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:
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.
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.
- 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.
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.
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.
