Scalable, optically-responsive human neuromuscular junction model reveals convergent mechanisms of synaptic dysfunction in familial ALS

Reviewer #1 (Public Review):

Summary:

The authors propose an improved neuro-muscle co-culture system to study ALS-related functional differences in human pluripotent stem cell lines.

Strengths:

A simple co-culture system with functional readout.

Weaknesses:

There are concerns about the lack of novelty, rigor, and clarity in the approach. The strength of the study is undermined by its reliance on transcription factors used more than a decade ago, low myocyte activity, and inadequate validation methods, such as the lack of single-cell transcriptome analysis and detailed neuromuscular synapse characterization. The evidence presented requires substantial validation through rigorous experimental approaches and resolution of the identified concerns for the study's findings to be considered significant and reliable.

Reviewer #2 (Public Review):

The manuscript by Chen et al from the group of Helen Miranda aims to describe an improved neuromuscular junction (NMJ) model to study synaptic dysfunction in several cases of familial ALS. Overall, the system described in the paper appears as a valid platform to study disease phenotypes with exciting results showing specific effects of GDNF on non-SOD1 ALS patient lines. The strength of the paper lies in the use of myotubes, and motor neurons derived from the same donor. However, the current study: (1) lacks a clear comparison of the current system with numerous previously described systems; (2) is limited by the number of repeat experiments in the study and (3) has no description of the synaptic phenotype observed in the study. These major points are discussed in more detail below.

Major points:

(1) In the introduction the authors state (p. 4): "Finally, recent human NMJ models have been established from PSCs by differentiating these cells into both skeletal muscles and motor neurons in 2D and 3D formats. These previous systems present a remarkable advancement to the studies of human NMJs, however, they require long NMJ formation and maturation time (40 to 60 days), which, restricts their sensitivity and scalability [42]"

In fact, a number of studies have described various in-vitro NMJ systems, with the same timeframes for NMJ formation. For example, in studies by Osaki et al, 2018, Sci Adv; Bellmann et al, 2019, Biomat; Demestre et al, 2015, Stem Cell Res; Badu-Mensah et al, 2022, Biomat (this is just an exemplar selection of the papers); NMJ formation was observed as early as 14 d in culture, in line with or at least slightly longer than reported by Chen et al. With the exception of the study by Osaki et al, all co-culture systems cited above are 2D-based. The authors need to expand on this further or provide a quantitative assessment of why their system is better compared to previously published models.

(2) Further, when comparing their results with other work it is hard to claim how the current system is (p. 5) "more reproducible, and offers a 6-fold increase in scalability compared to previous models [40-43]". The authors need to expand on this further.

(3) Although mentioned, there were no examples of the modularity of the system, which of course would strengthen the paper and help to uncover ALS mechanisms of synaptic formation, for example by combining WT myotubes and fALS motor neurons (see point 4 below). The authors should show how they would adapt to 96 well plate format to showcase the scalability of the system. Based on their data on the efficacy of synaptic formation (60 per 0.7 cm2 area), is further miniaturization allowed?

(4) A lot of a-bungarotoxin staining corresponds to AChR clusters that do not seem to be associated with muscle and do not form normal rings of clustering (pretzel-like) associated with the NMJ in vivo. This is seen clearly in Figure 3B and Figure 5B. Figures 3B and 5B only show low-magnification images which makes it difficult to assess the specificity of localization of the pre-/post-synaptic markers. The authors should clearly show the morphologies of the NMJs formed in WT and fALS lines at high magnification. In addition, the authors should show co-localization images for a-bungarotoxin and myosin-heavy chains to confirm the localization of the bungarotoxin signal on the myotubes.

In addition to that, the authors report that the number of functional synapses formed on a plate varies from 30 (fASL) to 60 (Ctrl) per 10,000 neurons spread over the 0.7 cm2 area (0.6%). How do the authors explain an extensive loss of a-bungarotoxin signal in Figure 5B the majority of which likely corresponds to AChR clusters that are formed outside of neuronal connections? Such clustering can be usually observed in immature co-cultures and in vivo prior to the innervation of myotubes. One explanation could be that myotubes derived from fALS PSC are less capable of synaptic formation. Noteworthy, a study of PSC-derived myotubes and motor neurons from PSC lines with various SOD1 mutations has already been published, but not cited by Chen et al (Badu-Mensah et al). Given the importance of those confounding factors, the authors should test cell-intrinsic (motor neuron-related) vs non-cell-intrinsic mechanisms by co-culturing healthy myotubes with fALS-derived motor neurons followed by NMJ quantification.

