The tail domain of neurofilament light chain accumulates in neuronal nuclei during oxidative injury

  1. Aleksandra Arsić
  2. Ivana Nikić-Spiegel

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Abstract

Neurofilament light chain (NFL), one of the major subunits of neuron-specific intermediate filaments, is an important component of the neuronal cytoskeleton. Mutations in the NEFL gene and disturbances of NFL turnover or transport are associated with the pathophysiology of various diseases of the nervous system. Also, NFL is released from neurons as a result of neuroaxonal damage and NFL levels in cerebrospinal fluid and blood have emerged as a biomarker of the progression of numerous neurological diseases. One common underlying factor in these diseases is oxidative stress. Although both NFL and oxidative injury play a role in the development of neurological diseases, the effect of oxidative stress on NFL at the cellular level has not been extensively studied. Here, by investigating the response of neurofilaments to in vitro oxidative injury, we discovered that NFL localizes in the nuclei of primary neurons. We show that this depends on calpain activity and determine that a proteolytic fragment of the tail domain, not the full-length NFL, is that which accumulates in the nuclei. Furthermore, we demonstrate that the recombinant NFL tail domain accumulates in the nuclei of neurons and neuroblastoma cells, even in the absence of oxidative injury. Finally, we show that the tail domain of NFL interacts with the DNA in the nuclei of living cells, suggesting that NFL plays a role in the regulation of gene expression in response to oxidative injury.

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    Reply to the reviewers

    Response to the reviewers

    Manuscript number: RC-2022-01407

    Corresponding author(s): Ivana, Nikić-Spiegel

    1. General Statements

    We would like to thank the reviewers for careful reading of our manuscript and for their insightful and useful comments. We are happy to see that the reviewers find these results to be of interest and significance. The way we understand reviewers’ reports, their main concerns can be roughly divided in following categories: 1) providing more quantitative data 2) interpretation of the Annexin V/PI assay 3) additional evidence for calpain involvement. We intend to address these experimentally or by modifying the text, as outlined below.

    2. Description of the planned revisions

    Reviewer #1

    Fig1A/B o SYTO 16 staining suggests slight reshaping of nucleus upon spermine NONOate, showing less blurry punctae. From the SYTO 16 profile, this should be quantifiable.

    By looking at the shown examples and the entire dataset, it appears to us as if neuronal nuclei are shrinking upon spermine NONOate treatment resulting in their less blurry appearance. We are not sure if this is what the reviewer is referring to, but this can also be quantified by measuring changes in neuronal nuclear size. We already have this data from the measurements shown in Fig4 and we intend to show it in the revised version of the manuscript. Line profile measurements are also possible, but the nuclear size quantification might be more suitable for this purpose.

    o There is a subset of neuron nuclei that are SYTO 16 positive. Please quantify the ratio

    We will use our existing dataset to quantify the ratio of NFL positive and SYTO16 positive nuclei.

    FigS1A o Show NeuN with Anti-NFL merged figures

    We will show merged NeuN and anti-NFL images, which might require rearrangement of the existing figures and figure panels. We will do this in the revised manuscript.

    FigS1C o Show quantification and timeline. I want to know whether there is also a plateau reached here.

    As the data shown in the FigS1C do not include NeuN staining, we will do additional experiments and perform proposed quantifications.

    FigS2A-F o Though the statements might be true, selecting one nucleus for a line profile as a statement for the whole dataset seems problematic. Average a larger number of unbiased selected nuclei profiles across multiple cultures to make a stronger statement, or a percentage of positive nuclei as in FigS1b.

    Corresponding images and line profiles are representative of the entire dataset. However, we agree with the reviewer that this is not obvious from the current manuscript version. Thus, to strengthen our findings, we intend to quantify the percentage of positive nuclei as in FigS1b. The only difference will be that instead of NeuN, we will use SYTO16 as a nuclear marker. The reason being that the existing datasets contain images of NFL and SYTO16 and not NeuN.

    FigS3 • There are no fluorescence profiles, no quantification

    As the reviewer suggests, we will quantify the ratio of NFL positive and SYTO16 positive nuclei, and include the quantifications in the revised manuscript.

    General statement: There do seem to be punctated patterns of non-nucleus accumulating NFL fragments. Can they be localized to any specific structure?

    We assume that the reviewer is referring to neuronal/axonal debris. They are present after injury but they do not colocalize with nuclear stains. We will address this in the revised manuscript.

    Fig1C-F • I find it too simplistic to categorize c+f and d+e together. There is a huge difference in the examples of nuclear localization between d and e. To not comment on their distinction (if that is consistent) is problematic. Also, since we don't see a merge with either NeuN or SYTO 16, reader quantification is difficult.

    We thank the reviewer for bringing this up. We will carefully check our entire dataset and we will update the figures and the text accordingly. We will also show the corresponding SYTO16 images, as the reviewer suggested.

    Would the microfluidic device construction allow for time to transport any axonally damaged fragments to the soma?

