Author response:
Reviewer #1 (Public review):
This manuscript presents an interesting exploration of the potential activation mechanisms of DLK following axonal injury. While the experiments are beautifully conducted and the data are solid, I feel that there is insufficient evidence to fully support the conclusions made by the authors.
In this manuscript, the authors exclusively use the puc-lacZ reporter to determine the activation of DLK. This reporter has been shown to be induced when DLK is activated. However, there is insufficient evidence to confirm that the absence of reporter activation necessarily indicates that DLK is inactive. As with many MAP kinase pathways, the DLK pathway can be locally or globally activated in neurons, and the level of DLK activation may depend on the strength of the stimulation. This reporter might only reflect strong DLK activation and may not be turned on if DLK is weakly activated. The results presented in this manuscript support this interpretation. Strong stimulation, such as axotomy of all synaptic branches, caused robust DLK activation, as indicated by puc-lacZ expression. In contrast, weak stimulation, such as axotomy of some synaptic branches, resulted in weaker DLK activation, which did not induce the puc-lacZ reporter. This suggests that the strength of DLK activation depends on the severity of the injury rather than the presence of intact synapses. Given that this is a central conclusion of the study, it may be worthwhile to confirm this further. Alternatively, the authors may consider refining their conclusion to better align with the evidence presented.
We wish to further clarify a striking aspect of puc-lacZ induction following injury: it is bimodal. It is either induced (in various injuries that remove all synaptic boutons), or not induced, including in injuries that spared only 1-2 remaining boutons. This was particularly evident for injuries that spared the NMJ on muscle 29, which is comprised of only a few boutons. In some instances, only a single bouton was evident on muscle 29. While our injuries varied enormously in the number of branches and boutons that were lost, we did not see a comparable variability in puc-lacZ induction. In the revision we will include additional images to better demonstrate this observation.
The reviewer (and others) fairly point out that our current study focuses on puc-lacZ as a reporter of Wnd signaling in the cell body. We consider this to be a downstream integration of events in axons that are more challenging to detect. It is striking that this integration appears strongly sensitized to the presence of spared synaptic boutons. Examination of Wnd’s activation in axons and synapses is a goal for our future work.
As noted by the authors, DLK has been implicated in both axon regeneration and degeneration. Following axotomy, DLK activation can lead to the degeneration of distal axons, where synapses are located. This raises an important question: how is DLK activated in distal axons? The authors might consider discussing the significance of this "synapse connection-dependent" DLK activation in the broader context of DLK function and activation mechanisms.
While it has been noted that inhibition of DLK can mildly delay Wallerian degeneration (Miller et al., 2009), this does not appear to be the case for retinal ganglion cell axons following optic nerve crush (Fernandes et al., 2014). It is also not the case for Drosophila motoneurons and NMJ terminals following peripheral nerve injury (Xiong et al., 2012; Xiong and Collins, 2012). Instead, overexpression of Wnd or activation of Wnd by a conditioning injury leads to an opposite phenotype - an increase in resiliency to Wallerian degeneration for axons that have been previously injured (Xiong et al., 2012; Xiong and Collins, 2012). The downstream outcome of Wnd activation is highly dependent on the context; it may be an integration of the outcomes of local Wnd/DLK activation in axons with downstream consequences of nuclear/cell body signaling. The current study suggests some rules for the cell body signaling, however, how Wnd is regulated at synapses and why it promotes degeneration in some circumstances but not others are important future questions.
For the reviewer’s suggestion, it is interesting to consider DLK’s potential contributions to the loss of NMJ synapses in a mouse model of ALS (Le Pichon et al., 2017; Wlaschin et al., 2023). Our findings suggest that the synaptic terminal is an important locus of DLK regulation, while dysfunction of NMJ terminals is an important feature of the ‘dying back’ hypothesis of disease etiology (Dadon-Nachum et al., 2011; Verma et al., 2022). We propose that the regulation of DLK at synaptic terminals is an important area for future study, and may reveal how DLK might be modulated to curtail disease progression. Of note, DLK inhibitors are in clinical trials (Katz et al., 2022; Le et al., 2023; Siu et al., 2018), but at least some have been paused due to safety concerns (Katz et al., 2022). Further understanding of the mechanisms that regulate DLK are needed to understand whether and how DLK and its downstream signaling can be tuned for therapeutic benefit.
