An optogenetic cell therapy to restore control of target muscles in an aggressive mouse model of amyotrophic lateral sclerosis

  1. J Barney Bryson
  2. Alexandra Kourgiantaki
  3. Dai Jiang
  4. Andreas Demosthenous
  5. Linda Greensmith

  • Curated by eLife

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    This fundamental study presents a method to restore muscle innervations in ALS mouse models using optogenetics. It is convincing that embryonic stem cell derived motor neurons can be transplanted into and applied to reinnervate the muscles in an ALS mouse model. The work will be of broad interest to researchers and medical biologists to develop new strategies for the treatment of neurodegenerative disorders resulting from denervated skeletal muscles.

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Abstract

Breakdown of neuromuscular junctions (NMJs) is an early pathological hallmark of amyotrophic lateral sclerosis (ALS) that blocks neuromuscular transmission, leading to muscle weakness, paralysis and, ultimately, premature death. Currently, no therapies exist that can prevent progressive motor neuron degeneration, muscle denervation, or paralysis in ALS. Here, we report important advances in the development of an optogenetic, neural replacement strategy that can effectively restore innervation of severely affected skeletal muscles in the aggressive SOD1 G93A mouse model of ALS, thus providing an interface to selectively control the function of targeted muscles using optical stimulation. We also identify a specific approach to confer complete survival of allogeneic replacement motor neurons. Furthermore, we demonstrate that an optical stimulation training paradigm can prevent atrophy of reinnervated muscle fibers and results in a tenfold increase in optically evoked contractile force. Together, these advances pave the way for an assistive therapy that could benefit all ALS patients.

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  1. Version published to 10.7554/elife.88250.3 on eLife
  2. Version published to 10.7554/elife.88250 on eLife
  3. Version published to 10.7554/elife.88250.2 on eLife
  4. eLife

    Author Response

    The following is the authors’ response to the original reviews.

    Reviewer #1 (Recommendations For The Authors):

    Major concerns:

    1. In lines 41-43, there seems to be some confusion about the terminology regarding "sporadic ALS". ALS is subdivided into familial and sporadic forms. Familial ALS simply indicates that the patient has a family history of ALS and presumably has a genetic predisposition for developing this disease. In many families, the identity of the mutation remains unknown. Sporadic ALS patients do not have a family history of this disease. However, this does not imply that they lack mutations that caused disease. In fact, 5-10% of these patients have the hexanucleotide repeat expansion in C9orf72. This mutation is also found in about 40% of familial ALS cases.

    We have now amended the manuscript to be more accurate in our description of underlying genetics of ALS. This changes to this section are as follows:

    Lines 39-47:

    "...The median survival time in ALS, from initial onset of symptoms to death, typically as a result of respiratory complications, is only 20-48 months Chiò et al. (2009) and ALS has an estimated global mortality of 30,000 patients per year Mathis et al. (2019).

    ALS is typically classified into either familial (fALS) or sporadic (sALS) forms of the disease, based on whether or not patients have an identified family history of the disease; between 5-10% of total ALS cases fall into the former category, fALS, with the remaining 90-95% consisting of sALS cases Mathis et al. (2019). To date, over 20 monogenic mutations that cause ALS have been identified, however these still only account for 45% of fALS cases and only 7% of sALS cases Mejzini et al. (2019)..."

    1. In Fig. 4-supplement 1, 7DD and 5DD are not defined. I assume one is the fast-firing and one is the slow-firing motor neurons. I am also a bit confused as to why the 5DD neurons produce greater muscle force than the 7DD neurons when electrically stimulated. It seems to suggest that there is some difference between the two types of neurons or the groups of mice used to test them.

