Drosophila neuromuscular junction indicate that many synaptic defects in unc-104-null mutants are mediated independently of Unc-104’s transport function, via the Wallenda (Wnd)/DLK MAP kinase axonal damage signaling pathway. Wnd signaling becomes activated when Unc-104’s function is disrupted, and leads to impairment of synaptic structure and function by restraining the expression level of active zone (AZ) and synaptic vesicle (SV) components. This action concomitantly suppresses the buildup of synaptic proteins in neuronal cell bodies, hence may play an adaptive role to stresses that impair axonal transport. Wnd signaling also becomes activated when pre-synaptic proteins are over-expressed, suggesting the existence of a feedback circuit to match synaptic protein levels to the transport capacity of the axon.The kinesin-3 family member Unc-104/KIF1A is required for axonal transport of many presynaptic components to synapses, and mutation of this gene results in synaptic dysfunction in mice, flies and worms. Our studies at the  Each nerve cell, or neuron, has a long nerve fiber – called an axon – that forms specialized sites for information exchange – called synapses – with other cells. Many molecules work at synapses to coordinate the exchange of information. These molecules are largely made in the central part of the neuron – known as the cell body – and are then transported along the axon to the synapses.The transport of these molecules is carried out by proteins known as molecular motors. One molecular motor, called KIF1A in humans and Unc-104 in fruit flies, is thought to be a major transporter of synaptic molecules. Mutations that hinder this molecular motor result in neurons failing to form synapses and, instead, synaptic components accumulate in the cell body. However, it was not clearif Unc-104 does actually carry all of the components needed to assemble synapses along axons, or if it influences synapse formation in another way.Now, Li, Zhang et al. report new evidence that supports the second of these two hypotheses. The experiments made use of fruit flies in which the gene for Unc-104 had been deleted, and revealed that inhibiting enzymes in a specific signaling pathway could reverse the synaptic problems caused by the loss of Unc-104. The signaling pathway, which is conserved between flies and humans, involves an enzyme that is called Wnd in flies and DLK in humans. The Wnd/DLK signaling pathway was previously known to regulate how neurons respond when their axons are damaged .Further investigation by Li, Zhang et al. revealed that signaling via the Wnd enzyme becomes triggered whenever the Unc-104 molecular motor is impaired. This activation correlates with the build-up of synaptic proteins in the cell body. Once activated, the pathway then reduces the total amount of synaptic proteins that the cell makes. This reduction matches the neuron’s reduced ability to transport them along the axon, and may help the neuron to adapt when axonal transport is impaired. However, the reduction in synaptic proteins also impaired the exchange of information at the synapses.These findings suggest how DLK could be behind problems with synapses in diseases in which transport along axons is impaired. These diseases include hereditary spastic paraplegia, which has been linked to mutations in human KIF1A, and may also include ALS and Alzheimer’s disease, which have recently been linked to DLK.DLK has received recent attention as a candidate drug target because it contributes to the deterioration of damaged neurons. These new findings further expand that interest by suggesting that inhibiting DLK may help neurons to maintain working synapses, which is more useful than simply preventing damaged neurons from dying. Synapse development, maintenance and plasticity involve highly orchestrated trafficking events in both pre and postsynaptic cells. In contrast to postsynaptic receptors, whose trafficking and organization has been studied extensively in many different synapse types , much leKif1a and its unc-104 orthologues in C. elegans and Drosophila  cause severe defects in synapse development. In Drosophila unc-104-null mutants, synaptic boutons fail to form, SV and AZ components fail to traffic to nascent synapses, and concomitantly, SV and AZ associated proteins accumulate in the cell body , unc-104-null mutants are severely defective in the formation of presynaptic boutons, fail to localize SVs to NMJ terminals and show strong reductions in AZ localization (3wnd single mutants) had no significant effect on bouton formation, AZ number, or presynaptic protein localization, but double mutants with unc-104-null alleles , gave a very informative phenotype: in null;wnd3/3unc-104 double mutants the synaptic bouton formation  or fos fully rescued the mEJP frequency defects -lacZ enhancer trap   . This in by RNAi , hence runc-104 hypomorphic mutant NMJs, which show increased numbers of synaptic branches and boutons which are smaller in size . Therefore, to further evaluate potential effects upon Wnd protein we utilized two different wnd-GFP fusion proteins. First, a MiMIC-wnd-GFP line  has been established as a sensitive method to detect Wnd protein change  with anti-Wnd antibodies, Wnd protein remained below the detection threshold in both wild type and GFP line , in whicd tagged . By IHC, mutants . Second,n change . This hi mutants . This co mutants , and thiKDwnd vesicles we observed no impairment in transport kinetics or flux in unc-104 mutants  lacked Brp . . However we observed this trend in total levels of VGlut . . We suspof VGlut . This maunc-104 mutants. This response may serve to counteract their buildup in neuronal cell bodies while causing the observed defects in synapse structure and function. In support of this, unc-104 mutations led to decreased expression of a vglut-DsRed transcriptional reporter in motoneurons, in a Wnd-dependent manner , and observed that NMJ synapse development progressed normally  Transport of SV precursors to synaptic terminals is likely a direct function, since the failure to deliver adequate VGlut, Syt1 and CSP persists in null;wndunc-104 double mutants, and the Unc-104 homologue KIF1A physically interacts with SV precursors .The kinesin-3 family member Unc-104/KIF1A is known to be an important mediator of synaptic development and maintenance: mutations in  defects . While tecursors . (2) Regunc-104 mutants, it is now worth considering whether it contributes to other phenotypes previously described for Unc-104 and its homologues in other species. These include impaired dendritic branching   , and her (SPG30) . The posunc-104 mutants? The mechanism(s) that lead to activation of Wnd and its DLK homologues are of general interest for their roles in axonal regeneration as well as degeneration and neuronal death. In addition to axonal injury .This line of reasoning leads to further consideration of Unc-104’s cargo. Our live imaging data do not support a simple model that Wnd is a cargo of Unc-104 . Known cunc-104 mutants, which accumulate presynaptic proteins in the neuronal cell body. We noticed a similar role for endogenous Wnd in wild type neurons during the onset of embryonic NMJ development . However we are limited in our ability to detect total changes in mRNA and protein levels from whole nerve cord preparations by the fact that the Wnd signaling pathway may not be acting in all cell types. It is also possible that additional post-transcriptional mechanisms, such as regulation of protein stability or translation, or bulk turnover via autophagy, factor into the regulation of presynaptic proteins by Wnd. A recent study has suggested that DLK may activate the integrated stress response pathway in mammalian neurons , RNAi lines: moody RNAi (control), Octβ2R RNAi , unc-104 RNAi , wnd RNAi (vdrc 103410 and vdrc 26910), Rab3 RNAi (TRiP BL31691 and BL34655), UAS-Dcr2 was a gift from Stephan Thor . Flies were raised at 25°C or 29°C (as indicated for certain RNAi knock-down) on standard Semidefined yeast-glucose media  was cloned into a plasmid derived from pCaSpeR-AUG-bGal  in PBS/HL3 solution for 3 min for Cac-GFP, 10 min for Brp and GluRIII/GluRIIC staining or 20 min for other antibody staining, followed by blocking in PBS with 0.1% Triton (PBT) containing 5% Normal Goat Serum (NGS) block for 30 min. Control and experimental animals were always dissected, fixed and stained in the same condition and imaged in parallel using identical confocal settings.unc-104-null alleles include P350/P350 and 52/52.Embryos were dissected, fixed and stained as described in . In brieAB_2314866), 1:200; ms anti-Synapsin (DSHB Cat# 3C11 (anti SYNORF1) Lot# RRID:AB_528479), 1:50; ms anti-CSP-1 (DSHB Cat# DCSP-1 (ab49) Lot# RRID:AB_2307340), 1:100; ms anti-DLG(DSHB Cat# 4F3 anti-discs large Lot# RRID:AB_528203), 1:1000; Rb anti-GluRIII (gift from Diantonio lab), 1:2500; Rb anti-SytI , 1:500; ms anti-lac-Z (DSHB Cat# 40-1a Lot# RRID:AB_528100), 1:100; Rb anti-Phospho-Smad1/5 ,1:100; Rb anti-DsRed , 1:1000; Rat anti-elav (DSHB Cat# Rat-Elav-7E8A10 anti-elav Lot# RRID:AB_528218), 1:50; A488 rabbit anti-GFP , 1:1000 and Alexa488/cy3/Alexa647 conjugated Goat anti-HRP (Jackson ImmunoResearch), 1:300. Rabbit anti-VGlut (gift from Diantonio lab), 1:10000, staining was carried as described in Primary antibody and secondary antibody incubations were conducted in PBT containing 5% NGS at 4°C overnight and at room temperature for 2 hr, respectively, with three 10 min washes in PBT after each antibody incubation. The following primary antibodies and dilutions were used: ms anti-Brp  to define the region of interest. For cell bodies, we selected the signal above a specified threshold, . To estimate the total VGlut or Brp level within a single motoneuron , scored for the presence or absence of an apposing AZ component (Brp or Liprin-α-GFP). To affirm that AZ components were indeed completely