(5) The authors present the advantage of optogenetic stimulation, but they only show the proof-of-principle and never really apply it to their studies. Specifically, with regard to Figure 6, are motor units derived from fALS PSCs incapable of being ontogenetically activated to the same extent as control motor units? Does the dysfunction stem from fALS motor neurons or fALS myotubes?

(6) Figures 6 B and C appear to be identical except for the addition of the GDNF effect on the fALS lines. This should all be put in one figure. The authors should also show whether GDNF-induced functional recovery is associated with recovery in the number of motor units or with merely synaptic function by quantifying the NMJ number in the presence of GDNF.

(7) Figure 5 and Figure 6. The authors only use one line per fALS mutation and their corresponding isogenic controls. They state that the n=6 for these experiments represents the technical replication of the experiment. These experiments should be performed at least n=3 times starting from neuronal differentiation, and not by seeding replicate wells representing a true replication of each experiment. This would significantly strengthen their argument that their method is robust and the results are easily reproducible.

(8) In the discussion the authors may want to mention that the lack of function of GDNF on the SOD1 lines may relate to the fact that SOD1 mutations do not lead to TDP43 pathology. Although speculative this suggests that in cases with TDP43 mutations (their data) or sporadic disease GDNF may be effective.

(9) Although beyond the scope of this paper, it would of course be interesting to see if sporadic forms of ALS had this same phenotype.

Author response:

eLife assessment

This is an important study describing a neuromuscular junction co-culture system using human cells that the authors use to study the synaptic consequences of ALS mutations. The data supporting the system are solid and show the value of using myotubes and motor neurons from the same donor. The study will be of interest to researchers who model neuromuscular junction disorders, however, the authors could more comprehensively compare and contrast their system with previous literature describing other similar models. There are also technical weaknesses that limit the interpretation of specific findings.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The authors propose an improved neuro-muscle co-culture system to study ALS-related functional differences in human pluripotent stem cell lines.

Strengths:

A simple co-culture system with functional readout.

We appreciate the recognition that this is a simplified co-culture system with a straight-forward functional evaluation.

Weaknesses:

There are concerns about the lack of novelty, rigor, and clarity in the approach. The strength of the study is undermined by its reliance on transcription factors used more than a decade ago, low myocyte activity, and inadequate validation methods, such as the lack of single-cell transcriptome analysis and detailed neuromuscular synapse characterization. The evidence presented requires substantial validation through rigorous experimental approaches and resolution of the identified concerns for the study's findings to be considered significant and reliable.

The muscle differentiation protocol used in our work is an adaptation of the Albini S, et al. Cell Rep. 2013. This protocol was selected due to its efficiency to differentiate skeletal muscles from pluripotent stem cells (PSCs). Modifications from the original publications were made in the plasmids (MYOD and BAF60C) used, such as the inclusion of selection genes, puromycin and blasticidin, to improve efficiency. Moreover, a criticism of the previously used overexpression system, especially overexpression of MYOD, is that it introduces artificial expression of this gene throughout muscle differentiation, when it is only supposed to be expressed early in myogenesis. Thus, the constructs used in our work are dox inducible, which enables us to control the expression of MYOD and restrict it to the first 48 hours. This protocol resulted in a highly efficient skeletal muscle differentiation, as noted in our manuscript. “The PSC-derived skeletal muscles were characterized by the presence of Desmin (DES) and Myosin Heavy Chain (MHC), and as early as day 8 of differentiation nearly 100% of the cells co-expressed these markers.” We agree with the reviewer that the myocyte activity identified in our work is lower compared to Albini et al. (2013), mostly explained by the modification we made to the method, from a 3D to a 2D culture. In Albini et al. (2013) the electrophysiological properties were assayed in skeletal myospheres (3D), which are known to improve contractility measurements. Conversely, in 2D cultures when the contractility intensifies the cells detach from the plate. Thus, a tight regulation of cell concentration for optimal maturation and formation of contractile skeletal muscle culture without premature detachment of the cells is required. We believe that single-cell or single-nuclei transcriptome analysis from the co-culture setting of two well-defined cell types might yield little value for method characterization, however, as part of a follow up study we are performing morphological NMJ characterization and applying single-nuclei transcriptome analysis in the fALS disease context to identify specific molecular mechanisms that result in synaptic dysfunction.

