ENGRAILED ‐1 transcription factor has a paracrine neurotrophic activity on adult spinal α‐motoneurons

  1. Mélanie Lebœuf
  2. Stephanie E Vargas‐Abonce
  3. Eugénie Pezé‐Hedsieck
  4. Edmond Dupont
  5. Lucia Jimenez‐Alonso
  6. Kenneth L Moya
  7. Alain Prochiantz

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Abstract

Several homeoprotein transcription factors transfer between cells and regulate gene expression, protein translation, and chromatin organization in recipient cells. ENGRAILED‐1 is one such homeoprotein expressed in spinal V1 interneurons that synapse on α‐motoneurons. Neutralizing extracellular ENGRAILED‐1 by expressing a secreted single‐chain antibody blocks its capture by spinal motoneurons resulting in α‐motoneuron loss and limb weakness. A similar but stronger phenotype is observed in the Engrailed‐1 heterozygote mouse, confirming that ENGRAILED‐1 exerts a paracrine neurotrophic activity on spinal cord α‐motoneurons. Intrathecal injection of ENGRAILED‐1 leads to its specific internalization by spinal motoneurons and has long‐lasting protective effects against neurodegeneration and weakness. Midbrain dopaminergic neurons express Engrailed‐1 and, similarly to spinal cord α‐motoneurons, degenerate in the heterozygote. We identify genes expressed in spinal cord motoneurons whose expression changes in mouse Engrailed‐1 heterozygote midbrain neurons. Among these, p62/SQSTM1 shows increased expression during aging in spinal cord motoneurons in the Engrailed‐1 heterozygote and upon extracellular ENGRAILED‐1 neutralization. We conclude that ENGRAILED‐1 might regulate motoneuron aging and has non‐cell‐autonomous neurotrophic activity.

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  1. Version published to 10.15252/embr.202256525
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    Reply to the reviewers

    RC-2022-01632

    Answers to referees

    First of all, we wish to thank the 3 referees for their careful evaluation of the manuscript. We see many issues that they have raised as legitimate and have tried to provide experimental or editorial answers. In contrast, some issues are presently addressed in the context of a future manuscript and we had rather not introduce these studies in the revised version.

    Below, one will find the answers and the description of the revisions already introduced in the revised manuscript (questions are recalled in blue italics).

    New and modified figures, plus not shown figures and tables are indicated in the text below but could not be pasted in the document and can be found in the Revision plan.

    Referee # 1

    Evidence, reproducibility and clarity

    They then delivered 86/8 and LSBio anti-En1 antibodies, that catch En1 in the cleft and prevent it from being captured by MNs.

    Perhaps we were not clear. We did not deliver the antibodies 86/8 and LSBio, we used them for western blots and immunohistochemistry (IHC) to identify EN1 and localize it. We delivered the third antibody, a single-chain anti EN1 antibody (scFvEN1), that captures extracellular EN1 and prevents it from being captured by MNs on the basis of the LSBio staining (Figure 4A-C).

    Finally, heterozygotes revealed also a degeneration in dopaminergic neurons within midbrain similar to the one observed in spinal MNs, along with an upregulation of SQTSM1/p62 gene/protein, a factor in MN ageing linked to the classical genes implicated in familial forms of ALS (SOD1, TDP-43, FUS, and C9ORF72).

    This is a fair comment/work description, that does not require answers.

    Significance

    *Major comments: *

    It is unclear why levels of intensity for RNAscope were not quantified, and qPCR was preferred for quantifications in Figure 1b. RNAscope is a technique that allows for spatial distribution analysis of the markers and their level of the expression. This data can be easily quantified utilizing the QuPath software which is open access. Same concerns apply to Figure 2a.

    Quantitative RT-PCR provides a quantitative measure of gene expression. Since only V1 interneurons (including, Renshaw cells) express EN1, we infer the spatial distribution, although not expression level cell by cell. Figure 2A is an actual counting at 4.5 months of En1+ cells and of Calbindin+ cells (Renshaw cells), both identified by RNAscope. Thus, it is clear that the number of En1-expressing cells (V1 interneurons) is not modified at 4.5 months when muscle weakness and death of aMNs are well advanced (around 70% of the aMNs that will eventually die, are already gone). Long-term survival of V1 interneurons is further demonstrated in Figure 2D (left panel) until 15.5 months, (see also below) whereas total En1expression is reduced by half. Quantification neuron by neuron of the amount of En1 transcribed (RNAscope) would indicate the variation, among interneurons, of En1 transcription in WT and mutant mice. This is interesting per se but would not modify the main information that these neurons do not die in the heterozygote and that En1 transcription does not decrease with time in both WT and mutant genotypes (at least until 15.5 months).

    *Antibodies should be validated utilizing a reporter mouse. En1cre mice are commercially available and can be crossed with reporters (TdTomato or YFP mice). Utilizing this tissue En1 antibodies can be easily validated. The EN1 antibody shown in Figure 1c seems unspecific, staining several neuronal populations in the spinal cord. *

    Indeed, antibody validation is extremely important. LSBio is commercial (CliniSciences), 86/8 was developed in the laboratory and fully characterized and used in previous studies (e.g. Alvarez-Fischer et al. Nature Neurosci. 14: 1260-1266, 2011; Rekaik et al. Cell Reports 13: 242-250, 2015; Blaudin de Thé et al. EMBO J. 37: e97374, 2018), scFv against EN1 was prepared from the 4G11 hybridoma (Developmental Hybridoma Bank, Iowa City, USA) and validated in previous studies (e.g. Wizenmann et al. Neuron 64: 355-366, 2009). In the present study, the two polyclonal were further validated inseveral ways.

