Pregnancy associated plasma protein-aa regulates endoplasmic reticulum-mitochondria associations

  1. Mroj Alassaf
  2. Mary Halloran

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Abstract

The endoplasmic reticulum (ER) and mitochondria form close physical associations to facilitate calcium transfer, thereby regulating mitochondrial function and dynamics. For neurons with high metabolic demands, such as sensory hair cells, precise regulation of ER-mitochondria associations is especially critical for cell survival. We previously identified the secreted metalloprotease Pregnancy associated plasma protein-aa (Pappaa) as a novel regulator of mitochondrial function in zebrafish lateral line hair cells (Alassaf et al., 2019). Here, we show that pappaa mutant hair cells exhibit excessive and abnormally close ER-mitochondria associations, suggesting increased ER-mitochondria calcium transfer. Indeed, we find that pappaa mutant hair cells are more vulnerable to pharmacological induction of ER-calcium release. Additionally, pappaa mutant hair cells display ER stress and dysfunctional downstream processes of the ER-mitochondria axis including mitochondrial fragmentation and autophagy. Together our results support a model in which Pappaa regulates mitochondrial function, at least in part, by regulating ER-mitochondria associations.

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  1. eLife

    ###This manuscript is in revision at eLife

    The decision letter after peer review, sent to the authors on August 5 2020, follows.

    Summary

    This current study builds on previous work from the same group published in eLife. This past work focused on the mechanism that renders lateral line hair cells of pappaa mutants more susceptible to the ototoxin neomycin. This work found that mitochondrial dysfunction was the underlying cause for neomycin susceptibility. This current study expands on the previous work and suggests that not only defects in mitochondria, but also the ER are involved in neomycin susceptibility. The authors use a variety of approaches including TEM, live imaging, pharmacology and RT-qPCR in their present study. Using TEM the authors show that mitochondria - ER associated are more numerous. Furthermore, similar to disrupting mitochondrial calcium pharmacologically, disrupting ER calcium also renders Pappaa-deficient hair cells more susceptible to neomycin. The authors suggest that this ER dysfunction manifests in several ways. They use live imaging to show that in pappaa mutants hair cells are unable to properly package neomycin into autosomes. In addition, via RT-qPCR they show that pappaa mutants have an increased unfolded protein response (UPR). Currently the relationship between all of these pathological issues is unclear, but this work does reveal additional mechanisms that could render loss of Pappaa detrimental to hair cell health. Although the work is well written and presented and statistically sound, there are several experiments that are needed to strengthen the claims presented in this study.

    Essential Revisions

    1. Location of TEM micrographs in hair cell. The morphology of organelles can vary based on location within the cell. For example, in hair cells the ER near the nucleus can be distinct from the ER present near the contacts made with efferent neurons or afferent neurons. (https://pubmed.ncbi.nlm.nih.gov/1430341/; https://physoc.onlinelibrary.wiley.com/doi/10.1113/jphysiol.2013.267914).

    Can the authors indicate what direction the sections (apical-basal or transverse) were taken, where in the hair cells are the sections were taken and how they determined this location?

    1. Quantification of mitochondrial fragmentation. It is clear from the TEM cross sections that the mitochondria in hair cells (Figure 3 A) are quite different between pappaa mutants and controls. Whether there are mitochondria or ER networks are present is not apparent from these TEM images. Nor is it entirely clear that the networks are fragmented. The authors use plugins developed for confocal imaging to estimate fragmentation base on circularity and area/perimeter measurement. It is unclear if these measurement translate to hair cells or TEM. In addition to fragmentation in TEM images, the fragmented mitochondria in pappaa mutants are also hard to see in the live, max-projected mitoTimer images.

    The mitochondrial networks and fragmentation may be clearer or be better quantified by acquiring super resolution images of hair cells labeled with mitoTracker. In addition, it is possible that the fragmentation may also be visible or more convincing in movies of Z-stacks of mitoTracker label compared to in the max-projected images provided.

    1. Examination of hair cell ER morphology. The previous work on Pappaa in zebrafish hair cells focused extensively on the mitochondria while the currently study the shifted the focus to the hair cell ER. While the ER-mito distances are convincing, a more wholistic picture of the amount or distribution of the ER in wildtype and mutant is lacking.