absent, confocal settings and brightness levels were optimized for the weakest signals in toneuron , we summpuc-lacZ level was measured within the nucleus region for motoneurons, selected by P-smad staining in the dorsal regions of A4-A8 in the ventral nerve cord.When imaging nerve cord, we used 0.8 μm step size for the z-stack and focused on the posterior and central nerve cord, corresponding to A4-A8. When imaging axons or the NMJ, we used 0.4 μm step size for z-stacks. Axonal segments were imaged 900 μm away from the nerve cord. NMJ images were collected at segment A3 for muscle 4 or  for muscle 26, 27 and 29, which are innervated by the SNc neuron. For live imaging analysis of GFP-wnd-KD transport, third instar larvae were dissected in the center of a circle reinforcement label (Avery) in HL3 solution  with 0.4rd brains were collected in PBS and homogenized for each sample. The following antibodies and dilutions were used: rb anti-Wnd 4–3 . To ensure that we could always recruit both axons, for each muscle we tested a range of stimulation voltage (1–70V) to find the threshold which triggered the largest response within the testing range. A stimulus slightly larger than this threshold was then used at a frequency of 0.2 Hz and duration of 1 ms for EJP measurements. Axon Laboratory software was used for acquisition and the Mini Analysis program (Synaptosoft Inc) was used for analysis of mEJP frequency and amplitude. Parameters for Mini Analysis were set as: 0.2 (threshold), 1 (area threshold), 30,000 , 40,000 (period to search a decay time), 40,000 (time before a peak for baseline), 20,000 (period to average a baseline), 0.6 (fraction of peak to find a decay time) and detect complex peak.Third instar larvae were dissected within 3 min in HL3 solution containing 0.65 mM Calcium at 22°C. Muscle 6 at segment A3 was located by the use of an OLYMPUS BX51WI scope with a 10x water objective and then recorded intracellularly with an electrode made of thick wall glass (1.2 mm x 0.69 mm) pulled by SUTTER PULLER P-97. Amplifier GeneClamp 500B and digitizer Digidata 1440A were used. The recording was only used if the resting potential was negative to −60 mV and muscle resistance was >5 mΩ. A GRASS S48 STIMULATOR was used to obtain a large range of stimulation voltage range (1–70V). We noticed that resting potential: corrected EJP = EJP/(1 f(EJP/Edriving force)), where Edriving force=Eresting potential-Ereversal potential and f = the membrane capacitance factor (Δt/τ). At the Drosophila NMJ, Ereversal potential is estimated around 0 mV, Eresting potential from our recordings is around −70 mV and f = 0.55 2/d2, where SD = standard deviation, d = effect size and Zα/2 or Zβ are constants when α = 0.5 and β = 80% , the sample size and biological replicate number was determined for comparison with previous studies  or one-way ANOVA followed by Tukey test (multiple groups). Normality of datasets for parametric/ANOVA was confirmed using the D’Agostino-Pearson-omnibus K2 test. p values smaller than 0.05 were considered statistically significant. All p values are indicated as *p<0.05, **p<0.01, and ***p<0.001 and ****p<0.0001. Data are presented as mean ±SEM. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your work entitled "The Wallenda/DLK kinase restrains the expression of pre-synaptic proteins according to their transport" for consideration by Li et al. present work investigating Wnd/DLK MAP kinase pathway regulation of expression levels of synaptic genes following disrupted axonal trafficking upon knockdown of Unc-104/Kif1a, a Kinesin 3 motor. Based on the exciting finding that loss of wnd suppresses the reduced presynaptic active zone (AZ) protein accumulation and associated functional deficits of unc-104 hypomorphic mutants, the authors propose a model in which the Wnd pathway 'senses' levels of presynaptic proteins in the cell body and acts as a brake on expression when levels exceed the ability of the cell to transport these proteins to synapses. The experiments are generally carefully done and individual experiments serve the purpose of supporting the genetic interaction. However, as a whole, the paper worryingly feels like a catalog of correlative connections, missing conceptual depth. A consistent term used throughout the paper is "modest effect" or "modest role", suggesting ambiguity of the observations and the conclusions. Some of the issues could be addressed by re-writing, others need additional data. Thus, while this is an interesting model, it is not fully supported by the data presented as detailed below.While some of our concerns can be addressed by better analysis and writing, others require significant experiments. We had prolonged discussions around whether these concerns can be addressed in two months and if done would we be persuaded. We feel that some suggested experiments which are critical to the thrust of the paper can be done in two months. Yet, when we asked if these concerns were addressed whether the model proposed and data presented would be acceptable despite 'mechanistic' gaps, we were not persuaded. In this situation, another round of review would not be correct.Reviewer #1:Li et al. present work investigating Wnd/DLK MAP kinase pathway regulation of expression levels of synaptic genes following disrupted axonal trafficking upon knockdown of Unc-104/Kif1a, a Kinesin 3 motor, and during development. Based on the exciting finding that loss of wnd suppresses the reduced presynaptic active zone (AZ) protein accumulation and associated functional deficits of unc-104 hypomorphic mutants, the authors propose a model in which the Wnd pathway 'senses' levels of presynaptic proteins in the cell body and acts as a brake on expression when levels exceed the ability of the cell to transport these proteins to synapses. While this is an interesting model, it is not fully supported by the data presented as detailed below.1) Support for their model:Drosophila ..A) The authors propose that unc-104 function can be divided into a transport role for SVs that is independent of Wnd and a Wnd/DLK pathway-dependent role in regulating AZ proteins. The differentiation between Brp and SV proteins is based on the finding that reduced synaptic levels of Brp in unc-104 nulls are suppressed by wnd but SV protein levels are not, in contrast to their findings in unc-104 hypomorphs. Simpler models should be considered. For example, the observation that wnd suppresses the loss of SV proteins at synapses in hypomorphs with residual Unc-104 activity but not nulls is more easily explained by the model that SV transport, but not Brp transport, is fully dependent on Unc-104, consistent with prior observations in B) The strongest evidence for activation of Wnd in Unc-104 mutants comes from the puc-LacZ experiments in unc-104 RNAi lines . Given tIn C) The evidence that Wnd regulates the levels of presynaptic protein levels and in particular that it acts at the level of transcription is somewhat inconsistent. Upon Wnd overexpression, the authors observed decreased protein levels at active zones as predicted. However, in wnd null embryos they see increased levels in some proteins at some stages, but decreases in other proteins . This and the absence of a consistent effect on the timing or expression, weakens their conclusion that wnd controls the levels and timing of expression of AZ and SV components.In Other major concerns:1) Much of the wnd, unc-104 interaction data was obtained using a previously published hypomorphic alleles of unc-104. Given their central role in this work, the authors should provide some information on the nature of these alleles. In the analysis of interactions between null mutants presented in 2) An electrophysiological analysis was conducted in hiw mutants (where Wnd levels are increased), but should be completed in wnd overexpression lines to more directly test the hypothesis that increased Wnd explains functional defects in unc-104 mutants. Here, the authors present the surprising result that wnd suppresses the physiological defect in hiw mutants. This is in contrast to results published in an earlier paper with overlapping authors . They suggest the difference may be due to the use of different wnd alleles, but this needs to be addressed experimentally given the importance of wnd suppression phenotypes to this work and the high impact of the previously published result that is called into question.3) Given that synaptic function is fully suppressed in hypomorphs, it is surprising that locomotion and viability show little improvement. This disconnect should be further addressed by evaluating the relevant allelic combinations, not just RNAi. Relatedly, how is there so much movement in double null mutants  when the4) It was cumbersome to parse through the data as presented in 21 figures  including 2 separate model figures. Reviewer #2:In "The Wallenda/DLK kinase restrains the expression of pre-synaptic proteins according to their Transport" Drs. Collins, Rasse and colleagues reveal an unexpected novel role for Unc-104 in regulating synaptic development and bouton composition in a manner not directly related to its long-range transport functions.Synaptic vesicle precursor proteins and active zone proteins are synthesized in neuronal cell bodies and transported to synapses. Kinesin motors (Unc-104) transport SV precursors. AZ components are reduced at the synaptic terminals of the unc-104 mutants, but their transport has never been shown to be driven directly by unc-104. Through an exhaustive set of experiments (using diverse mutant and RNAi combinations), the authors show that defects in unc-104 lead to the activation of the Wnd signaling pathway (via Bsk and Fos), which in a cell-autonomous manner regulates AZ protein levels in neuronal cell bodies and synaptic terminals. Suppressing Wnd signaling in unc-104 strong hypomorphs or in RNAi knockdowns significantly