A non-human primate model of Amyotrophic Lateral Sclerosis

Reviewer #1 (Public review):

Summary:

The authors have used a macaque (two animals only) to follow the migration of 'seeded' TDP43 protein in neuronal pathways - thus mimicking the spread of ALS in the human CNS. Previous experiments in rodents failed to demonstrate this, posing interesting and important biological differences, possibly related to the UMN-LMN system in higher order apes and humans.

Strengths:

An important step forward.

Weaknesses:

No weaknesses were identified by this reviewer. Only 2 animals were used, but that is appropriate given the sensate status of the macaque. In the opinion of this reviewer, the results are entirely convincing.

Reviewer #2 (Public review):

Summary:

There are astonishingly few papers trying to reproduce the process of initiation and spreading that Braaks studies have suggested and postulated. The authors should be applauded for pioneering such a difficult experiment. They overexpressed the TDP-43 protein in the motor neuron pool of the brachioradialis muscle and showed that by this technique, motor neurons in this pool died, and the muscle got denervated. They had evidence of a spreading process from the spinal cord to the cortex, demonstrated by showing widespread deposits of phosphorylated TDP-43 bilaterally in the cervical cord and the motor cortex. By their experiment, they created a dying-backwards model, not a model of corticofugal spread, like that shown by Braak. No muscle weakness was observed, not even in the brachioradialis.

Strengths:

The strength of this innovative study is the fact that this spreading experiment uses the phylogenetically young connectome of primates (macaques). They also made the thought-provoking observation of spreading from the cord to the motor cortex, not the corticofugal spread model observed by Heiko Braak. This is thought-provoking because this enables the observer to compare their model with the findings in humans.

Weaknesses:

The following aspects are not a weakness but need to be better explained for the interested reader - and potentially improved in future studies for which the authors laid the foundation:

(1) Why do the authors use the brachioradialis motor neuron pool to overexpress TDP-43? More is known about other muscles and how they are embedded in the motor connectome of primates. Why not the biceps brachii or the hand extensors or - even better - the small muscles of the hand? These are known to be strongly monosynaptically connected with the motor cortex. The authors should explain this. I am unclear if there was a specific reason which I did not see or understand. In my view, the brachioradialis is not the best representative of the primate connectome, for example, to examine this model and compare it with the corticofugal spread.

(2) In the Braaks experiment, only (seemingly soluble) non-phoshorylated TDP-43 "crossed" synapses. Phosphorylated TDP-43 did not do this. The authors of this study saw phosphorylated TDP43 in motor neurons and the cortex. Is there any potential explanation for how it crosses synapses? If it really does, there is an obvious difference to the human situation which needs to be emphasized and explained (in the future).

(3) There were significant deposits of phosphorylated TDP-43 in oligodendrocytes in humans. Whilst I understand that one experiment cannot solve every question - I am curious about whether the authors saw anything in oligodendrocytes?

(4) Which was the pattern of damage? Of course, this pattern is not likely to have a monosynaptic pattern - like in humans........but was there a pattern? Did it have a physiologically meaningful basis? Was there any relation to the corticofugal monosynaptic pattern? What are the differences? The authors speak of "multiple waves". Does this mean that if this were a corticofugal model, for example, oculomotor neurons would also degenerate?

Reviewer #3 (Public review):

Summary:

In this paper by Jones and colleagues, a non-human primate model is described in which wild-type TDP-43 is expressed in the cervical spinal cord. This gave rise to loss of motor neurons in the ventral horn at that level in the cervical spinal cord. MRI of the muscles allowed to see increased intensity in the mostly affected brachioradialis muscle, suggesting this muscle becomes denervated. At the neuropathological level, TDP-43 and pTDP-43 staining in the cytoplasm is increased, not only at the specific level of the cervical spinal cord, but also at a distance.

Strengths:

A clear strength is the state-of-the art focal expression of the TDP-43 transgene at a focal site in the cervical spinal cord. This is achieved by combining a general expression of a flipped loxP flanked TDP-43 vector using AAV9 intrathecal administration, followed by an intramuscular AAV2 hSyn CRE-TdTomato vector in the brachioradialis muscle in order to induce focal recombination and expression of TDP-43 in motor neurons innervating this muscle on one side.

