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  1. Author Response

    Reviewer #2 (Public Review):

    Wu Yang et al. investigated how exophers (large vesicles released from neuronal somas) are degraded. They find that the hypodermal skin cells surrounding the neuron break up the exophers into smaller vesicles that are eventually phagocytosed. The neuronal exophers accumulate early phagosomal markers such as F-actin and PIP2, and blocking actin assembly suppressed the formation of smaller vesicles and the clearance of neuronal exophers. They show the smaller vesicles are labeled with various markers for maturing phagosomes, and inhibiting phagosome maturation blocked the breakdown of exophers in to smaller vesicles. Interestingly, they discover that GTPase ARF-6, effector SEC-10/Exocyst, and the phagocytic receptor CED-1 in the hypodermis are required for efficient production of exophers by neurons.

    Strength

    The study clearly demonstrates that exophers are eliminated via hypodermal cellmediated phagocytosis. Exophers are broken down into smaller vesicles that accumulate phagocytic markers, and inhibiting this process shows that exophers are not resolved. The paper does a thorough examination of various markers and mutants to demonstrate this process.

    The hypodermal cells not only engulf these small vesicles, but they also play a role in the formation of exophers. Exopher production is reduced when ARF-6, SEC-10, or CED-1 are knocked down in the hypodermis. This is intriguing because phagocytosis is a critical step in the final elimination of cells, but in this unique situation, it appears that the neuron fails to extrude the exopher without phagocytes.

    Weakness

    Non-professional phagocytes engulfing cell corpses and many other types of cellular debris (e.g. degenerating axons) have been shown in multiple systems and the observations here are not surprising. Many of the markers used in the study are wellestablished phagocytic markers and do not bring forward a new technological advance.

    What's interesting is that the breakdown of exophers into smaller vesicles and eventual clearance follows a different sequence of events than macrophages. Exophers appear to undergo phagosomal fission before interacting with lysosomes. This would be difficult to appreciate by a general reader.

    While the paper has strengths, it appears that the message is not clear. The title suggests that the reader will learn about how ARF-6 and CED-1 control exopher extrusion. Although this observation is intriguing and maybe the main point of the paper, there does not appear to be a substantial amount of data to support this claim. The only data to back this up is in the final figure and the majority of the paper is focused on how hypodermal cells phagocytose exophers.

    The title has been revised.

    To show exopher secretion is dependent on the hypodermal cells-

    1. Could authors induce exopher production through other means? And test any involvement of CED-1? For example, authors note exopher production increases under stress conditions including expression of mutant Huntingtin protein. It would be intriguing if loss of CED-1 would be sufficient to block or reduce exopher production in that context and would highlight an exciting role for phagocytic cell types.

    We interpreted this question as an inquiry into whether the neuron intrinsic exopher inducer was relevant to reliance on hypodermal interaction for exophergenesis, given our use of aggregating mCherry as the inducer. Unfortunately, our Huntingtin expressor lines now display high levels of transgene silencing, precluding their use in this experiment. To address this concern, we switched to a low toxicity GFP expressing transgene from the Chalfie lab, uIs31[Pmec17::GFP]. We found that arf-6 mutations suppressed exophers in this background as effectively as they did in previous mCherry experiments, indicating that our results are not dependent upon the particular transgene marking the touch neurons, or the specific protein they express (Fig 6E).

    1. It is not clear if the CED-1 localization to the exopher is due to CED-1 expression during phagocytosis or is it involved in the extrusion. Perhaps the basal level of CED-1 is important for the extrusion but the strong expression is important for recognition of the exopher.

    In the experiments we performed we used a constitutively expressed hypodermisspecific CED-1::GFP to show localization to exophers, so the recruitment of CED1::GFP in hypodermal membranes to the site where the neighboring neuron is producing an exopher is not caused by changes in expression, but rather is more likely to reflects protein recruitment. We now point this out more explicitly in the text. Added text: “Since the hypodermal CED-1DC::GFP we used is constitutively expressed, we attribute the exopher surrounding CED-1DC::GFP signal to CED-1 recruitment by exopher-surface signals."

    1. While the data with ttr-52 and anoh-1 alleles is compelling, do we know that exophers actually expose PS? Especially since at a certain point, the exopher is still attached to the neuronal soma. Is PS still exposed by exopher in CED-1 background?

    We are also very interested in this. Unfortunately, we have had difficulty obtaining sufficient MFGE8 PS-biosensor expression in the adult to test this question directly.

    1. What is the fate of a neuron that is unable to produce exophers? Could one look at lifespan of ALMR neuron in CED-1, ARF-6 or Sec-10 allele (potentially with specificity to hypodermis)?

    To address this question we measured the function of the mechanosensory touch neurons, using the classic gentle touch response assay in mCherry expressing animals, comparing controls to arf-6 and ced-1 mutants. For both arf-6 and ced-1 alleles, we found reduced response to gentle touch in older adults (Ad10), indicating a deficit in neuronal function. These results are consistent with exopher production maintaining neuronal health into old age, but interpretation is limited since neither ced-1 or arf-6 act specifically in exophergenesis and therefore also affect the animals in additional ways. Currently, there are no known genetic perturbations that act specifically in exophergenesis, so there is no better approach to do the analysis. We had already published similar results in our 2017 Nature paper that first described exophers, showing that gentle touch response is better preserved in a touch neuron HttQ128::CFP strain that produced a touch neuron exopher than in the same mutant background in which the touch neurons that had not produced an exopher.

