Author Response
The following is the authors’ response to the original reviews.
eLife assessment
This important study provides a framework bearing on the role of Eph-Ephrin signaling mechanisms in the clinically condition of amyotrophic lateral sclerosis. It provides compelling evidence for the roles of glial cells in this condition. This novel astrocyte-mediated mechanism may help identify future therapeutic targets.
Drs. Huang and Zaidi: Thank you for considering this revision of our manuscript for potential publication in eLife. We have addressed the excellent comments of the two reviewers, including the addition of new data. We have included detailed response-to-reviewer comments below to address each specific point, and we have highlighted all the changes in the manuscript text (using a red font color) made in response to these comments. Based on the reviewers’ critiques, we feel our re-working of the manuscript has made for a greatly improved study.
Reviewer #1 (Public Review):
In the manuscript by Urban et al., the authors attempt to further delineate the role which non-neuronal CNS cells play in the development of ALS. Toward this goal, the transmembrane signaling molecule ephrinB2 was studied. It was found that there is an increased expression of ephrinB2 in astrocytes within the cervical ventral horn of the spinal cord in a rodent model of ALS. Moreover, the reduction of ephrinB2 reduced motoneuron loss and prevented respiratory dysfunction at the NMJ. Further driving the importance of ephrinB2 is an increased expression in the spinal cords of human ALS individuals. Collectively, these findings present compelling evidence implicating ephrinB2 as a contributing factor towards the development of ALS.
We thank Reviewer #1 for the very helpful critique. We address each of the specific comments below (in the “Recommendations for the Authors” section of this Response to Reviewer Comments document), and have made changes to the manuscript based on these excellent points.
Reviewer #2 (Public Review):
The contribution of glial cells to the pathogenesis of amyotrophic lateral sclerosis (ALS) is of substantial interest and the investigators have contributed significantly to this emerging field via prior publications. In the present study, authors use a SOD1G93A mouse model to elucidate the role of astrocyte ephrinB2 signaling in ALS disease progression. Erythropoietin-producing human hepatocellular receptors (Ephs) and the Eph receptor-interacting proteins (ephrins) signaling is an important mediator of signaling between neurons and non-neuronal cells in the nervous system. Recent evidence suggests that dysregulated Eph-ephrin signaling in the mature CNS is a feature of neurodegenerative diseases. In the ALS model, upregulated Eph4A expression in motor neurons has been linked to disease pathogenesis. In the present study, authors extend previous findings to a new class of ephrinB2 ligands. Urban et al. hypothesize that upregulated ephrinB2 signaling contributes to disease pathogenesis in ALS mice. The authors successfully test this hypothesis and their results generally support their conclusion.
Major strengths of this work include a robust study design, a well-defined translational model, and complementary biochemical and experimental methods such that correlated findings are followed up by interventional studies. Authors show that ephrinB2 ligand expression is progressively upregulated in the ventral horn of the cervical and lumbar spinal cord through pre-symptomatic to end stages of the disease. This novel association was also observed in lumbar spinal cord samples from postmortem samples of human ALS donors with a SOD1 mutation. Further, they use a lentiviral approach to drive knock-down of ephrinB2 in the central cervical region of SOD1G93A mice at the presymptomatic stage. Interestingly, in spite of using a non-specific promoter, authors note that the lentiviral expression was preferentially driven in astrocytes.
Since respiratory compromise is a leading cause of morbidity in the ALS population, the authors proceed to characterize the impact of ephrinB2 knockdown on diaphragm muscle output. In mice approaching the end stage of the disease, electrophysiological recordings from the diaphragm muscle show that animals in the knock-down group exhibited a ~60% larger amplitude. This functional preservation of diaphragm function was also accompanied by the preservation of diaphragm neuromuscular innervation. However, it must be noted that this cervical ephrinB2 knockdown approach had no impact on disease onset, disease duration, or animal survival. Furthermore, there was no impact of ephrinB2 knockdown on forelimb or hindlimb function.
