Mechanical force of uterine occupation enables large vesicle extrusion from proteostressed maternal neurons

Reviewer #1 (Public Review):

Summary:
The authors sought to understand the stage-dependent regulation of exophergenesis, a process thought to contribute to promoting neuronal proteostasis in C. elegans. Focusing on the ALMR neuron, they show that the frequency of exopher production correlates with the timing of reproduction. Using many genetic tools, they dissect the requirements of this pathway to eventually find that occupancy of the uterus acts as a signal to induce exophergenesis. Interestingly, the physical proximity of neurons to the egg zone correlates with exophergenesis frequency. The authors conclude that communication between the uterus and proximal neurons occurs through the sensing of mechanic forces of expansion normally provided by egg occupancy to coordinate exophergenesis with reproduction.

Strengths:
The genetic data presented is thorough and solid, and the observation is novel.

Weaknesses:
The main weakness of the study is that the detection of exophers is based on the overexpression of a fluorescent protein in touch neurons, and it is not clear whether this process is actually stimulated in wild-type animals, or if neurons have accumulated damaged proteins in relatively young day 2 animals.

Reviewer #2 (Public Review):

Summary:
This paper reports that mechanical stress from egg accumulation is a biological stimulus that drives the formation of extruded vesicles from the neurons of C. elegans ALMR touch neurons. Using powerful genetic experiments only readily available in the C. elegans system, the authors manipulate oocyte production, fertilization, embryo accumulation, and egg-laying behavior, providing convincing evidence that exopher production is driven by stretch-dependent feedback of fertilized, intact eggs in the adult uterus. Shifting the timing of egg production and egg laying alters the onset of observed exophers. Pharmacological manipulation of egg laying has the predicted effects, with animals retaining fewer eggs having fewer exophers and animals with increased egg accumulation having more. The authors show that egg production and accumulation have dramatic consequences for the viscera, and moving the ALMR process away from eggs prevents the formation of exophers. This effect is not unique to ALMR but is also observed in other touch neurons, with a clear bias toward neurons whose cell bodies are adjacent to the filled uterus. Embryos lacking an intact eggshell with reduced rigidity have impaired exopher production. Acute injection into the uterus to mimic the stretch that accompanies egg production causes a similar induction of exopher release. Together these results are consistent with a model where stretch caused by fertilized embryo accumulation, and not chemical signals from the eggs themselves or egg release, underlies ALMR exopher production seen in adult animals.

Strengths:
Overall, the experiments are very convincing, using a battery of RNAi and mutant approaches to distinguish direct from indirect effects. Indeed, these experiments provide a model generally for how one would methodically test different models for exopher production. The paper is well-written and easy to understand. I had been skeptical of the origin and purpose of exophers, concerned they were an artefact of imaging conditions, caused by deranged calcium activity under stressful conditions, or as evidence for impaired animal health overall. As this study addresses how and when they form in the animal using otherwise physiologically meaningful manipulations, the stage is now set to address at a cellular level how exophers like these are made and what their functions are.

Weaknesses:
Not many. The experiments are about as good as could be done. Some of the n's on the more difficult-to-work strains or experiments are comparatively low, but this is not a significant concern because of the number of different, complementary approaches used. The microinjection experiment in Figure 7 is very interesting, there are missing details that would confirm whether this is a sound experiment.

Reviewer #3 (Public Review):

Summary:
In this paper, the authors use the C. elegans system to explore how already-stressed neurons respond to additional mechanical stress. Exophers are large extracellular vesicles secreted by cells, which can contain protein aggregates and organelles. These can be a way of getting rid of cellular debris, but as they are endocytosed by other cells can also pass protein, lipid, and RNA to recipient cells. The authors find that when the uterus fills with eggs or otherwise expands, a nearby neuron (ALMR) is far more likely to secrete exophers. This paper highlights the importance of the mechanical environment in the behavior of neurons and may be relevant to the response of neurons exposed to traumatic injury.

Strengths:
The paper has a logical flow and a compelling narrative supported by crisp and clear figures.

The evidence that egg accumulation leads to exopher production is strong. The authors use a variety of genetic and pharmacological methods to show that increasing pressure leads to more exopher production, and reducing pressure leads to lower exopher production. For example, egg-laying defective animals, which retain eggs in the uterus, produce many more exophers, and hyperactive egg-laying is accompanied by low exopher production. The authors even inject fluid into the uterus and observe the production of exophers.

Weaknesses:
The main weakness of the paper is that it does not explore the molecular mechanism by which the mechanical signals are received or responded to by the neuron, but this could easily be the subject of a follow-up study.

I was intrigued by this paper, and have many questions. I list a few below, which could be addressed in this paper or which could be the subject of follow-up studies.

- Why do such a low percentage of ALMR neurons produce exophers (5-20%)? Does it have to do with the variability of the proteostress?
- Why does the production of exophers lag the peak in progeny production by 24-48 hours? Especially when the injection method produces exophers right away?
- As mentioned in the discussion, it would be interesting to know if PEZO-1/PIEZO is required for uterine stretching to activate exophergenesis. pezo-1 animals accumulate crushed oocytes in the uterus.