The dynamic interplay between ATP/ADP levels and autophagy sustain neuronal migration in vivo

  1. Cedric Bressan
  2. Alessandra Pecora
  3. Dave Gagnon
  4. Marina Snapyan
  5. Simon Labrecque
  6. Paul De Koninck
  7. Martin Parent
  8. Armen Saghatelyan

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Abstract

Cell migration is a dynamic process that entails extensive protein synthesis and recycling, structural remodeling, and considerable bioenergetic demand. Autophagy is one of the pathways that maintain cellular homeostasis. Time-lapse imaging of autophagosomes and ATP/ADP levels in migrating cells in the rostral migratory stream of mouse revealed that decreases in ATP levels force cells into the stationary phase and induce autophagy. Pharmacological or genetic impairments of autophagy in neuroblasts using either bafilomycin, inducible conditional mice, or CRISPR/Cas9 gene editing decreased cell migration due to the longer duration of the stationary phase. Autophagy is modulated in response to migration-promoting and inhibiting molecular cues and is required for the recycling of focal adhesions. Our results show that autophagy and energy consumption act in concert in migrating cells to dynamically regulate the pace and periodicity of the migratory and stationary phases to sustain neuronal migration.

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  1. Version published to 10.7554/elife.56006 on eLife
  2. eLife

    ###Reviewer 2

    This work investigates the role of autophagy in the migration of neuroblasts in the forebrain of adult mice. The authors provide evidence that activity of the autophagic pathway is related to the ratio of the migratory / stationary phase. They also provide evidence that activity of the autophagolysosomal pathway is related to the ATP/ADP levels and that autophagy targets the focal adhesion molecule paxilin. Autophagy is emerging as a central pathway in the regulation of neuronal development and the manuscript adds interesting and new evidence to this concept. Overall I consider this an important and well designed study.

    Major Comments

    1. In order to support the central notion that ATP/ADP levels control the autophagolysosomal turnover of paxilin, which in turn regulates migration the authors should investigate the localization and expression of paxilin in their experiments using the analysis that was applied in figure 3.

    2. The data presented in Figure 1-4 is sound and directly related to the central point of the paper, i.e., that ATP/ADP levels control the autophagolysosomal turnover of paxilin. The data presented in Figure 5 is circumstantial and except for showing that different pathways that have been linked to migration processes (not specifically migration of neuroblasts in the adult RMS) modulate expression of autophagy-related proteins. The data does not contribute to the key message of the paper and I would suggest to remove the data entirely.

    3. I am convinced that the paper carries an important and novel message but the order of how the results are presented seems not ideal to me. I believe that the order: analysis of autophagolysosomal activity in relation to migratory phases, analysis of metabolism with a focus on ATP/AMP ratios, and finally analysis of Paxilin as a potential target of Autophagy would be more stringent and convincing.

    4. Data presentation: in many instances the authors provide sample images of only one experimental condition e.g. Fig 2 M-O, R, W. While this may provide an impression of how the data was collected I think that it would be more convincing if the authors provided example images of all experimental conditions to illustrate differences. In addition the figure legend for Figure 2 M-O does not clarify which/ whether the sample images are from WT or mutant mice.

    5. The authors write that "...An Atg5 deficiency led to the accumulation of neuroblasts in the RMS close to the SVZ (582.7 {plus minus} 72.5 cell/mm2 in WT mice vs. 846.7 {plus minus} 72.7 cell/mm2 in cKO mice; p<0.05, n = 4 animals per group), with an accompanying decrease in the density of neuroblasts in the rostral RMS (RMS of the OB) and the OB. The graphs in figure 2P do not support the statement. that the density of neuroblasts in the rostral RMS and the OB are lower in ATG5 KO conditions. Please correct the statement and provide an explanation of how the numbers of neuroblasts can be stable if a higher number is observed in the more caudal portions of the RMS.

    6. The authors use CRISPR/Cas9 to knockout Atg12, if possible I would like to ask the authors to confirm the loss of Atg12 protein.

    7. The authors use the RFP GFP LC3 reporter which allows estimation of autophagic flux in vivo. In their analyses of autophagolysosomal activity (Figure 1I) they only estimate the RFP punctae. determining changes in autophagolysosomal activity would be stronger and more convincing if the authors performed the GFP+RFP/RFP punctae ratio.

  3. eLife

    ###Reviewer 1

    In the last few years the role of ATP metabolism at the synapse and in neuron development has become a topic of growing interest for the field of neuroscience. Autophagy is a prominent cellular process that has important roles in axon degeneration, cell death and nervous system disease. Importantly, the role of autophagy in neuron development has been less heavily studied than in disease contexts.

    The authors provide extensive quantitative datasets clearly indicating that genetic or pharmacological inhibition of autophagy results in reduced migration from the SVZ to the olfactory bulb. Indeed, a strength of the study is evidence showing that genetic effects on multiple autophagy components and AMPK (which activates autophagy) affect neuron migration. Another high point is extensive, quantitative time-lapse analysis of neuron migration in acute slice. This ex vivo approach is informative and makes for a compelling case regarding the role of autophagy in neuron migration from the SVZ to the olfactory bulb. While some in vivo data is provided more evidence on this front would further strengthen the study.