We thank Reviewer #2 for the very helpful critique. We address each of the specific comments below, and have made changes to the manuscript based on all of these excellent points.
The major limitation of the manuscript as currently written is the conclusion that the preservation of diaphragm output following ephrinB2 knockdown in SOD1 mice is mediated primarily (if not entirely) by astrocytes. The authors present convincing evidence that a reduction in ephrinB2 is observed in local astrocytes (~56% transduction) following the intraspinal injection of the lentivirus. However, the proportion of cell types assessed for transduction with the lentivirus in the spinal cord was limited to neurons, astrocytes, and oligodendrocyte lineage cells. Microglia comprise a large proportion of the glial population in the spinal grey matter and have been shown to associate closely with respiratory motor pools. This cell type, amongst the many others that comprise the ventral gray matter, have not been investigated in this study. Thus, the primary conclusion that astrocytes drive ephrinB2-mediated pathogenesis in ALS mice is largely correlative.
This is an excellent point. While the majority of transduced cells were astrocytes, we did not identify the lineage of a portion of the transduced cells, which could consist of cell types such as microglia, endothelial cells and others, some of which have been linked to ALS pathogenesis. Nevertheless, we find that the cells expressing high levels of ephrinB2 in ventral horn of SOD1G93A mice are all astrocytes (as seen in Figure 1O-Q), strongly suggesting – though not definitively demonstrating – that astrocyte ephrinB2 is the pathogenic source in this model (even if our viral transduction did not solely target astrocytes).
In the revised version of the manuscript, we now include an extensive paragraph in the Discussion section dedicated to this point.
Importantly, we have toned down our conclusion by modifying the title by removing “…in spinal cord astrocytes…”. We changed the title from “EphrinB2 knockdown in spinal cord astrocytes preserves diaphragm innervation in a mutant SOD1 mouse model of ALS" to “EphrinB2 knockdown in cervical spinal cord preserves diaphragm innervation in a mutant SOD1 mouse model of ALS”.
Further, it is interesting to note that no other functional outcomes were improved in this study. The C3-C5 region of the spinal cord consists of many motor pools that innervate forelimb muscles. CMAP recordings conducted at the diaphragm are a reflection of intact motor pools. This type of assessment of neuromuscular health is hard to re-capitulate in the kind of forelimb task that is being employed to test motor function (grip strength). Thus, it would be interesting to see if CMAP recordings of forelimb muscles would capture the kind of motor function preservation observed in the diaphragm muscle.
We did perform forelimb grip strength analysis on these animals and found no effect of focal ephrinB2 knockdown. However, this functional assay is impacted more by distal forelimb muscle groups controlled by motor neuron pools located at more caudal locations of the spinal cord (i.e. low cervical and high thoracic), likely explaining the lack of effect on grip strength.
Unfortunately, we did not perform this CMAP recording on forelimb muscle, and these mice have all already been sacrificed. We have added discussion of this point to the revised manuscript.
On a similar note, the functional impact of increased CMAP amplitude has not been presented. An increase in CMAP amplitude does not necessarily translate to improved breathing function or overall ventilation. Thus, the impact of this improvement in motor output should be clearly presented to the reader.
This is a very important point. While CMAP recording is a powerful assay of functional innervation of diaphragm muscle by phrenic motor neurons, it does not directly measure respiratory function. There are assays to test outcomes such as ventilatory behavior and gas exchange (e.g. whole-body plethysmography; blood gas measurements, etc.). We did not however perform these analyses. Respiratory function involves contribution of a number of other muscle groups, and these muscles are innervated by various motor neuron pools located across a relatively-large expanse of the CNS neuraxis. As we focally targeted ephrinB2 knockdown to only a small area, we would not expect effects on these other functional assays, which is why we restricted our testing to CMAP recording since this can be used to specifically study the phrenic motor neuron pool (and can be combined with detailed histological analyses in the cervical enlargement and at the diaphragm NMJ).