    We have now defined these terms and the amended figure legend now reads as follows:

    "(A) Fast-firing motor neurons (produced using a 7-day differentiation protocol thus labelled as “7DD”) or slow-firing ChR2+ motor neurons (produced using a 5-day differentiation protocol thus labelled as “5DD”) were engrafted in age matched SOD1G93A mice… Our expectation was that fast-firing motor neurons, which normally innervate larger numbers (>100) of stronger fast-twitch muscle fibres per motor unit would elicit significantly greater contractile force when optically stimulated, compared to slow-firing motor neurons that innervate small numbers (<10) of weaker, slow-twitch muscle fibres per motor unit. Surprisingly, our data did not show any difference when using grafts consisting of fast-firing motor neurons, versus slow-firing motor neurons, at least in response to optical stimulation. The factors underlying this surprising result, and the apparent discrepancy between electrically-evoked muscle contractions in nerves that had bene engrafted with either fast or slow firing motor neurons, are likely to be highly complex; we hope to further explore this as part of a separate follow up study."

    1. Along those lines, do these two subpopulations of motor neurons innervate the same set of muscle fibers? More generally, are certain types of muscle fibers preferentially innervated by this approach? Answering these questions could point to additional ways to enhance the effectiveness of this treatment approach. This should be discussed.

    This point is partially addressed in our response to Point 2 above, but to further extrapolate: certainly, the phenotype of individual muscle fibres is largely dictated by the firing properties of the motor neuron that innervates it. Slow-twitch muscle fibres tend to produce less contractile force but are more fatigue resistant, whereas fast-twitch muscle fibres produce more force but fatigue rapidly. There is evidence that expression of the chemorepellent molecule ephrin-A3 prevents the inappropriate innervation of slow-twitch muscle fibres by fast-firing motor neurons, which express the cognate receptor EphA8 [PMID: 26644518]. Importantly, fast-firing motor neurons are preferentially susceptible to disease mechanisms in ALS and the fast-twitch muscle fibres that they innervate are therefore more likely to undergo denervation and atrophy. Surprisingly, in this study we clearly show that grafts consisting of slow-firing motor neurons are able to innervate all regions of the triceps surae muscle group, including the normally exclusively fast-twitch superficial regions of the gastrocnemius and the exclusively slow-twitch soleus muscle. This finding strongly suggests that the normal developmental pairing of motor neuron and muscle fibre properties is not essential in this therapeutic context. Indeed, the use of more disease-resistant slow-firing motor neurons may provide some advantages. Again, we hope to be able to further explore this relationship in forthcoming follow-up studies.

    1. The authors state that exercise programs are likely to accelerate disease progression. This is not supported by the current body of clinical data. In fact, current guidelines are for moderate (not strenuous) exercise, and mouse studies have demonstrated a protective effect of moderate exercise on disease progression.

    We apologise for the lack of clarity on this point, as it was not our intention to imply that voluntary exercise accelerates disease progression. We have now amended the manuscript to specify “ENS-based exercise programs” to avoid any confusion.

    1. It is unclear what the experimental endpoint is. Page 25 defines it as 135 days of age, but ranges are given the figure legends, suggesting that some other criteria were used. It also seems unclear at what determined the age at which each animal was treated since they were also not treated at the same age.

    We hope that our response in the Public Reviews section above has fully addressed this point.

    1. I am a little confused by Figure 5 - figure supplement 5, panel D. Why do the authors give specific p-values here but not in the other panels? The sample sizes in D are very low, in some cases with only 1 animal in a group, and performing statistical tests under these conditions seems futile. The statistical power is nearly zero.

    For the purposes of consistency, we have now replaced the specific p-values in panel D with “ns”. The low n-values for the MUNE analysis data is due to the extremely difficult nature of identifying the contribution of individual motor units to the total muscle contractile response, when the maximal muscle force is extremely weak. In the absence of optical stimulation training, the extremely weak force elicited by acute optical stimulation precluded our ability to separate out the contribution of individual motor units and, often, in animals where this was not possible, we did not always perform electrically-evoked MUNE analysis. Unfortunately, we are not currently in a position to increase the n-values for this component of the study. Our ongoing research to enhance the amplitude of the muscle response to optical stimulation will hopefully help to more clearly address this in the future.

    1. One concern about this approach is that the procedure could accelerate the denervation of the target muscle. Figure 5 - figure supplement 6, panel B, indicates a significant reduction in force on the ipsilateral side relative to the contralateral side, at least under electrical stimulation of the nerve. This would be consistent with the hypothesis that the procedure does enhance disease progression in the treated limb. Is there a reduction in voluntary motor activity in these animals, such as in grip strength or the position of the foot while walking?