    While this is an interesting, high-quality study, the authors do not introduce an important body of prior literature on autophagy in neuron migration (Peng et al, 2012 JBC; Petri et al, 2017 EMBO; Gstrein et al, Nat Neuro 2017; Li et al, 2019 Cereb Cortex). As a result, it is unclear to the reviewer whether this is a significant step forward for the field, or a further valuable study solidifying the role of autophagy in neuron migration. At this point, the reviewer leans towards the latter view point. Below, are further details on this issue and several suggestions the reviewer hopes will improve what is a very nice piece of science.

    Major Comments

    1. The Gstrein paper is a very important piece of prior work, but is buried in the Discussion. This needs to be brought up in the introduction and noted appropriately. The manuscript also does not cite two other important papers showing that changes in autophagy can affect neuron migration in the olfactory bulb in adults in vivo (Petri et al, 2017 EMBO), and that molecular perturbations that affect autophagy impact neuronal migration in the cerebral cortex in vivo (Peng et al, 2012 JBC). Further recent work has shown that altered autophagy accompanies impaired neuronal migration in vivo in the cerebral cortex (Li et al, 2019 Cereb Cortex) and in vitro in a neuronal cell line (Li et al, 2019 Front Endocrinol). Placing the existing study's contribution more carefully and thoroughly within the context of this prior body of work on autophagy in neuron migration at the onset of the paper is critical.

    The attempted selling point of conflicting roles for autophagy in cell migration based on other cell-based studies and non-neuronal tissues is not particularly helpful and distracts from a major issue: There are already multiple studies indicating that autophagy affects neuron migration in vivo, and it is unclear how this work represents a major advance.

    1. The introduction does not comment on the role of ATP in neuron development and function; this has been an area of intense study in recent years. This type of background would be helpful for framing the context of the findings here.

    2. In vivo data indicating that autophagy influences neuron migration from SVZ to olfactory bulb is very important. Perhaps the reviewer is mistaken, but it seems like only Figure 2P shows quantitation of vivo data. This indicates loss of Atg5 results in increased cell numbers in the RMS. This an extremely important point. Hence, more evidence that other pharmacological or genetic manipulations of autophagy change cell density/migration in vivo would be valuable.

    The authors state: "with an accompanying decrease in the density of neuroblasts in the rostral RMS (RMS of the OB) and the OB". This is not supported by necessary statistical analysis, and looks likely to be insignificant. Statistics should be run here and commentary adjusted accordingly.

    1. In Figure 4B and C, quantitation of the ATP biosensor Perceval is shown. The authors claim a 20 fold change during migration. However, the Perceval ratio goes from 1.01 to 0.99 during one migration step and then 1.01 to 0.97 in the second step (Fig 4B). How is this a 20x change? To the contrary, this seems like quite a modest decrease in ATP ratio.

    How does this ratio change in a positive control where ATP production is reduced by impairing glycolysis or mitochondrial function?

    How does the role of ATP production by glycolysis versus mitochondrial stores influence migration?

    Presentation of data as a % change in Figure 4C is not ideal and gives the impression of artificially exaggerated effect sizes. Statistics are also notably absent from Figure 4C which makes a critical, quantitative point about migration and ATP consumption.

    1. The study emphasizes the point that AMPK senses changes in ATP levels and also activates autophagy. Both of these concepts are well known. Thus, pharmacologically blocking a known activator of autophagy like AMPK and showing effects on cell migration further supports the idea that autophagy is required for cell migration. This does not tie together that changes in ATP levels are affecting autophagy and, therefore, migration. Is there a way to directly manipulate ATP levels and then looks for impacts on both autophagy and migration? Is there a way to alter AMPK activation by ATP changes?

    2. In Figure 5D, treatments that increase and reduce the speed of migration show the same effects on autophagy as assessed by LC3II levels. How do the authors explain this? Wouldn't one expect opposing outcomes? Is this correct?

    3. Links between paxillin and cell migration are correlative, and not particularly convincing. Does reducing or increasing paxillin function affect migration? Does triggered specific heightened degradation/turnover of paxillin affect migration?

  4. eLife

    ##This manuscript is in revision at eLife

    The decision letter after peer review, sent to the authors on March 30, 2020, follows.

    Summary

    In this work you uncover the complex interplay between autophagy and energy consumption to regulate the pace and periodicity of the migratory and stationary phases in a prototypic model of migration in adult brain (the SVZ-OB). Both reviewers considered your work important as you provided evidence that 1) activity of the autophagic pathway is related to the ratio of the migratory / stationary phase, 2) activity of the autophagolysosomal pathway is related to the ATP/ADP levels, and 3) autophagy targets paxillin, a focal adhesion protein that is the direct target of LC3II.

    Essential Revisions

    The original reviews are attached below. Most of the major points can be addressed with minimal new experiments, but may require reanalysis of data or samples you already have. Based on these reviews, it is essential to address the following key points:

    1. Deepen the analysis of paxillin localization and expression
    2. Confirm the impact of Atg5 on the density of neuroblasts in the RMS (increased?) and the OB (unchanged?)
    3. Confirm the loss of Atg12 protein,
    4. Quantify autophagolysosomal activity (Figure 1I) by analyzing GFP+RFP/RFP punctae ratio

    In addition, while the science is strong, claims regarding the advance over previous studies should be toned down, since there is existing literature showing roles of autophagy in neuronal migration. The paper needs rewriting to accurately place your work in the context of prior research in the field. Specific recommendations include : i) rewrite the Introduction and Discussion to cite the literature appropriately: (ii), the second referee suggests that your Results could be presented in a different way in order to make better use of your data.

  5. Version published to 10.1101/2020.04.02.022228v1 on bioRxiv