reduces the defects in AZ at the synapse, defects in synaptic transmission, larval mobility and viability, but only partly rescues SV protein levels. Furthermore, puckered expression and axonal regeneration, both of which require enhanced Wnd signaling, are enhanced in unc-104 mutants.Crucially, through genetic interaction studies with null mutants of unc-104 (as opposed to hypomorphs) the authors revealed two separable functions for unc-104: one for trafficking SV associated proteins to the synapse  and one for activating the Wnd pathway, which in unc-104 mutants lead to the decrease of multiple synaptic proteins.eLife, but there are several points that the authors could address based on their current approaches:While these represent important, transformative, and convincing advances in our understanding of unc-104 and Wnd/DLK signaling in synapse formation, the one weakness of the paper is that it does not uncover HOW Unc-104 regulates Wnd signaling, or how Wnd signaling constrains synaptic protein levels. Filling this gap is likely beyond the scope of the current work and not necessary to merit publication in 1) It is important for the authors to quantify Wnd-MiMIC levels in the neuronal cell bodies  The authors present hiw data but do not address hiw function in their model or discussion. The authors should measure hiw RNA and/or protein levels in Unc-104 mutants to ask if hiw is contributing to the phenotype. If hiw protein (but not RNA) is downregulated in the unc-104 mutant, it might suggest that autophagy-mediated degradation of Hiw , and perhaps other synaptic proteins, underlies the regulatory mechanism by which unc-104 constrains synaptic protein levels in response to cell body accumulation of cargo. This could be simply tested by evaluating the amount and distribution of an autophagy flux marker such as ATG8-GFP-mCherry.3) The observation that ectopic overexpression of Brp, SytI and VGlut leads to increased puc-lacZ reporter signal begs the question whether this also triggers changes in Wnd (or hiw) levels or localization, as these results could provide more insight into the mechanism via which Unc-104 deficits lead to Wnd activation.Reviewer #3:This paper describes a genetic interaction between the Unc-104 motor protein and the Wnd DLK kinase. The authors presented impressive amount of data from synapse cell biology to physiology with an attempt to conclude that Wnd activation is sensitive to disruption of unc-104 mediated transport of some axonal components. The measurement for Wnd activity mostly relied on induction of a puc-LacZ reporter in the soma, and morphological changes of NMJ terminals. While experiments are generally carefully done, individual experiment serves the purpose of supporting the genetic interaction. However, as a whole, the paper feels like a catalog of correlative connections, missing conceptual depth. A consistent term used throughout the paper is "modest effect" or "modest role", suggesting ambiguity of the observations and the conclusions. Some of the issues could be addressed by re-writing, others need additional data.JNK and FOS, also suppress unc-104 lethality? In subsection “Wnd inhibits synapse assembly independently of cargo transport downstream of Unc-104” the final sentence of the second paragraph seems to state that "most or all" unc-104; wnd mutants actually die. Is the suppression of lethality rather minor?Their observation that Wnd suppresses the lethality of unc-104 mutants is the key observation led to this study. But it is not clear whether this suppression is directly caused at the level of presynaptic transport. Does inhibition of Wnd downstream factors, In axon regeneration analysis in unc-104 mutants, the observed effects should be rescued using wild type unc-104.Subsection “Activation of the Wnd signaling pathway in neurons is sufficient to impair presynaptic structure and synaptic transmission”: first paragraph, the authors state "ectopic activation of Wnd should mimic the unc-104 mutant phenotypes". But where is Wnd normally activated?In the same section, second paragraph: The authors made a comment about the result in They talked about similarities of how loss of Hiw or of Unc-104 affect Wnd function. An important experiment should include a double mutant study of Hiw and unc-104.[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your letter of appeal has been considered by a Senior Editor in discussion with the reviewers, and we are prepared to consider a revised submission with no guarantees of acceptance.Thank you for choosing to send your work entitled "The Wallenda/DLK kinase restrains the expression of pre-synaptic proteins according to their transport" for consideration at eLife.While we are happy to consider a revised manuscript, you will see that we have some serious concerns that need careful attention. We see merit in the author's rebuttal letter that the most concerns can be addressed with rewriting and some additional experiments and key issues addressed. The overall impact of the work, if these points are well-addressed, can be strong, since it provides a fundamentally new model for how unc-104 contributes to neuronal function, by activating an axonal injury pathway. However, the data and model also need to be explained more clearly to be digestible by a broad readership such as at In response to the concerns raised about inconsistent results in their wnd loss-of-function analysis, the authors are dismissive, imploring us to "Note that our model is NOT that Wnd functions as a major restraint on these proteins during normal conditions or during development," yet their subheading describing these results is "Wnd restrains the expression of presynaptic proteins at early stages of synaptogenesis." In a second example, the authors dismiss concerns about their analysis of MiMIC-GFP instead of endogenous Wnd levels by simply stating that the Wnd antibody does not work well for immunohistochemistry without addressing the fact that they have previously published analogous experiments using their Wnd antibody for immunohistochemistry . The authors may want to pay particular attention to these points.The authors should measure puc-LacZ expression in additional unc-104 mutants. Also, rescue of additional phenotypes aside from NMJ morphology and active zone apposition to PSD  by BSK and FOS would provide strong support for the model. However, I was not concerned that the Mimic tool showed an increase in Wnd while the western blot did not, since the Mimic tool accounts for changes in subcellular distribution – this point can be addressed better by the authors in the manuscript. However, it is absolutely essential that the MIMIC phenotype be quantified and use of the Kinase Dead explained in the text.The effect of wnd on the timing and expression of presynaptic proteins needs to be much more clearly explained, as discussed in the rebuttal. As it stands this section is very distracting and confusing. The authors should also clarify their model for how synaptic protein levels are regulated in the unc-104 mutants . The main point of the paper does not hinge on a particular model of synaptic protein level control, but the authors do need to clarify their explanations, either in the text or with more experiments.Wnd null mutants alone should be included in Electrophysiological analysis (and synaptic vesicle protein levels) in wnd overexpression lines would provide strong corroboration that increased Wnd explains functional defects in unc-104 mutants. Addressing the discrepancy with previous results (Collins 2006) on Wnd suppression of the hiw phenotype will be also be important.On the other hand, experiments to address the cellular function of hiw would be nice but are not essential to the main interesting points of the paper. If the authors have the data or a more clear model of the role of hiw, it would be helpful to include, but we don't think it's necessary.The rescue of the axon regrowth phenotype are not critical for the main conclusions of the paper and could take a lot of time.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by K VijayRaghavan as the Senior Editor and the Reviewing Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Restraint of presynaptic protein levels by Wnd/DLK signaling mediates synaptic defects associated with the kinesin-3 motor Unc-104" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.As you can see your manuscript needs only a few minor points that need to be addressed. Please do that speedily. We can then accept the revised manuscript after an editorial examination.Reviewer #1:In their revised manuscript, the authors have been very responsive to reviewer suggestions. The logical flow of the manuscript is significantly improved and experimental concerns directly addressed.I have one question regarding the addition of wnd null data to Reviewer #2:The manuscript is much improved with the suggested experiments, reorganization, and rewriting. My concerns have been adequately addressed and I think that this is a very interesting, well-executed, and thought-provoking study. [Editors’ note: the author responses to the first round of peer review follow.]Reviewer #1:Li et al. present work investigating Wnd/DLK MAP kinase pathway regulation of expression levels of synaptic genes following disrupted axonal trafficking upon knockdown of Unc-104/Kif1a, a Kinesin 3 motor, and during development. Based on the exciting finding that loss of wnd suppresses the reduced presynaptic active zone (AZ) protein accumulation and associated functional deficits of unc-104 hypomorphic mutants, the authors propose a model in which the Wnd pathway 'senses' levels of presynaptic proteins in the cell body and acts as a brake on expression when levels exceed the ability of the cell to transport these proteins to synapses. While this is an interesting model, it is not fully supported by the data