    We hope that this important point has been satisfactorily addressed in the Public Reviews section. Unfortunately, we did not undertake any behavioural analysis relating to voluntary motor function of the engrafted (or contralateral) hindlimbs, which may have provided useful data to address this point. As described above, the most likely explanation for this finding is due to physical nerve damage caused by the intraneural injection procedure; in our efforts to refine our strategy and move it towards clinical translation, we will take this into consideration in our future research.

    1. Based on Fig. 6D, it seems that the vast majority of innervated NMJs at endpoint are innervated by cells from the graft. And yet, electrical stimulation evokes substantially greater muscle force. This may suggest that optical control of engrafted motor neurons will not yield enough force for routine tasks or that the few remaining endogenous motor neurons are much more effective at generating force. These potential limitations and ways to overcome them should be discussed.

    There appears to be a slight misunderstanding, since our aim here was to sample a sufficiently powered number of motor end-plates innervated by YFP+ for statistical analysis. To do this we specifically chose regions of interest containing at least 1 YFP+ NMJ and the adjacent muscle fibres were included at random, whatever their innervation status. Had we sampled regions of interest at random, we would have been likely to capture only a very few YFP+ terminal as they occupy a very small volume of the total muscle section and the maximum scanning area for each high-resolution z-confocal stack is relatively small, so we feel that this selection was warranted.

    Minor comments:

    1. The donor mouse strain should be described as 129S1/SvImJ.

    We have now corrected this.

    1. The first time the supplementary figures show up in the manuscript, they seem to have two titles each, such as "Figure 1-figure supplement 1. (Figure 4 - figure supplement 1)". The second seems to be the correct one.

    This was caused by an issue with the Latex template, which has now been resolved.

    1. PCB is not defined the first time it is used (page 8, line 332).

    We have now defined this term on first use: printed circuit board (PCB)

    1. CNI is not defined in the text (page 12, line 432).

    We have now defined this abbreviation at the first usage on Page 4, Line 158

    1. Some of the fonts on the graphs are very small, such as Fig. 5J.

    We have increased the font size as much as possible for Fig. 5.

    1. Figure 6 - figure supplement 1 does not include a key to indicate which antigens are stains and which color refers to which antigen. This is also needed for the videos.

    We have now included a key on this figure supplement to indicate the relevant antigens and stain and we have also done the same for the videos.

    1. Video 5 seems to indicate that there is a dead zone in the back of the chamber. Does this raise any concerns about the consistency of training from animal to animal?

    This is an extremely astute observation. However, the intermittent activation of the implantable LED devices is not due to a dead zone; rather, it is due to the orientation of the power receiving coil within the device and it’s alignment with the resonance frequency chamber that transmits the power to the device. As the animals move around, and particularly when they rear up, the power receiving coil occasionally becomes misaligned and fails to receive sufficient power to activate the LED. Since the pulses are delivered every 2 seconds, for 1 hour per day, we feel that the animals, on average, receive sufficient numbers of pulses to implement the training regimen. Indeed, we feel that the results speak for themselves.

  5. eLife

    eLife assessment

    This fundamental study presents a method to restore muscle innervations in ALS mouse models using optogenetics. It is convincing that embryonic stem cell derived motor neurons can be transplanted into and applied to reinnervate the muscles in an ALS mouse model. The work will be of broad interest to researchers and medical biologists to develop new strategies for the treatment of neurodegenerative disorders resulting from denervated skeletal muscles.

  6. eLife

    Reviewer #1 (Public Review):

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder leading to the loss of innervation of skeletal muscles, caused by the dysfunction and eventual death of lower motor neurons. A variety of approaches have been taken to treat this disease. With the exception of three drugs that modestly slow progression, most therapeutics have failed to provide benefit. Replacing lost motor neurons in the spinal cord with healthy cells is plagued by a number of challenges, including the toxic environment, inhibitory cues that prevent axon outgrowth to the periphery, and proper targeting of the axons to the correct muscle groups. These challenges seem to be well beyond our current technological approaches. Avoiding these challenges altogether, Bryson et al. seek to transplant the replacement motor neurons into the peripheral nerves, closer to their targets. The current manuscript addresses some of the challenges that will need to be overcome, such as immune rejection of the allograft and optimizing maturation of the neuromuscular junction.

