Axon death signalling in Wallerian degeneration among species and in disease

Axon loss is a shared feature of nervous systems being challenged in neurological disease, by chemotherapy or mechanical force. Axons take up the vast majority of the neuronal volume, thus numerous axonal intrinsic and glial extrinsic support mechanisms have evolved to promote lifelong axonal survival. Impaired support leads to axon degeneration, yet underlying intrinsic signalling cascades actively promoting the disassembly of axons remain poorly understood in any context, making the development to attenuate axon degeneration challenging. Wallerian degeneration serves as a simple model to study how axons undergo injury-induced axon degeneration (axon death). Severed axons actively execute their own destruction through an evolutionarily conserved axon death signalling cascade. This pathway is also activated in the absence of injury in diseased and challenged nervous systems. Gaining insights into mechanisms underlying axon death signalling could therefore help to define targets to block axon loss. Herein, we summarize features of axon death at the molecular and subcellular level. Recently identified and characterized mediators of axon death signalling are comprehensively discussed in detail, and commonalities and differences across species highlighted. We conclude with a summary of engaged axon death signalling in humans and animal models of neurological conditions. Thus, gaining mechanistic insights into axon death signalling broadens our understanding beyond a simple injury model. It harbours the potential to define targets for therapeutic intervention in a broad range of human axonopathies.

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Comments to the Author
The title of this review suggests a broad overview of the molecular mechanisms of the major forms of axonal degeneration with a special focus on different species and diseases. However, in the present manuscript only Wallerian degeneration (WD) is discussed in detail while other forms of axonal degeneration are not introduced. WD is not put in a broader context. Moreover, the role of axonal degeneration (or WD in particular) in disease is described very incompletely on only 1 page. Therefore either the title should be adjusted to e.g. "Axon death signalling in Wallerian degeneration among different species" or the manuscript should be significantly expanded to include other forms of axonal degeneration with the respective molecular mechanisms and a much broader and more thorough discussion about the role of axonal degeneration in disease.
WD is only one form of axonal degeneration (others including acute axonal degeneration, dying back axonal degeneration, focal axonal degeneration, retraction, pruning etc.). This should be made very clear and either the other forms of axonal degeneration should be discussed in detail or the article should be merely focussed on WD.
WD affects only the distal part of the axon after a lesion but not the remaining proximal part that is still connected to the soma. In many cases, especially after neurotrauma, it is a necessary prerequisite for later axonal regeneration that the distal part of the degenerating axon is cleared. It is therefore rather the remaining proximal axon stump that should be stabilised as it is still connected to the soma and might be the origin of axonal regeneration. WD in this case is a necessary physiological process that should not be inhibited.
In line with this view, several studies have shown that Wld-S counteracts and significantly delays axonal regeneration. These studies should also be discussed here (e.g. overview/perspective: Tang, Cells 2019 "Why is NMNAT Protective against Neuronal Cell Death and Axon Degeneration, but Inhibitory of Axon Regeneration?" and many others).
Autophagy has been described as an important pathophysiological mediator of WD and should thus be discussed in the review (e.g. Wang et al., Sci Adv 2019 "Rapid depletion of ESCRT protein Vps4 underlies injury-induced autophagic impediment and Wallerian"; Wakatsuki et al., JCB 2017 "GSK3B-mediated phosphorylation of MCL1 regulates axonal autophagy to promote Wallerian degeneration").
For the discussion of calcium as a "death mediator", the following studies should be cited as they have nicely proven the mechanism in mammals in vivo: Kerschensteiner et al., Nat Med 2005 "In vivo imaging of axonal degeneration and regeneration in the injured spinal cord." and Knöferle et al., PNAS 2010 "Mechanisms of acute axonal degeneration in the optic nerve in vivo".
The involvement of calpain in axonal degeneration has also been demonstrated in mammals in vivo and here CRMP2 could be identified as a major downstream target: Zhang et al., Sci Rep 2016 "Calpain-mediated cleavage of collapsin response mediator protein-2 drives acute axonal degeneration". This should be included.