Reviewer #2 (Public review):
Summary:
The authors study a panel of sparsely labeled neuronal lines in Drosophila that each form multiple synapses. Critically, each axonal branch can be injured without affecting the others, allowing the authors to differentiate between injuries that affect all axonal branches versus those that do not, creating spared branches. Axonal injuries are known to cause Wnd (mammalian DLK)-dependent retrograde signals to the cell body, culminating in a transcriptional response. This work identifies a fascinating new phenomenon that this injury response is not all-or-none. If even a single branch remains uninjured, the injury signal is not activated in the cell body. The authors rule out that this could be due to changes in the abundance of Wnd (perhaps if incrementally activated at each injured branch) by Wnd, Hiw's known negative regulator. Thus there is both a yet-undiscovered mechanism to regulate Wnd signaling, and more broadly a mechanism by which the neuron can integrate the degree of injury it has sustained. It will now be important to tease apart the mechanism(s) of this fascinating phenomenon. But even absent a clear mechanism, this is a new biology that will inform the interpretation of injury signaling studies across species.
Strengths:
(1) A conceptually beautiful series of experiments that reveal a fascinating new phenomenon is described, with clear implications (as the authors discuss in their Discussion) for injury signaling in mammals.
(2) Suggests a new mode of Wnd regulation, independent of Hiw.
Weaknesses:
(1) The use of a somatic transcriptional reporter for Wnd activity is powerful, however, the reporter indicates whether the transcriptional response was activated, not whether the injury signal was received. It remains possible that Wnd is still activated in the case of a spared branch, but that this activation is either local within the axons (impossible to determine in the absence of a local reporter) or that the retrograde signal was indeed generated but it was somehow insufficient to activate transcription when it entered the cell body. This is more of a mechanistic detail and should not detract from the overall importance of the study
We agree. The puc-lacZ reporter tells us about signaling in the cell body, but whether and how Wnd is regulated in axons and synaptic branches, which we think occurs upstream of the cell body response, remains to be addressed in future studies.
(2) That the protective effect of a spared branch is independent of Hiw, the known negative regulator of Wnd, is fascinating. But this leaves open a key question: what is the signal?
This is indeed an important future question, and would still be a question even if Hiw were part of the protective mechanism by the spared synaptic branch. Our current hypothesis (outlined in Figure 4) is that regulation of Wnd is tied to the retrograde trafficking of a signaling organelle in axons. The Hiw-independent regulation complements other observations in the literature that multiple pathways regulate Wnd/DLK (Collins et al., 2006; Feoktistov and Herman, 2016; Klinedinst et al., 2013; Li et al., 2017; Russo and DiAntonio, 2019; Valakh et al., 2013). It is logical for this critical stress response pathway to have multiple modes of regulation that may act in parallel to tune and restrain its activation.
Reviewer #3 (Public review):
Summary:
This manuscript seeks to understand how nerve injury-induced signaling to the nucleus is influenced, and it establishes a new location where these principles can be studied. By identifying and mapping specific bifurcated neuronal innervations in the Drosophila larvae, and using laser axotomy to localize the injury, the authors find that sparing a branch of a complex muscular innervation is enough to impair Wallenda-puc (analogous to DLK-JNK-cJun) signaling that is known to promote regeneration. It is only when all connections to the target are disconnected that cJun-transcriptional activation occurs.