Reviewer #2 (Public Review):

The manuscript by Chen et al from the group of Helen Miranda aims to describe an improved neuromuscular junction (NMJ) model to study synaptic dysfunction in several cases of familial ALS. Overall, the system described in the paper appears as a valid platform to study disease phenotypes with exciting results showing specific effects of GDNF on non-SOD1 ALS patient lines. The strength of the paper lies in the use of myotubes, and motor neurons derived from the same donor. However, the current study: (1) lacks a clear comparison of the current system with numerous previously described systems; (2) is limited by the number of repeat experiments in the study and (3) has no description of the synaptic phenotype observed in the study. These major points are discussed in more detail below.

We appreciate the recognition that “the system described in the paper appears as a valid platform to study disease phenotypes with exciting results showing specific effects of GDNF on non-SOD1 ALS patient lines” and the careful evaluation of our work. We plan to address the points raised by this reviewer in the revision.

Major points:

(1) In the introduction the authors state (p. 4): "Finally, recent human NMJ models have been established from PSCs by differentiating these cells into both skeletal muscles and motor neurons in 2D and 3D formats. These previous systems present a remarkable advancement to the studies of human NMJs, however, they require long NMJ formation and maturation time (40 to 60 days), which, restricts their sensitivity and scalability [42]"

In fact, a number of studies have described various in-vitro NMJ systems, with the same timeframes for NMJ formation. For example, in studies by Osaki et al, 2018, Sci Adv; Bellmann et al, 2019, Biomat; Demestre et al, 2015, Stem Cell Res; Badu-Mensah et al, 2022, Biomat (this is just an exemplar selection of the papers); NMJ formation was observed as early as 14 d in culture, in line with or at least slightly longer than reported by Chen et al. With the exception of the study by Osaki et al, all co-culture systems cited above are 2D-based. The authors need to expand on this further or provide a quantitative assessment of why their system is better compared to previously published models.

Indeed, there are previous publications that have described neuromuscular junctions (NMJs) in cocultures of iPSC-derived skeletal muscles and motor neurons. Some of the publications mentioned above did show NMJ formation within ~20ish days, albeit with several caveats such as culture heterogeneity, i.e. 50% motor neuron differentiation efficiency. We agree with the reviewer that this needs to be expanded and clarified, and we will address this concern in the revision.

(2) Further, when comparing their results with other work it is hard to claim how the current system is (p. 5) "more reproducible, and offers a 6-fold increase in scalability compared to previous models [40-43]".

The authors need to expand on this further.

This is an important aspect of this work, and we believe that our protocol offers a higher reproducibility due to, at least partially, the homogeneity of the starting cultures of iPSC-derived skeletal muscles and iPSC-derived motor neurons, and that the direct 2D co-culture approach is more suitable for miniaturization compared to 3D cultures or microfluidic chamber devices. Thus, we will expand on this idea in the revision.

(3) Although mentioned, there were no examples of the modularity of the system, which of course would strengthen the paper and help to uncover ALS mechanisms of synaptic formation, for example by combining WT myotubes and fALS motor neurons (see point 4 below). The authors should show how they would adapt to 96 well plate format to showcase the scalability of the system. Based on their data on the efficacy of synaptic formation (60 per 0.7 cm2 area), is further miniaturization allowed?

We appreciate the points raised by the reviewer. The “mix-and-match” approach to co-culture wild-type and affected iPSC-derived skeletal muscles with iPSC-derived motor neurons is a main focus of our lab and an advantage to protocols like ours, where cells are differentiated independently and later co-cultured together; however, a comprehensive characterization of various mix-match combinations is beyond the scope of this Tools and Resources article. Since the initial submission of this manuscript, we have extensively optimized the scalability of the co-cultures from the initial 0.7 cm2 to 0.32 cm2 (96-well plates). Further miniaturization is also being optimized to 0.136 cm2 (384-well plates). This point will be clarified in the revision.