If the authors really wish to discuss the role of WD in neurological diseases, this part needs to be substantially expanded. Also negative results of therapeutic interventions in WD should be discussed, e.g. the negative effects on axonal regeneration and negative results in MS models (e.g. Singh et al., 2017 "Relationship of acute axonal damage, Wallerian degeneration, and clinical disability in multiple sclerosis"). Also differential effects on axon and soma should be taken into account (e.g. Beirowski Eur J Nsc 2008 "The WldS gene delays axonal but not somatic degeneration in a rat glaucoma model"). Decision letter (RSOB-19-0118.R0)

15-Jul-2019
Dear Professor Neukomm We are pleased to inform you that your manuscript RSOB-19-0118 entitled "Axon death signaling in species and disease" has been accepted by the Editor for publication in Open Biology. The reviewer has recommended publication, but also suggest some minor revisions to your manuscript. Therefore, we invite you to respond to the comments and revise your manuscript.
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Data accessibility section To ensure archived data are available to readers, authors should include a 'data accessibility' section immediately after the acknowledgements section. This should list the database and accession number for all data from the article that has been made publicly available, for instance: Referee: Comments to the Author(s) The title of this review suggests a broad overview of the molecular mechanisms of the major forms of axonal degeneration with a special focus on different species and diseases. However, in the present manuscript only Wallerian degeneration (WD) is discussed in detail while other forms of axonal degeneration are not introduced. WD is not put in a broader context. Moreover, the role of axonal degeneration (or WD in particular) in disease is described very incompletely on only 1 page. Therefore either the title should be adjusted to e.g. "Axon death signalling in Wallerian degeneration among different species" or the manuscript should be significantly expanded to include other forms of axonal degeneration with the respective molecular mechanisms and a much broader and more thorough discussion about the role of axonal degeneration in disease.
WD is only one form of axonal degeneration (others including acute axonal degeneration, dying back axonal degeneration, focal axonal degeneration, retraction, pruning etc.). This should be made very clear and either the other forms of axonal degeneration should be discussed in detail or the article should be merely focussed on WD.
WD affects only the distal part of the axon after a lesion but not the remaining proximal part that is still connected to the soma. In many cases, especially after neurotrauma, it is a necessary prerequisite for later axonal regeneration that the distal part of the degenerating axon is cleared. It is therefore rather the remaining proximal axon stump that should be stabilised as it is still connected to the soma and might be the origin of axonal regeneration. WD in this case is a necessary physiological process that should not be inhibited.
In line with this view, several studies have shown that Wld-S counteracts and significantly delays axonal regeneration. These studies should also be discussed here (e.g. overview/perspective: Tang, Cells 2019 "Why is NMNAT Protective against Neuronal Cell Death and Axon Degeneration, but Inhibitory of Axon Regeneration?" and many others). The involvement of calpain in axonal degeneration has also been demonstrated in mammals in vivo and here CRMP2 could be identified as a major downstream target: Zhang et al., Sci Rep 2016 "Calpain-mediated cleavage of collapsin response mediator protein-2 drives acute axonal degeneration". This should be included.
If the authors really wish to discuss the role of WD in neurological diseases, this part needs to be substantially expanded. Also negative results of therapeutic interventions in WD should be discussed, e.g. the negative effects on axonal regeneration and negative results in MS models (e.g. Singh et al., 2017 "Relationship of acute axonal damage, Wallerian degeneration, and clinical disability in multiple sclerosis"). Also differential effects on axon and soma should be taken into account (e.g. Beirowski Eur J Nsc 2008 "The WldS gene delays axonal but not somatic degeneration in a rat glaucoma model"). Author's Response to Decision Letter for (RSOB-19-0118.R0)

02-Aug-2019
Dear Professor Neukomm We are pleased to inform you that your manuscript entitled "Axon death signaling in Wallerian degeneration aming species and in disease" has been accepted by the Editor for publication in Open Biology.
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Point-by-point response to the reviewer's comments (manuscript RSOB-19-0118)
We would like to thank the reviewer for comments, suggestions and concerns. Below, we will respond point-by-point to each comment raised by the reviewer. In the manuscript as well as in this document, changes are highlighted in red. In this document, the grey text serves as help for orientation.