Overall, this is a thorough and well-performed investigation of the mechanism of spared-branch influence on axon injury signaling. The findings on control of wnd are important because this is a very widely used injury signaling pathway across species and injury models. The authors present detailed and carefully executed experiments to support their conclusions. Their effort to identify the control mechanism is admirable and will be of aid to the field as they continue to try to understand how to promote better regeneration of axons.
Strengths:
The paper does a very comprehensive job of investigating this phenomenon at multiple locations and through both pinpoint laser injury as well as larger crush models. They identify a non-hiw based restraint mechanism of the wnd-puc signaling axis that presumably originates from the spared terminal. They also present a large list of tests they performed to identify the actual restraint mechanism from the spared branch, which has ruled out many of the most likely explanations. This is an extremely important set of information to report, to guide future investigators in this and other model organisms on mechanisms by which regeneration signaling is controlled (or not).
Weaknesses:
The weakest data presented by this manuscript is the study of the actual amounts of Wallenda protein in the axon. The authors argue that increased Wnd protein is being anterogradely delivered from the soma, but no support for this is given. Whether this change is due to transcription/translation, protein stability, transport, or other means is not investigated in this work. However, because this point is not central to the arguments in the paper, it is only a minor critique.
We agree and are glad that the reviewer considers this a minor critique; this is an area for future study. In Supplemental Figure 1 we present differences in the levels of an ectopically expressed GFP-Wnd-kinase-dead transgene, which is strikingly increased in axons that have received a full but not partial axotomy. We suspect this accumulation occurs downstream of the cell body response because of the timing. We observed the accumulations after 24 hours (Figure S1F) but not at early (1-4 hour) time points following axotomy (data not shown). Further study of the local regulation of Wnd protein and its kinase activity in axons is an important future direction.
As far as the scope of impact: because the conclusions of the paper are focused on a single (albeit well-validated) reporter in different types of motor neurons, it is hard to determine whether the mechanism of spared branch inhibition of regeneration requires wnd-puc (DLK/cJun) signaling in all contexts (for example, sensory axons or interneurons). Is the nerve-muscle connection the rule or the exception in terms of regeneration program activation?
DLK signaling is strongly activated in DRG sensory neurons following peripheral nerve injury (Shin et al., 2012), despite the fact that sensory neurons have bifurcated axons and their projections in the dorsal spinal cord are not directly damaged by injuries to the peripheral nerve. Therefore it is unlikely that protection by a spared synapse is a universal rule for all neuron types. However the molecular mechanisms that underlie this regulation may indeed be shared across different types of neurons but utilized in different ways. For instance, nerve growth factor withdrawal can lead to activation of DLK (Ghosh et al., 2011), however neurotrophins and their receptors are regulated and implemented differently in different cell types. We suspect that the restraint of Wnd signaling by the spared synaptic branch shares a common underlying mechanism with the restraint of DLK signaling by neurotrophin signaling. Further elucidation of the molecular mechanism is an important next step towards addressing this question.
Because changes in puc-lacZ intensity are the major readout, it would be helpful to better explain the significance of the amount of puc-lacZ in the nucleus with respect to the activation of regeneration. Is it known that scaling up the amount of puc-lacZ transcription scales functional responses (regeneration or others)? The alternative would be that only a small amount of puc-lacZ is sufficient to efficiently induce relevant pathways (threshold response).
While induction of puc-lacZ expression correlates with Wnd-mediated phenotypes, including sprouting of injured axons (Xiong et al., 2010), protection from Wallerian degeneration (Xiong et al., 2012; Xiong and Collins, 2012) and synaptic overgrowth (Collins et al., 2006), we have not observed any correlation between the degree of puc-lacZ induction (eg modest, medium or high) and the phenotypic outcomes (sprouting, overgrowth, etc). Rather, there appears to be a striking all-or-none difference in whether puc-lacZ is induced or not induced. There may indeed be a threshold that can be restrained through multiple mechanisms. We posit in figure 4 that restraint may take place in the cell body, where it can be influenced by the spared bifurcation.
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