    Yes, the construction of the microfluidic devices allows the transport of axonal proteins back to the soma. Based on our experiments, it seems that damaged NFL from the axonal compartment could be contributing to the accumulation of NFL fragments in the nuclei. However, this contribution seems to be minimal as we cannot detect nuclear NFL upon the injury of axons alone. Alternatively, it could be that the processing of axonal NFL fragments proceeds differently if neuronal bodies are not injured and that this is the reason we don’t detect the NFL nuclear accumulation upon injury of axons alone. We will discuss this in the revised manuscript.

    Fig2C+D • The statement ".... no annexin V was detected on the cell membrane" needs to be shown more clearly

    We will modify figures to address this comment.

    Please provide merged AnnexinV/PI images

    We will modify figures to address this comment.

    The conclusion about 2D, that nuclear accumulated NFL overlaps with PI is not supported by the example image shown. There are plenty of PI positive spots that are not NFL positive and even several NFL positive ones that do not have a clear PI staining. Please quantify and then show a very clear result in order to be able to suggest necrosis as the underlying process.

    We are not sure if we understand the reviewer’s concern correctly. We will try to clarify it here and in the revised text. If necessary, we will tone down our conclusion, but the reason why not all of PI positive spots are NFL positive is most likely due to the fact that not all injured nuclei are NFL positive. We quantified in FigS1 that up to 60% of nuclei under injury conditions show NFL accumulations. That is why we are not surprised to see some PI positive/NFL negative nuclei. And the fact that there are some NFL positive nuclei which appear to be PI negative is most likely related to the fact that the PI binding is affected. In addition, upon closer inspection of NFL and PI panels in Fig2d it can be observed that NFL positive nuclei are also PI positive, albeit with a lower PI fluorescence intensity. We will modify the figure to show this clearly in the revised manuscript.

    FigS5 C+D • If the case is made that nitric oxide damage induces necrosis, then why is it that the AnnexinV example of Staurosporine exposure (which induces apoptosis) looks similar to that of nitric oxide damage in Fig2d and necrosis induction with Saponin looks very different?

    We thank the reviewer for bringing this up. We will try to clarify this in the revised manuscript. Regarding the specific questions, the most likely explanation why staurosporine treated neurons look similar to the ones treated with spermine NONOate is that in the late stages of apoptosis cell membrane ruptures and allows for the PI to label nuclei. This is probably the case here as illustrated by the nucleus in the middle of the image (FigS5c) that shows the fragmentation characteristic for the apoptosis. This is not happening in early apoptotic cells due to the presence of an intact plasma membrane. On the other hand, the reason why saponin treated cultures look different compared to spermine NONOate is that membranes are destroyed by saponin so that the PI can enter the cell. For that reason, there could have not been any AnnexinV binding to the membrane which would correspond to the AnnexinV signal of spermine NONOate treated neurons. As we will discuss below, we did not try to mimic spermine NONOate-induced injury with saponin treatment. Instead this was a control condition for PI labeling and imaging. We also used a rather high concentration of saponin which probably destroyed all the membranes which was not the case with spermine NONOate treatment. We intend to do additional control experiments to address this.

    Additionally, does necrosis induction with Saponin also cause NFL fragment accumulation in the nucleus? Please show a co-staining of them. Also, the authors want to make a claim about reduce PI binding in NFL accumulated necrotic cells. In these examples, the intensity of the nuclear stain of PI with Saponin looks dimmer than with Staurosporine. Are the color scalings similar? It might be that the necrotic process itself causes reducing binding of PI and is not related to the presence of NFL.

    With regards to this question, it is important to note that Annexin V and PI imaging was done in living cells. To obtain the corresponding anti-NFL signal as shown in Fig 2c,d we had to fix the neurons, perform immunocytochemistry and identify the same field of view. We tried to do the same procedure after saponin treatment (Supplementary Figure 5d) but the correlative imaging was very difficult due to the detachment of neurons from the coverslip after the saponin treatment. For this reason, we could not identify the same field of view co-stained with NFL. However, other fields of view did not show NFL fragment accumulation. This could also be the consequence of the high saponin concentration that we used as we discuss above. We have also noticed the reduced intensity of PI binding in the nuclei of saponin-treated neurons. However, if the necrotic process itself reduces the binding of PI to the DNA, then all of the neurons treated with spermine NONOate would have an equally low PI signal. In our experiments, only the nuclei which contained NFL accumulations had a low PI signal, while the signal of NFL-negative nuclei was higher (as shown in Fig2d). We would also like to point out again that the saponin treatment was our control of the PI’s ability to penetrate cells and bind the DNA, as well as our imaging conditions, and not the control of the necrotic process itself. This is the reason why we didn’t go into details about neuronal morphology and NFL localization upon saponin treatment. We thank the reviewer for pointing this out since it prompted us to reevaluate what we wrote in the corresponding paragraph of the manuscript. We realized that the confusion might stem from our explanation of the AnnexinV/PI assay controls in the lines 196-198 (“Additional control experiments in which neurons were treated with 10 μM staurosporine (a positive control for induction of apoptosis) or with 0.1% saponin (a positive control for induction of necrosis) confirmed the efficiency of the annexin V/PI assay (Supplementary Fig. 5c,d).”). We will modify this portion of the text to clearly state that staurosporine and saponin treatments were controls of the AnnexinV and PI binding to their respective targets and not of the apoptosis/necrosis process. When it comes to the saponin treatment, our intention was only to permeabilize the membranes in order to allow PI penetration and DNA binding and not to induce necrosis or to mimic the effect of the spermine NONOate. We also intend to perform experiments with lower concentration of saponin to try to address this experimentally in addition to the text modifications.