presented as detailed below.We appreciate that the reviewer recognizes the novelty and strength of our findings and the model we propose. With a careful and much appreciated eye on the data, the reviewer summarizes below some concerns about individual observations. We are optimistic that our explanations and clarifications described here, along with some adjustments and clarifications in the writing, can resolve this reviewer’s concerns.1) Support for their model:A) The authors propose that unc-104 function can be divided into a transport role for SVs that is independent of Wnd and a Wnd/DLK pathway-dependent role in regulating AZ proteins.This statement is not precise for what we propose. Please see the clarifications below.The differentiation between Brp and SV proteins is based on the finding that reduced synaptic levels of Brp in unc-104 nulls are suppressed by wnd but SV protein levels are not, in contrast to their findings in unc-104 hypomorphs.unc-104; wnd double mutants compared to unc-104 mutants alone, we see a major increase in total levels  for both AZ and SV proteins. This is evident for both unc-104-hypomorph .unc-104 mutants. However it is not the full picture, since this explanation does not account buildup of both AZ and SV components in the cell bodies of unc-104-null; wnd and unc-104-hypomorph;wnd double mutants  The strongest evidence for activation of Wnd in Unc-104 mutants comes from the puc-LacZ experiments in unc-104 RNAi lines . Given tunc-104 mutants is an important part of the paper. We show this for two independent RNAi lines, which show similar phenotypes to the multiple hypomorphic alleles (bris and O3.1) used in this paper across other assays. The suggestion to show this for additional alleles is do-able. We already have puc-lacZ combined into unc-104-hypomorph lines so we think it would be straight-forward to include this additional data.We agree that evidence for Wnd’s activation in the unc-104-hypomorph mutant and RNAi lines we see synaptic over-branching phenotypes that are hallmark of Wnd activation, and these are dependent upon Wnd and downstream JNK and Fos signaling components , and also for unc-104 RNAi knockdown in neurons . More importantly, under all these different conditions, when activation of the Wnd signaling is reversed/suppressed via wnd RNAi-knockdown or in wnd, unc-104 double mutants, the cell body accumulation is dramatically enhanced.We see cell body accumulations of presynaptic proteins in multiple hypomorph and null Upon reviewing the manuscript after reading the reviewer’s queries, we realize it would be helpful to provide more background information about the different ‘hypomorph’ alleles, and clarify/explain in more detail how all of the alleles show similar synaptic phenotypes and relationships with Wnd to that of simple knock-down of Unc-104 with RNAi.In contrast to the multiple mutations that reduce Unc-104 function, mutations that impair kinesin-1 function show accumulations of presynaptic proteins within segmental nerves rather than in the cell bodies, and also do not significantly induce the puc-lacZ reporter .In To examine Wnd protein levels, we employed multiple approaches . The WndThe observation that a global increase in endogenous Wnd levels was not detected by Western Blot may benefit from some further clarification and discussion. The Western Blots are necessarily carried out with extracts made from dissected larval CNS/brains, pooled in entirety. We cannot isolate individual neurons for this analysis. We’ve observed that the activation of Wnd and the regulation of presynaptic proteins by Wnd appears most striking in motoneurons. The potential specificity to motoneurons makes it harder to detect simple biochemical evidence for the relationship using standard biochemical methods from whole larval brains.C) The evidence that Wnd regulates the levels of presynaptic protein levels and in particular that it acts at the level of transcription is somewhat inconsistent. Upon Wnd overexpression, the authors observed decreased protein levels at active zones as predicted. However, in wnd null embryos they see increased levels in some proteins at some stages, but decreases in other proteins . This and the absence of a consistent effect on the timing or expression, weakens their conclusion that wnd controls the levels and timing of expression of AZ and SV components.loss-of-function phenotypes in an uninjured background should be mild. With an original motivation to understand whether there is also a role for Wnd during normal synaptic development, we examined these phenotypes carefully during development. We found that the wnd loss-of-function phenotypes are indeed mild. However, strikingly, we did notice some elevation in VGlut and Syt protein co-incident with the onset of expression for these proteins. Note that our model is NOT that Wnd functions as a major restraint on these proteins during normal conditions or during development. Rather, Wnd restrains these proteins when it is substantially activated. In normal conditions this may occur only at extremely low levels to keep total synaptic protein levels in balance.An important but overlooked point here is that Wnd signaling is normally highly restrained in wild type, uninjured animals. If its role is primarily to respond to injury or stress, then it is expected that The fact that we did not observe an increase in Brp does not negate the model. Very little is currently known about how the expression of AZ and SV proteins are regulated developmentally –this is an interesting topic for future studies. It is likely that multiple mechanisms exist for different proteins, and that Brp levels are not sufficiently high during these stages of normal development to detect a role for Wnd in uninjured animals.In As discussed above for point B, studies with whole brain extracts  are likely to miss the cell specific regulation which is occurring in motoneurons. It is a future direction to profile the responses in motoneurons specificially. This requires some additional technical innovations since cell sorting causes axotomy, which activates the Wnd pathway on its own.Other major concerns:1) Much of the wnd, unc-104 interaction data was obtained using a previously published hypomorphic alleles of unc-104. Given their central role in this work, the authors should provide some information on the nature of these alleles.wnd mutants for multiple hypomorphic allele combinations of unc-104 mutants , and also when Unc-104 protein levels are simply reduced by RNAi-knockdown. So we think that any manipulation that impairs Unc-104’s basic function leads to activation of Wnd signaling.We agree and can provide further clarification in the text. An important clarification point is that we see similar suppression by In the analysis of interactions between null mutants presented in wnd mutants alone at the embryonic stages in We have indeed included detailed analysis of Have the authors analyzed dominant suppression of unc-104 phenotypes by wnd, which would strongly support the conclusion that the two genes function in a linear pathway?unc-104 phenotypes . We feel that experiments with wnd heterozyogotes would not really add much more to this paper, especially since it is not clear in our data that removal of one copy of wnd leads to a 50% reduction of Wnd levels in all cell types.We’ve observed that partial knockdown of Wnd signaling by RNAi can suppress unc-104 and wnd mutants have opposite loss-of-function phenotypes, so we’re not sure what we would learn from a double heterozygote.The reviewer may instead be referring to double-heterozygote experiments, which can be helpful for revealing relationships in a linear pathway via their sensitivity to gene dosage of both factors. This kind of analysis is more do-able when both genes have similar loss-of-function phenotypes. However 2) An electrophysiological analysis was conducted in hiw mutants (where Wnd levels are increased), but should be completed in wnd overexpression lines to more directly test the hypothesis that increased Wnd explains functional defects in unc-104 mutants. Here, the authors present the surprising result that wnd suppresses the physiological defect in hiw mutants. This is in contrast to results published in an earlier paper with overlapping authors . They suggest the difference may be due to the use of different wnd alleles, but this needs to be addressed experimentally given the importance of wnd suppression phenotypes to this work and the high impact of the previously published result that is called into question.wnd over-expression could further and more simply support this point. These experiments should be complete-able in a relatively short time frame, so we will aim to add this data to the revised manuscript. If we can indeed include this data, then we may then want to move the hiw data to supplemental data, where the partial differences with the 2006 study are less distracting.The main point to communicate in hiw mutants (and unc-104 mutants) show mild reductions in quantal content, they are significantly reduced for mini frequency. The observation that wnd mutations suppress hiw’s defect in mini frequency are actually conserved between both our observations and the previous 2006 study. The single difference is that the 2006 study did not reveal a suppression of the defect in quantal content, while ours does. We would indeed like to understand this better – resolving the differences will require repeating exactly the same allele combinations in our own conditions for comparison. This is do-able and we are willing to do it. The main point of this would be to provide further context for previous observations in the field rather than to further validate the observations we have made here in this paper .While 3) Given that synaptic function is fully suppressed in hypomorphs, it is surprising that locomotion