Another strength is the non-human primate background, which is much closer to the human situation.

Weaknesses:

Given the complexity and cost of the model, the n is very low.

The design of the experiments and the results shown about the toxicity induced by this focal TDP-43 expression do not allow us to conclude that it is a good model for ALS for several reasons. It is not clear that the TDP-43 overexpression results in spreading weakness or in spreading motor neuron loss. The neuropathological changes described suggest that there is a kind of stress response, which extends to regions away from the site of primary damage, but more is needed to provide convincing evidence that there is spreading of disease pathology reminiscent of human ALS.

Reviewer #4 (Public review):

Summary:

In this manuscript, the authors present data describing the development of a model of ALS in rhesus macaques. They use a viral intersectional model to overexpress TDP-43 in a population of motor neurons and then study the spread of the pathology about 7 months later. They demonstrate that both the cervical spinal cord and motor cortex (new and old M1) are full of TDP-43, suggesting that the pathology spreads from the single motor pool to presumably related neurons.

Strengths:

This is a super-important study in two main ways:

(1) This could be the birth of a really important model, one that is really needed for making progress in understanding ALS and the development of therapeutics. There are shortfalls with all the rodent models. Models dependent on cell cultures are superb for understanding cell-autonomous processes, but miss out on connectivity, particularly the long-range connectivity. Organoids may ultimately prove to be beneficial, but they would need cortex, spinal cord, and muscle, and translatability from them is not assured. So a NHP model is needed, and this may be it. Furthermore, the Methods are meticulously described and will undoubtedly facilitate reproducibility.

(2) The concept of the spread of pathology has been proposed for some time, I think, based initially on the detailed clinical observations of Ravits and colleagues. The authors have looked at this directly and provide supporting evidence for this interesting hypothesis. They show spread locally and contralaterally in the spinal cord (although a figure would be nice) and to the motor cortex.

Taking only these 2 points into account is more than sufficient for me to be enthusiastic about this work.

Weaknesses:

I'd like to make a couple of points that if addressed, could, in my view, help the authors strengthen this work.

(1) We don't know how many MNs were transduced by the rAAV. There was no tdTom expression, for whatever reason. The authors show an image of a control experiment with a single MN transduced, but there should be a red motor pool, at least in the control experiments. The impression that I get is that very few were transduced, and, in my mind, this makes the findings even more interesting - maybe you don't need many "starter" MNs.

(2) Continuing on this point, this leads the authors to conclude that all BR MNs have died. They support this by the reduced MN count (see point 3). Firstly, do we know how many BR MNs there are in the rhesus macaque, and does the reduction seen correspond to this number? Secondly, and more importantly, the muscle looks normal on MRI at 28 weeks - it does not look like a denervated muscle. The authors state that it has maybe been reinnervated, but by what, if all the BR MNs are dead? This does not seem like a plausible explanation to me. Muscle histology, NMJs, and fibre typing would have been useful to understand what's going on with the MNs. (And electrophysiology would have been wonderful, but beyond the scope of this study.)

(3) Some MN biologists, like me, fuss a lot about how to count MNs, which is almost as difficult as counting the number of angels on the head of a pin. Every method has its problems. Focusing on the two methods here: (a) ChAT immunohistochemistry is pretty good in healthy states, but we don't know what happens to ChAT expression in different diseases, particularly when you have a new model. If its expression is decreased, then it is not a good marker for MNs; (b) Identifying MNs based on the size and morphology of neurons in the ventral horn is also insufficient. For example, ~30% of neurons in a typical pool are small gamma MNs, and a significant proportion (depending on the muscle) of the remainder will be small alpha MNs. So what one is counting is, at best, the large alpha MNs, not all the MNs in a pool. And in ALS, it's these largest MNs that are affected at the earliest stages. The small ones might be fine. So results will be skewed. (Hence, it would be interesting to see if the muscle had a higher proportion of Type I fibres after being reinnervated by S-type MNs.)