  2. eLife assessment

    This manuscript will be of interest to a wide range of cell biologists interested in understanding cell-cell communication. The discovery that an engulfing cell can control the extrusion and degradation of large vehicles from its target cell is important and intriguing. The authors present compelling data that show that exophers (large neuronal extrusions proposed to discard toxic cargo) are taken up by adjacent hypodermal cells, split into smaller fragments, and eventually degraded by lysosome fusion. The authors identify a number of small GTPases and accessory components, as well as the phagocytic receptor (CED-1) and the likely eat-me signal (phosphatidylserine).

  3. Reviewer #1 (Public Review):

    In this manuscript, Wang et al provide a pathway required for the production and degradation of exophers - large neuronal extrusions proposed to discard toxic cargo. Exophers were fairly recently described by this group and have now been observed in mammalian neurons, suggesting a broad importance in neuronal health. How exophers were disposed of by surrounding tissues was not known. Here, the authors identify a pathway required for exopher degradation into small debris (starry night), and intriguingly, genes proposed to be required in the degrading cells (hypodermis) for exopher production in neurons.

    Strengths of the manuscript include significant new insights into a problem that had not been investigated in mechanistic detail, and the combined use of genetics and cell biology to sort genes into pathways involved in exopher production and degradation. Several differences are found between exopher and cell corpse disposal, highlighting the importance of the study. The findings should be of interest to a broad audience.

  4. Reviewer #2 (Public Review):

    Wu Yang et al. investigated how exophers (large vesicles released from neuronal somas) are degraded. They find that the hypodermal skin cells surrounding the neuron break up the exophers into smaller vesicles that are eventually phagocytosed. The neuronal exophers accumulate early phagosomal markers such as F-actin and PIP2, and blocking actin assembly suppressed the formation of smaller vesicles and the clearance of neuronal exophers. They show the smaller vesicles are labeled with various markers for maturing phagosomes, and inhibiting phagosome maturation blocked the breakdown of exophers in to smaller vesicles. Interestingly, they discover that GTPase ARF-6, effector SEC-10/Exocyst, and the phagocytic receptor CED-1 in the hypodermis are required for efficient production of exophers by neurons.

    Strength
    The study clearly demonstrates that exophers are eliminated via hypodermal cell-mediated phagocytosis. Exophers are broken down into smaller vesicles that accumulate phagocytic markers, and inhibiting this process shows that exophers are not resolved. The paper does a thorough examination of various markers and mutants to demonstrate this process.

    The hypodermal cells not only engulf these small vesicles, but they also play a role in the formation of exophers. Exopher production is reduced when ARF-6, SEC-10, or CED-1 are knocked down in the hypodermis. This is intriguing because phagocytosis is a critical step in the final elimination of cells, but in this unique situation, it appears that the neuron fails to extrude the exopher without phagocytes.

    Weakness

    Non-professional phagocytes engulfing cell corpses and many other types of cellular debris (e.g. degenerating axons) have been shown in multiple systems and the observations here are not surprising. Many of the markers used in the study are well-established phagocytic markers and do not bring forward a new technological advance.

    What's interesting is that the breakdown of exophers into smaller vesicles and eventual clearance follows a different sequence of events than macrophages. Exophers appear to undergo phagosomal fission before interacting with lysosomes. This would be difficult to appreciate by a general reader.

    While the paper has strengths, it appears that the message is not clear. The title suggests that the reader will learn about how ARF-6 and CED-1 control exopher extrusion. Although this observation is intriguing and maybe the main point of the paper, there does not appear to be a substantial amount of data to support this claim. The only data to back this up is in the final figure and the majority of the paper is focused on how hypodermal cells phagocytose exophers.

    To show exopher secretion is dependent on the hypodermal cells-

    1. Could authors induce exopher production through other means? And test any involvement of CED-1? For example, authors note exopher production increases under stress conditions including expression of mutant Huntingtin protein. It would be intriguing if loss of CED-1 would be sufficient to block or reduce exopher production in that context and would highlight an exciting role for phagocytic cell types.
    2. It is not clear if the CED-1 localization to the exopher is due to CED-1 expression during phagocytosis or is it involved in the extrusion. Perhaps the basal level of CED-1 is important for the extrusion but the strong expression is important for recognition of the exopher.
    3. While the data with ttr-52 and anoh-1 alleles is compelling, do we know that exophers actually expose PS? Especially since at a certain point, the exopher is still attached to the neuronal soma. Is PS still exposed by exopher in CED-1 background?
    4. What is the fate of a neuron that is unable to produce exophers? Could one look at lifespan of ALMR neuron in CED-1, ARF-6 or Sec-10 allele (potentially with specificity to hypodermis)?

  5. Reviewer #3 (Public Review):

    In this paper, the authors examine the fate of exophers ejected from C. elegans neurons overexpressing a presumably aggregated mCherry protein. They show that exophers are taken up by adjacent hypodermal cells, split into smaller fragments, and eventually degraded by lysosome fusion. They identify a number of small GTPases and accessory components, as well as the phagocytic receptor (CED-1) and the likely eat-me signal (phosphatidylserine).

    The manuscript follows up on previous exopher work from some members of the current collaboration, and provides a detailed analysis of exopher fate, that will likely be useful for understanding similar events in other settings. The studies are well done, the images and data are convincing, and the interpretations are generally appropriate.