    In the WBs we compared ventral midbrain (VMB) and spinal cord (SC) tissues and found similar patterns. Strong evidence for antibody specificity is immunostaining extinction with the antigen and with absence of first antibody, which we carried out.

    We have now used LSBio and 86/8 to perform a WB on spinal cord (SC) and ventral midbrain (VMB) extracts with or without the first antibody and we find that the absence of first antibody fully eliminates band staining. The western has been introduced in the revised manuscript in place of the cross immunoprecipitation.

    Finally, we have quantified EN1 in the aMNs of the heterozygote at 3 months (before cell death), showing that EN1 content is decreased by approximately 2-fold (LSBio antibody) in both a and gMNs with no change in neuron number. This result demonstrating that EN1 is diluted by approximately twofold (concentration per neuron when all neurons are still present), in addition to further validating the antibody, is itself interesting and has been introduced in the revised manuscript as Supp. Fig. 1A.

    Regarding the staining in other neuronal populations, there is always some background, in particular in the tissue treatment conditions used for RNAscope. Furthermore, given the large number and wide distribution of V1 interneurons (Fig. 1A), we cannot preclude that EN1 is present at a low concentration in the extracellular space and in several cell types (discussed in Fig. 9 of the manuscript). This does not weaken the main conclusion that it primarily accumulates in MNs which do not express En1 (RNAscope).

    *Investigations of En1 expression in motor neurons from already available omics data sets would support the idea that En1 is expressed in motor neurons. *

    The En1 locus is silent in MNs. Microdissection of MNs and proteomic analysis would not be definitive since the interneurons that produce EN1 are in close vicinity of the MNs and since some protein is necessarily present in the extracellular space (where it is trapped by scFvEN1), making contamination unavoidable.

    Differentiation between Gamma and Alpha motor neurons should be performed using specific markers as Err3, Wnt7a or NeuN.

    This is a possible way to do the distinction, but size criterion in Cresyl violet is supported in the literature (Wu et al. Journal of Biological Chemistry, 287: 27335-27344, 2012; Dutta et al. Experimental Neurology, 309: 193-204, 2018). In our study, it is further validated by the demonstration that, in 9-month-old animals, the results obtained (cell number and specific death of large neurons >300µm2, but not of intermediate size ones 200-299µm2) are replicated by counting ChAT-stained neuron (Figure 2C). It is of particular interest that the number of medium size neurons (also ChAT-positive medium size MNs) does not increase when the number of large size (Cresyl and ChAT-positive) neurons decreases, thus precluding a “shrinkage effect”. Most importantly, the size criterion (Cresyl violet) allows us not to be mistaken by a possible down-regulation of markers in the mutant, independently of cell survival. We provide for the reviewer (Revision plan) but not for publication, the evolution with time of the number of neurons based on size (above 200 µm2) showing clearly that at 15.5 months the large population (>300 µm2) is decreased in the En1-Het, with very little change for neurons between 200 and 300 µm2, and certainly not an increase which would be expected if shrinkage occurred.

    We were indeed surprised by this finding and a plausible explanation is that a lower metabolic activity makes interneurons less sensitive to stress than aMNs which have to “fuel” long axons and high firing rates (not the case for gMNs). We propose this explanation in the discussion and make it clearer in our revised version. We agree that it is speculative and that the point raised by the reviewer is very interesting. We hope to address this in the future and have discussed this point.

    Since the cells do not die, we did not look for signs of apoptosis.

    We analyze lumbar sections from L1 to L5 as now indicated in the methods section in the manuscript

    The set of experiments reported in Figure 4 is of difficult interpretation without showing the actual presence of extracellular En1, that could be assessed with protein detection or RNAscope.

    This is another interesting suggestion, but we think that it will be difficult to distinguish low extracellular staining due to EN1 diffusion from some unspecific background. Since the scFvEN1 is secreted by astrocytes, it necessarily neutralizes extracellular EN1, resulting in a decrease in the MN content of the protein. This is an experiment with high specificity since the same scFv harboring a Cysteine to Serine point mutation that prevents EN1 recognition (no disulfide bound formation between the light and heavy chains) does not block EN1 capture by MNs (Fig. 4C for IHC and quantifications).

    As for extracellular EN1 mRNA identified by RNAscope, we hesitate to embark on the idea as mRNAs are likely secreted in insufficient amounts to be identified, even by RNAscope. The results that we have (no En1 visible by RNAscope in MNs, loss of EN1 in MNs following extracellular scFvEN1 activity, and preferential addressing of injected EN1 to MNs) demonstrate EN1 capture by MNs. Indeed, we cannot completely preclude the transfer of tiny amounts (escaping RNAscope detection in MNs) of En1 mRNA (for example, through extracellular vesicles), but we plead for not considering this hypothesis in the present paper. However, if the reviewer wishes, the possibility can be introduced in the discussion.

    Referee 2

    Evidence, reproducibility and clarity

    In general, most of the experiments shown in this study are well done and convincing. However, the data on p62 upregulation appear correlative and do not allow any conclusions about the mechanism and function how EN-1 modulates motoneuron survival and function. In addition, this study is not very precise on the mechanisms how motoneurons degenerate in this model so that there are only limited insights into the way how EN-1 acts on motoneurons in a physiological manner and under pathophysiological conditions.