    Fig3d • Please show similarly scaled images from controls for proper comparison

    We will show similarly scaled images of the control neurons so that they can be properly compared. They were initially not scaled the same for visualization purposes, but we will modify this in the revised manuscript.

    How do the authors scale the degree and kinetics of induced damage between application of hydrogen peroxide/CCCP and glutamate toxicity? Does glutamate toxicity take longer to affect the cell, not allowing enough time to accumulate NFL fragments in the nucleus?

    It is challenging to scale the degree and kinetics of induced damage with different stressors. That is why we did not intend to do this. Instead we set different injury conditions based on the published literature. That is why can only speculate when it comes to this. In this regard, it can be that the glutamate toxicity takes “longer” to affect the cells even though it is very difficult to compare them on a timescale, especially when considering different mechanisms of action. We will discuss this limitation in the revised manuscript.

    Fig4B • Some groups (like NO and NO + emricasan) have much larger numbers of close to 0 intensity, compared to the control group. Why?

    We were wondering the same when we analyzed the data. The fact that our nuclear fluorescence intensity analysis picked up NFL signal in control neurons which had no nuclear NFL accumulation made us realize that the intensity measured in the nuclei of control group comes entirely from the out of focus fluorescence – from neurofilaments in cell bodies, dendrites and axons (an example can be seen in the FigS6). That is why we presented the corresponding data with a cut-off value based on the control signal (as mentioned in lines 238-240). Since the oxidative injury causes NFL degradation (not only in neuronal soma, but also neuronal processes), the overall fluorescence intensity of the NFL immunocytochemical staining is reduced in injured neurons. We can see that in all of our images. Consequently, there is no contribution of out of focus fluorescent signal to the measured fluorescence intensity in the majority of nuclei. Due to that, the nuclei without NFL accumulation (at least 40% of injured nuclei) will appear to have a close to 0 intensity of the fluorescent signal. We will discuss and clarify this additionally in the revised manuscript.

    Please add the ratio of above/below threshold (50/50 obviously in controls)

    We will update the figure in the revised manuscript.

    The description of the CTCF value calculation seems a little... muddled? Several parameters are described whereas "integrated density" is not even used. Why not simply mean intensity of nuclear ROI-mean intensity of background ROI?

    We included the integrated density in the description since it is measured together with the raw integrated density and can also be used for the CTCF value calculation. However, since we didn’t use it for the CTCF calculation, we will remove it from the corresponding section of the manuscript. We calculated the CTCF value instead of calculating mean intensity of the nuclear ROI - mean intensity of the background ROI, since the CTCF value also takes into account the area of the ROI and not just the mean intensity.

    Also, please tell me if the areas for nuclear ROIs change, as I noted for Fig1A/B

    We will include this information in the revised manuscript.

    To make sure that one of the 3 experimental repeats didn't skew the results, please show the median fluorescence intensity for each individual experiment to clarify that the supposed effect is repeated across experiments.

    We have already noticed that in the earliest of the three experiments overall fluorescence intensity was higher, but this was consistent across all the experimental groups and did not skew the results or affect the overall conclusion. However, we will double-check this and revise the figure.

    From the text "...and due to the NFL degradation during injury...": this seems to contradict the process? Either the NFL fragment accumulates in the nucleus or it is degraded during injury. And isn't the degradation through calpain what supposedly allows this fragment of NFL to go to the nucleus in the first place? I reckon that the authors are possibly trying to reconcile why there are many close-to-0 intensity nuclei in the NO and NO + emricasan groups, but I don't feel the explanation given here fits.

    As we tried to explain in our response above, we think that the overall degradation of neurofilaments in neurons affects the fluorescence intensity originating from the out of focus neurofilaments. Therefore, the nuclei without NFL accumulation in injured conditions have a close to 0 fluorescence intensity. Additionally, we think that this is not an either/or situation, but that both degradation and nuclear accumulation of NFL happen simultaneously. We also think that degradation of axonal NFL and the transport of its tail domain to the soma will at least partially contribute to the accumulation in the nucleus. In any case, degradation and nuclear accumulation seem to be differentially regulated in individual neurons, as some of them show nuclear NFL accumulation and some not. Furthermore, calpain and other mechanisms could also cause NFL degradation up to the point at which these fragments can no longer be recognized by the anti-NFL antibody leading to the loss of signal. We will try to clarify this in the revised version of the manuscript.

    Fig5 • Does the distribution of this GFP in B match any of the various antibody stainings of different NFL fragments? Perhaps this is still a valid fragment of NFL, just not picked up by any AB?

