A phosphoswitch at acinus-serine437 controls autophagic responses to cadmium exposure and neurodegenerative stress

  1. Nilay Nandi
  2. Zuhair Zaidi
  3. Charles Tracy
  4. Helmut Krämer

  • Curated by eLife

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    Evaluation Summary:

    Initial insights were provided by yeast genetic experiments into the mechanisms of starvation-induced autophagy. Since malfunctioning of this process is involved in numerous diseases, there is a need for further understanding the role and regulation of autophagy in different physiological settings. This work convincingly shows that a newly identified phosphatase controls basal levels of autophagy via regulation of phospho-acinus levels and reveals how cadmium intoxication triggers a neuroprotective autophagic response in the popular animal model Drosophila.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

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Abstract

Neuronal health depends on quality control functions of autophagy, but mechanisms regulating neuronal autophagy are poorly understood. Previously, we showed that in Drosophila starvation-independent quality control autophagy is regulated by acinus (acn) and the Cdk5-dependent phosphorylation of its serine 437 (Nandi et al., 2017). Here, we identify the phosphatase that counterbalances this activity and provides for the dynamic nature of acinus-serine 437 (acn-S437) phosphorylation. A genetic screen identified six phosphatases that genetically interacted with an acn gain-of-function model. Among these, loss of function of only one, the PPM-type phosphatase Nil (CG6036), enhanced pS437-acn levels. Cdk5-dependent phosphorylation of acn-S437 in nil 1 animals elevates neuronal autophagy and reduces the accumulation of polyQ proteins in a Drosophila Huntington’s disease model. Consistent with previous findings that Cd 2+ inhibits PPM-type phosphatases, Cd 2+ exposure elevated acn-S437 phosphorylation which was necessary for increased neuronal autophagy and protection against Cd 2+ -induced cytotoxicity. Together, our data establish the acn-S437 phosphoswitch as critical integrator of multiple stress signals regulating neuronal autophagy.

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  1. Version published to 10.7554/elife.72169 on eLife
  2. eLife

    Evaluation Summary:

    Initial insights were provided by yeast genetic experiments into the mechanisms of starvation-induced autophagy. Since malfunctioning of this process is involved in numerous diseases, there is a need for further understanding the role and regulation of autophagy in different physiological settings. This work convincingly shows that a newly identified phosphatase controls basal levels of autophagy via regulation of phospho-acinus levels and reveals how cadmium intoxication triggers a neuroprotective autophagic response in the popular animal model Drosophila.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    Following up on their previous work characterizing acinus as an autophagy regulator, the authors have now identified a PPM-type phosphatase named nil that mediates the dephosphorylation of acinus-S437 (which they have previously shown to be phosphorylated by Cdk5/p35). This new regulatory step is shown not only important for basal autophagy levels and for clearing a pathological polyQ repeat protein, but it also mediates a neuroprotective autophagy response during cadmium intoxication because cadmium inhibits this phosphatase. The methods are up to the standards of the field and properly support the conclusions. The in situ dephosphorylation assay is not as common as using fully recombinant proteins (or perhaps phospho-acinus co-IP-d from lysates) in an in vitro reaction, but it certainly supports that it is nil that dephosphorylates acinus. This work significantly adds to our knowledge of the regulation of autophagy and especially how intoxication by the heavy metal cadmium triggers protective autophagy to promote survival of the animals.

  4. eLife

    Reviewer #2 (Public Review):

    Autophagy is a key process in maintaining neuronal health. In their previous studies, authors identified role of Acinus in promoting basal autophagy and provided evidence for Cdk5 to phosphorylate Acinus at S437 to modulate its stability and subsequentially regulate basal level of autophagy. They further demonstrated enhancement of the Cdk5/Can-pS437 pathway in response to polyQ protein accumulation.

    In the current study, the authors identified PPM-type phosphatase Nilkatha (Nil) crucial for controlling phospho-switch on Acn-S437. Nil counteracts Cdk5-p35 kinase complex dependent phosphorylation of Acn-S437. The authors demonstrated Acn-S437 phospho-switch as a key integrator of multiple stress signals including Cd2+ induced cytotoxicity and polyQ protein accumulation. Overall the findings are interesting and important. The conclusions are mostly supported by the data presented in this work, but some aspects of data analysis would be stronger if they were extended.

    1. Throughout the study the authors demonstrated increase in autophagic flux either with an increase in Atg8a-II to Atg8a-I ratio or an increase in Atg8a puncta with or without inhibiting lysosomal acidification and degradation with chloroquine. While these methods hint towards increase in autophagic flux, it's not entirely clear if the cargo is indeed cleared up. It might have been worth doing p62 staining/western blots in addition to the Atg8a staining/western blots to validate that the cargo is indeed degraded. A decrease in p62 corresponding to an increase in Atg8a-II to Atg8a-I ratio would imply increase autophagic flux.

    2. Increased Acn-S437 phosphorylation elevates the basal, starvation-independent autophagy (Nandi et. al. 2017). Here, authors demonstrate that Nil regulates Acn-S437 phosphorylation in nucleus as well as has a potential involvement in regulating components of endo-lysosomal or autophagic trafficking. Authors also demonstrate that Nil1 null mutants have increased autophagic flux. It is however not clear if the the increased autophagic flux is a result of Acn-S437 phosphorylation or a result of Nil's interaction with autophagy pathway or a combined effect of both?

    3. The authors demonstrate that Acn-pS437 is necessary for autophagic response to Cd2+ exposure. It is however not clear if Acn-pS437 is sufficient for autophagic response to Cd2+ exposure. I would have included a phenotype rescue experiment here.

    4. The authors also predict that Acn-pS437 induced autophagy in nil1 mutants helped the animals to cope with low levels of Cd2+ induced oxidative stress. I would have included blocked autophagy in nil1 mutants to validate this observation.

    5. The authors observed an altered pattern with flw knockdown and larval lethality with mts knockdown. The authors provide an additive effect on Yorkie activity rather than direct effects on Acn as a possible explanation. Since exploring these observations further are not particularly useful for this paper, nevertheless it is an interesting result and it might still be worth discussing these observations in discussion.

  5. Version published to 10.1101/2021.07.27.453988v1 on bioRxiv