    This criticism is justified, at least in part, as we agree that p62 upregulation is correlative. However, the fact that the neutralization of extracellular EN1 by the scFv increases p62 expression, is in favor of a causative link. The increase is also seen at 3 months in the En1-Het when all aMNs are still present but not after, which is interesting because, due to aMNs death, surviving MNs receive more EN1, information provided below and now introduced and discussed in the revised manuscript (Supp. Fig. 1B).

    As for p62, and as also mentioned by referee 3, Fig. 8 is very hard to follow and we propose to simplify it to make the message clearer:

    We have revised Fig. 8C, D in which we focus exclusively on SQTSM1/p62 mean expression (see revision plan)

    A second information is that a difference in mean p62 expression between WT and Het is seen only at 3 months in aMNs. For aMNs, we propose that this is due to the fact that they are very sensitive to EN1 dosage (in contrast with gMNs which do not die in the En1-Het). At 3 months, aMNs have only half of their normal EN1 content. Later, at 4.5 months 75% of the aMNs bound to die are already dead (Fig. 2D) and the remaining neurons receive more EN1 (even more so at 9 months), as could be measured (see above Supp. Fig. 1B). We thus can propose an accelerated aging of aMNs at 3 months due to both EN1 decrease and high metabolic activity (higher than in gMNs).

    In the case of the scFv, scFvEN1, but not the mutated version induces enhanced mean p62 expression in the 80% surviving aMNs and in gMNs at 7 months (low aMN death in this model, see Fig. 4F). As can be seen also in a newly added figure (Supp. Fig. 2) that has been introduced in the revised manuscript and is shown below, 7-month-old scFv animals and 3- to 3.5-month-old En1-Het have similar phenotypes. This mild scFv phenotype (a-MN death and muscle strength loss) in 7-month-old mice in spite of a huge loss in the EN1 content of MNs (Fig. 4C) suggests that the En1-Het phenotype is not entirely due to the decrease in EN1 transport from V1 interneurons to MNs (see discussion and Fig. 9).

    It remains true that we have voluntarily decided not to examine in depth the molecular mechanisms allowing EN1 to exert its protective activity, a decision that we would like to defend and maintain.

    A first reason is that in previous papers on mesencephalic dopaminergic (mDA) neurons (Alvarez-Fischer et al. Nature Neurosci. 14: 1260-1266, 2011; Rekaik et al. Cell Reports 13: 242-250, 2015; Blaudin de Thé et al. EMBO J. 37: e97374, 2018), we evaluated several mechanisms involved in EN1 neurotrophic activity and we did not want this study to be a duplication of studies done on a different neuronal population, even if mechanisms might differ in part, between aMNs and mDA neurons. What has interested us more is that, in the two cases, age is an important factor in the unveiling of the degeneration phenotype (mDA neurons start dying at 1.5 months and aMNs at 3 months). It is because of this similarity that we performed the bioinformatic study that has led us to SQTSM1/p62. In this context, it is of interest that mean SQTSM1/p62 expression (variability of expression between neurons is not discussed in the revised version) increases with age in the wild type, thus can be seen as an age marker. It allows us to propose that EN1 extracellular neutralization and the loss of one En1 allele, that increases mean SQTSM1/p62 expression accelerate aging.

    A second reason is that the study is oriented toward a possible use of EN1 as a therapeutic protein. This orientation also has to do with the focus on SQTSM1/p62. Indeed, there are probably many pathways downstream of EN1, but in the bioinformatic analysis of genes differentially regulated in WT and En1-Het mDA neurons and also expressed in MNs, SQTSM1/p62 is the only one that interacts with the 4 genes mutated in the major ALS familial forms. In addition, SQTSM1/p62 mutations have been observed in ALS patients (References 41 to 45 in the manuscript).

    Finally, the most important point is that the main message of this paper is the discovery of a non-cell autonomous EN1 activity in the spinal cord and of its ability to travel between V1 interneurons and MNs. This specificity best explained by a targeting signal that we have identified is at the basis of the specific addressing to MNs of EN1 intrathecally injected, which also has implications for its potential therapeutic use.

    Specific points of criticism

      • In Fig. 2a, the authors show that EN-1-positive interneurons are not reduced at 4.5 months in the spinal cord. No data are shown for later time points such as 9 months, the corresponding stage when motoneuron loss is observed, or at 16 months which corresponds to the data shown in Fig.1. The argument that there is no reduction of V1 interneurons between 4.5 months and 16 months because there is no decrease of EN-1 expression between 4.5 and 16 months, as shown in Fig. 1b is not convincing. EN-1 expression could change in individual cells, thus compensating for the loss. Data on numbers of EN-1-positive cells at 9 and 16 months should be included, and a potential autocrine effect of EN-1 on V1 interneurons, as observed in midbrain dopaminergic neurons, characterized in more detail. * Fig. 2A illustrates the absence of interneuron loss at 4.5 months, but this set of data is completed by those of Fig. 2D that demonstrate the maintenance of V1 interneuron number until 15.5 months, at least. It can be noted that, in contrast with interneurons, aMNs at 4.5 months have experienced massive cell death (70% approx. of total aMN death at 15.5 months). As a whole, data of Fig. 2 demonstrate that the number of small neurons (100-199 µm2) and intermediate size neurons (200-299 µm2) does not change with age, at least through 15.5 months. This is in strong contrast with large aMNs (>300 µm2). As already explained in our answers to referee 1, size is an excellent marker for the identification of neuronal subtypes and the analysis of survival (See answers to referee 1, justifying the use of neuron size).
    • In Fig. 2e, the authors present data on loss of muscle strength between 4.5 and 15.5 months. They conclude that this reflects gradual neuromuscular strength loss. Since neuromuscular endplates have a very high safety factor, they can maintain full function even if transmitter release is reduced by more than 80%. Therefore, the loss of muscle strength seems to reflect the progressive loss of presynaptic terminals at neuromuscular endplates, rather than a gradual loss of neuromuscular strength. *

    We apologize for the semantic confusion. What is measured is a progressive loss of muscle strength due to the progressive loss of presynaptic terminals and not a gradual loss of neuromuscular strength. This is now modified throughout the revised text.

