Author response:
The following is the authors’ response to the previous reviews.
Reviewer #1 (Public review):
This paper examines the role of MLCK (myosin light chain kinase) and MLCP (myosin light chain phosphatase) in axon regeneration. Using loss-of-function approaches based on small molecule inhibitors and siRNA knockdown, the authors explore axon regeneration in cell culture and in animal models from central and peripheral nervous systems. Their evidence shows that MLCK activity facilitates axon extension/regeneration, while MLCP prevents it.
Major concerns:
(1) In the title, authors indicate that the observed effects from loss-of-function of MLCK/MLCP take place via F-actin redistribution in the growth cone. However, there are no experiments showing a causal effect between changes in axon growth mediated by MLCK/MLCP and F-actin redistribution.
Thank you for your comments. We revised the title of our manuscript to “MLCK/MLCP regulates mammalian axon regeneration and redistributes the growth cone F-actin”. (line 3)
(2) The author combines MLCK inhibitors with Bleb (Figure 6), trying to verify if both pairs of inhibitors act on the same target/pathway. MLCK may regulate axon growth independent of NMII activity. However, this has very important implications for the understanding not only on how NMII works and affects axon extension, but also in trying to understand what MLCP is doing. One wonders if MLCP actions, which are opposite of MLCK, also independent of NMII activity? The authors, in the discussion section, try to find an explanation for this finding, but I consider it fails since the whole rationale of the manuscript is still around how MLCK and MLCP affect NMII phosphorylation.
Thank you for your comments. Although both MLCK and MLCP regulate the activity of NMII, it has been reported that they also govern domain-specific spatial control of actin-based motility in the growth cone. Specifically, MLCK activity is essential for arc translocation and retrograde flow within the P domain, while MLCP appears to specifically modulate arc movement and associated myosin II contractility in the T zone and C domain (Ref). Therefore, it is proposed that the regulatory mechanisms of MLCK and MLCP are highly complex during the process of axon growth.
[Ref]：Xiao-Feng Zhang, Andrew W Schaefer, Dylan T Burnette, Vincent T Schoonderwoert, Paul Forscher. Rho-dependent contractile responses in the neuronal growth cone are independent of classical peripheral retrograde actin flow. Neuron. 2003 Dec 4;40(5):931-44.
What follows is a discussion of the merits and limitations of different claims of the manuscript in light of the evidence presented.
(1) Using western blot and immunohistochemical analyses, authors first show that MLCK expression is increased in DRG sensory neurons following peripheral axotomy, concomitant to an increase in MLC phosphorylation, suggesting a causal effect (Figure 1). The authors claim that it is common that axon growth-promoting genes are upregulated. It would have been interesting at this point to study in this scenario the regulation of MLCP.
We thank Reviewer for the positive comment on our manuscript.
(2) Using DRG cultures and sciatic nerve crush in the context of MLCK inhibition (ML-7) and down-regulation, authors conclude that MLCK activity is required for mammalian peripheral axon regeneration both in vitro and in vivo (Figure 2). In parallel, the authors show that these treatments affect as expected the phosphorylation levels of MLC.
The in vitro evidence is of standard methods and convincing. However, here, as well as in all other experiments using siRNAs, no Control siRNAs were used. Authors do show that the target protein is downregulated, and they can follow transfected cells with GFP. Still, it should be noted that the standard control for these experiments has not been done.
Thank you for your comments. We utilized scrambled siRNA as a control. I sincerely apologize for the oversight in the manuscript; although we mentioned that scrambled siRNA was used as a control in the figure legends, we failed to clearly articulate this important information in the methods section. We have revised the manuscript accordingly. (line 87, line 549, line, line 562, line 568).
(3) The authors then examined the role of the phosphatase MLCP in axon growth during regeneration. The authors first use a known MLCP blocker, phorbol 12,13-dibutyrate (PDBu), to show that is able to increase the levels of p-MLC, with a concomitant increase in the extent of axon regrowth of DRG neurons, both in permissive as well as non-permissive substrates. The authors repeat the experiments using the knockdown of MYPT1, a key component of the MLC-phosphatase, and again can observe a growth-promoting effect (Figure 3).
The authors further show evidence for the growth-enhancing effect in vivo, in nerve crush experiments. The evidence in vivo deserves more evidence and experimental details (see comment 2). A key weakness of the data was mentioned previously: no control siARN was used.