  7. eLife

    Reviewer #2 (Public Review):

    The authors provide convincing evidence that optogenetic stimulation of ChR2-expressing motor neurons implanted in muscles effectively restore innervation of severely affected skeletal muscles in the aggressive SOD1 mouse model of ALS, and concluded that this method can be applied to selectively control the function of implicated muscles, which was supported by convincing data presented in the paper.

  8. Version published to 10.21203/rs.3.rs-1970365/v3 on Research Square
  9. Version published to 10.7554/elife.88250.1 on eLife
  10. eLife

    Author Response

    Reviewer #1 (Public Review):

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder leading to the loss of innervation of skeletal muscles, caused by the dysfunction and eventual death of lower motor neurons. A variety of approaches have been taken to treat this disease. With the exception of three drugs that modestly slow progression, most therapeutics have failed to provide benefit. Replacing lost motor neurons in the spinal cord with healthy cells is plagued by a number of challenges, including the toxic environment, inhibitory cues that prevent axon outgrowth to the periphery, and proper targeting of the axons to the correct muscle groups. These challenges seem to be well beyond our current technological approaches. Avoiding these challenges altogether, Bryson et al. seek to transplant the replacement motor neurons into the peripheral nerves, closer to their targets. The current manuscript addresses some of the challenges that will need to be overcome, such as immune rejection of the allograft and optimizing maturation of the neuromuscular junction.

    Bryson et al. begin by examining the survival of mESC-derived motor neurons allografted into SOD1 mice. The motor neurons, made on a 129S1/SvImJ, were transplanted into the tibial nerve of SOD1 mice on a C57BL/6J background. Without immunosuppression, most cells were lost between 14 and 35 days, suggesting an immune response had eliminated them. Tacrolimus prevented cell loss, but it also inhibited innervation of the muscle. It also uncovered the tumorigenic potential of contaminating pluripotent cells. In contrast, immunosuppression using H57-597, an antibody targeting T-cell receptor beta, prevented graft rejection while permitting some innervation of muscle. Pretreatment of the cells with mitomycin-C eliminated pluripotent cells, preventing tumor formation. The authors noted that this combination only innervated ~10% of endplates, likely due to the fact that the implanted motor neurons are not active.

    The authors then began the process of optimizing the cells themselves, using measurements taken in late-stage SOD1 mice. Fast-firing and slow-firing populations of neurons were first compared. Using optical stimulation, these two cell types appeared to be similar. The authors opted to use slow-firing neurons in the subsequent experiments. Recognizing that neuromuscular junction (NMJ) innervation and maintenance are dependent on motor neuron activity, implantable optical stimulators were also evaluated. 14 days after transplanting the cells, optical stimulation training was initiated for one hour each day. This training led to a nearly 13-fold increase in force generation, although this still remained well below the force generated by electrical stimulation. The enhanced innervation also prevented the atrophy of muscle fibers caused by denervation.

    Overall, the data for the function of the implanted cells are convincing. The dCALMS technique that the authors have developed is quite interesting and will likely be applicable to analyze muscles for other therapeutics. The identification of calcineurin inhibitors as inhibitors of reinnervation will also be important for the development of other cell-based therapeutics for ALS.

    This is an excellent summary of the state of the field of ALS therapy development and provides a clear rationale for our novel therapeutic strategy, in the near-complete vacuum of conventional treatment options for patients suffering from this devastating disorder. We are delighted that the Reviewer clearly appreciated the value of our alternative therapeutic strategy and found our supporting data to be convincing, as well as drawing attention to the dCALMS technique, which we agree could be of significant value in the investigation of other therapeutic strategies aimed at restoring muscle innervation. We are extremely grateful for the Reviewer’s diligence in assessing our manuscript.