Importantly, this is why we chose to use “preserves diaphragm innervation” in the manuscript title, as opposed to wording such as “preserves diaphragm function” in the title. In addition, have added this point to the Discussion section in the revised manuscript.
Further, to the best of my knowledge, expression of Eph (or EphB) receptors has not been explicitly shown at the phrenic motor pool. It is thus speculative at best that the mechanism that the authors suggest in preserving diaphragm function is in fact mediated through Eph-EphrinB2 signaling at the phrenic motor pool. This aspect of the study would warrant a deeper discussion.
We address this important comment with multiple pieces of data showing that Eph receptors are expressed in the phrenic motor neuron pool. EphrinB2 binds and activates EphBs, as well as EphAs such as EphA4. Importantly, previous work has linked expression of EphA4 in motor neurons to the rate of ALS progression (Van Hoecke, et al. Nature Medicine. 2012). Consistent with these studies, single-nucleus RNAseq on mouse cervical spinal cord shows that alpha motor neurons of cervical spinal cord express various EphA and EphB receptors (http://spinalcordatlas.org/; Blum et al., Nature Neuroscience, 2021; Alkaslasi et al., Nature Communications, 2021). In addition, this dataset identifies a phrenic motor neuron-specific marker (ErbB4); when we specifically look at the expression profile of only the ErbB4-expressing alpha motor neurons, the data reveal that phrenic motor neurons express a number of EphA and EphB receptors, including EphA4.
To validate expression specifically of EphA4, we performed IHC for phosphorylated EphA4 (a marker of activated EphA4) on C3-C5 spinal cord sections from SOD1G93A mice injected with shRNAephrinB2 or control vector. We find that large ventral horn neurons are positive for phosphorylated EphA4. The ventral horn at these cervical spinal cord levels includes motor neuron pools in addition to just phrenic motor neurons; therefore, this result by itself does not conclusively show that phrenic motor neurons express EphA4, though they likely do since we find EphA4 expression in most ventral horn neuron cell bodies in C3-C5. A representative image is included in Supplemental Figure 1.
In the revised manuscript, we added a paragraph to the Discussion section to address this important comment from the reviewer, including describing these data on Eph receptor expression.
Lastly, although authors include both male and female animals in this investigation, they do not have sufficient power to evaluate sex differences. Thus, this presents another exciting future of investigation, given that ALS has a slightly higher preponderance in males as compared to females.
As the reviewer notes, our studies are under-powered with respect to examining possible sex-specific effects. We now include a brief discussion of this issue in the revised manuscript.
In summary, this study by Urban et al. provides a valuable framework for Eph-Ephrin signaling mechanisms imposing pathological changes in an ALS mouse model. The role of glial cells in ALS pathology is a very exciting and upcoming field of investigation. The current study proposes a novel astrocyte-mediated mechanism for the propagation of disease that may eventually help to identify potential therapeutic targets.
Recommendations for the authors: please note that you control which revisions to undertake from the public reviews and recommendations for the authors.
Both reviewers were enthusiastic about your paper. Reviewer (1) had some technical queries (see his/her items 2 and 4). Reviewer (2) had some questions about principles (items 1 and 2) with the remaining points being technical queries.
We have addressed all comments of both reviewers. We detail our responses in this Response to Reviewer Comments document and have made the associated modifications to the revised manuscript.
Reviewer #1 (Recommendations For The Authors):
Questions and/or Recommendations:
There is convincing evidence that there is increased expression of ephrinB2 over time in the mouse model of ALS. Is there a corresponding increase in astrocytes in this animal model?
We previously published data showing quantification of astrocyte numbers within the spinal cord of this same SOD1G93A mouse model. Specifically, we performed this quantification in the ventral horn of the lumbar spinal cord following disease onset. We found that there was a modest increase in the number of GFAP+ astrocytes at this location and disease time point.