and viability show little improvement. This disconnect should be further addressed by evaluating the relevant allelic combinations, not just RNAi. Relatedly, how is there so much movement in double null mutants  when theActually, neither of these results are surprising.unc-104 mutants and is responsible for many interesting synaptic phenotypes) are separable from a primary function for Unc-104 in transporting SVs . Perhaps the strong difference in bar graphs in For the unc-104 mutants.We worry based on these points and also the summary statement that these ‘modest’ or ‘inconsistent’ effects upon lethality and motility were a primary criticism for the paper. Therefore it is important to clarify that these effects are consistent with the model we present in the paper . The ‘modest’ rescue of lethality should not diminish the importance of our findings, which identify specific cellular roles for Wnd in 4) It was cumbersome to parse through the data as presented in 21 figures  including 2 separate model figures.We appreciate the efforts from the reviewer to evaluate all of the data, and their feedback has helped us see how we might be able to improve the writing to make the paper less cumbersome to read. It has been over 7 years of hard efforts between our two labs to reach our current understanding of the surprising relationship between Unc-104 and Wnd. There are many more data that are not included in this paper. We will work further on the writing to better communicate how the individual experiments build upon one another to fit into the big picture story.We appreciate this point. It is important to consider the levels in the different cellular compartments and to assess differences in total levels accounting for all of the compartments. This comparison is our goal for It also looks like the effect of wnd loss of function on Brp levels is very different between wnd mutants in 3/3wnd and 3/df,wnd respectively). Since the 3/3wnd animals are maintained in the same strain background there is a chance to accumulate genetic modifiers of the phenotype in the background. We prefer to carry out all of our studies with two different alleles crossed to one another to be certain the phenotype is indeed due to the mutation we are manipulating.The Reviewer #2:[…] While these represent important, transformative, and convincing advances in our understanding of unc-104 and Wnd/DLK signaling in synapse formation, the one weakness of the paper is that it does not uncover HOW Unc-104 regulates Wnd signaling, or how Wnd signaling constrains synaptic protein levels. Filling this gap is likely beyond the scope of the current work and not necessary to merit publication in eLife, but there are several points that the authors could address based on their current approaches:We are glad that reviewer 2 shares the same point of view with us in both strengths and one weakness. We also agree that this weakness is beyond the scope and not necessary for our current work to be published. The reviewer’s suggestions listed below are fair and we are ready to address all of these points.1) It is important for the authors to quantify Wnd-MiMIC levels in the neuronal cell bodies  leads to lethality and technical complications. We can also add additional information about the Wnd-kinase-dead (KD) transgene, which has been used in previous studies to uncover biologically important mechanisms of regulation . See also our discussion to reviewer 1’s point B, above.2) The authors present hiw data but do not address hiw function in their model or discussion. The authors should measure hiw RNA and/or protein levels in Unc-104 mutants to ask if hiw is contributing to the phenotype. If hiw protein (but not RNA) is downregulated in the unc-104 mutant, it might suggest that autophagy-mediated degradation of Hiw , and perhaps other synaptic proteins, underlies the regulatory mechanism by which unc-104 constrains synaptic protein levels in response to cell body accumulation of cargo. This could be simply tested by evaluating the amount and distribution of an autophagy flux marker such as ATG8-GFP-mCherry.unc-104 mutants. Based on several observations thus far (some in the manuscript and some yet unpublished), we think that loss of Hiw function is unlikely to explain or mediate the activation of Wnd in unc-104 mutants.These interesting suggestions center around the goal of understanding whether Hiw, a major known regulator of Wnd, plays a role in Wnd’s activation in hiw and unc-104 mutants upon the levels and localization of Wnd are strikingly different: in hiw mutants, the levels of Wnd are strongly elevated throughout the animal and this elevated Wnd protein is observed to localizes to synapses and neuropil  The effects of 2) hiw mutants show a strong delay in axonal degeneration . However we found no delay to degeneration in unc-104 mutants (unpublished).unc-104 is knocked down (unpublished).3) Western blot results did not show an obvious reduction in Hiw protein level when atg7 and knock-down of atg13 or Fip200 in neurons, which are critical for different stages of autophagy , did not alter Wnd signaling using puc-lacZ as a reporter (unpublished). This runs a bit counter to predications suggested by the Shen et al., 2009 study. So more needs to be done to sort this out and this is a topic for future work.The potential role of autophagy is an interesting topic to further understand. Thus far we have found that null mutations in 3) The observation that ectopic overexpression of Brp, SytI and VGlut leads to increased puc-lacZ reporter signal begs the question whether this also triggers changes in Wnd (or hiw) levels or localization, as these results could provide more insight into the mechanism via which Unc-104 deficits lead to Wnd activation.eLife’. We have some additional ideas on this question that we want to carry out in future studies.This is a good suggestion but a negative or mild result could occur simply because the effect of overexpressing a single pre-synaptic protein does not activate the pathway to the same extent, so would not be useful in ruling out a model. We appreciate that the reviewer acknowledges that filling the gap of HOW Unc-104 regulates Wnd signaling ‘is likely beyond the scope of the current work and not necessary to merit publication in Reviewer #3:This paper describes a genetic interaction between the Unc-104 motor protein and the Wnd DLK kinase. The authors presented impressive amount of data from synapse cell biology to physiology with an attempt to conclude that Wnd activation is sensitive to disruption of unc-104 mediated transport of some axonal components.This statement reflects some confusion between our major conclusions in the paper with models and ideas that we raise. We propose an idea that Wnd pathway activation is sensitive to build-up of Unc-104’s cargos. However this is the speculative idea in the paper, which is framed as a model for thinking about the data as a whole, combined with other observations in the field. It is not the primary goal of the paper to reach this idea as a ‘conclusion’.The measurement for Wnd activity mostly relied on induction of a puc-LacZ reporter in the soma, and morphological changes of NMJ terminals.unc-104 null mutants  and the other reviews (as discussed above) are all readily addressable, and do not detract from the major conclusions and impact of this paper.While the genetic interaction between Unc-104 and Wnd is very strong, there are indeed several results in the paper that show “modest” effects, and we also presented several negative data. However all of these data are helpful for reaching our understanding of the relationship between Unc-104 and Wnd. Importantly, all of these “modest” effects are consistent with the model we propose, and none are essential for the primary conclusions of the paper. Reasons for “modest effects are discussed in points C and 3 from reviewer 1. Due to the misunderstandings reflected in the comments, we plan in revision to do a better job with the writing to make these reasons clear up front, so that is easier for readers to follow the expectations for individual experiments and how they build upon one another.Their observation that Wnd suppresses the lethality of unc-104 mutants is the key observation led to this study. But it is not clear whether this suppression is directly caused at the level of presynaptic transport. Does inhibition of Wnd downstream factors, JNK and FOS, also suppress unc-104 lethality? In subsection “Wnd inhibits synapse assembly independently of cargo transport downstream of Unc-104” the final sentence of the second paragraph seems to state that "most or all" unc-104; wnd mutants actually die. Is the suppression of lethality rather minor?There is a significant misunderstanding here: the suppression of lethality data is NOT a ‘key observation’ in this study! Instead, the key observations that led to and are fundamental to our study are the striking suppression of synaptic phenotypes  suggested that unc-104 mutants show enhanced axonal outgrowth phenotypes in C. elegans PVD neurons. This may be consistent with the enhanced axonal regeneration phenotype we have observed here. However, because this is not essential data for the conclusions of our study, we hope the reviewers agree to go forward without the rescue data for the unc-104 regeneration phenotype.While this experiment is do-able, it would not add much to the paper. We included the enhanced regeneration as further evidence that the Wnd pathway is activated in unc-104 mutants.In the revision, we plan put the regeneration data into the supplemental data, and instead bring the synaptic overgrowth data from supplemental data  into theSubsection “Activation of the Wnd signaling pathway in neurons is sufficient to impair presynaptic structure and synaptic transmission”: first paragraph, the authors state "ectopic activation of Wnd should