    However, there are some issues that should be addressed. These include some common misconceptions about ALS. While ALS is split into familial and sporadic forms based on the presence or absence of a family history of the disease, mutations in the known ALS-associated genes are found in both forms [1]. The authors also state that exercise programs are likely to accelerate degeneration in ALS. This is incorrect. Moderate exercise is part of the current guidelines for treating ALS, and mouse studies have demonstrated a therapeutic effect of moderate exercise [2]. Regarding the experimental design, there are some important details missing. The animals do not appear to have been operated on at the same age, and the criteria for when to perform the operation were not described. A similar problem exists for when the animals were determined to reach endpoint [3]. The authors also do not seem to address a potential pitfall of this approach: acceleration of the disease process. Indeed, some of the data comparing the ipsilateral side to the contralateral side suggest that the implantation of the cells and/or the light source increase the denervation of the muscle [4]. Finally, there is a fairly large difference between the motor output provided by optical stimulation relative to electrical stimulation. It is currently unclear what level needs to be reached to provide an effective response in the intact animal. Thus, it is difficult to determine if the level of reinnervation that this study has achieved will be sufficient to improve a patient's quality of life [5].

    The Reviewer raises some extremely important points and highlights some additional constructive issues where more clarity is required (numbered 1-5 above). We have attempted to address each of these points in order to strengthen the key message of our study and the integrity of our manuscript:

    1. The Reviewer is absolutely correct in highlighting that causative mutations in identified genes occur in both sporadic and familial forms of ALS and that this classification simply reflects whether or not there is a known family history of the disease (which can also encompass a spectrum of disorders including frontotemporal degeneration). We will revise our manuscript in order to be more accurate and provide clarity on this important point.

    2. Regarding the potential acceleration of muscle denervation, we specifically state that the use of electrical nerve stimulation (ENS) to artificially evoke muscle contraction has been shown to accelerate denervation of the diaphragm muscle in clinical trials aimed at maintaining respiratory function in ALS patients, which significantly shortened life-expectancy. It was not our intention to imply that moderate voluntary exercise, as opposed to artificial “ENS-based” muscle stimulation programmes, could accelerate muscle denervation. Indeed, the negative side-effects of ENS that we highlighted provide a clear rationale for developing a safer alternative to artificially control muscle function once innervation by endogenous motor neurons progressively deteriorates in ALS patients; specifically, our selection of optogenetic nerve stimulation (ONS), which is highly selective to the engrafted light-sensitive motor neurons, recruits motor units in correct physiological order and avoids rapid muscle fatigue potentially overcomes the safety concerns associated with ENS.

    Importantly, unchecked disease progression means that complete paralysis of almost all muscles will eventually occur, due to loss of upper or lower motor neurons and accompanying muscle denervation, which would eventually preclude the ability of ALS patients to undertake voluntary exercise programmes, or even activities of daily life. Our approach is aimed at overcoming this inevitable loss of voluntary muscle control and onset of complete paralysis by providing a safe and effective method of artificially maintaining control of targeted muscles that would otherwise become completely paralyzed, as well as preventing their irreversible atrophy.

    To avoid the possibility that readers may infer that we are suggesting voluntary exercise programs accelerate degeneration in ALS and to provide additional clarity, we will revise the manuscript to stress that we specifically refer to “ENS-based” exercise programmes in relation to acceleration of muscle denervation.

    1. Regarding our experimental design, the congenic B6.SOD1G93A mouse model of ALS is an extremely well-characterized model, with a highly consistent timeframe of disease phenotype manifestation and progression. In order to maximize the translational value of our study, we selected an early post-symptom onset timepoint (95d +/- 4.6 days) that mirrors a time at which human ALS patients would be likely to benefit from the therapeutic strategy: in the vast majority of cases, it is not possible to treat humans until a diagnosis of ALS has been confirmed, which can often take up to 12 months from first presentation. Importantly, ALS patients in the final stages of disease progression would be unlikely to be suitable for this therapy, due to irreversible muscle atrophy, which would preclude the ability of the engrafted motor neurons to form functionally useful connections. Indeed, our strategy is to engraft the replacement motor neurons prior to severe muscle atrophy occurs, so that they are in place to compensate and take over the function of endogenous lower motor neurons as they progressively degenerate and paralysis ensues. In so doing, the replacement motor neurons could prevent the irreversible atrophy of targeted muscles through ONS-based exercise programmes and thereby indefinitely extend the ability of targeted muscles to perform functionally useful movements.