    • More detailed data on NMJ morphology should be included. How does EN-1 modulate neuromuscular endplates? Is EN-1 located at neuromuscular endplates after being taken up from motoneurons? Even if the mechanism is indirect, via upregulation of p62 under conditions when EN-1 signaling is reduced, does this situation lead to enhanced localization of p62 at neuromuscular endplates? *

    We do not see expression of En1 mRNA or the presence of EN1 protein at the level of the endplate (Supp. Fig. 3 in revision plan)

    • The data shown in Fig. 3 on changes in NJM morphology appear incomplete and not convincing. As SV2a is not a good marker for changes in presynaptic compartments since it does not allow conclusions on how many synaptic vesicles are released, additional markers for presynaptic active zones such as Bassoon, Piccolo, Munc-13 should be studied. The analysis of fully occupied endplates appears arbitrary, and the differences are relatively small. Additional EM pictures and quantitative analyses of active zone proteins in the presynaptic compartment would help to support the argument of the authors that presynaptic compartments degenerate before cell bodies are lost in EN-1 +/- mice. *

    SV2a and NF staining (it is not only SV2a) at the level of endplates identified by a-Bungarotoxin labeling has been used in a large number of studies (Wahlin et al. J. Comp. Neurol. 506: 822-837, 2008; Hasting et al. Scientific Reports 10: 1-13, 2020; Yahata et al. J. Neurosci. 29: 6276-6284, 2009 ; Jones et al. Cell Reports 21: 2348-2356, 2017) Our goal was not to document the loss of synaptic activity through the use of the three suggested markers, Bassoon, Piccolo and Munc-13. Doing it would force us to initiate experiments taking several months to prepare the material and do a quantitative analysis in the models of EN1 loss of function (En1-Het) and neutralization (scFv), plus rescue by EN1. Nor do we wish to initiate a novel collaboration to produce a quantitative ultrastructural study. We see the latter morpho-functional studies beyond the scope of the manuscript and wish to be given the possibility to present them in a separate study (see below in “Description of the experiments that the authors prefer not to carry out”).

    The distinction between fully occupied, partially occupied and denervated endplates is not arbitrary and we apologize for not having sufficiently described the methodology. As illustrated in modified Fig. 3 and explained in Material and Methods, a fully innervated endplate is defined as an endplate in which 80% or more of the green pixels (a-BGT) are covered by a red pixel (SV2a), a partially one is between 20 and 80% and a denervated one below 20% coverage. Thus at 9 months and later ages, close to 30% of the endplates are either partially innervated or denervated. In fact, it is more likely that they are partially innervated since the number of AChR clusters does not change (totally denervated clusters normally dissolve). The 80% threshold for fully innervated was selected to give a margin of security, and it is likely that the percentage of 25 to 30% of partially innervated endplates is an underestimation.

    In the Revision plan is presented a table with the calculations and modified Figure 3.

    We agree that we were not clear enough in our description and that it may have given the impression that the differences were relatively small. We think that retrograde degeneration is strongly supported by a loss of muscle strength that parallels the decrease in fully occupied endplates (a-BGT, NF, SV2a) and precedes aMN loss by more than 1 month. We have recently contacted an electrophysiology group to establish a collaboration that will allow us to follow functional changes at the level of the spinal cord and of the neuromuscular junction and we see the experiments proposed by the reviewer as complementary to these physiological approaches. Yet, we do not want to ignore the opinion of the reviewer and mention it in the conclusion, on the basis of his/her comment.

    • The authors present evidence for a glycosaminoglycan (GAG) binding domain that appears responsible for uptake of EN-1 into motoneurons. However, it is unclear into which cellular compartment EN-1 is taken up after GAG binding on motoneurons. The authors propose this could be an alternative pathway to conventional endosomal uptake. How can the EN-1 that is taken up into cells exert transcriptional effects in motoneurons? As a minimum, more data on the subcellular distribution of endocytosed EN-1 should be included to support current hypotheses and to close the gap from cellular uptake to transcriptional regulation. *

    The question is justified since we did not recall until page 12 of the Discussion that EN1 is, as most tested homeoprotein transcription factors, captured by a mechanism distinct from endocytosis. While not yet fully understood, the process involves the formation of inverted micelles that allow for direct targeting to the cytoplasm and from there to the nucleus thanks to the NLS. We now mention in the introduction that EN1 transfer and HP transfer is based on unconventional secretion and internalization processes.

    • The differences in p62 expression with age in WT and EN-1 +/- mice as shown in Fig. 8c are not convincing. First, the p = 0.0499 and p = 0.0536 values for differences at 3-4 months of age appear borderline, and it is unclear what the dispersion analysis that is shown really means. Moreover, the question remains how a potential dysregulation of p62 then affects NMJ morphology and function. Is this change in p62 also detectable in presynaptic compartments? *

    We agree that p values in the range of 0.05 are not extremely high and this is due to the heterogeneity in SQTSM1/p62 expression, that reflects that of MN populations, and induces a high variance. We also agree that this figure is too complicated and a simplified version has been proposed above (see answers to reviewer 1). To summarize, Fig. 8C shows that in WT animals, with no aMN death (grey) the level of SQTSM1/p62 expression in aMNs and gMNs increases between 3 and 4.5 months and between 4.5 months and 9 months, with significances varying between pThe new Fig. 8 panel D (please see above, answers to referee 1) now includes the results obtained with the scFvs. A phenotype comparison between the two models (En1-Het and scFvEN1) has been introduced in Supp. Fig. 2 (see above).