(4) Statistics. These are complex experiments looking at the spread of a disease. The experimental unit is therefore the monkey, n=2. In each monkey, multiple sections are analysed, which are key technical replicates and often summative. For example, do we care about the average cell number in Figures 4D, E, 5 I, J or 6G, H, or rather the total cell number? Do the error bars mean anything? To be clear, I am by no means minimising the importance of the overall convincing findings. But I do not think this statistical analysis is particularly meaningful.

Author response:

Public Reviews:

Reviewer #1 (Public review): 

Summary: 

The authors have used a macaque (two animals only) to follow the migration of 'seeded' TDP43 protein in neuronal pathways - thus mimicking the spread of ALS in the human CNS. Previous experiments in rodents failed to demonstrate this, posing interesting and important biological differences, possibly related to the UMN-LMN system in higher order apes and humans. 

Strengths: 

An important step forward. 

Weaknesses: 

No weaknesses were identified by this reviewer. Only 2 animals were used, but that is appropriate given the sensate status of the macaque. In the opinion of this reviewer, the results are entirely convincing. 

Reviewer #2 (Public review): 

Summary: 

There are astonishingly few papers trying to reproduce the process of initiation and spreading that Braaks studies have suggested and postulated. The authors should be applauded for pioneering such a difficult experiment. They overexpressed the TDP-43 protein in the motor neuron pool of the brachioradialis muscle and showed that by this technique, motor neurons in this pool died, and the muscle got denervated. They had evidence of a spreading process from the spinal cord to the cortex, demonstrated by showing widespread deposits of phosphorylated TDP-43 bilaterally in the cervical cord and the motor cortex. By their experiment, they created a dying-backwards model, not a model of corticofugal spread, like that shown by Braak. No muscle weakness was observed, not even in the brachioradialis. 

Strengths: 

The strength of this innovative study is the fact that this spreading experiment uses the phylogenetically young connectome of primates (macaques). They also made the thought-provoking observation of spreading from the cord to the motor cortex, not the corticofugal spread model observed by Heiko Braak. This is thought-provoking because this enables the observer to compare their model with the findings in humans. 

Weaknesses: 

The following aspects are not a weakness but need to be better explained for the interested reader - and potentially improved in future studies for which the authors laid the foundation: 

(1) Why do the authors use the brachioradialis motor neuron pool to overexpress TDP-43? More is known about other muscles and how they are embedded in the motor connectome of primates. Why not the biceps brachii or the hand extensors or - even better - the small muscles of the hand? These are known to be strongly monosynaptically connected with the motor cortex. The authors should explain this. I am unclear if there was a specific reason which I did not see or understand. In my view, the brachioradialis is not the best representative of the primate connectome, for example, to examine this model and compare it with the corticofugal spread. 

The brachioradialis muscle was chosen primarily for reasons of animal welfare; our concern when designing the experiments was that the muscle we chose for injection might become very wasted and weak before the experiment had been completed. If we had injected a hand muscle, this would have affected manipulation, feeding and grooming behaviours, whereas had we injected biceps brachii or forearm extensors, this would have affected more important behaviours requiring strength for body support in the home cage (e.g. climbing, swinging, etc.). The advantage of choosing brachioradialis is that there is some functional redundancy; in macaques, compared to biceps brachii, brachioradialis has a relatively minor role in elbow flexion and supination of the forearm. We therefore reasoned that there should be physiological compensation for any weakness in brachioradialis, and thus minimal effects on normal behaviour.

A secondary practical consideration was the importance of good quality MR imaging of the injected muscle and the positioning of the focussing coil; because of the physical constraints related to the monkey sitting in our narrow-bore scanner, the forearm muscles were the optimal choice. 

With reference to the ‘primate connectome’, whilst hand muscles are known to have strong cortico-motoneuronal connections, we have shown previously that monosynaptic corticomotoneuronal connections are as strong in muscles innervated by the deep radial nerve (like brachioradialis) as in intrinsic hand muscles (Witham et al, 2016).

Finally, for the purposes of these experiments, all we required was a method for inoculating TDP-43 into a motor neuron pool within the spinal cord, without direct surgical trauma to the spinal cord. Our aim was to test the hypothesis that extracellular TDP-43 is sufficient to cause spreading neuronal changes in macaque, similar to those observed in human ALS/MND; our aim was not to replicate the actual pattern of human MND observed clinically.

