mitochondrial parameters is limited. The authors found only mild changes when worms were exposed to 2,3-Pentanedione and no changes with butanol. However, these results are not further discussed in the context of the overall conclusions of the study. Moreover, the role of serotoninergic neurotransmission in the development of this phenotype was not tested in this condition. How do the authors know the specificity of these odorants to target AWC neurons? We recommend that the authors further discuss the relevance of these findings and their relation to the different AWC neuronal population (ON vs OFF).

• Data in Figure 4 points rather to a partial effect of pdr-1/Parkin depletion on mtUPR induction rather than total dependency on pdr-1. We think that the direct comparison between AWC(-) and pdr-1(gk448);AWC(-) should be the important one here.

• We also miss the experiment in which mtUPR induction was evaluated in AWC(-)/atfs-1 mutants. We may understand if the authors take it for granted that mtUPR induction cannot happen under those conditions, but then it should discussed in the manuscript.

Minor comments:

• The assays used to measure mitochondrial function (OCR) and mitochondrial DNA levels (mtDNA) are suitable, but additional complementary assays or techniques could strengthen the conclusions. For example, assessing mitochondrial membrane potential, mitochondrial morphology, or specific enzymatic activities related to mitochondrial function would provide a more comprehensive view of mitochondrial health.

• The method for nematode synchronization (bleaching and egg isolation) may introduce variability in the experiments, as synchronization methods can sometimes lead to variations in the developmental stage of the worms. The use of age-synchronized populations may provide more consistent results.

• The use of pharmacological treatments like FUDR to inhibit germline proliferation can have off-target effects. A discussion of these potential effects and control experiments to account for them would be valuable.

• Details about the conditions of cell culture, such as ‌the duration of culture should be provided to ensure reproducibility.

• Using Seahorse assays to measure mitochondrial function is standard, but these assays can be sensitive to experimental conditions. Information about the assay conditions (for example, substrates used) and quality control measures taken would provide more clarity.

• While stereoscopes are useful for observing C. elegans, more advanced imaging techniques like confocal microscopy or automated image analysis might provide more precise and quantitative data, especially when studying subtle changes in fluorescence.

• There was something odd in “in metabolic flux” in the following sentence: “The unfolded protein response of the mitochondria (UPRMT) is upregulated upon infection by many pathogens and in metabolic flux, and pathogenic infection and metabolic byproducts are a present hazard in consuming nutrients.”

• Figure 1: TOF is only defined in methods. Would be great to have it defined here as well.

• Figure 1: The details in figure legend are not enough to fit the p-values with the corresponding comparison in the plot. One way to solve this issue is by directly including the values in the plot or by establishing a clear convention, such as left-to-right.

• Authors found that mtUPR was induced in AWC(-) mutants with and without FUDR. However, data seem to point to a blunted effect on mtUPR induction in FUDR+-treated worms. This aspect is not discussed by the authors.

• In the experiment where ceh-36p::HisCl worms where treated with HA, we missed another condition in which control worms where treated with HA and mitochondrial parameters were evaluated.

• Same experiment with HA, the difference in hsp-6::gfp fluorescence seems less pronounced in Fig S1D compared to Fig1B - is this related to the timeline as mentioned above i.e. if the authors had analyzed animals at a later timepoint after AWC loss, would the difference be bigger?

• Supplemental Figure 1G. mtDNA quantification was measured in n=1 worms. We think this has not enough power to conclude that there is a difference. Is there any particular reason to measure in n=1 worms? Moreover, there is a typo in figure legend text (says “F”).

• We think that the difference between SCV and DCV neurotransmission would be better placed in the text when the data of Figure 2 is reported.

• Data in Figure 2 shows that unc-13 and unc-31 mutants already present a decrease in mitochondrial function and content. It would be interesting to have a discussion about how these data could be confounding when studying these parameters in these mutants with AWC-ablated neurons.

• Figure 3D - how do the authors explain the significant mtDNA increase between serotonin-deficient AWC KO (even higher than N2 WT) and serotonin-deficient animals?

• When reporting data about PDR-1 dependency, Parkin was not introduced before. A comment on why parkin is evaluated might be useful.

• The data that support resistance to infection seem robust. However, we think it would be relevant to have non-infected control worms of all experimental groups to better understand the effect of P. aeruginosa infection on these worms.

Comments on reporting:

• Generally applicable for most figure panels with statistical results - If more than 2 experimental groups are shown in one graph, the authors should do an ANOVA analysis instead of t-tests.

• Please, consider adding scale bars to the images

• Some p values are not shown, for example. For example, in figure 2, the comparison stated between AWC vs unc-13/unc-31; AWC is not shown. Also applies to other figures

• No image of worms in Figure 1H.

• Figure 1E. We think y-axis should state Fold-change, and not log2 fold change, if WT is assigned to “1”

• The authors have not provided precise information regarding the sample sizes used in each experiment. Adequate statistical power relies on having an appropriate sample size, so it's crucial to ensure that the sample sizes are adequate to detect meaningful effects. One clear example in survival data on Figure 5.

• There is no information provided in the preprint regarding the availability of the raw data to validate and reproduce the results.

• The preprint could benefit from more detailed reporting of statistical methods and information regarding data availability.

Suggestions for future studies:

• While the study provides intriguing insights into the role of AWC neurons in mitochondrial dynamics and pathogen resistance, the specific mechanisms connecting serotonin signaling and mitophagy machinery to these outcomes could benefit from more detailed mechanistic investigations.

• Explore the potential influence of the gut microbiome on the interaction between the AWC neurons, serotonin signaling, and mitochondrial dynamics. Investigate whether specific bacterial species or metabolites play a role in this regulatory process.

• Investigate whether the findings from the study have any clinical relevance. Explore whether manipulating serotonin signaling or mitochondrial dynamics could be a potential therapeutic approach for mitochondrial-related diseases or metabolic disorders in humans.

• Extend the study to other model organisms or cell culture systems to assess the conservation of the observed effects of serotonin signaling on mitochondrial dynamics. Investigate whether similar mechanisms exist in mammals or other species.

Competing interests

The author declares that they have no competing interests.