    We have no evidence that EN1 modulates the SQTSM1/p62 promoter directly. The identification of this gene as a target (not necessarily a direct target) of EN1 comes from the bioinformatic analysis described in the manuscript and we were intrigued by the interaction with the 4 main familial ALS mutations and the existence of families with SQTSM1/p62 mutations. This is what led us to analyze its expression in our two models of EN1 loss of function. Although the En1-Het mouse is not an ALS model, the results support the idea that EN1 could be used as a therapeutic protein in several familial and even sporadic forms of the disease. The latter hypothesis is now being tested on MNs derived from iPSCs (sporadic patients, fALS and isogenic variants, and healthy controls). If the data lend weight to our hypothesis, as collaborative and in-house preliminary data suggest, then a complete analysis of EN1 targets in human MNs will be undertaken. Again, we really think that this is out of the scope of this study.

    For Fig. 8, we fully agree that it can give headaches and we apologize. Moreover, it induces wrong interpretations (mean intensity increases with age and dispersion between 4.5 and 9 months has a calculated p__Referee #3__

    Evidence, reproducibility and clarity

    Nevertheless, the connection between EN-1 and p62 is not well developed by the data presented and future readers may be left with many questions regarding how EN-1 and p62 are related (e.g. direct interaction? transcriptional regulation?), whether p62 is indeed the mediator of EN-1 trophic effects, or the significance of the increased levels of p62 for motoneuron disease

    The reviewer is right and we have tried to better explain and to simplify. Please see responses to referees 1 and 2.

    *Figure 1C: There appears to be EN1 immunoreactivity (green) in several areas of the spinal cord, including dorsal regions. Can the authors clarify what that labeling could be representing? *

    Unfortunately, there is always some background staining, in particular in the tissue treatment conditions appropriate for RNAscope. Furthermore, given the large number and wide distribution of V1 interneurons (Fig. 1A), we cannot preclude that EN1 is present at a low concentration in the extracellular space and in several cell types (now represented in Fig. 9). This does not weaken the main conclusion that it primarily accumulates in MNs which do not express En1 (RNAscope).

    *Figure 1D: These immunoprecipitation results lack a negative control with irrelevant antibody to confirm that the band shown it's being recognized specifically by the antibodies reacting with the blot. *

    Please see the response to reviewer 1 above with the Western blot and the absence of staining on a WB in absence of first antibody (86/8 or LSBio).

    F*igure 1E: The intensity of the EN1 labeling in MNs, much stronger than in V1 interneurons, is intriguing, given that MNs do not express engrailed-1 mRNA. One would have expected the opposite. It may help here if it was possible to show that immunoreactivity in MNs is diminished in the het mutant mouse. *

    We also were surprised by this intensity higher in MNs than in V1 interneurons, as if the protein was exported rapidly towards the target neurons. We have done the experiment proposed by the referee, found a twofold (approx.) immunoreactivity reduction in En1-Het MNs (see above Supp. Fig. 2A in answers to referee 2). This supplemental figure has been introduced in the revised version. The experiment was done at 3 months when no MN death has yet occurred. Later the neurons “replenish” with EN1, probably because they do not have to share the limited supply with the dead ones (see above answers to referee 2 and Supp. Fig. 2B).

    *Figure 2D: There are a few possible problems with these data and their interpretation. First, this reviewer feels that 5 neurons (y-axis) is a rather small number. Are these 5 neurons per what area? From how many mice? I did not find that information in the figure legend. A larger area should be quantified so that we get numbers that are more robust. Second, such differences could also be due to hypotrophy of the MNs, namely, that MN number is the same but they are smaller. *

    The differences cannot be attributed to hypotrophy. A first reason is that, at 9 months, the Cresyl violet and ChAT staining give the same results for medium size and large neurons (Fig. 2C). Furthermore, when one counts the cells throughout 15.5 months, the decrease in the number of large neurons is not compensated by an increase in the number of medium size or small ones. The reasoning and a graph, not intended for publication can be found in answers to referee 1.

    *Figure 3A: It would be useful that the authors explain how these AChR clusters were defined, visualized and counted. I could not find this information in the Methods. Perhaps this could be done by showing an alpha-BTX image illustrating the clusters. *

    We fully agree that the procedure was not well explained and we have introduced a correction in the Material and Methods section. For more details, please see answers to referee 2.

    *Figure 3B: As each adult endplate is only innervated by one MN, one would have expected fewer clusters and/or endplates, if indeed MNs are missing in this mouse, rather than endplates that are partially occupied. This could be clarified a bit more explicitly. *

    This is true and the ambiguity takes its origin in insufficient explanation of how fully innervated, partially innervated and denervated endplates were defined. Please see above and also in answers to reviewer 2. Modifications have been introduced in the text and in Fig. 3. The referee is right, the absence of change in the number of AChR clusters suggests that there are very few fully denervated endplates and that what is defined as such in the analysis corresponds to partially innervated endplates (see above). This is now discussed in the text.

    Figure 6B: Would not be better to do this with a virus, like in the case of the antibody? A more robust effect on MN survival may be attainable and thus strengthen the concept.