These points will be addressed in a revised version of the manuscript. 

(2) In the Braaks experiment, only (seemingly soluble) non-phoshorylated TDP-43 "crossed" synapses. Phosphorylated TDP-43 did not do this. The authors of this study saw phosphorylated TDP43 in motor neurons and the cortex. Is there any potential explanation for how it crosses synapses? If it really does, there is an obvious difference to the human situation which needs to be emphasized and explained (in the future). 

To clarify, there was no evidence of phosphorylated TDP-43 crossing synapses. It is more likely that excess non-phosphorylated TDP-43 crossed synapses, and that this then subsequently led to TDP-43 phosphorylation.  

(3) There were significant deposits of phosphorylated TDP-43 in oligodendrocytes in humans. Whilst I understand that one experiment cannot solve every question - I am curious about whether the authors saw anything in oligodendrocytes? 

We have not looked at this.

(4) Which was the pattern of damage? Of course, this pattern is not likely to have a monosynaptic pattern - like in humans........but was there a pattern? Did it have a physiologically meaningful basis? Was there any relation to the corticofugal monosynaptic pattern? What are the differences? The authors speak of "multiple waves". Does this mean that if this were a corticofugal model, for example, oculomotor neurons would also degenerate? 

The description of ‘multiple waves’ in paragraph 2 of the discussion section is entirely hypothetical, based on the assumption that there are different mechanisms by which TDP-43 spreads through the nervous system, from slow local spread by diffusion to more rapid long-range axonal spread to widely separated regions. For the neuropathological staging analysis, we therefore looked at different brain regions (hypoglossal nuclei, reticular formation, inferior olives, frontal cortex, temporal cortex and hippocampal formation). This analysis only showed loss of motor neurons in the spinal cord ipsilateral to the side of the muscle injections, in segments consistent with the location of brachioradialis motoneurons. We did not demonstrate a ‘pattern of damage’ as described in humans in our experiments because this is a pre-symptomatic pre-clinical model, with no established ‘damage’ from each wave. We speculate that this is because animals were terminated too early in the disease process.

However, whilst there was no established neuronal degeneration outside the cervical spinal cord, the observation that there were more pTDP-43 positive Betz cells in left (contralateral to the brachioradialis injection) New M1 than Old M1 (see Figure 6I and J) would support spread via monosynaptic connections to motoneurons; New M1 is where most monosynaptic cortico-motoneuronal connections originate.

Reviewer #3 (Public review): 

Summary: 

In this paper by Jones and colleagues, a non-human primate model is described in which wild-type TDP-43 is expressed in the cervical spinal cord. This gave rise to loss of motor neurons in the ventral horn at that level in the cervical spinal cord. MRI of the muscles allowed to see increased intensity in the mostly affected brachioradialis muscle, suggesting this muscle becomes denervated. At the neuropathological level, TDP-43 and pTDP-43 staining in the cytoplasm is increased, not only at the specific level of the cervical spinal cord, but also at a distance. 

Strengths: 

A clear strength is the state-of-the art focal expression of the TDP-43 transgene at a focal site in the cervical spinal cord. This is achieved by combining a general expression of a flipped loxP flanked TDP-43 vector using AAV9 intrathecal administration, followed by an intramuscular AAV2 hSyn CRE-TdTomato vector in the brachioradialis muscle in order to induce focal recombination and expression of TDP-43 in motor neurons innervating this muscle on one side. 

Another strength is the non-human primate background, which is much closer to the human situation. 

Weaknesses: 

Given the complexity and cost of the model, the n is very low. 

As is common in most studies in non-human primates, we have carried out all statistical analysis within one animal (e.g. the comparison of motoneuron numbers between left and right cord). We then show that results are reproducible in two animals. Although the number of animals is lower than in a typical rodent study, we see this as an advantage of the model, adhering to the 3Rs principle of ‘reduction’.

The design of the experiments and the results shown about the toxicity induced by this focal TDP-43 expression do not allow us to conclude that it is a good model for ALS for several reasons. It is not clear that the TDP-43 overexpression results in spreading weakness or in spreading motor neuron loss. The neuropathological changes described suggest that there is a kind of stress response, which extends to regions away from the site of primary damage, but more is needed to provide convincing evidence that there is spreading of disease pathology reminiscent of human ALS. 

