A non-human primate model of Amyotrophic Lateral Sclerosis
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eLife Assessment
This fundamental study provides a major contribution to our understanding of Amyotrophic Lateral Sclerosis (ALS) pathogenesis by utilizing a primate model that overcomes the historical limitations of rodent paradigms. By demonstrating the retrograde and trans-synaptic spread of pathological TDP-43 from the periphery to the spinal cord and motor cortex, the authors propose a new model for the disease spreading. The evidence supporting these findings is compelling, characterized by rigorous post-mortem histological observations. This work will be of profound interest to neuroscientists and translational researchers seeking to decode the mechanisms of systemic disease progression in ALS.
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Abstract
Approximately 97% of patients with amyotrophic lateral sclerosis (ALS) have cytoplasmic mislocalization and aggregation of the ubiquitous nuclear protein, TDP-43. Current rodent models of this disease fail to replicate the progressive motor weakness and characteristic histopathology, possibly because of fundamental neuroanatomical and genetic differences between rodents and humans. In this study, the TDP-43 protein was overexpressed in the motor neuron pool of the brachioradialis muscle unilaterally in two six-year-old female rhesus macaques, using an intersectional genetics approach involving infection with genetically modified adeno-associated virus. Magnetic resonance images demonstrated delayed signal hyperintensities limited to the injected brachioradialis that persisted for 6-7 weeks, consistent with motor neuron degeneration and denervation of the targeted muscle. At post-mortem, the virus-mediated focal protein overexpression event was found to induce widespread deposits of pathological phosphorylated TDP-43 throughout the cervical spinal cord and motor cortex bilaterally, indicating an ALS-like spread of proteinopathy from the transfection site.
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eLife Assessment
This fundamental study provides a major contribution to our understanding of Amyotrophic Lateral Sclerosis (ALS) pathogenesis by utilizing a primate model that overcomes the historical limitations of rodent paradigms. By demonstrating the retrograde and trans-synaptic spread of pathological TDP-43 from the periphery to the spinal cord and motor cortex, the authors propose a new model for the disease spreading. The evidence supporting these findings is compelling, characterized by rigorous post-mortem histological observations. This work will be of profound interest to neuroscientists and translational researchers seeking to decode the mechanisms of systemic disease progression in ALS.
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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.
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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:
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?
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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 …
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.
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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 …
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.
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