    This would be another interesting experiment and we are presently exploring this possibility (with preliminary results). The choice of the virus and of the promoters is very important. We are comparing several AAVs, including AAV2, AAV2-TT (which diffuses better) and AAV8. For the promoter, we do not want to express within MNs as the imported protein might have special properties, associated with import. V1 interneurons would be best, but we have to verify if this does not modify V1 physiology. Astrocyte is another option, but with a similar pitfall. This means that we have a long way to go before proposing a “gene therapy” approach.

    In addition, in the context of future clinical studies, we were eager, on the basis of the long-lasting activity of the protein already observed in the mesencephalic dopaminergic neurons (Alvarez-Fischer et al. Nature Neurosci. 14: 1260-1266, 2011; Rekaik et al. Cell Reports 13: 242-250, 2015; Blaudin de Thé et al. EMBO J. 37: e97374, 2018), to try a protein therapy in the spinal cord. Interestingly, the effects are also long-lasting in the spinal cord, (12 weeks in the mouse before a second injection is needed) and, according to contacted physicians, intrathecal injections, every second month or even more frequently, could be envisaged in the human. In that case, protein injection is possibly advantageous for the following reasons:

    (i) viral particles can travel far and we do not know what would be the side effects.

    (ii) the protein is short-lived but specifically addressed to MNs (thanks to the presence of EN1 binding sites at their surface), thus minimizing the issues associated with permanent expression and side effects.

    (iii) EN1 is a natural protein normally secreted and the immune system might not be solicited as much as with viral approaches.

    *Figure 7A: The protein seems to be mainly in the cytoplasm of those cells (nuclei are dark and unlabeled), which is also unusual for a transcription factor that functions in the nucleus. Also surprising that the protein is gone in 3 days, but has effects over 24 weeks. Any explanation for that? *

    The protein is imported and is thus both in the cytoplasm where it exerts an effect on protein translation (Brunet et al. Nature 438: 94-98, 2005; Alvarez-Fischer et al. Nature Neurosci. 14: 1260-1266, 2011; Yoon et al. Cell 148: 752-764, 2012) and in the nucleus where it exerts its transcriptional and “epigenetic activity (see below for the latter). In fact, different antibodies and fixation procedures can favor cytoplasmic or nuclear staining. When nuclear, the dark point at the center, probably the nucleolus is less stained.

    Two images illustrating this point are shown in the revision plan.

    For the second part of the question, three days are sufficient for a long-lasting activity. This was also observed in the midbrain where the protein restores the epigenetic marks jeopardized by an acute oxidative stress (Rekaik et al. Cell Reports 13: 242-250). This has led to the hypothesis that EN1 has an important action at the level of the structure of the heterochromatin, thus a long-lasting “epigenetic” activity. We are presently working on the latter effects on the chromatin structure using human MNs derived from iPSCs (patients and control).

    *Figure 7B: It's not clear what the blue and red bars mean, as this is not explained in the legend. Also, the y-axis says "%Chat+" suggesting they are counting MNs, but in the text they talk about EN-1 capture. If the latter, the y-axes should indicate % EN-1 over Chat, or something like that. In general, better figure legends would improve the experience of the reader. *

    In this experiment, we wanted to test the presence of a GAG-binding domain in EN1. To test its potential role in EN1 internalization and localization, we co-injected or not the RK-EN1 with hEN1 protein. Then, we counted the percentage of MNs (%ChAT+) which contain, or not, the hEN1 protein (hEN1+ in red or hEN1- in blue), allowing us to verify if the RK-EN1 alters the internalization of the hEN1 protein. So yes, we are looking at the capture of EN1 by the MNs with or without the RK-peptide (or control peptides). We have modified the text to make the point clearer.

    *Statistical analyses: In principle, comparisons of data obtained in studies that involved two variable parameters (such as time and genotype/treatment) should be weighted by a 2-way ANOVA test, which is more stringent since more conditions are being tested simultaneously. Usually a t-test is reserved for a pairwise comparison in an experiment involving only two conditions of the same variable. *

    The reviewer is correct. The two-way ANOVA is explained in the Statistical analyses section of the Methods. The analyses were carried out and the results listed in the legends for Figs 2, 3, 4, 6 and Supp. Fig. 1.

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    Referee #3

    Evidence, reproducibility and clarity

    This is an interesting and provocative manuscript reporting non-cell autonomous trophic activities of a homeobox protein, a concept pioneered by Dr. Prochiantz since many years ago. The study involves a significant amount of experimental work and the authors are to be congratulated by the scope and ambition of their study. Given previous studies by this laboratory on EN-1 functions in midbrain dopaminergic neurons, the concept advanced in the present paper is not entirely novel, although it is indeed interesting to find EN-1 activities in motoneurons; these were unexpected. Given that this is a non-cell-autonomous effect (EN-1 is made and released by neurons adjacent to MNs), it would have been interesting to explore the conditions under which EN-1 synthesis, release and effects are regulated, whether by lesion, degeneration, etc. But that may be something the authors wish to leave for a future report. It is welcome that an effort was put into trying to mechanistically understand how these trophic effects are mediated. This reviewer understands that this is a major undertaking. Nevertheless, the connection between EN-1 and p62 is not well developed by the data presented and future readers may be left with many questions regarding how EN-1 and p62 are related (e.g. direct interaction? transcriptional regulation?), whether p62 is indeed the mediator of EN-1 trophic effects, or the significance of the increased levels of p62 for motoneuron disease. In its present form, this paper will be welcome, if nothing else by the provocative ideas that it advances. For this, it clearly deserves to be published in a good journal (whatever that means these days). Here below are a few questions and suggestions which the authors may want to take into consideration.