mimic the unc-104 mutant phenotypes". But where is Wnd normally activated?wnd loss-of-function phenotypes during stages of embryonic development in Please see our discussion above for reviewer 1’s point C. All of our data, combined with other data in the field for Wnd/DLK function in flies and worms, suggest its activity is restrained to very low levels in ‘normal’ wild type uninjured animals. Most previous work in the field has focused on Wnd/DLK’s role in injured neurons (when it is activated). There has previously been no supporting data (in flies or worms) to indicate that Wnd also functions in uninjured animals. In this present study we explored this question more deeply, through a careful analysis of In the same section, second paragraph: The authors made a comment about the result in Please refer to our discussion of this for reviewer 1’s point 2 above. Importantly, and similarly to the other points, this point is addressable and is also not essential for the main conclusions of this paper.They talked about similarities of how loss of Hiw or of Unc-104 affect Wnd function. An important experiment should include a double mutant study of Hiw and unc-104.hiw; unc-104 double mutants to hiw and unc-104 single mutants, which have very different phenotypes for both levels and localization. Because of the major transport defects in unc-104 mutants, it is unlikely that Wnd localization and levels in the double mutant would resemble the hiw single mutant. Instead it is quite likely that one would observe increased levels of Wnd (due to loss of hiw function) AND an in increase in levels of Wnd in the cell body which occurs in the unc-104 mutants. This kind of data would not help us distinguish whether Unc-104 and Hiw regulate Wnd through a common pathway or parallel pathways.We believe that this suggested experiment is to help in determining whether Hiw and Unc-104 regulate Wnd through the same or distinct pathways. However a consideration of the potential outcomes makes it unlikely that the experiment will yield new information. One would need to compare Wnd levels and localization in the However to address the general question we could include some more discussion about potential relationships between Hiw and Unc-104. See our discussion for reviewer 2’s point 2. However this relationship is a discussion point that is not essential for the major conclusions of this paper.hiw; unc-104 double mutant is an important experiment for this paper. Instead, it would be reasonable to include the discussion to reviewer 2’s point 2 above in the published review and rebuttal section for eLife. This would be a great format for the field to see these additional points discussed, which are side-points for the main points of the paper.So, in summary for this point we respectfully disagree that analysis of the [Editors' note: the author responses to the re-review follow.]While we are happy to consider a revised manuscript, you will see that we have some serious concerns that need careful attention. We see merit in the author's rebuttal letter that the most concerns can be addressed with rewriting and some additional experiments and key issues addressed. The overall impact of the work, if these points are well-addressed, can be strong, since it provides a fundamentally new model for how unc-104 contributes to neuronal function, by activating an axonal injury pathway. However, the data and model also need to be explained more clearly to be digestible by a broad readership such as at eLife.We appreciate the opportunity to address the points raised in the review. We have now provided the additional requested data, itemized below.In addition, the questions raised in the first round of review helped us see how the writing and manuscript organization could be improved so that a broader audience could follow the narrative and the significance of our findings. To accomplish this we made changes in the order of data  presentation, and changes in text throughout the paper including the Abstract, Title, and Results. The accompanying ‘track changes’ version of the manuscript highlights the significant changes since the first version.In response to the concerns raised about inconsistent results in their wnd loss-of-function analysis, the authors are dismissive, imploring us to "Note that our model is NOT that Wnd functions as a major restraint on these proteins during normal conditions or during development," yet their subheading describing these results is "Wnd restrains the expression of presynaptic proteins at early stages of synaptogenesis."We appreciate this point, and apologize that the previous attempt to explain our thinking came off as dismissive. In our revision we have worked to communicate our interpretation of results more carefully.unc-104 mutants by down-regulating the expression levels of SV and AZ protein components. However this is a pathological context, in unc-104 mutants. We wondered whether Wnd signaling ever plays a similar role in wild type animals.The principal finding in the paper is that Wnd signaling accounts for synaptic defects in Previous studies have suggested that while Wnd/DLK signaling becomes activated in injured neurons, it is normally highly restrained in uninjured animals , and, in contrast to its essential role in responses to axonal injury, roles for Wnd in developmental axonal outgrowth or synapse formation have not been previously described . Importantly, the function of the Wnd signaling pathway had not previously been examined at the critical developmental stages of synapse formation, due to the technical challenges of dissecting late-stage embryos (whose diameter is approximately the size of two human hairs). The late-stage embryo dissection skills that we mastered for this project allowed us to probe this question carefully with the results presented in wnd mutations upon SV protein expression are indeed modest in comparison to the dramatic effects of wnd in unc-104 mutants. However they are nevertheless interesting to report since they suggest that Wnd signaling also restrains synaptic protein levels during development in wild type, uninjured animals.Our findings in  indicateAccording to the modified text, we changed the subheading of this section to “Wnd delays the expression of SV proteins during early stages of synapse development”, and we have made large revisions in writing for this section.In a second example, the authors dismiss concerns about their analysis of MiMIC-GFP instead of endogenous Wnd levels by simply stating that the Wnd antibody does not work well for immunohistochemistry without addressing the fact that they have previously published analogous experiments using their Wnd antibody for immunohistochemistry . The authors may want to pay particular attention to these points.hiw mutants, when Wnd protein is elevated. However in wild type as well as unc-104 mutant animals, we were unable to detect a reproducible IHC signal above background in our current conditions using these antibodies. While some misfortune may be potentially attributed to poor antibody storage conditions, we consider the difference between hiw mutants  and unc-104 mutants  to be meaningful.As the reviewers pointed out, the Wnd antibodies developed in  could successfully detect a robust and Wnd-specific signal by both immunohistochemistry (IHC) and western blot (WB) in For the additional reagents used to examine Wnd protein, we have added some additional explanation into the manuscript text. That section is quoted here:unc-104 mutant animals (data not shown). […] This highly expressed transgene also revealed an increase in cell body localized Wnd in unc-104 mutants  with anti-Wnd antibodies, Wnd protein remained below the detection threshold in both wild type and  mutants .”The authors should measure puc-LacZ expression in additional unc-104 mutants.puc-lacZ measurements for additional unc-104 alleles (O3.1/170 and bris/170) in puc-lacZ expression  by BSK and FOS would provide strong support for the model.wnd mutations in rescue of the severely impaired spontaneous synaptic release (mEJP frequency) in unc-104 mutants  also mimic wnd mutations in the accumulation of SV and AZ proteins in the cell body of unc-104 mutants . The main point of the paper does not hinge on a particular model of synaptic protein level control, but the authors do need to clarify their explanations, either in the text or with more experiments.As agreed by the reviewer, the major effect we observed by IHC on presynaptic protein expression occurs in motoneurons. Since the RT-PCR data was obtained from the entire larval CNS, this data is limited in sensitivity and utility for making conclusions about any changes in mRNA levels in motoneurons. Therefore we have removed this data  to autophagy . It will be interesting to determine whether translation and/or autophagy contribute to the regulation of presynaptic proteins by Wnd signaling.”Wnd null mutants alone should be included in unc-104-null phenotype) is now unc-104;wnd-null mutant embryos) is now wnd single mutants resemble wild type animals.Our revision includes these controls side-by-side in the figures for comparison. Due to our reorganization the previous However, though phenotypes in transheterozygotes can be informative, lack of a dominant transheterozygote phenotype might just reflect gene dose requirements, and so we did not put as much weight on that particular experiment.bris/P350;wnd3/+unc-104) and found that heterozygous mutations in wnd fail to dominantly suppress the unc-104 defects. Since this observation neither proves nor negates the overarching model, we decided not to include this data.We have examined synaptic defects of transheterozygotes  in wnd overexpression lines would provide strong corroboration that increased Wnd explains functional defects in unc-104 mutants.This was a great suggestion – this experiment provides more