(4) A lot of a-bungarotoxin staining corresponds to AChR clusters that do not seem to be associated with muscle and do not form normal rings of clustering (pretzel-like) associated with the NMJ in vivo. This is seen clearly in Figure 3B and Figure 5B. Figures 3B and 5B only show low-magnification images which makes it difficult to assess the specificity of localization of the pre-/post-synaptic markers. The authors should clearly show the morphologies of the NMJs formed in WT and fALS lines at high magnification. In addition, the authors should show co-localization images for a-bungarotoxin and myosin-heavy chains to confirm the localization of the bungarotoxin signal on the myotubes.

In addition to that, the authors report that the number of functional synapses formed on a plate varies from 30 (fASL) to 60 (Ctrl) per 10,000 neurons spread over the 0.7 cm2 area (0.6%). How do the authors explain an extensive loss of a-bungarotoxin signal in Figure 5B the majority of which likely corresponds to AChR clusters that are formed outside of neuronal connections? Such clustering can be usually observed in immature co-cultures and in vivo prior to the innervation of myotubes. One explanation could be that myotubes derived from fALS PSC are less capable of synaptic formation. Noteworthy, a study of PSCderived myotubes and motor neurons from PSC lines with various SOD1 mutations has already been published, but not cited by Chen et al (Badu-Mensah et al). Given the importance of those confounding factors, the authors should test cell-intrinsic (motor neuron-related) vs non-cell-intrinsic mechanisms by co-culturing healthy myotubes with fALS-derived motor neurons followed by NMJ quantification.

The iPSC-derived skeletal muscle cultures were plated as a monolayer and even though the abungarotoxin staining does not show the pretzel-like shape NMJs, similar to other in vitro NMJ protocols (Badu-Mensah et al, Biomat 2023; Pereira et al., Nat Commun 2021; Uzel et al., Sci Adv 2016), abungarotoxin does show association with the muscles. For quantification purposes we omitted the MHC staining to decrease background, however we will include it in the revision in response to the reviewer’s concern.

We agree with the reviewer that the suggested approaches would yield insight into disease mechanism but are beyond the scope of this method development study. In fact, we are very excited about our follow up study pursuing a more in-depth analysis of cell-autonomous vs non-cell autonomous pathogenesis to understand the NMJ dysfunction in fALS. We apologize that the “Badu-Mensah et al” work was not included, this was our oversight and will be added in the revision.

(5) The authors present the advantage of optogenetic stimulation, but they only show the proof-ofprinciple and never really apply it to their studies. Specifically, with regard to Figure 6, are motor units derived from fALS PSCs incapable of being ontogenetically activated to the same extent as control motor units? Does the dysfunction stem from fALS motor neurons or fALS myotubes?

We agree that these are important questions to be addressed and are actively pursuing these experiments as part of the natural follow up investigation from the present Tools and Resources article.

(6) Figures 6 B and C appear to be identical except for the addition of the GDNF effect on the fALS lines. This should all be put in one figure. The authors should also show whether GDNF-induced functional recovery is associated with recovery in the number of motor units or with merely synaptic function by quantifying the NMJ number in the presence of GDNF.

We will combine Figures 6B and 6C in the revision. Our follow up study also includes the interrogation of the mechanism through which GDNF rescues fALS NMJ dysfunction.

(7) Figure 5 and Figure 6. The authors only use one line per fALS mutation and their corresponding isogenic controls. They state that the n=6 for these experiments represents the technical replication of the experiment. These experiments should be performed at least n=3 times starting from neuronal differentiation, and not by seeding replicate wells representing a true replication of each experiment. This would significantly strengthen their argument that their method is robust and the results are easily reproducible.

We will clarify that the technical replicates originated from independent differentiations in the revision.

(8) In the discussion the authors may want to mention that the lack of function of GDNF on the SOD1 lines may relate to the fact that SOD1 mutations do not lead to TDP43 pathology. Although speculative this suggests that in cases with TDP43 mutations (their data) or sporadic disease GDNF may be effective.

We appreciate this suggestion and will highlight this as possible inclusion criteria for GDNF treatment in the discussion of our revised version of the manuscript.

(9) Although beyond the scope of this paper, it would of course be interesting to see if sporadic forms of ALS had this same phenotype.

We agree with the reviewer and we hope to include iPSC derived NMJs from sporadic ALS patients in a future study.