    Figure 1C: There appears to be EN1 immunoreactivity (green) in several areas of the spinal cord, including dorsal regions. Can the authors clarify what that labeling could be representing?

    Figure 1D: These immunoprecipitation results lack a negative control with irrelevant antibody to confirm that the band shown it's being recognized specifically by the antibodies reacting with the blot.

    Figure 1E: The intensity of the EN1 labeling in MNs, much stronger than in V1 interneurons, is intriguing, given that MNs do not express engrailed-1 mRNA. One would have expected the opposite. It may help here if it was possible to show that immunoreactivity in MNs is diminished in the het mutant mouse.

    Figure 2D: There are a few possible problems with these data and their interpretation. First, this reviewer feels that 5 neurons (y-axis) is a rather small number. Are these 5 neurons per what area? From how many mice? I did not find that information in the figure legend. A larger area should be quantified so that we get numbers that are more robust. Second, such differences could also be due to hypotrophy of the MNs, namely, that MN number is the same but they are smaller.

    Figure 3A: It would be useful that the authors explain how these AChR clusters were defined, visualized and counted. I could not find this information in the Methods. Perhaps this could be done by showing an alpha-BTX image illustrating the clusters.

    Figure 3B: As each adult endplate is only innervated by one MN, one would have expected fewer clusters and/or endplates, if indeed MNs are missing in this mouse, rather than endplates that are partially occupied. This could be clarified a bit more explicitly.

    Figure 6B: Would not be better to do this with a virus, like in the case of the antibody? A more robust effect on MN survival may be attainable and thus strengthen the concept.

    Figure 7A: The protein seems to be mainly in the cytoplasm of those cells (nuclei are dark and unlabeled), which is also unusual for a transcription factor that functions in the nucleus. Also surprising that the protein is gone in 3 days, but has effects over 24 weeks. Any explanation for that? Figure 7B: It's not clear what the blue and red bars mean, as this is not explained in the legend. Also, the y-axis says "%Chat+" suggesting they are counting MNs, but in the text they talk about EN-1 capture. If the latter, the y-axes should indicate % EN-1 over Chat, or something like that. In general, better figure legends would improve the experience of the reader.

    Statistical analyses: In principle, comparisons of data obtained in studies that involved two variable parameters (such as time and genotype/treatment) should be weighted by a 2-way ANOVA test, which is more stringent since more conditions are being tested simultaneously. Usually a t-test is reserved for a pairwise comparison in an experiment involving only two conditions of the same variable.

    Significance

    see above

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    Referee #2

    Evidence, reproducibility and clarity

    Engrailed-1 does not act only in a cell-autonomous way in neural development, but also has non-cell-autonomous functions. These functions depend on the release of this homeoprotein which has been characterized in much detail by previous work of this group. In this paper, they show that EN-1 is expressed in spinal V1 interneurons, both on the RNA and on the protein level. In spinal motoneurons, EN-1 protein but not RNA is detected. Neutralization of extracellular EN-1 with a secreted antibody apparently blocks transfer from these interneurons to motoneurons and causes motoneuron disease symptoms. A similar phenotype is also observed in EN-1 +/- mice. Most importantly, the authors also demonstrate that intrathecal injection of EN-1 into EN-1 +/- mice restores loss of muscle strength and prevents motoneuron death. The authors also show that the autophagy modulator SQTSM1/p62 is expressed at elevated levels in EN-1 +/- mice and in mice after injection of the EN-neutralizing antibody. Since p62 expression also seems to be increased in general during aging in motoneurons, the authors conclude that EN-1 from spinal V1 interneurons is a regulator of motoneuron aging. In general, most of the experiments shown in this study are well done and convincing. However, the data on p62 upregulation appear correlative and do not allow any conclusions about the mechanism and function how EN-1 modulates motoneuron survival and function. In addition, this study is not very precise on the mechanisms how motoneurons degenerate in this model so that there are only limited insights into the way how EN-1 acts on motoneurons in a physiological manner and under pathophysiological conditions.

    Specific points of criticism:

    1. In Fig. 2a, the authors show that EN-1-positive interneurons are not reduced at 4.5 months in the spinal cord. No data are shown for later time points such as 9 months, the corresponding stage when motoneuron loss is observed, or at 16 months which corresponds to the data shown in Fig.1. The argument that there is no reduction of V1 interneurons between 4.5 months and 16 months because there is no decrease of EN-1 expression between 4.5 and 16 months, as shown in Fig. 1b is not convincing. EN-1 expression could change in individual cells, thus compensating for the loss. Data on numbers of EN-1-positive cells at 9 and 16 months should be included, and a potential autocrine effect of EN-1 on V1 interneurons, as observed in midbrain dopaminergic neurons, characterized in more detail.
    2. In Fig. 2e, the authors present data on loss of muscle strength between 4.5 and 15.5 months. They conclude that this reflects gradual neuromuscular strength loss. Since neuromuscular endplates have a very high safety factor, they can maintain full function even if transmitter release is reduced by more than 80%. Therefore, the loss of muscle strength seems to reflect the progressive loss of presynaptic terminals at neuromuscular endplates, rather than a gradual loss of neuromuscular strength.
    3. More detailed data on NMJ morphology should be included. How does EN-1 modulate neuromuscular endplates? Is EN-1 located at neuromuscular endplates after being taken up from motoneurons? Even if the mechanism is indirect, via upregulation of p62 under conditions when EN-1 signaling is reduced, does this situation lead to enhanced localization of p62 at neuromuscular endplates?
    4. The data shown in Fig. 3 on changes in NJM morphology appear incomplete and not convincing. As SV2a is not a good marker for changes in presynaptic compartments since it does not allow conclusions on how many synaptic vesicles are released, additional markers for presynaptic active zones such as Bassoon, Piccolo, Munc-13 should be studied. The analysis of fully occupied endplates appears arbitrary, and the differences are relatively small. Additional EM pictures and quantitative analyses of active zone proteins in the presynaptic compartment would help to support the argument of the authors that presynaptic compartments degenerate before cell bodies are lost in EN-1 +/- mice.
    5. The authors present evidence for a glycosaminoglycan (GAG) binding domain that appears responsible for uptake of EN-1 into motoneurons. However, it is unclear into which cellular compartment EN-1 is taken up after GAG binding on motoneurons. The authors propose this could be an alternative pathway to conventional endosomal uptake. How can the EN-1 that is taken up into cells exert transcriptional effects in motoneurons? As a minimum, more data on the subcellular distribution of endocytosed EN-1 should be included to support current hypotheses and to close the gap from cellular uptake to transcriptional regulation.
    6. The differences in p62 expression with age in WT and EN-1 +/- mice as shown in Fig. 8c are not convincing. First, the p = 0.0499 and p = 0.0536 values for differences at 3-4 months of age appear borderline, and it is unclear what the dispersion analysis that is shown really means. Moreover, the question remains how a potential dysregulation of p62 then affects NMJ morphology and function. Is this change in p62 also detectable in presynaptic compartments?
    7. Is there any molecular evidence that EN-1 modulates the p62 gene promoter directly? What is the argument to assume that increase in SQTSM1/p62 expression and dispersion is an indicator of aging? The mean intensity, if I understand Fig. 8c correctly, does not significantly increase, it is only the dispersion that changes. In general, the data shown in Fig. 8c are hard to read and interpret. For example, in the right panel, the difference between the dispersion in 4.5 and 9 month old EN +/- mice is indicated as p = 0.06, but marked with 4 stars. The presentation of these data should be changed to make them clearer.

    Referees cross-commenting

    I agree with all comments from the other reviewers

    Significance

    This study expands previous work of the authors, in particular work that has been performed and published on the effects of EN-1 on mesencephalic dopaminergic neurons. If adequately revised, it could make an interesting contribution to the general understanding how spinal V1 interneurons act on funcitonality and survival of spinal motoneurons.

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    Referee #1

    Evidence, reproducibility and clarity

    The present work focuses on Engrailed 1 (En1), a homeoprotein that is expressed in spinal V1 interneurons that connect to α-motoneurons (MNs). The authors studied its role in neuromuscular strength and MN retention and loss with the aid of different approaches. First, they studied its expression in spinal cord with RNAscope, a novel ISH method that makes possible to detect biomarkers that would be otherwise difficult to study with traditional ISH techniques. They then delivered 86/8 and LSBio anti-En1 antibodies, that catch En1 in the cleft and prevent it from being captured by MNs; moreover, they used a heterozygotic En1 mouse model to reduce En1 levels. The behavioral assessment, studied with grip strength, inverted grip test and hindlimb extensor reflex, showed motor alterations, paralleled by α-MNs loss and with an even stronger phenotype in the heterozygotic mice. This phenotype, however, appeared weeks before the MN loss, so they used NMJ assessment to determine what it seems to be a retrograde degeneration. En1 administered intrathecally was effectively internalized by the MNs and led to a long-term amelioration of the motor impairments and renervation of the NMJ, that needed to be boosted after 12 weeks for a stable therapeutic effect. Finally, heterozygotes revealed also a degeneration in dopaminergic neurons within midbrain similar to the one observed in spinal MNs, along with an upregulation of SQTSM1/p62 gene/protein, a factor in MN ageing linked to the classical genes implicated in familial forms of ALS (SOD1, TDP-43, FUS, and C9ORF72). They authors did not observe degeneration in V1 interneurons. They conclude that En1 might have a role in regulating MN ageing in degenerative motor disorders.

    Significance

    Overall, the manuscript is well written however, some of the data appears too preliminary for publication. While the potential beneficial effect of En1 intrathecal administration looks promising and worth of publication, it is difficult to understand the mechanism of action. Some of the results are puzzling and require further investigations.

    Major comments:

    It is unclear why levels of intensity for RNAscope were not quantified, and qPCR was preferred for quantifications in Figure 1b. RNAscope is a technique that allows for spatial distribution analysis of the markers and their level of the expression. This data can be easily quantified utilizing the QuPath software which is open access. Same concerns apply to Figure 2a.

    Antibodies should be validated utilizing a reporter mouse. En1cre mice are commercially available and can be crossed with reporters (TdTomato or YFP mice). Utilizing this tissue En1 antibodies can be easily validated. The EN1 antibody shown in Figure 1c seems unspecific, staining several neuronal populations in the spinal cord.

    Investigations of En1 expression in motor neurons from already available omics data sets would support the idea that En1 is expressed in motor neurons.

    Differentiation between Gamma and Alpha motor neurons should be performed using specific markers as Err3, Wnt7a or NeuN.

    How can the authors explain the lack of loss of En1 interneurons in the En1-Het mice? Do spinal En1 interneurons show any signs of apoptosis (e.g., cleaved caspase 3 marker)? Which levels of the spinal cord were used for interneuron quantifications? Segments between L1 and L3 would be preferable.

    The set of experiments reported in Figure 4 is of difficult interpretation without showing the actual presence of extracellular En1, that could be assessed with protein detection or RNAscope.

  6. Version published to 10.1101/2022.08.16.504081 on bioRxiv