direct evidence than the analysis of hiw phenotypes (which is now moved to supplementary data). We provided these analysis in the revision, in Quoting from subsection “Activation of the Wnd signaling pathway in neurons is sufficient to impair presynaptic structure and synaptic transmission”:wnd alone in motoneurons resulted in cell-autonomous presynaptic defects that are comparable to unc-104 mutants. […]Taken together with the rescue of unc-104 synaptic defects by wnd mutations  on Wnd suppression of the hiw phenotype will be also be important.bris/P350unc-104 and ΔNhiw mutants by ~70% and ~80%, respectively, and this defect was fully suppressed by mutations in wnd as well as downstream signaling components bsk and fos . We noted that ΔNhiw, which is a true null allele  showed a slightly lower quantal content than ND8hiw . […] These observations suggest that the mechanism of Wnd signaling activation in unc-104 mutants is unlikely to occur via Hiw.”shot) bears more resemblance to unc-104 mutants. ). Another recent study suggests that shot, unc-104, and tau mutants may influence a similar pathway . This suggests a possibility that unc-104 and shot mutants  may share a similar mechanism of Wnd signaling activation.Interestingly, the activation of Wnd signaling in mutants for the spectroplakin shortstop (The rescue of the axon regrowth phenotype are not critical for the main conclusions of the paper and could take a lot of time.unc-104-hypomorph/null mutants and in independent RNAi lines for unc-104 (We appreciate this point. We observed enhanced axonal growth after injury both in  unc-104 . HoweverReviewer #1:In their revised manuscript, the authors have been very responsive to reviewer suggestions. The logical flow of the manuscript is significantly improved and experimental concerns directly addressed.I have one question regarding the addition of wnd null data to Good point – for the embryo data we needed to dissect and fix independently but these were fixed for the same time points and imaged in parallel with identical settings. We have modified the quoted text to be more accurate by replacing “same dish/slide” with “same condition”. See subsection “Immunocytochemistry”.


Dosidicus gigas). Axoplasm extracted from these axons was found to contain ribosomal RNAs, ~8000 messenger RNA species, many encoding the translation machinery, membrane proteins, translocon and signal recognition particle (SRP) subunits, endomembrane-associated proteins, and unprecedented proportions of SRP RNA . While these components support endoplasmic reticulum-dependent protein synthesis, functional assessment of a newly synthesized membrane protein in axolemma of an isolated axon is technically challenging. Ion channels are ideal proteins for this purpose because their functional dynamics can be directly evaluated by applying voltage clamp across the axon membrane. We delivered in vitro transcribed RNA encoding native or Drosophila voltage-activated Shaker KV channel into excised squid giant axons. We found that total K+ currents increased in both cases; with added inactivation kinetics on those axons injected with RNA encoding the Shaker channel. These results provide unambiguous evidence that isolated axons can exhibit de novo synthesis, assembly and membrane incorporation of fully functional oligomeric membrane proteins.Local translation of membrane proteins in neuronal subcellular domains like soma, dendrites and axon termini is well-documented. In this study, we isolated the electrical signaling unit of an axon by dissecting giant axons from mature squids ( Consistently, large number of mRNA transcripts have been reported in these compartments36.Neurons are morphologically compartmentalized cells. Their cell bodies possess the nucleus and organelles to synthesize proteins, many of which are transported to axons and dendrites. Nonetheless, cumulative evidence has suggested that local protein translation is essential35, the number of unique transcripts in axons are now believed to be in the thousands; and likely many are actively translated locally37. In this study, we aimed to obtain a transcriptome using axoplasm, exclusively derived from the giant axon of the Humboldt squid (Dosidicus gigas). These axons are known to be formed by fusion of axons originating from cell bodies residing in the stellate ganglion38 that gives rise to a large electrical signal transmission unit, normally larger than 1.0 mm in diameter 21, NFPC (NF-protocadherin)24 in axon growth cones has been found to influence axon navigation and growth in cell culture, suggesting that these locally synthesized proteins are functional. Similarly, Spencer et al.44 have shown that local translation of conopressin receptor in Aplysia axons could alter the membrane excitability properties in response to externally added conopressin. In the present study, we examined whether membrane proteins, newly synthesized by isolated squid giant axons, are functional. Because Dosidicus’ axons have a large diameter, we can utilize them for direct RNA delivery inside the axoplasm. Thus, we injected squid giant axons with in vitro synthesized RNA encoding heterologous Drosophila Shaker KV channel or native D. gigas KV1.1. We then monitored the incorporation of newly synthesized ion channels into axon membrane by voltage clamp, which allowed us to study directly, the function of these membrane proteins. In both cases we found an increase in potassium currents across the axolemma. Furthermore, we observed that local synthesis of functional heterologous Shaker KV channel, which inactivates with fast kinetics, carried new discernable functional properties to the squid axon system. Since KV channels are known to undergo post-translational processing and oligomerization in endomembranous compartments prior to membrane-targeting46, our study unambiguously demonstrated that new KV channel subunits can be de novo translated, folded and hetero-tetramerized into functional ion channels that were targeted to axolemma likely using the axoplasm’s resident endomembranous machinery.Synthesis of membrane proteins such as ALCAM (activated leukocyte cell adhesion molecule)D. gigas. For comparison, bioanalyzer profiles of total RNA from Stellate ganglion which contains the cell bodies of the giant axon, and Schwann cells which surround the giant axon are shown in Fig. D. gigas axoplasm, and subjecting them to deep sequencing (RNA-Seq) using Hi-Seq. 2000 RNA-seq platform (Illumina). Upon performing quality control, ~95% reads were found to have a PHred score > 30. Since the reference genome sequence for D. gigas is not available, sequence reads from the RNA-seq were used to perform a de novo assembly to generate sequence contigs, referred to as components, which would represent the “squid axonal transcriptome”. This assembly generated 86,518 sequence components with N50 length of 756 nts and ~40% GC content. For each component generated, ORFs were predicted and blastx characterized against Swiss-prot database allowing the identification of open reading frames (ORFs) corresponding to 7820 unique genes (Supplementary Table de novo assembly. Numbers of mapped reads per kilobase per million reads (RPKM) were used to estimate the relative abundance of individual axoplasmic ORFs  RNA. These sequences  upon alignment to human SRP (hSRP) RNA  present on the ER membrane which would interact with the Sec61 complex (translocon) to translocate the protein into the ER co-translationally, in a GTP-dependent manner. The squid axonal transcriptome generated in this study was found to contain mRNAs encoding SRP54, SRP68 and SRP72 which bind to SRP RNA, as well as the ER resident membrane proteins SRα, SRβ and Sec61α  at ~300 nt in the bioanalyzer profile Fig. ; unlike ple Fig. . This sain vitro-transcribed cRNA encoding for Shaker KV channel. In addition to being an exogenous membrane protein for squid, these channels are known to display a distinctive and unique fast inactivation which is absent in squid KV channels expressed natively. cRNA was injected along the axon’s length with a Hamilton syringe (0.5 μl) supplied with a needle gauge 32  and one outside (Ext. Plate) they would represent the ionic current that originates exclusively at the axolemma under voltage controlled between the inside (Int.) and outside (Ext.) of the axon. Figure + current recording obtained about 50 min. after cRNA injection, which was the approximate time required to mount an axon in the voltage clamp chamber. The ionic current observed was in response to a voltage step to + 80 mV from a holding potential of −70 mV. The characteristics of this initial K+ current were indistinguishable from those carried by native squid KV channels . In contrast to the initial current, these new channels were found to exhibit a profound fast inactivation process. As expected, those channels that inactivated, were seen to recover from it with a relatively slow time constant  domain57. Since D. gigas KV1.1 and Shaker KV channel T1 domain sequences were found to be highly similar  or the mixture (blue). Interestingly, heterotetrameric channels were seen to recover from inactivation much more slowly . Thus, the changes in kinetics of inactivation clearly demonstrated that Squid KV1.1 can indeed co-assemble with Shaker KV to form functional heterotetrameric channels.Fast inactivation of Shaker Klar Fig. , it is plar Fig. . Xenopuswly Fig.  with a t59, as it can provide new mechanistic insights for maintenance of proper axonal function. Here we have shown that the axoplasm of squid giant axon contains a transcriptome representative of a compartment with membrane protein synthesis and trafficking capabilities. Moreover, we have demonstrated not just the presence, but also functional characteristics of membrane proteins, specifically ion channels, newly synthesized within isolated axons.Much of the axonal electrical signal