Comments to the Author(s) The title of this review suggests a broad overview of the molecular mechanisms of the major forms of axonal degeneration with a special focus on different species and diseases. However, in the present manuscript only Wallerian degeneration (WD) is discussed in detail while other forms of axonal degeneration are not introduced. WD is not put in a broader context. Moreover, the role of axonal degeneration (or WD in particular) in disease is described very incompletely on only 1 page. Therefore either the title should be adjusted to e.g. "Axon death signalling in Wallerian degeneration among different species" or the manuscript should be significantly expanded to include other forms of axonal degeneration with the respective molecular mechanisms and a much broader and more thorough discussion about the role of axonal degeneration in disease.
The focus of this review lies in axon death signaling in Wallerian degeneration (WD). In the first part of the manuscript, we defined WD as a two-step process consisting of axon death and glial clearance. As the reviewer correctly indicated, we specifically discuss axon death in WD, while other forms of axon degeneration weren't in the main focus of this review. We therefore adjusted the title to:

Axon death signaling in Wallerian degeneration among species and in disease
We would like to keep "disease" in the title, for the following two reasons: 1) Wld S serves as a golden standard in the field of WD. Since its discovery, Wld S has been thoroughly used in a broad range of models with neurological conditions (C. elegans, Drosophila and mouse). If axon degeneration occurs in the absence of injury, and can be blocked by over-expression of Wld S , they undergo Wallerian-like degeneration. A beneficial outcome mediated by over-expression of Wld S led to the conclusion that the signaling pathway of WD (axon death) might be engaged in those axons. An important aspect of Wld S is dosage. The stronger it is over-expressed, the better it protects, which suggests that levels of Wld S dictate whether there might be a beneficial outcome. To date, multiple reviews have already discussed how attenuated axon death signaling in WD (that is, over-expression of Wld S ) might be beneficial in axons in a broad range of diseased and injured nervous systems, and we included (Conforti et al. 2014, Nature Neuroscience) in the chapter "6. Axon death signaling in disease". This was the main reason for us to emphasize in the manuscript that Wld S will not be covered as representative of the WD signaling pathway in disease.
2) Since the discovery of dSarm/Sarm1 in 2012, several labs started to investigate the role of Sarm1 in the diseased and injured nervous system. Contrary to the gain-of-function situation in Wld S , loss of dSarm/Sarm1 has a much more potent attenuation phenotype than Wld S (Gilley et al., 2017, Cell Reports). This is why we focused on Sarm1, which, as indicated above, offers 1 page of insights into the diseased and injured nervous system in our review. Importantly, we also emphasize that Sarm1 -/knockout mice are not protected in a model of ALS (SOD1 G93A), while Wld S offers modest extension of life span. Given that Nmnat/Nmnat2 (Wld S ) controls dSarm/Sarm1 activation, this is unexpected. And it shows that over-expression of Wld S might do something to axons which is unrelated to axon death signaling in WD.

Appendix A
We therefore believe that Wld S should not be the sole reference for axon death signaling in WD. For this reason, we mainly focused on Sarm1 in mouse models of neurological conditions. WD is only one form of axonal degeneration (others including acute axonal degeneration, dying back axonal degeneration, focal axonal degeneration, retraction, pruning etc.). This should be made very clear and either the other forms of axonal degeneration should be discussed in detail or the article should be merely focused on WD.
We added a new section which clarifies that among the different forms of axon degeneration, we will focus on axon death signaling in WD: To date, distinct morphological modes of axon degeneration have been observed and underlying molecular mechanisms described [10]. Among them are dying back axon degeneration, retraction, axosome shedding, focal axonal degeneration induced by growth factor withdrawal and pruning, to name a few. Axon degeneration can also be triggered through axonal injury (axotomy), which is probably one of the simplest models to study how axons execute their own destruction. Identified by and named after Augustus Waller, Wallerian degeneration (WD) is… … Over recent years, significant advancements broadened our understanding of axon death signaling in WD.
WD affects only the distal part of the axon after a lesion but not the remaining proximal part that is still connected to the soma. In many cases, especially after neurotrauma, it is a necessary prerequisite for later axonal regeneration that the distal part of the degenerating axon is cleared. It is therefore rather the remaining proximal axon stump that should be stabilised as it is still connected to the soma and might be the origin of axonal regeneration. WD in this case is a necessary physiological process that should not be inhibited.