As already noted in our response to Reviewer 2 (point 1), animal welfare is an important consideration when designing these complex experiments in primates. We could not therefore justify allowing the animals to survive until extensive wasting and weakness were evident, recapitulating the human disease. 

The model developed in these experiments is therefore a pre-symptomatic pre-clinical model, in which animals are terminated before pathology leading to widespread motor neuron loss is evident. At post mortem we do have evidence of motor neuron loss in the segments supplying brachioradialis (C4-C8).

Stress of various forms, including blunt trauma (e.g. Anderson et al, 2021), stab/electrode insertion injury (e.g. Zambusi et al, 2022), chemical (e.g. arsenite) exposure (e.g. Huang et al, 2024), or hypoxia (Marcus et al, 2021) can result in pathological nucleocytoplasmic translocation of TDP-43. In our model, there was no direct trauma to the brain or spinal cord ante mortem, excluding one major cause of tissue stress. Hypoxia during the process of euthanasia is possible, but we would expect there would not be enough time before death for this to manifest as TDP-43 translocation. In the literature TDP-43 translocation due to stress is diffuse; we have demonstrated that in our model the TDP-43 pathology is not diffuse but selective. For example, there was no evidence of disease in the oculomotor nuclei; in the primary motor cortex (M1) there are significantly more pathological changes in the evolutionarily younger ‘NewM1’ compared to the neighbouring ‘OldM1’.

It is therefore improbable that our findings could be explained by ‘a kind of stress response’. Our findings are better explained by spread of the TDP-43 protein.

Reviewer #4 (Public review): 

Summary: 

In this manuscript, the authors present data describing the development of a model of ALS in rhesus macaques. They use a viral intersectional model to overexpress TDP-43 in a population of motor neurons and then study the spread of the pathology about 7 months later. They demonstrate that both the cervical spinal cord and motor cortex (new and old M1) are full of TDP-43, suggesting that the pathology spreads from the single motor pool to presumably related neurons. 

Strengths: 

This is a super-important study in two main ways: 

(1) This could be the birth of a really important model, one that is really needed for making progress in understanding ALS and the development of therapeutics. There are shortfalls with all the rodent models. Models dependent on cell cultures are superb for understanding cell-autonomous processes, but miss out on connectivity, particularly the long-range connectivity. Organoids may ultimately prove to be beneficial, but they would need cortex, spinal cord, and muscle, and translatability from them is not assured. So a NHP model is needed, and this may be it.

Furthermore, the Methods are meticulously described and will undoubtedly facilitate reproducibility. 

(2) The concept of the spread of pathology has been proposed for some time, I think, based initially on the detailed clinical observations of Ravits and colleagues. The authors have looked at this directly and provide supporting evidence for this interesting hypothesis. They show spread locally and contralaterally in the spinal cord (although a figure would be nice) and to the motor cortex. 

Taking only these 2 points into account is more than sufficient for me to be enthusiastic about this work. 

Weaknesses: 

I'd like to make a couple of points that if addressed, could, in my view, help the authors strengthen this work. 

(1) We don't know how many MNs were transduced by the rAAV. There was no tdTom expression, for whatever reason. The authors show an image of a control experiment with a single MN transduced, but there should be a red motor pool, at least in the control experiments. The impression that I get is that very few were transduced, and, in my mind, this makes the findings even more interesting - maybe you don't need many "starter" MNs. 

Unfortunately, we cannot know how many motoneurons were transduced.

However, the reviewer may be correct, that it is actually only a small fraction of the brachioradialis pool. This is supported by the evidence for rather focal denervation seen on MRI.

(2) Continuing on this point, this leads the authors to conclude that all BR MNs have died. They support this by the reduced MN count (see point 3). Firstly, do we know how many BR MNs there are in the rhesus macaque, and does the reduction seen correspond to this number? Secondly, and more importantly, the muscle looks normal on MRI at 28 weeks - it does not look like a denervated muscle. The authors state that it has maybe been reinnervated, but by what, if all the BR MNs are dead? This does not seem like a plausible explanation to me. Muscle histology, NMJs, and fibre typing would have been useful to understand what's going on with the MNs. (And electrophysiology would have been wonderful, but beyond the scope of this study.) 