    This could be accomplished either using a transgenic line that labels the ER or a KDEL antibody (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4007406/).

    1. It qualitatively appears that pappaa mutant hair cells are taking up a greater quantity of fluorescent Neo faster than WT i.e. the fluorescent intensity is greater in more hair cells. Did the authors quantify Neo-TR uptake?

    2. Specificity of the pharmacological treatments. The authors perform numerous pharmacological experiments to disturb ER calcium. The authors suggest that that their pharmacological manipulations trigger hair cell death due to the alteration in the interplay between ER/mito calcium in hair cells. What concentration of either of these drugs does it take to kill WT hair cells? Dose-response curves comparing WT and mutant would help support the idea that hair-cell death observed is a direct effect of the drugs on hair-cell ER-mitochondria calcium signaling.

    Pharmacology is non-cell autonomous and the authors do not present evidence that these compounds specifically impact hair cell ER or mitochondrial calcium. Alternatively, these compound could impact supporting cell ER (https://elifesciences.org/articles/52160) as well as the ER in the innervating afferent or efferent neurons.

    More direct evidence show that hair cell mitochondria or ER calcium (measurements using mitoGCaMP such as in the previous study) are impacted by these treatments would make the author's claims more compelling.

    1. The disconnect between IGFR1 and results in the current study. The identify and location of IGFR1 and the IGFBP are still undefined in this system and therefore it remains unclear exactly how IGRR1 or Pappaa impact sensory hair cells. In previous work on pappaa mutants (enhanced startle response, defects in photoreceptor synapse formation, defects in hair cell mitochondria) the role of IGFR1 in these processes was validated. In the current study, the link with IGFR1 is implied throughout.

    It is true that the relationship between IGFR1 and Pappa is well characterized and that currently the only known substrates of Pappa are IGFBPs. Despite this work, it is still possible that given the range of phenotypes in pappaa mutants, that Pappa has other protein substrates that have not yet been identified, or other has biological functions unrelated to the IGF system.

    To verify IGFR1 in this current study the authors could use NB1-31772 to stimulate IGF1 bioavailability and test whether this rescues either the autophagy or UPR defects in pappaa mutants. Being able to rescue these phenotypes also makes the study more compelling.

    1. The authors state that there is not more spontaneous hair cell death in pappaa mutants compared to controls (line 443). Previous work has shown in zebrafish that Usher mutants (cdh23, ush1c, myo7a) also have an early UPR (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4007406/). Similar to pappaa mutants usher mutants have the same # of hair cells compared to controls, indicating no spontaneous hair cell death. But interestingly Usher mutants do have more TUNEL positive hair cells compared to controls, indicating that more hair cells in Usher mutants are in the process of apoptosis. Based on this new finding implicating the UPR response in pappaa mutants, could pappaa mutants, similar to hair cells in Usher mutants be more fragile (neomycin susceptible) as they are more likely to be in the process of apoptosis? A TUNEL label in pappaa mutants could reveal this. In addition, this paper on UPR in Usher mutant hair cells could be a useful paper to add to the discussion.

    2. Line 445-451: "Together, these findings suggest that Pappaa may regulate ER-mitochondria associations by promoting ER homeostasis. It is important to note that the ER and mitochondria are engaged in a constant feedback loop." This line of reasoning seems rather circular, considering that the previous study showed Pappaa regulates mitochondrial function. If mitochondrial function is impaired, it seems likely that ER homeostasis would be disrupted as well.

    3. Methods: Overall, the methods section needs more detail. All experiments that were not previously performed by the author or the author's lab should have a concise description of what the authors did next to the reference (e.g. fish were imaged under Lab-Tek Chambered Coverglass (Fisher Scientific) where they were immobilized under a nylon mesh and two stainless-steel slice hold-downs (Warner Instruments) per Stawicki et al, 2014) A detailed description of how individual pappaa170 larvae used in experiments were genotyped is needed. A comprehensive description of how mitochondrial circularity was measured using the "mitochondrial morphology" plug-in in ImageJ is needed.

    4. Statistics: how did the authors determine the power of the experiments were sufficient to avoid Type I and Type II error?

  2. Version published to 10.1101/2020.06.09.142505 on bioRxiv