Thank you for your comments. As mentioned above, we used scramble siRNA as control in vivo experiment as well.
(4) In the next set of experiments (presented in Figure 4) authors extend the previous observations in primary cultures from the CNS. For that, they use cortical and hippocampal cultures, and pharmacological and genetic loss-of-function using the above-mentioned strategies. The expected results were obtained in both CNS neurons: inhibition or knockdown of the kinase decreases axon growth, whereas inhibition or knockdown of the phosphatase increases growth. A main weakness in this set is that drugs were used from the beginning of the experiment, and hence, they would also affect axon specification. As pointed in Materials and Method (lines 143-145) authors counted as "axons" neurites longer than twice the diameter of the cell soma, and hence would not affect the variable measured. In any case, to be sure one is only affecting axon extension in these cells, the drugs should have been used after axon specification and maturation, which occurs at least after 5 DIV.
Thank you for your comments. We acknowledge that the early administration of drugs can lead to unintended effects on neuronal polarization and axon formation. However, in line with our previous publication, we focused exclusively on measuring the longest length of the axon. To quantify axon length, we selected neurons exhibiting an axonal process exceeding twice the diameter of their cell body and measured the longest axon from 100 neurons for each condition (Ref 1, Ref 2). Consequently, we believe that drug administration at the onset of cell culture influences axon formation; however, it does not significantly affect the drug's impact on axon length.
[Ref 1]: Chang-Mei Liu, Rui-Ying Wang, Saijilafu, Zhong-Xian Jiao, Bo-Yin Zhang, Feng-Quan Zhou. MicroRNA-138 and SIRT1 form a mutual negative feedback loop to regulate mammalian axon regeneration. Genes Dev. 2013 Jul 1;27(13):1473-83.
[Ref 2]: Eun-Mi Hur, Saijilafu, Byoung Dae Lee, Seong-Jin Kim, Wen-Lin Xu, Feng-Quan Zhou. GSK3 controls axon growth via CLASP-mediated regulation of growth cone microtubules. Genes Dev. 2011 Sep 15;25(18):1968-81.
(5) In Figure 7, the authors a local cytoskeletal action of the drug, but the evidence provided does not differentiate between a localized action of the drugs and a localized cell activity.
We appreciate the reviewer’s insightful comments and have revised our title to “MLCK/MLCP Regulates mammalian axon regeneration and redistributes growth cone F-actin.” Furthermore, we have made corresponding revisions to the manuscript (line31, line 73).
References:
(1) Eun-Mi Hur 1, In Hong Yang, Deok-Ho Kim, Justin Byun, Saijilafu, Wen-Lin Xu, Philip R Nicovich, Raymond Cheong, Andre Levchenko, Nitish Thakor, Feng-Quan Zhou. 2011. Engineering neuronal growth cones to promote axon regeneration over inhibitory molecules. Proc Natl Acad Sci U S A. 2011 Mar 22;108(12):5057-62. doi: 10.1073/pnas.1011258108.
(2) Garrido-Casado M, Asensio-Juárez G, Talayero VC, Vicente-Manzanares M. 2024. Engines of change: Nonmuscle myosin II in mechanobiology. Curr Opin Cell Biol. 2024 Apr;87:102344. doi: 10.1016/j.ceb.2024.102344.
(3) Karen A Newell-Litwa 1, Rick Horwitz 2, Marcelo L Lamers. 2015. Non-muscle myosin II in disease: mechanisms and therapeutic opportunities. Dis Model Mech. 2015 Dec;8(12):1495-515. doi: 10.1242/dmm.022103.
Reviewer #2 (Public review):
Summary:
Saijilafu et al. demonstrate that MLCK/MLCP proteins promote axonal regeneration in both the central nervous system (CNS) and peripheral nervous system (PNS) using primary cultures of adult DRG neurons, hippocampal and cortical neurons, as well as in vivo experiments involving sciatic nerve injury, spinal cord injury, and optic nerve crush. The authors show that axon regrowth is possible across different contexts through genetic and pharmacological manipulation of these proteins. Additionally, they propose that MLCK/MLCP may regulate F-actin reorganization in the growth cone, which is significant as it suggests a novel strategy for promoting axonal regeneration.
Strengths:
This manuscript presents a wide range of experimental models to address its hypothesis and biological question. Notably, the use of multiple in vivo models significantly enhances the overall validity of the study.
