Transthyretin Promotes Axon Growth via Regulation of Microtubule Dynamics and Tubulin Acetylation
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Evaluation Summary:
In their manuscript, Eira et al. investigate the role of transthyretin in promoting axon elongation by modulating microtubule dynamics. The data point to a possible role of transthyretin in regulating microtubule dynamics by modulating tubulin acetylation levels during axon outgrowth. With additional support to strengthen this conclusion, the paper will be of interest to those in the neurodevelopment, neurodegeneration, and microtubule cytoskeleton fields.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript.The reviewers remained anonymous to the authors.)
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Abstract
Transthyretin (TTR), a plasma and cerebrospinal fluid protein, increases axon growth and organelle transport in sensory neurons. While neurons extend their axons, the microtubule (MT) cytoskeleton is crucial for the segregation of functional compartments and axonal outgrowth. Herein, we investigated whether TTR promotes axon elongation by modulating MT dynamics. We found that TTR KO mice have an intrinsic increase in dynamic MTs and reduced levels of acetylated α-tubulin in peripheral axons. In addition, they failed to modulate MT dynamics in response to sciatic nerve injury, leading to decreased regenerative capacity. Importantly, restoring acetylated α-tubulin levels of TTR KO dorsal root ganglia (DRG) neurons using an HDAC6 inhibitor is sufficient to completely revert defective MT dynamics and neurite outgrowth. In summary, our results reveal a new role for TTR in the modulation of MT dynamics by regulating α-tubulin acetylation via modulation of the acetylase ATAT1, and suggest that this activity underlies TTR neuritogenic function.
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Evaluation Summary:
In their manuscript, Eira et al. investigate the role of transthyretin in promoting axon elongation by modulating microtubule dynamics. The data point to a possible role of transthyretin in regulating microtubule dynamics by modulating tubulin acetylation levels during axon outgrowth. With additional support to strengthen this conclusion, the paper will be of interest to those in the neurodevelopment, neurodegeneration, and microtubule cytoskeleton fields.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript.The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
TTR, present in plasma and CSF, promotes axon regeneration. To try to understand this effect, the authors demonstrated that neurons cultured from TTR knockout mice have an increase in microtubule dynamics and decrease in microtubule acetylation as well as morphological shortcomings - all of which are restored by an HDAC6 inhibitor. These observations link the effects of TTR on axon regeneration to microtubules. The work is interesting, well done and provides novel information. However, the work is limited by the fact that there is no mechanistic information to understand how TRR elicits its effects on microtubule acetylation/dynamics and also by the fact that the restoration of the phenotype with HDAC6 inhibition is only on morphological parameters in culture. An in vivo regeneration model would have made …
Reviewer #1 (Public Review):
TTR, present in plasma and CSF, promotes axon regeneration. To try to understand this effect, the authors demonstrated that neurons cultured from TTR knockout mice have an increase in microtubule dynamics and decrease in microtubule acetylation as well as morphological shortcomings - all of which are restored by an HDAC6 inhibitor. These observations link the effects of TTR on axon regeneration to microtubules. The work is interesting, well done and provides novel information. However, the work is limited by the fact that there is no mechanistic information to understand how TRR elicits its effects on microtubule acetylation/dynamics and also by the fact that the restoration of the phenotype with HDAC6 inhibition is only on morphological parameters in culture. An in vivo regeneration model would have made this study stronger.
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Reviewer #2 (Public Review):
This study provides some interesting observations regarding the potential function of TTR on MT dynamics and axonal growth. Given the role of TTR on the pathology of peripheral nerves in peripheral neuropathies, investigating the role of TTR on axonal health could provide insights into how TTR is toxic to peripheral neurons. However, this study falls short of definitively showing that TTR is responsible for MT dynamic enhancements and neurite outgrowth, as TTR is not expressed on TTR KO neurons to test for rescue of these effects. Critically, a major issue in this manuscript, is with regards to the rigor of the data presented. The main issue is the lack of data ascertaining the use of the same subtype cells for the in cellulo analyses throughout the manuscript, i.e., the lack of …
Reviewer #2 (Public Review):
This study provides some interesting observations regarding the potential function of TTR on MT dynamics and axonal growth. Given the role of TTR on the pathology of peripheral nerves in peripheral neuropathies, investigating the role of TTR on axonal health could provide insights into how TTR is toxic to peripheral neurons. However, this study falls short of definitively showing that TTR is responsible for MT dynamic enhancements and neurite outgrowth, as TTR is not expressed on TTR KO neurons to test for rescue of these effects. Critically, a major issue in this manuscript, is with regards to the rigor of the data presented. The main issue is the lack of data ascertaining the use of the same subtype cells for the in cellulo analyses throughout the manuscript, i.e., the lack of identification/characterization as DRG neurons to show whether the cells the study uses are DRGs or in fact neurons altogether, and if they are DRG neurons, what type of DRG neurons are used to ascertain that comparisons are made between/within the same subtypes. Thus, data collected on plated neurons throughout the study would have to be done on equivalent DRG neurons. Providing this characterization is key because it is well-established that there are many subtypes of DRG neurons, with distinctively different properties that might vary widely with regards to TTR function, and an important question is whether TTR might be acting differentially on different DRG neuronal subtypes. Equally important, several conclusions are not sufficiently supported by data provided, including the conclusion the lack of MT severing based on the unchanged levels of enzymes that sever MTs, a result for which there could be other interpretations. Furthermore, there are inconsistencies in the data presentation, and lack of data (numbers of replicates are missing throughout).
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Reviewer #3 (Public Review):
In the present study, the authors address the underlying mechanisms of TTR neuritogenic role in DRG neurons. They showed that TTR increased microtubule dynamics in the distal end of growing axons but had no impact on microtubule dynamics in the axon shaft. TTR KO mice had a reduced level of α-tubulin acetylation and an intrinsic increase of dynamic microtubules in uninjured nerves, and failed to modulate dynamic microtubules in injured nerves in response to sciatic nerve injury. The use of an HDAC6 inhibitor to increase acetylated tubulin level restored dynamic microtubules and neurite outgrowth of TTR KO neurons. These findings aid our understanding in the roles of TTR during axon growth and regeneration. However, systematic detection is required for changes in α-tubulin acetylation level and microtubule …
Reviewer #3 (Public Review):
In the present study, the authors address the underlying mechanisms of TTR neuritogenic role in DRG neurons. They showed that TTR increased microtubule dynamics in the distal end of growing axons but had no impact on microtubule dynamics in the axon shaft. TTR KO mice had a reduced level of α-tubulin acetylation and an intrinsic increase of dynamic microtubules in uninjured nerves, and failed to modulate dynamic microtubules in injured nerves in response to sciatic nerve injury. The use of an HDAC6 inhibitor to increase acetylated tubulin level restored dynamic microtubules and neurite outgrowth of TTR KO neurons. These findings aid our understanding in the roles of TTR during axon growth and regeneration. However, systematic detection is required for changes in α-tubulin acetylation level and microtubule dynamics after TTR treatment and TTR KO. Furthermore, lacking specific manipulation of α-tubulin acetylation level and microtubule dynamics to rescue the phenotype is also a shortcoming of current study.
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