Mitochondrial stress in GABAergic neurons non-cell autonomously regulates organismal health and aging

Reviewer #1 (Public Review):

Summary:

The authors utiilze the model organism C. elegans to interrogate cell non-autonomous signaling between GABAergic neurons and somatic tissues. They demonstrate that RNAi of isp-1 or spg-7 in GABAergic neurons leads to lifespan extension and improved healthspan (by resistance to paraquat or heat stress), which are dependent on the transcription factor daf-16/FOXO3a.

Strengths:

The authors are clear and straightforward in their study. They examine the healthspan of C. elegans at days 3, 6, and 9 to give a wide perspective on how the phenotypes changes with aging. They use two methods to specifically knockdown isp-1 or spg-7 in GABAergic neurons: (1) a previously published rde-1 mutant that has rde-1 and sid-1 restored only in GABAergic neurons and (2) a novel model uses a sid-1 mutant that makes dsRNA of isp-1 or spg-7 in GABAergic neurons. They use multiple methods to examine healthspan. They identified daf-16/FOXO3a as the mechanism of their phenotype and ruled out other transcription factors. The authors do not use FUdR in their studies, which is known to confound experiments.

Weaknesses:

(1) Incomplete validation of GABAergic knockdown. The study relies on the specific knockdown of isp-1 or spg-7 in GABAergic neurons, but in the opinion of this reviewer, the authors do not adequately validate their models to demonstrate GABAergic specificity. For the previously published rde-1 mutant model, a simple validation of specific knockdown of GFP in GFP-labeled GABAergic neurons should be included. They should also show that GFP RNAi would not be effective in knocking down intestinal GFP, for example.

Their second model is poorly explained and not validated and this reviewer could not find similar previously published models of its kind. This model claims that dsRNA of isp-1 was made in the GABAergic neurons of a sid-1 mutant, but no evidence is shown to support this claim. The authors point to changes in phenotypes such as lifespan extension and reduced lipofuscin in the intestines as proof that knockdown is occurring in the GABAergic neurons, but this is indirect evidence. Rigorous validation of this model is needed, especially if it is the first model of its kind.

(2) Lifespan. The control lifespans using the rde-1 mutants are very short-lived and no explanation for this is provided (eg. Figure 1D, E). The authors use two RNAis in their lifespan with daf-16 and isp-1. For their controls, they should use empty vector mixed with isp-1, not only isp-1 RNAi.

(3) Cell non-autonomous effects. The claims that GABAergic mitochondrial dysfunction have effects on somatic tissues is weak. More specific tests on somatic stress resistance are warranted for their claims. Better quality images of intestinal mitochondria are needed. Examining additional tissues, such as muscle, would also strengthen their claims. For example, they could examine muscle mitochondria and determine if muscle strength is improved in their models.

(4) Dependence on daf-16/FOXO3a. The authors show that loss of daf-16 reverses the lifespan and healthspan effects in their model. Next, they show that loss of daf-16 reverses the effects of isp-1 in the intestines and in the germline. However, they only show the daf-16 mutant data and not the positive control (EV and isp-1 alone), which should be included. Furthermore, the phenotypes they examine are only a subset of somatic phenotypes, and this reviewer would be more convinced with the additional controls and with more parameters examined.

Reviewer #2 (Public Review):

Summary:

In this work, the authors show that GABAergic neurons play a role in sensing mitochondrial stress and regulating organismal aging. Thus, disrupting the mitochondrial mitochondria function in GABAergic neurons induces resistance to thermal and paraquat stresses, promotes longevity, and affects reproduction. This mechanism is regulated by the iron-sulfur subunit of complex III of the mitochondrial electron transport chain, ISP-1, and a mitochondrial quality control m-AAA protease, SPG-7, which in turn requires DAF-16/FoxO activity in GABAergic neurons.

Strengths:

A strength of this work is that the authors identify the specific site where mitochondrial stress promotes health and longevity, i.e., GABAergic neurons. In addition, the paper corroborates the findings with the appropriate experiments. How neuronal regulation of mitochondrial function impacts systemic health and aging is of interest to cell biology and neuroscience fields.

Weaknesses:

The entire paper is based on tissue-specific RNAi in GABAergic neurons, which was achieved using two different conditions of RNAi (although not for all experiments). However, multiple studies have shown deficiencies in the tissue-specific RNAi in C. elegans, especially for the rde-1(ne219) mutant used in this study. Therefore, it is necessary to repeat critical experiments by rescuing the isp-1 or spg-7 mutants in GABAergic neurons. Additionally, it is clear in the paper that perturbing mitochondrial function requires DAF-16/FoxO activity in GABAergic neurons to promote longevity, yet the downstream cellular pathways are not described.

Reviewer #3 (Public Review):

Summary:

This manuscript describes RNAi depletion of isp-1 or spg-7 in the GABAergic neurons of C. elegans leads to: lifespan extension; increased resistance to paraquat oxidative stress and heat stress; decreased brood size and mitotic germ cell numbers in the gonad and increased DNA aggregates in the oocytes; increased mitochondrial membrane potential, ATP levels, mitochondrial mass, mitochondrial DNA copies, mitochondrial DNA polymerase gamma polg-1 levels, and decreased ROS levels. The authors further show that daf-16 is necessary for GABAergic depletion of isp-1 mediated lifespan extension, stress resistance, increased mitochondrial membrane potential, mitochondrial mass and DNA copies, and decreased brood size. Unc-25 for GABA synthesis, unc-31 for neuropeptide secretion, and flp-13 neuropeptide are all in the same pathway of isp-1 RNAi in GABAergic neurons for lifespan extension and stress resistance.

Strengths:

The topic is interesting and relatively novel in terms of GABAergic mitochondrial dysfunction. The data provided support the conclusions well.

Weaknesses:

The mechanistic evidence needs to be improved substantially.