To clarify, we did not conclude that all brachioradialis motor neurons had died, rather that all transfected brachioradialis motor neurons pool had died. As noted above, when these cells die and the muscle is denervated, the MRI signal changes occupy only a small volume of the muscle and are transient. We would not expect to see long-term MRI changes in muscle anatomy after this limited denervation-reinnervation event. 

Analysis of muscle histology, including fibre typing, is outwith the scope of this initial paper reporting the model; we hope that this will form the basis of a future publication.

(3) Some MN biologists, like me, fuss a lot about how to count MNs, which is almost as difficult as counting the number of angels on the head of a pin. Every method has its problems. Focusing on the two methods here: (a) ChAT immunohistochemistry is pretty good in healthy states, but we don't know what happens to ChAT expression in different diseases, particularly when you have a new model. If its expression is decreased, then it is not a good marker for MNs; (b) Identifying MNs based on the size and morphology of neurons in the ventral horn is also insufficient. For example, ~30% of neurons in a typical pool are small gamma MNs, and a significant proportion (depending on the muscle) of the remainder will be small alpha MNs. So what one is counting is, at best, the large alpha MNs, not all the MNs in a pool. And in ALS, it's these largest MNs that are affected at the earliest stages. The small ones might be fine. So results will be skewed. (Hence, it would be interesting to see if the muscle had a higher proportion of Type I fibres after being reinnervated by S-type MNs.) 

This is an interesting point, and we agree that each method used to quantify MN number carries its own limitations. The problem of MN identification is heightened in a MND-like pathological state, especially when considering evidence of reduced ChAT activity in spinal motoneurons in end-stage disease in post mortem human samples (Oda et al, 1995), and more recent evidence from Casas et al. (2013), who demonstrated early presymptomatic reduction in ChAT expression in SOD1G93A mice. It is important to note that this was a modest reduction, not complete abolition of signal (76% of control levels). ChAT immunoreactivity was still present and motor neurons were still identifiable as ChAT-positive at this pre-clinical stage of disease. As counts in our study were performed based on detecting ChAT in cells, it seems unlikely that we would miss cells. However, we cannot rule this out. If indeed this did occur, it would mean that the reduced motoneuron counts which we observed reflect not only cell death, but also profound motoneuron dysfunction which is presumably the proximal precursor to cell death.

We acknowledge that size-based criteria applied to ChAT-positive neurons will preferentially capture large alpha motor neurons, and that gamma motor neurons and small alpha motor neurons are likely underrepresented in our counts. Our counts therefore reflect the large alpha motor neuron population rather than the total motor neuron pool. We believe that this is not a critical limitation in the context of the present study. Large alpha motor neurons are the population of primary pathological interest in ALS and related MND, being the earliest and most severely affected subtype. The selective vulnerability of fast-fatigable large alpha motor neurons in ALS is well established, and their preferential loss is the defining feature of disease progression in both human post mortem tissue and rodent models (Lalancette-Hébert et al., 2016). In this respect, our size threshold selects for precisely the population whose degeneration is most relevant to the disease phenotype we are modelling. 

We intend to include comments on these important points in the revised version of the manuscript.

In response to the final point regarding muscle histology and proportions of Type I fibres, as stated above, reporting of muscle histology, including fibre typing, is planned for a separate publication.

(4) Statistics. These are complex experiments looking at the spread of a disease. The experimental unit is therefore the monkey, n=2. In each monkey, multiple sections are analysed, which are key technical replicates and often summative. For example, do we care about the average cell number in Figures 4D, E, 5 I, J or 6G, H, or rather the total cell number? Do the error bars mean anything? To be clear, I am by no means minimising the importance of the overall convincing findings. But I do not think this statistical analysis is particularly meaningful. 

Here, the experimental unit is the tissue slice, mounted on a slide for histological analysis, and not the monkey. All statistical comparisons are made within a single animal. We then show that the findings can be replicated in two animals, both of which show significant results. This is standard approach taken in primate neuroscience, given the need to reduce animal numbers to the minimum consistent with producing convincing results.