    Although the initial graft optimization component of this study, including the tacrolimus trial, was performed across a variety of disease stages (commencing between 57-101 days of age), once we identified the H57-596 monoclonal antibody as an effective means to promote graft survival (without interfering with target muscle innervation), all subsequent grafts were initiated at an early symptom onset timepoint: 95.7 ± 4.6 days for slow-firing motor neuron grafts and 106.8 ± 7.2 days for fast-firing motor neuron grafts. Transgenic SOD1G93A mice were specifically bred for this study and due to complexities of coordinating stem cell differentiation and motor neuron production, optical stimulation device production and access to surgical facilities, with timed matings set up 3-4 months in advance, we feel that this age range was acceptable and doesn’t detract from the findings of our study.

    Similarly, we made every effort to ensure that experimental end-point was consistent, at 133 ±8 days for all grafts involving H57-597 administration, which reflects translationally-relevant late-stage disease progression. Since the physiological experiments performed as part of this study are extremely time-consuming, it was necessary to stagger the experimental end-point over several days. Again, we feel that this range is acceptable and still reflects a consistent, translationally-relevant timepoint. Importantly, since the experimental paradigm tested in this study was aimed at individually targeted muscles, which would have been unlikely to have an effect on disease duration or survival, we did not feel that it was ethically justifiable to allow the B6.SOD1G93A mice to approach end-stage disease (which occurs at an average age of 150 days of age in this model).

    In the interests of full transparency, the age at which treatment commenced and the experimental end-point for every animal used in this study is reported in Supplementary Tables 2 and 3.

    1. The Reviewer raises an extremely pertinent question, regarding whether the engrafted motor neurons themselves, or the implanted stimulation device, may accelerate the progressive loss of innervation of targeted muscles by endogenous motor neurons, in light of our data that shows decreased force evoked by electrical stimulation of ipsilateral (engrafted) versus contralateral (control) muscles. It is worth noting that supramaximal electrical nerve stimulation, used to evoke maximal muscle force, should activate both endogenous and engrafted motor neurons, therefore the combined activation of both populations would be expected to result in a summative (greater) contractile response. The fact that we see the converse is unlikely to be due to an accelerated loss of endogenous motor innervation as a result of the engrafted cells, but is much more likely to be caused by physical nerve damage during the surgical engraftment process: we used a customized Hamilton syringe with a 29G needle to manually inject the cells into the targeted nerve branches, which has an outer diameter of 330μm whilst the diameter of the tibial nerve in an adult mouse is approximately 400μm. This is likely to have led to damage of the endogenous motor (and potentially sensory) axons that may have diminished regenerative capacity due to ongoing disease mechanisms. Fortunately, there is significant scope to refine the engraftment procedure by using smaller gauge needles (potentially made of more flexible materials), bespoke injection systems that can deliver the cells at a controlled rate and micromanipulators that avoid can avoid nerve damage caused by excessive movement of the needle within the nerve. Importantly, the significantly greater scale of human nerves, compared to murine nerves targeted in this study, would also be a significant advantage in terms of physically delivering the cells in ALS patients.

    2. The Reviewer’s final comment is entirely justified given that, even in the best cases following optical stimulation training of engrafted SOD1G93A mice, optical stimulation still evoked less contractile force than supramaximal electrical stimulation. The likely reasons for this are complex: there is almost certainly scope to further optimize the optical stimulation training paradigm, which could result in reinforcement of the de novo neuromuscular junctions formed between the engrafted motor neurons and targeted muscle fibres; it is possible that the expression level of the channelrhodopsin-2 protein at the cell surface may require optimization in order to reliably initiate action potentials in the engrafted motor neurons – development of newer channelrhodopsin variants may resolve this potential issue, whilst providing additional advantages (such as enabling transcutaneous stimulation) at the same time. Finally, the maximum contractile response of the triceps surae muscle elicited by optical stimulation that we observed was approximately 13g, which equates to approximately 50% of the body mass of an adult SOD1G93A mouse. Although this is only approximately 10% of the maximal contractile force of a wild-type triceps surae muscle, this would almost certainly provide the ability to perform functionally useful motor tasks if it could be reproduced in ALS patients, particularly if large numbers of targeted muscles could be controlled in a coordinated manner, something that we are actively working on.