    The GFP signal in B appears rather homogenous and it does not match any of the various antibody stainings of different NFL fragments. As the reviewer points out, this could also be a valid fragment of NFL fused to GFP that none of our antibodies is recognizing. We will clarify this in the revised manuscript.

    "... and was indistinguishable from the full277 length NFL-GFP." Based on what parameters?

    We will clarify this in the revised text, but we meant in terms of overall neurofilament network and cell appearance, which is commonly used to test the effect of NFL mutations.

    The authors claim that b is different from d, but I am not convinced. I would like to see a time dependent curve from multiple cells showing a differential change in nuclear and cytosolic GFP signal.

    As we also wrote in the manuscript, in the majority of neurons that were monitored during injury we were not able to detect an increase in the GFP fluorescence intensity in the nucleus. This is what prompted further experiments with NFL(ΔA461–D543)-FLAG. We will clarify this additionally in the revised manuscript and perform line profile intensity measurements to show the difference in nuclear and cytosolic GFP signal.

    Secondly, the somatic GFP intensity for NFL increases for full length NFL-GFP. How is this explained, if it is only a separation of NFL and GFP? If anything, GFP should float away. And if the answer is that NFL is recruited to the nucleus, you showed that inhibition of calpain activity partially prevents that. So, if calpain activity is necessary for the transport of NFL to the nucleus, then wouldn't it also cut the GFP from NFL before it reaches the nucleus?

    We thank the reviewer for bringing this up and we apologize for the confusion. This can be explained by the fact that the images were scaled in a way that the GFP signal over time could still be seen easily (i.e. differently across different time points which we unfortunately forgot to mention in the figure legend). In the revised manuscript, we will either scale the images the same or we will alternatively show the displayed grey values in individual panels.

    Fig6 • It is recommended to overlap the transfected cells with a stain for endogenous NFL to show that despite the absence of the FLAG-tag, there is still NFL.

    We did not overlap the anti-NFL with anti-FLAG and SYTO16 staining, due to the space constraint and the intent to clearly show the overlap of FLAG and SYTO16 signals in the merged images above the graphs. However, the line profile intensity measurements were done in all three channels and show that despite the absence of FLAG, there is still NFL in the nucleus (Fig6b), or that both FLAG and NFL are present in the nucleus (Fig6d, NFL signal shown in gray). However, as this is not obvious and can easily be overlooked, we will show the endogenous NFL staining overlap in the revised version of the manuscript.

    Fig7 • „ ...all disrupted neurofilament assembly...": this sounds like the staining for native NFL supposedly shows a distortion due to a dominant negative effect of the expression of these constructs? Please clarify.

    Yes, we were referring to the disruption of neurofilament assembly due to a dominant negative effect of the expression of NFL domains. We will clarify this in the revised version of the manuscript.

    Discussion: • The authors show that after overepression of the head domain only, it possibly passively diffuses into the nucleus even in the absence of oxidative injury. However, it seems to be suggested as well that the head domain would not be freely floating around if it wouldn't be for increased calpain activity as a result of oxidative injury in the first place. Therefore, a head domain fragment localized in the nucleus would still more prominently happen upon oxidative injury and interact with DNA through prior identified putative DNA interaction sites from Wang et al. Please comment.

    That is correct. Upon injury and calpain cleavage, it is conceivable that a fragment containing the NFL head domain would also be present in the cell and could potentially diffuse to the nucleus and interact with the DNA. However, by staining injured neurons with an antibody that recognizes amino acids 6-25 of the NFL head domain, we were not able to detect an NFL signal in the nucleus (FigS2a,b). It could be that either the NFL head domain does not localize in the nuclei upon injury, or that the fragment localizing in the nucleus does not contain amino acids 6-25 of the NFL head domain. As the putative DNA-binding sites described by Wang et al involve 7 amino acids located in the first 25 residues of the NFL head domain, we would expect to detect it with the aforementioned antibody. However, as that was not the case we speculated that the interaction of NFL and DNA occurs differently in living cells, as opposed to the test tube conditions utilized by Wang et al. We will comment and clarify this in the revised version of the manuscript.

    • Reviewer #2*
    • Major Comments:
    1. The initial data presented in the paper is good, does response of oxidative damage with proper controls, testing the antibodies to NF-L and etc. (Fig. 1-Fig. 4). *

    We thank the reviewer for their positive feedback.

    1. The evidence for calpain involvement in NF-L cleavage during oxidative damage is missing. Provide the evidence for full length NF-L construct and deletion mutants transfected into cells by immunoblot for cleavage of NF-L, perform nuclear and cytoplasmic extract preparations and show that enrichment of the tagged cleaved NF-L fragment in nuclear fraction.

    We thank the reviewer for their comments and suggestions. Since we saw in our microscopy experiments that calpain inhibition reduced the accumulation of NFL in the nucleus, and since it is known that NFL is a calpain substrate (Schlaepfer et al., 1985; Kunz et al., 2004 and others), we did not perform additional experiments to confirm the involvement of calpain in NFL degradation during injury. However, to strengthen our findings, we intend to perform the suggested experiments and include the results in the revised manuscript.