We thank Reviewer for the positive comment on our manuscript.
Weaknesses:
- The authors previously published that blocking myosin II activity stimulates axonal growth and that MLCK activates myosin II. The present work shows that inhibiting MLCK blocks axonal regeneration while blocking MLCP (the protein that dephosphorylates myosin II) produces the opposite effect. Although this contradiction is discussed, no new evidence has been added to the manuscript to clarify this mechanism or address the remaining questions. Critical unresolved questions include: what happens to myosin II expression when both MLCK and MLCP are inhibited? If MLCK/MLCP are acting through an independent mechanism, what would that mechanism be?
- In the discussion, the authors mention the existence of two myosin II isoforms with opposing effects on axonal growth. Still, there is no evidence in the manuscript to support this point.
- It is also unclear how MLCK/MLCP acts on the actin cytoskeleton. The authors suggest that proteins such as ADF/cofilin, Arp 2/3, Eps8, Profilin, Myosin II, and Myosin V could regulate changes in F-actin dynamics. However, this study provides no experimental evidence to determine which proteins may be involved in the mechanism.
Thank you for your comments. Axon growth is an exceptionally intricate process, facilitated by the coordinated regulation of gene expression in the soma, axonal transport along the shaft, and the assembly of cytoskeletal elements and membrane proteins at the growth cone. In this paper, our results primarily demonstrate that MLCK/MLCP plays a crucial role in regulating mammalian axon regeneration and redistributing F-actin within the growth cone; however, we did not investigate which specific proteins act downstream of MLCK/MLCP during axon regeneration.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
- A title more suitable for the evidence shown can be: MLCK/MLCP regulates mammalian axon regeneration and redistributes the growth cone F-actin.
Thank you for your comments. We revised the title of our manuscript to“MLCK/MLCP regulates mammalian axon regeneration and redistributes the growth cone F-actin” (line 3).
-In figure 3, It would be useful to indicate in the figure legend, that the red arrow is pointing to a suture that was performed during surgery to mark clearly the injury site.
Thank you for your comments. We revised Figure 3 legend that indicates the red arrow is pointing to a suture that was performed during surgery to mark clearly the injury site (line 571-572).
- The following is a concern raised in the previous round, and that the response by the authors was so complete and accurate that I consider it would be useful to include it in the discussion section.
Thank you for your comments. We included those contents in the discussion section of our revised manuscript (line 348-354, line 355-359).
The author combines MLCK inhibitors with Bleb (Figure 6), trying to verify if both pairs of inhibitors act on the same target/pathway. The rationale is wrong for at least two reasons.
a- Because both lines of evidence point to contrasting actions of NMII on axon growth, one approach could never "rescue" the other.
Reply by authors in R1：If MLCK regulates axon growth through the activation of Myosin, the inhibitory effect of ML-7 (an MLCK inhibitor) on axon growth might be influenced by Bleb, a NMII inhibitor. However, our findings reveal that the combination of Bleb and ML-7 does not alter the rate of axon outgrowth compared to ML-7 alone. This suggests that the roles of ML-7 and Bleb in axon growth are independent. It means MLCK may regulate axon growth independent of NMII activity.
b- Because the approaches target different steps on NMII activation, one could never "prevent" or rescue the other. For example, for Bleb to provide a phenotype, it should find any p-MLC, because it is only that form of MLC that is capable of inhibiting its ATPase site. In light of this, it is not surprising that Bleb is unable to exert any action in a situation where there is no p-MLC (ML-7, which by inhibiting the kinase drives the levels of p-MLC to zero, Figure 4A). Hence, the results are not possible to validate in the current general interpretation of the authors. (See 'major concern').
Reply by authors in R1: The reported mechanism of blebbistatin is not through competition with the ATP binding site of myosin. Instead, it selectively binds to the ATPase intermediate state associated with ADP and inorganic phosphate, which decelerates the phosphate release. Importantly, blebbistatin does not impede myosin's interaction with actin or the ATP-triggered disassociation of actomyosin. It rather inhibits the myosin head when it forms a product complex with a reduced affinity for actin. This indicates that blebbistatin functions by stabilizing a particular myosin intermediate state that is independent of the phosphorylation status of myosin light chain (MLC).
[Ref] Kovács M, Tóth J et al. Mechanism of blebbistatin inhibition of myosin II. J Biol Chem. 2004 Aug 20;279(34):35557-63.