    Reviewer #2 (Public Review):

    The authors provide convincing evidence that optogenetic stimulation of ChR2-expressing motor neurons implanted in muscles effectively restores innervation of severely affected skeletal muscles in the aggressive SOD1 mouse model of ALS, and conclude that this method can be applied to selectively control the function of implicated muscles. This was supported by convincing data presented in the paper.

    This is an interesting paper providing new/improved optogenetic methods to restore or improve muscle strength in ALS. In general, it is of high significance in both the techniques and concept, and the paper was well written. The evidence supporting the conclusions is convincing, with rigorous muscle tension physiological analysis, and nerve and muscle histology and image analysis. The work will be of broad interest to medical biologists on muscle disorders.

    One weak point is that proper control experiments were not clearly presented - these could be shown in the paper. For example, one control experiment with only YFP but no ChR2 expression with optogenetic stimulation should be performed, following similar procedures and analysis applied to the ChR2-transduced animals.

    We are extremely grateful for the Reviewer’s expert appraisal of our manuscript and we are delighted to hear that they found our study to be highly significant, of broad interest and that our supporting evidence for this novel therapeutic approach was convincing and rigorous.

    Regarding the inclusion of suggested control experiments, we have extensive negative results data from physiological recordings of muscles in response to optical stimulation in animals where the engrafted motor neurons were rejected (prior to our identification of a 100% effective immunosuppression regimen). This clearly revealed that, in the absence of ChR2-expressing motor neurons, optical stimulation does not elicit any response from the target muscle. However, we do not feel that inclusion of this negative data, which is entirely predictable, would have strengthened the findings of our study. Similarly, if we had engrafted motor neurons that only express YFP, we would have been unable to elicit any muscle contractile activity in response to optical stimulation. As a control, this may have some value in determining the ability of motor neurons derived from other cell lines that do not express ChR2 to survive and innervate target muscles but we don’t feel that the additional work would get us closer to achieving our ultimate goal of using motor neuron replacement in combination with optogenetic stimulation to restore/maintain muscle function in ALS patients. Moreover, the complex and iterative process of developing the cell line used in this study (reported in detail in our previous study) would make it extremely difficult to produce a suitable control stem cell line expressing only YFP. Having said that, we are actively in the process of developing new, more sophisticated human and mouse stem cell lines, using more translationally-relevant gene targeting methods to stably knock-in a variety of updated channelrhodopsin variants that may have superior properties for our approach. This will be reported in follow up study/studies as we feel that it goes well beyond the scope of the current study.

  11. eLife

    eLife assessment

    This fundamental study presents a method to restore muscle innervations in ALS mouse models using optogenetics. It is convincing that embryonic stem cell derived motor neurons can be transplanted into and applied to reinnervate the muscles in an ALS mouse model. The work will be of broad interest to researchers and medical biologists to develop new strategies for the treatment of neurodegenerative disorders resulting from denervated skeletal muscles.

  12. eLife

    Reviewer #1 (Public Review):

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder leading to the loss of innervation of skeletal muscles, caused by the dysfunction and eventual death of lower motor neurons. A variety of approaches have been taken to treat this disease. With the exception of three drugs that modestly slow progression, most therapeutics have failed to provide benefit. Replacing lost motor neurons in the spinal cord with healthy cells is plagued by a number of challenges, including the toxic environment, inhibitory cues that prevent axon outgrowth to the periphery, and proper targeting of the axons to the correct muscle groups. These challenges seem to be well beyond our current technological approaches. Avoiding these challenges altogether, Bryson et al. seek to transplant the replacement motor neurons into the peripheral nerves, closer to their targets. The current manuscript addresses some of the challenges that will need to be overcome, such as immune rejection of the allograft and optimizing maturation of the neuromuscular junction.