    1. Show calpain activation during oxidative damage by performing alpha-Spectrin immunoblots identify calpain specific 150-kda Spectrin and caspase specific 120-kDa fragment generation in these cells. Also, calpain activation can be measured by MAP2 level alteration and p35 to p25 conversion. Without this evidence it's very hard to believe if the calpain activity is increased or decreased during oxidative damage and these markers are altered by using calpain inhibitors.

    To confirm the calpain activation, we intend to perform anti-alpha spectrin and/or anti-MAP2 blots in lysates of control and injured neurons and include the results in the revised manuscript.

    1. The premise that NF proteins are absent in cell bodies and present only in axons is not correct. It has been demonstrated by multiple investigators that NFs are present in the perikaryon and dendrites of many types of neurons (Dahl, 1983, Experimental Cell Research)., Dr. Ron Liem's group showed NF protein expression in cell bodies of dorsal root ganglion cells (Adebola et ., 2015, Human Mol Genetics) and also showed N-terminal antibodies for NF-L, NF-M and NF-H stain rat cerebellar neuronal cell bodies and dendrites (Kaplan et al., 1991, Journal of Neuroscience Research) when NFs are less phosphorylated. (Schlaepfer et al., 1981, Brain Research) show staining of cell bodies of cortex and dorsal root ganglion cell bodies with NF antibody Ab150, and Yuan et al., 2009 in mouse cortical neurons with GFP tagged NF-L.

    We are not sure what the reviewer is referring to since we cannot find a corresponding section in which we claim that NF proteins are absent in cell bodies. We wrote the following “Anti-NFL antibody staining of neurons treated with the control compound showed the expected neurofilament morphology, that is, a strong fluorescence intensity in axons and lower intensity in cell bodies and dendrites (Fig. 1a)” in our results section (lines 119-121), but the claim we were trying to make there was that NF proteins are particularly abundant in axons. We will clarify this in the revised manuscript.

    1. Quantifying NF-L signal or tagged NF-L fragment signals in the cell body by ICC has many problems and making conclusions. It's extremely difficult to have control over levels of proteins in transfected overexpression models and comparing two or three different constructs with each other by ICC. Not every cell expresses same levels of protein in transfected cells and quantifying it by ICC again has a major problem. This can be addressed if there are stable lines that express equal levels of protein in all cells that comparisons can be made. Under thesese circumstances validation of the hypothesis presented in the study has no strong direct evidence to demonstrate that calpain is activated and NF-L fragment translocate to the nucleus.

    We agree that the results from overexpression-based experiments should be interpreted with caution as levels of expression vary between the cells. We intend to discuss this in the revised manuscript. However, we find it difficult to experimentally address this comment since we are not sure which specific experiments the reviewer is referring to. With regards to this, we would like to emphasize that most of the initial experiments in which we observed NFL accumulation in the nuclei of injured neurons were based on the ICC labeling of endogenous NFL and didn’t involve its overexpression. This includes labeling of endogenous NFL in various types of neurons, comparing the effects of different types of oxidative injury, as well as testing the effects of calpain inhibition on the observed nuclear accumulation (Figures 1-4; Supplementary Figures 1-6). We later resorted to the overexpression experiments in primary neurons (Figures 5-7; Supplementary Figure 7, 10) to gain more information about the identity of NFL fragment which was detected in the nucleus. Due to the low transfection efficiency of primary neurons, we performed an additional set of overexpression experiments in neuroblastoma ND7/23 cells (Figure 8; Supplementary Figures 8,9) and obtained similar results in a higher number of cells. We agree that having stable cell lines which e.g. express same levels of NFL domains would be a more elegant approach and we intend to make them for our follow-up studies, however the generation of said stable cell lines might be beyond the scope of this revision. Furthermore, looking at our data with overexpression of NFL domains in ND7/23 cells (Supplementary Figure 8,9), it appears to us as if different domains are rather homogenously expressed in different cells. While the expression levels might vary, it seems that they all show the same trend when it comes to their localization (which was the main point of those experiments).

    1. The interpretation that NF-L preventing DNA labeling cells is misinterpretation. NFs have very long half-life compared to other proteins. Due to oxidative damage, DNA is degraded in the cells but NFs that have very long half-life you see as NFs rings in the dead cells. So, NFs do not prevent DNA labeling, but DNA or chromatin is degraded in dead cells.

    We thank the reviewer for their useful insight. DNA degradation could certainly be the reason why we observe a lower fluorescence intensity of the propidium iodide fluorescence in the nuclei of injured neurons. We intend to discuss this in the revised manuscript. However, if the DNA degradation is the only reason for the lower PI fluorescence intensity, then the PI fluorescence intensity would be the same in all injured nuclei. In our experiments, we saw the reduced PI fluorescence intensity in nuclei that contained NFL accumulations and not in other nuclei. Additionally, we observed a reduction of SYTO16 fluorescent labeling of nuclei which contained accumulations of the NFL tail domain, even in the absence of oxidative injury. Due to these reasons we speculated that NFL accumulation in the nucleus might hinder nuclear dyes from interacting with the DNA. But this is only a speculation and we will try to clarify this further in the revised manuscript including alternative explanations.