transmission function has been shown to be manifested at its membrane. The possibility that axons can synthesize membrane proteins locally has been under extensive investigationD. gigas offered us an opportunity to examine ~8–10 cm of a singular unmyelinated electrical signaling unit of an axon, utilized immediately after dissection from a freshly captured adult animal 30, whereas such mRNAs were largely depleted from mouse DRGs axons when analyzed using deep sequencing35. Notably, we found transcripts encoding membrane proteins such as Na+/K+-ATPase subunits, glutamate receptors, ion channels and transporters in the squid axonal transcriptome  complex with the SRP complex has been shown to be critical for co-translational transport of these polypeptides into ER. The SRP complex, via its interaction with the SRP receptor (SR), has been reported to deliver the RNC complex onto a translocon, formed by Sec61 and associated proteins in eukaryotic ER membrane39. While polysomes64 and smooth ER65 have been reported in squid giant axons, it has not been determined whether RNC complexes in axons can bind to ER membranes and deliver the new polypeptide inside the ER for protein folding, modification and assembly. Our RNA-Seq studies showed that the D. gigas giant axons contain RNA species representative of the molecular machinery required for RNC delivery to ER during membrane protein synthesis. Firstly, we found enriched amounts of SRP RNA in D. gigas giant axons . Secondly, we detected mRNAs encoding protein subunits of SRP complex. Thirdly, substantial amounts of transcripts encoding translocon-forming Sec61α subunit were found present in the RNA obtained from squid giant axons. In addition, a variety of structural and functional ER protein-encoding mRNAs were found in the transcriptome -anchored placental alkaline phosphatase21 or monitoring changes in membrane excitability due to signaling through newly synthesized conopressin receptor in Aplysia axons44, offered indirect ways to assay function of an integral membrane protein. Therefore, direct measurement of a membrane protein in a mature axon’s electrical signaling compartment has persisted to be technically challenging.The functionality of axonal membrane protein synthesis machinery can be validated by direct measurement of function of newly synthesized membrane proteins at the axolemma of excised axons. Several studies have utilized different strategies to monitor the synthesis of membrane proteins in axons. In D. gigas KV1.1 (homologous) and Drosophila Shaker KV channel (heterologous). Measurement of current flow and kinetics under voltage clamp provided a direct readout of the function of both these integral membrane proteins. Shaker channel mRNAs are known to get translated as monomeric subunits whose assembly into functional tetrameric channels requires processing in the ER-Golgi system67. Therefore, a positive current signal from a functional Shaker channel in axolemma would serve as a strong indication of the presence of a functional endomembrane machinery which can be used for membrane protein processing. In addition, Shaker KV channel has been shown to exhibit distinct inactivation kinetics, which were absent in squid KV1.1 channel under voltage clamp68. Different kinetics of the two channels were used in our experiments to distinguish the heterologous channel from the homologous one. Because of the placement of the electrodes across the axolemma, only the signal originating from the ion channels incorporated in the axolemma, without contamination from either intra-axonal membranes or surrounding Schwann cells, could be recorded with high confidence. When isolated squid giant axons were injected with in vitro synthesized RNA encoding homologous D. gigas KV channel, an increase in K+ current was observed . RNA purification was carried out using PureLink RNA Mini kit (Ambion) as per manufacturer’s protocol . ~3 μg total RNA was obtained from the axoplasm. Total RNA from all samples were analyzed using Agilent Bioanalyzer 2100. RNA from all samples were run using the RNA 6000 Nano kit.Squid rRNA sequences were found to be similar to mouse rRNA sequences. Therefore, RiboZero rRNA Removal kit (Human/Rat/Mouse) (Illumina) was used for rRNA depletion of 1 μg axonal sample. The depleted RNA was run using RNA 6000 Pico kit in the Agilent Bioanalyzer 2100.Poly(A)-enriched libraries were constructed for one axoplasm sample and one axon sample using Truseq Stranded mRNA RNA Library Prep kit (Illumina) according to manufacturer’s protocol. Ribo-depleted library was separately constructed using the Truseq Stranded Total RNA with Ribo-Zero Globin kit (Illumina), according to manufacturer’s protocol with the following alteration: the ribo-depleted library was not subjected to fragmentation before sequencing. This was done to ensure enrichment of signal from species under peak (iii) over and above the longer RNA.https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), adaptor clipped using the “fastx_clipper” tool (http://hannonlab.cshl.edu/fastx_toolkit/commandline.html#fastx_clipper_usage), hard quality-trimmed (-Q33 -f 18 -l 123) using the “fastx_trimmer” tool (http://hannonlab.cshl.edu/fastx_toolkit/commandline.html#fastx_trimmer_usage), and dynamic quality-trimmed (-Q33 -q 20) using the “fastq_quality_filter” tool . Intact read-pairs remaining post clipping and trimming were subset from orphan reads using common awk commands (http://www.grymoire.com/Unix/Awk.html).All libraries prepared were paired-end sequenced 125 bp per read via HiSeq. 2000 (Illumina). Target sequencing depth was >120 M read pairs for each of the poly(A)-enriched libraries and >50 M read pairs for the ribo-depleted library. Post sequencing, reads per library were quality inspected using the “FastQC” tool (https://www.qiagenbioinformatics.com/products/clc-genomics-workbench/) using select parameters . Component sequences that returned were then passed to the “Find Open Reading Frames (ORFs)” tool supported in the CLC Genomics Workbench using select parameters . Identified ORFs per component were then characterized by “blastx” (https://www.ncbi.nlm.nih.gov) using reviewed protein sequences only found in the “Swiss-Prot” database of “UniProt” (http://www.uniprot.org/); unreviewed sequences were not used. Annotation of ORFs included the assignment of the “Uniprot” accession with the highest returned “Score” by “blastx” having an “E-value” <1e-6. ORFs were not annotated if the “E-value” returned by blastx was >= 1e-6. These annotations represented the orthologs of proteins detected from RNA-seq data. Post annotation, enumerated expression per ORF in “Reads Per Kilobase Million” mapped reads units, or “RPKM”, was calculated after performing map back of the intact read pairs per poly(A)-enriched library against the components. Map back was accomplished using the “Map Reads to Reference” tool supported in the CLC Genomics Workbench in conjunction with select parameters .Intact read pairs generated after quality control from the two poly(A)-enriched libraries were combined and passed to the “De Novo Assembly” tool supported in the CLC Genomics Workbench (https://github.com/trinityrnaseq/trinityrnaseq/wiki) under select parameters  for de novo assembly and Bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) for map back . Component sequences were blastn ) characterized.Intact read pairs obtained from the ribo-depleted library were used to generate a separate independent set of components using Trinity . Supplementary files, including the component sequences per assembly and ORF blast annotations, are available for download at th percentile amongst the predicted axonal ORFs was determined. The Uniprot accession numbers for all these proteins were submitted for core analysis using Ingenuity Pathway Analysis software (Qiagen). The categories representing various molecular and cellular functions were considered for further analysis. The software provides a p-value depending on the number of focus genes in the dataset and total number of genes enlisted for the category in the database. This p-value was used as a representation to determine the cellular processes that are crucial for axonal function.A dataset of unique proteins which had an RPKM value within the top 25D. gigas KV1.1 channel was subcloned in a pGemSqHE vector in which the 5’ and 3’ UTR sequences of the squid Na+/K+-ATPase beta subunit flank the channel’s coding region. These UTR regions were selected because they belong to one of the most abundant mRNA species of a membrane protein in the axoplasm’s sample. Shaker KV channel was subcloned in pGemHE vector. cDNAs were linearized with NheI and RNAs were synthesized using T7 RNA polymerase (Ambion). RNA was injected in a solution containing (mM): 5 GTP sodium, 5 ATP magnesium, 50 K glutamate, 50 KPO4, 5 K phospho-arginine, 5 K Hepes and 5 Mg Hepes (pH~ 7.5–8). Axons were mounted in a three-compartment chamber  or higher when both constructs were mixed. Defolicullated oocytes were obtained from Ecocyte Bioscience  and microinjected with 50 nl of RNA mix containing 5–10 ng of Shaker Kv RNA, 25–50 ng of D. gigas Kv1.1 RNA or the mix of both. After injection, oocytes were incubated at 17 °C in ND96 medium. Electrophysiological experiments were performed 1–2 days after injection. The oocytes’ vitelline layer was manually removed. Excised inside-out patches were obtained with borosilicate pipettes of 2 MΩ. Current acquisition was performed with a Digidata 1322 A , an Axopatch 200b patch clamp amplifier (Molecular Devices) and Clampex Software (Molecular Devices). The Bath solution contained (in mM) 160 KCl, 0.5 MgCl2, 1 EGTA and 10 HEPES (pH 7.4). The pipette solution contained (in mM) 150 NaCl, 10 KCl, 3CaCl2, 1 MgCl2 and 10 HEPES (pH 7.4). All recordings were made at room temperature.Same RNAs used in axons were injected into Supplementary Table S1