In line with this view, several studies have shown that Wld-S counteracts and significantly delays axonal regeneration. These studies should also be discussed here (e.g. overview/perspective: Tang, Cells 2019 "Why is NMNAT Protective against Neuronal Cell Death and Axon Degeneration, but Inhibitory of Axon Regeneration?" and many others).
We are aware of the overview/perspective mentioned by the reviewer (Tang, Cells 2019) and other related reviews. In this particular review/perspective, there are multiple studies covered that show how the proximal axon (connected to the soma) induces stress-responses, and ultimately engages axon regeneration. We are also aware that attenuated axon death signaling harbors inhibitory features required for axons to regenerate. However, we believe that axon regeneration would be a topic for a separate review. We therefore avoided discussing the proximal axon (e.g. axon regeneration), and focused specifically on the distal from the soma separated axon (e.g. axon degeneration).
Moreover, the message of this review is not to suggest that WD, as a necessary physiological process, should be inhibited after injury in the distal, from the soma separated axon. Rather, it provides an overview of essential genes required for axon death signaling in WD.
Autophagy has been described as an important pathophysiological mediator of WD and should thus be discussed in the review (e.g. Wang et al., Sci Adv 2019 "Rapid depletion of ESCRT protein Vps4 underlies injury-induced autophagic impediment and Wallerian"; Wakatsuki et al., JCB 2017 "GSK3Bmediated phosphorylation of MCL1 regulates axonal autophagy to promote Wallerian degeneration").
We agree with the reviewer's comment. In fact, some work was already mentioned and cited under chapter "4.8. Other mediators". We significantly expanded this paragraph which now reads as follows: So far, axon death mediators were discussed whose modification results in a robust attenuation. . The precise interaction of these mediators with the signaling cascade remains to be determined, either due to tissue specific phenotypes or the involvement of whole organelles.
For the discussion of calcium as a "death mediator", the following studies should be cited as they have nicely proven the mechanism in mammals in vivo: We added both references under chapter "4.1. Ca 2+ ": As mentioned above, a local initial influx of Ca 2+ right after axotomy from the site of injury is observed both in invertebrate and vertebrate models in vivo and in vitro [24,27,50,51]. The first, rapid shortterm Ca 2+ wave precedes the lag phase ( Figure 2). The disassembly of severed axons is initiated by high extra-axonal Ca 2+ concentrations: if the extracellular environment is devoid of Ca 2+ (by adding EGTA, or in a medium lacking Ca 2+ ), or when voltage-gated Ca 2+ channels are inhibited, the fragmentation of severed axons is significantly delayed ( Figure 3A) [26,50,[52][53][54].
The involvement of calpain in axonal degeneration has also been demonstrated in mammals in vivo and here CRMP2 could be identified as a major downstream target: Zhang et al., Sci Rep 2016 "Calpain-mediated cleavage of collapsin response mediator protein-2 drives acute axonal degeneration". This should be included.
As mentioned above, we significantly expanded the chapter "4.8. Other mediators". One CRMP2 reference was already included in our manuscript (Kinoshita et al., 2019), we now added a second reference (Zhang et al., 2016), both of which are highlighting CRMP2: So far, axon death mediators were discussed whose modification results in a robust attenuation. . The precise interaction of these mediators with the signaling cascade remains to be determined, either due to tissue specific phenotypes or the involvement of whole organelles. Figure 1 does not include the events of acute axonal degeneration and of possible dying back axonal degeneration of the proximal axon. This should at least be mentioned in the legend. Also autophagy as a major player in axonal degeneration and clearing of axons should be included in the figure. It would be desirable to include NMNAT as the major molecular player in WD in the figure.
As mentioned above, in this review, we would like to focus on the distal, from the soma separated axon. Therefore, we avoided the proximal axon, including the discussion of a possible dying back mechanism after injury.
Moreover, rather than focusing on specific molecules or programs in Figure 1 (e.g. Nmnat/Nmnat2 or autophagy, respectively), we wanted to provide a broad overview about WD, that is the axonal intrinsic axon death signaling cascade, and the glial extrinsic clearance machinery. Furthermore, Nmnat/Nmant2 is discussed in details later in the manuscript, and indicated in Figure 3.