    Minor comments:

    1. In the introduction on page 4 reference is missing for NF transport, aggregation and perikaryal accumulation (on line 93).

    We will add a reference to the revised manuscript.

    1. The statement in discussion on page 14 line 454 for Zhu et al., 1997 study is not accurate. It should be modified to sciatic nerve crush not spinal cord injury.

    We will correct this mistake in the revised manuscript.

    1. What is the size of the calpain cleaved NF-L tail domain? If you perform immunoblots on cell extracts treated with oxidative agents one would know it.

    We will perform immunoblots on cell lysates and incorporate the corresponding results in the revised manuscript.

    1. Authors could make their conclusions clear. This is particularly true for the experiments in Figure 4 panels c and d. It is very difficult to understand the conclusions of the experiments. First state the expectation and then described whether the expectation is true or different.

    We will do as the reviewer suggested in the revised manuscript.

    1. The ICC images are at extremely low magnification. They should be shown at 100x or 120x so that details of the cell body and the nucleus can be seen.

    Our intention was to show larger fields of view and wherever appropriate insets, but we will try to improve this in the revised manuscript by either zooming in, cropping or adding additional insets with individual cell bodies and nuclei. In general, images were taken with an optimal resolution/pixel size in mind for any of the used objectives (60x/1.4 NA or 100x/1.49 NA) and we can easily modify our figure panels to show more details.

    1. Oxidative damage leads to beaded accumulation of NF-L in neurites and axons. Authors should address this issue.

    We will discuss this in the revised manuscript.

    1. The combination treatment of the inhibitors (last 3 sets of the Fig. 4 b) has no statistical significance should be removed.

    Actually, these differences were statistically significant (Supplementary Table 1). For clarity and as described in the figure legend (line 516: “The most relevant significant differences are indicated with an asterisk”) we showed only a subset of them on the graph, but we will change this in the revised manuscript.

    1. Why only two antibodies recognize cleaved NF-L? If the antibodies at directed at tail region, they should recognize it unless the phosphorylated tail at Ser473 may inibit the antibody binding. In that case NF-L Ser473 specific antibody (EMD Millipore: MABN2431) may be used to test this idea.

    This is a very good point that we also wonder about. Even if all antibodies are directed at tail region, exact epitopes are not described for all of them. That makes it also difficult for us to understand and speculate on this. However, we have already ordered the new antibody as suggested by the reviewer and we will experimentally test it.

    **Referees cross-commenting**

    I agree with the reviewer#1 about presenting the quantification data for the indicated figures to make conclusions strong and see how much of variation is there among sampled cells.

    As discussed in our response to reviewer #1, we will provide additional quantifications.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    4. Description of analyses that authors prefer not to carry out

    Reviewer #2, major comment 7. Authors could do chromatin immunoprecipitation (chip) analysis to identify NF-L binding sites on chromatin and perform gel shift assays to show NF-L tail domain binding to specific consensus DNA sequences.

    We thank the reviewer for their suggestion. We are very interested in performing additional experiments and identifying the NFL binding sites on the DNA (either by chromatin immunoprecipitation or DamID-seq) and we intend to perform these experiments as soon as possible. Unfortunately, at the moment we do not have the expertise to perform such experiments in our lab. Instead, this type of follow-up project requires establishing a collaboration which is beyond the scope of this revision.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The authors hypothesize that Neurofilament-L subunit of NFs participates in oxidative medicated damage by calpain activation and cleavage of its C-terminal region, translocation to nucleus and activation of toxicity driven gene expression. Authors used cell culture system, NF-L gene transfections and immunocytochemistry to make their conclusions.

    Major Comments:

    1. The initial data presented in the paper is good, does response of oxidative damage with proper controls, testing the antibodies to NF-L and etc. (Fig. 1-Fig. 4).
    2. The evidence for calpain involvement in NF-L cleavage during oxidative damage is missing. Provide the evidence for full length NF-L construct and deletion mutants transfected into cells by immunoblot for cleavage of NF-L, perform nuclear and cytoplasmic extract preparations and show that enrichment of the tagged cleaved NF-L fragment in nuclear fraction.
    3. Show calpain activation during oxidative damage by performing alpha-Spectrin immunoblots identify calpain specific 150-kda Spectrin and caspase specific 120-kDa fragment generation in these cells. Also, calpain activation can be measured by MAP2 level alteration and p35 to p25 conversion. Without this evidence it's very hard to believe if the calpain activity is increased or decreased during oxidative damage and these markers are altered by using calpain inhibitors.
    4. The premise that NF proteins are absent in cell bodies and present only in axons is not correct. It has been demonstrated by multiple investigators that NFs are present in the perikaryon and dendrites of many types of neurons (Dahl, 1983, Experimental Cell Research)., Dr. Ron Liem's group showed NF protein expression in cell bodies of dorsal root ganglion cells (Adebola et ., 2015, Human Mol Genetics) and also showed N-terminal antibodies for NF-L, NF-M and NF-H stain rat cerebellar neuronal cell bodies and dendrites (Kaplan et al., 1991, Journal of Neuroscience Research) when NFs are less phosphorylated. (Schlaepfer et al., 1981, Brain Research) show staining of cell bodies of cortex and dorsal root ganglion cell bodies with NF antibody Ab150, and Yuan et al., 2009 in mouse cortical neurons with GFP tagged NF-L.
    5. Quantifying NF-L signal or tagged NF-L fragment signals in the cell body by ICC has many problems and making conclusions. It's extremely difficult to have control over levels of proteins in transfected overexpression models and comparing two or three different constructs with each other by ICC. Not every cell expresses same levels of protein in transfected cells and quantifying it by ICC again has a major problem. This can be addressed if there are stable lines that express equal levels of protein in all cells that comparisons can be made. Under thesese circumstances validation of the hypothesis presented in the study has no strong direct evidence to demonstrate that calpain is activated and NF-L fragment translocate to the nucleus.
    6. The interpretation that NF-L preventing DNA labeling cells is misinterpretation. NFs have very long half-life compared to other proteins. Due to oxidative damage, DNA is degraded in the cells but NFs that have very long half-life you see as NFs rings in the dead cells. So, NFs do not prevent DNA labeling, but DNA or chromatin is degraded in dead cells.
    7. Authors could do chromatin immunoprecipitation (chip) analysis to identify NF-L binding sites on chromatin and perform gel shift assays to show NF-L tail domain binding to specific consensus DNA sequences.

    Minor comments:

    1. In the introduction on page 4 reference is missing for NF transport, aggregation and perikaryal accumulation (on line 93).
    2. The statement in discussion on page 14 line 454 for Zhu et al., 1997 study is not accurate. It should be modified to sciatic nerve crush not spinal cord injury.
    3. What is the size of the calpain cleaved NF-L tail domain? If you perform immunoblots on cell extracts treated with oxidative agents one would know it.
    4. Authors could make their conclusions clear. This is particularly true for the experiments in Figure 4 panels c and d. It is very difficult to understand the conclusions of the experiments. First state the expectation and then described whether the expectation is true or different.
    5. The ICC images are at extremely low magnification. They should be shown at 100x or 120x so that details of the cell body and the nucleus can be seen.
    6. Oxidative damage leads to beaded accumulation of NF-L in neurites and axons. Authors should address this issue.
    7. The combination treatment of the inhibitors (last 3 sets of the Fig. 4 b) has no statistical significance should be removed.
    8. Why only two antibodies recognize cleaved NF-L? If the antibodies at directed at tail region, they should recognize it unless the phosphorylated tail at Ser473 may inibit the antibody binding. In that case NF-L Ser473 specific antibody (EMD Millipore: MABN2431) may be used to test this idea.

    Significance

    The study has very high significance. The results obtained with proper experimentation great implications in understanding how NF proteins are degraded in the cells and how these degraded fragments would alter neurodegeneration during oxidative stress and other conditions.

    Referees cross-commenting

    I agree with the reviewer#1 about presenting the quantification data for the indicated figures to make conclusions strong and see how much of variation is there among sampled cells.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The manuscript presented by Arsić and Nikić-Spiegel investigates a physiological consequence when neurons in vitro are exposed to oxidative stress injury, specifically a supposed interaction of the tail subdomain of the neurofilament light chain (NFL), after cleavage of the full NFL protein by calpain.

    General comments:

    The conclusions the authors draw from individual non-quantified example images are sometimes seen to be too simplistic when the shown examples ask for a more thorough investigation, especially when specific merged images are not available. It is highly recommended that the authors use the available data to come to more comprehensive answers across the entire acquired dataset. This for instance happens only in figures 4 and 8 and should be extended to other figures as well. There is not necessarily doubt about the author's general claims, but convincing the reader requires showing the variability and effect size of the entire group beyond a single selected example.

    If these more thorough quantifications continue to support the author's claims, then I find no objections for publication of this data.

    Specific comments:

    Fig1A/B

    • SYTO 16 staining suggests slight reshaping of nucleus upon spermine NONOate, showing less blurry punctae. From the SYTO 16 profile, this should be quantifiable.
    • There is a subset of neuron nuclei that are SYTO 16 positive. Please quantify the ratio

    FigS1A

    • Show NeuN with Anti-NFL merged figures

    FigS1C

    • Show quantification and timeline. I want to know whether there is also a plateau reached here.

    FigS2A-F

    • Though the statements might be true, selecting one nucleus for a line profile as a statement for the whole dataset seems problematic. Average a larger number of unbiased selected nuclei profiles across multiple cultures to make a stronger statement, or a percentage of positive nuclei as in FigS1b.

    FigS3

    • There are no fluorescence profiles, no quantification

    General statement:

    There do seem to be punctated patterns of non-nucleus accumulating NFL fragments. Can they be localized to any specific structure?