[ Lepore et al. Selective ablation of proliferating astrocytes does not affect disease outcome in either acute or chronic models of motor neuron degeneration. Experimental Neurology. 211 (2): 423-32, 2008. ]
One could speculate that the increase in ephrinB2 expression we observe across the ventral horn in the mutant SOD1 mice was solely due to this modest increase in astrocyte number. However, this is highly unlikely to be the case, as in wild-type mice and in mutant SOD1 mice prior to disease onset astrocytes (and all other cell types) express very low levels of ephrinB2. Throughout disease course in these mutant SOD1 mice, the ephrinB2 expression level in individual astrocytes dramatically increases (including across most or all astrocytes), suggesting that the total increase in ephrinB2 expression across the ventral horn was not due to just this modest increase in astrocyte numbers but was instead due to the dramatically elevated eprhinB2 expression in most/all astrocytes. We have added this point to the Discussion section in the revised manuscript.
It would help the reviewer and readers to show a lower magnification image of Figure 2, panels O and P to demonstrate the reduction of ephrin B2 in the ventral horns.
We have added the lower magnification images to Figure 2.
It is commended that not all data was "positive". Figure 4 especially shows some of the limitations of eprhinB2 knockdown. This provides a realistic image - strengths and limitations - of this approach. Very well done!
Thank you! In future work, we could employ alternative vector-based strategies to restrict transduction/knockdown to only astrocytes. With such experiments, it’s possible that the impact of ephrinB2 knockdown would not be the same, if ephrinB2 actions in non-astrocytes also plays a role in disease pathogenesis. We have added discussion of this same point to the revised manuscript in response to a similar comment above from Reviewer #2.
Reviewer comment 4: Fig 6 - if possible can you please add demographic (age/sex) with each band?
We have added this information to the Legend. For aesthetic reasons, we chose not to add this information directly to the figure itself and instead included all of this information for each sample/band in the Legend.
Reviewer #2 (Recommendations For The Authors):
Overall, the manuscript addresses a novel aspect of the role of astrocytes in mediating ALS pathogenesis. I commend the authors for a well thought-out and clearly presented study. However, a few concerns limit the enthusiasm and deserve attention to improve the clarity of the report.
The biggest limitation of this study is that microglia or other cell types (endothelial cells) have not been explored in this study. They constitute a big proportion of cell types in the spinal cord and to conclude that only astrocytes mediate ephrinB2 signaling in the ALS model would be a stretch without the proper stains.
Please see our comments above to address this same point from Reviewer #2.
A clear premise for the investigation of EphrinB2 ligands has not been presented in the introduction. The authors provide a good background on the emerging role of EphEphrin interactions in neurodegenerative diseases. But it is unclear how the authors landed on this sub-class of ephrins.
We have added this premise to the Introduction section of the revised manuscript. In published work, ephrinB2 has been shown to be upregulated in reactive astrocytes and to be involved in disease pathogenesis in other neurological disease models (e.g. traumatic spinal cord injury).
There are several acronyms that have not been defined in the manuscript, e.g. GPI.
We now define “GPI” and all other abbreviations in the revised manuscript.
While the authors state that males and females had been included in the study, their individual n's for various outcomes have not been presented in the results section. Further, n's are missing from the figure legends, which will aid the clarity of the presentation. Further, please clarify the ages of the mice in the methods section.
(1) We now provide the n’s for males versus females for all analyses in the figure legends. (2) We also now include the total n for each experimental condition in all of the figure legends. (3) We also now state the ages of the mice for the various analyses in the Methods section.
It appears that several statistical interactions have been summarized in the results section but inconsistently reported on figures.
We now provide the exact n’s for each analysis in all figure legends. We include all of the details of the statistical analysis in the text of the Results section and do not include this text in the Legends; we do this for all figures to maintain consistency.
I presume that when the authors write "the number of neurons with somal diameter greater than 200 μm and with an identifiable nucleolus was determined", the 200 was a typo. Mouse motor neurons do not have a diameter of 200 μm and perhaps the authors meant an area of 200μm2?
We have corrected this: 200 μm2
Authors should consider adding a quantification for the human tissue immunoblots.
We have added the quantification of these human tissue data for ephrinB2.
