Optical control of ERK and AKT signaling promotes axon regeneration and functional recovery of PNS and CNS in Drosophila

  1. Qin Wang
  2. Huaxun Fan
  3. Feng Li
  4. Savanna S. Skeeters
  5. Vishnu Krishnamurthy
  6. Yuanquan Song
  7. Kai Zhang

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Abstract

Neuroregeneration is a dynamic process synergizing the functional outcomes of multiple signaling circuits. Channelrhodopsin-based optogenetics shows feasibility of stimulating neural repair but does not pin down specific signaling cascades. Here, we utilized optogenetic systems, optoRaf and optoAKT, to delineate the contribution of the ERK and AKT signaling pathways to neuroregeneration in live Drosophila larvae. We showed that optoRaf or optoAKT activation not only enhanced axon regeneration in both regeneration competent and incompetent sensory neurons in the peripheral nervous system, but also allowed temporal tuning and proper guidance of axon regrowth. Furthermore, optoRaf and optoAKT differ in their signaling kinetics during regeneration, showing a gated versus graded response, respectively. Importantly in the central nervous system, their activation promotes axon regrowth and functional recovery of the thermonociceptive behavior. We conclude that non-neuronal optogenetics target damaged neurons and signaling subcircuits, providing a novel strategy in the intervention of neural damage with improved precision.

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  1. eLife

    ###This manuscript is in revision at eLife

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

    Summary

    This study uses ontogenetic probes to activate the Raf and Akt pathways. This approach represents a powerful system to dissect signaling outcomes of these pivotal enzymes. Light-controlled activation of these two relevant signals is used to assess their ability to mediate axonal pathfinding, branching, and regeneration. The optogenetic tools allow for an examination of the sufficiency of either Akt or ERK pathway activation in these processes, in both PNS and CNS neurons. The result that both pathways play significant yet distinct roles in these processes is important and is of wide interest.

    Several major and minor reservations were brought up by the reviewers. The major issues are to define more fully the upstream events that activate Raf-1 and Akt and the need for additional controls for the optogenetic probes. The kinetics of activation, as well as cell localization, requires attention. Another request is to examine if there is convergence in the two pathways. The major concerns are described below.

    Essential Revisions

    1. This study assumes that the tools trigger signaling pathways independently of upstream (neurotropic) signaling. However, whether these tools require some upstream signaling remain incompletely addressed. For example, activation of Raf1 requires upstream activation by kinases phosphorylating the N-terminal region (Y341 and S338). The phosphorylation of S338 is a commonly used read-out for Raf-1 activation (and mutants at this position show no activation). It would be very informative to examine the status of pS338 in optoRaf and to compare the optoRaf to a mutant S338A version, at least in Hek293 cells. Because these phosphorylations are linked to Raf dimerization, these studies would provide insight into whether Raf dimerization is required or possible in this context.

    2. It would be also helpful to include more specificity controls for Raf vs. Akt signaling in *Drosophila *neurons to ensure the signals directly go to cells where the functional assessments are being conducted.

    3. The kinetic experiments are interesting but somewhat incomplete, and it is unclear what the takeaway from these experiments should be. Importantly, it is not known how different pulsed light patterns translate temporally to signaling. It seems that from the data in figure 1, it is possible that in neurons patterns may maintain a constant activation of the pathway. Additional controls looking at the extent of signaling in neurons with these paradigms would be really helpful.

    Minor Points

    a) Recruiting optoAkt to the membrane does not make it independent of upstream PI3-K signaling, as PH-domain-containing kinases such as PDK1 are essential for Akt activation (by phosphorylating Akt on T308). Again, activation-specific phosphorylations on T308 could verify whether PI3K are involved in optoAkt function.

    b) In one experiment, the authors tested the functional outcome of combining Raf and Akt activation, but it would be helpful if these were done in other experimental paradigms as well. Are these signaling pathways semi-redundant functionally or additive and able to further enhance the extent of regeneration? In this regard, what are the obstacles to utilizing optoRaf and optoAkt concurrently? Would synergism be expected?

    c) The study suggests a lack of cross-talk between the two pathways. Given the ability of each pathway to achieve some regeneration on its own, the authors should discuss whether these pathways might eventually converge on common downstream effectors.

    d) Given previous genetic studies, it is a bit surprising that Raf signaling plays a more pronounced effect than Akt in regeneration. It would be helpful in the authors could comment on this in the discussion, not just in the context of Erk/Akt but also the broader regeneration literature.

    e) The Discussion points out the role of neurotrophic factor signaling, which is upstream of Raf and Akt. It should be acknowledged there is an absence of NGF family members and their receptors in Drosophila. This does not negate the results in the manuscript, but the significance of the findings should be clarified.

  2. Version published to 10.1101/2020.05.15.098251 on bioRxiv