    Fig1C-F

    • I find it too simplistic to categorize c+f and d+e together. There is a huge difference in the examples of nuclear localization between d and e. To not comment on their distinction (if that is consistent) is problematic. Also, since we don't see a merge with either NeuN or SYTO 16, reader quantification is difficult.
    • Would the microfluidic device construction allow for time to transport any axonally damaged fragments to the soma?

    Fig2C+D

    • The statement ".... no annexin V was detected on the cell membrane" needs to be shown more clearly
    • Please provide merged AnnexinV/PI images
    • The conclusion about 2D, that nuclear accumulated NFL overlaps with PI is not supported by the example image shown. There are plenty of PI positive spots that are not NFL positive and even several NFL positive ones that do not have a clear PI staining. Please quantify and then show a very clear result in order to be able to suggest necrosis as the underlying process.

    FigS5 C+D

    • If the case is made that nitric oxide damage induces necrosis, then why is it that the AnnexinV example of Staurosporine exposure (which induces apoptosis) looks similar to that of nitric oxide damage in Fig2d and necrosis induction with Saponin looks very different?
    • Additionally, does necrosis induction with Saponin also cause NFL fragment accumulation in the nucleus? Please show a co-staining of them. Also, the authors want to make a claim about reduce PI binding in NFL accumulated necrotic cells. In these examples, the intensity of the nuclear stain of PI with Saponin looks dimmer than with Staurosporine. Are the color scalings similar? It might be that the necrotic process itself causes reducing binding of PI and is not related to the presence of NFL.

    Fig3d

    • Please show similarly scaled images from controls for proper comparison
    • How do the authors scale the degree and kinetics of induced damage between application of hydrogen peroxide/CCCP and glutamate toxicity? Does glutamate toxicity take longer to affect the cell, not allowing enough time to accumulate NFL fragments in the nucleus?

    Fig4B

    • Some groups (like NO and NO + emricasan) have much larger numbers of close to 0 intensity, compared to the control group. Why?
    • Please add the ratio of above/below threshold (50/50 obviously in controls)
    • The description of the CTCF value calculation seems a little... muddled? Several parameters are described whereas "integrated density" is not even used. Why not simply mean intensity of nuclear ROI-mean intensity of background ROI?
    • Also, please tell me if the areas for nuclear ROIs change, as I noted for Fig1A/B
    • To make sure that one of the 3 experimental repeats didn't skew the results, please show the median fluorescence intensity for each individual experiment to clarify that the supposed effect is repeated across experiments.
    • From the text "...and due to the NFL degradation during injury...": this seems to contradict the process? Either the NFL fragment accumulates in the nucleus or it is degraded during injury. And isn't the degradation through calpain what supposedly allows this fragment of NFL to go to the nucleus in the first place? I reckon that the authors are possibly trying to reconcile why there are many close-to-0 intensity nuclei in the NO and NO + emricasan groups, but I don't feel the explanation given here fits.

    Fig5

    • Does the distribution of this GFP in B match any of the various antibody stainings of different NFL fragments? Perhaps this is still a valid fragment of NFL, just not picked up by any AB?
    • "... and was indistinguishable from the full277 length NFL-GFP." Based on what parameters?
    • The authors claim that b is different from d, but I am not convinced. I would like to see a time dependent curve from multiple cells showing a differential change in nuclear and cytosolic GFP signal.
    • Secondly, the somatic GFP intensity for NFL increases for full length NFL-GFP. How is this explained, if it is only a separation of NFL and GFP? If anything, GFP should float away. And if the answer is that NFL is recruited to the nucleus, you showed that inhibition of calpain activity partially prevents that. So, if calpain activity is necessary for the transport of NFL to the nucleus, then wouldn't it also cut the GFP from NFL before it reaches the nucleus?

    Fig6

    • It is recommended to overlap the transfected cells with a stain for endogenous NFL to show that despite the absence of the FLAG-tag, there is still NFL.

    Fig7

    • „ ...all disrupted neurofilament assembly...": this sounds like the staining for native NFL supposedly shows a distortion due to a dominant negative effect of the expression of these constructs? Please clarify.

    Discussion:

    •  The authors show that after overepression of the head domain only, it possibly passively diffuses into the nucleus even in the absence of oxidative injury. However, it seems to be suggested as well that the head domain would not be freely floating around if it wouldn't be for increased calpain activity as a result of oxidative injury in the first place. Therefore, a head domain fragment localized in the nucleus would still more prominently happen upon oxidative injury and interact with DNA through prior identified putative DNA interaction sites from Wang et al. Please comment.

    Significance

    This in vitro study, despite its acknowledged caveats, can provide novel support for the claim that calpain induced cleavage of the NFL may play a role in downstream gene expression in order to regulate a neural response upon oxidative injury. Further investigation into this topic may provide further understand of physiological gene expression through interaction with cleavage products as well as yield possible therapeutic targets for pathological conditions. This study therefore may be of interest to a broad audience.

  4. Version published to 10.1101/2022.03.03.481279v1 on bioRxiv