    Bryson et al. begin by examining the survival of mESC-derived motor neurons allografted into SOD1 mice. The motor neurons, made on a 129S1/SvImJ, were transplanted into the tibial nerve of SOD1 mice on a C57BL/6J background. Without immunosuppression, most cells were lost between 14 and 35 days, suggesting an immune response had eliminated them. Tacrolimus prevented cell loss, but it also inhibited innervation of the muscle. It also uncovered the tumorigenic potential of contaminating pluripotent cells. In contrast, immunosuppression using H57-597, an antibody targeting T-cell receptor beta, prevented graft rejection while permitting some innervation of muscle. Pretreatment of the cells with mitomycin-C eliminated pluripotent cells, preventing tumor formation. The authors noted that this combination only innervated ~10% of endplates, likely due to the fact that the implanted motor neurons are not active.

    The authors then began the process of optimizing the cells themselves, using measurements taken in late-stage SOD1 mice. Fast-firing and slow-firing populations of neurons were first compared. Using optical stimulation, these two cell types appeared to be similar. The authors opted to use slow-firing neurons in the subsequent experiments. Recognizing that neuromuscular junction (NMJ) innervation and maintenance are dependent on motor neuron activity, implantable optical stimulators were also evaluated. 14 days after transplanting the cells, optical stimulation training was initiated for one hour each day. This training led to a nearly 13-fold increase in force generation, although this still remained well below the force generated by electrical stimulation. The enhanced innervation also prevented the atrophy of muscle fibers caused by denervation.

    Overall, the data for the function of the implanted cells are convincing. The dCALMS technique that the authors have developed is quite interesting and will likely be applicable to analyze muscles for other therapeutics. The identification of calcineurin inhibitors as inhibitors of reinnervation will also be important for the development of other cell-based therapeutics for ALS.

    However, there are some issues that should be addressed. These include some common misconceptions about ALS. While ALS is split into familial and sporadic forms based on the presence or absence of a family history of the disease, mutations in the known ALS-associated genes are found in both forms. The authors also state that exercise programs are likely to accelerate degeneration in ALS. This is incorrect. Moderate exercise is part of the current guidelines for treating ALS, and mouse studies have demonstrated a therapeutic effect of moderate exercise. Regarding the experimental design, there are some important details missing. The animals do not appear to have been operated on at the same age, and the criteria for when to perform the operation were not described. A similar problem exists for when the animals were determined to reach endpoint. The authors also do not seem to address a potential pitfall of this approach: acceleration of the disease process. Indeed, some of the data comparing the ipsilateral side to the contralateral side suggest that the implantation of the cells and/or the light source increase the denervation of the muscle. Finally, there is a fairly large difference between the motor output provided by optical stimulation relative to electrical stimulation. It is currently unclear what level needs to be reached to provide an effective response in the intact animal. Thus, it is difficult to determine if the level of reinnervation that this study has achieved will be sufficient to improve a patient's quality of life.

  13. eLife

    Reviewer #2 (Public Review):

    The authors provide convincing evidence that optogenetic stimulation of ChR2-expressing motor neurons implanted in muscles effectively restores innervation of severely affected skeletal muscles in the aggressive SOD1 mouse model of ALS, and conclude that this method can be applied to selectively control the function of implicated muscles. This was supported by convincing data presented in the paper.

    This is an interesting paper providing new/improved optogenetic methods to restore or improve muscle strength in ALS. In general, it is of high significance in both the techniques and concept, and the paper was well written. The evidence supporting the conclusions is convincing, with rigorous muscle tension physiological analysis, and nerve and muscle histology and image analysis. The work will be of broad interest to medical biologists on muscle disorders.

    One weak point is that proper control experiments were not clearly presented - these could be shown in the paper. For example, one control experiment with only YFP but no ChR2 expression with optogenetic stimulation should be performed, following similar procedures and analysis applied to the ChR2-transduced animals.

  14. Version published to 10.21203/rs.3.rs-1970365/v2 on Research Square
  15. Version published to 10.21203/rs.3.rs-1970365/v1 on Research Square