Dominant α-tubulin mutations rescue tauopathy neurodegenerative phenotypes in C. elegans
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eLife Assessment
Using a genetic screen in C. elegans, Benbow et al., identify mutations in alpha-tubulin genes that suppress Tau-induced neurodegenerative phenotypes. The results solidly support the authors' claim that the tubulin mutants protect against neurodegeneration without altering tau aggregation and hyperphosphorylation. While precise mechanisms of protection by tubulin mutants remain to be established, the results are valuable for understanding the underlying cellular mechanisms of Tauopathies and for the development of therapeutic interventions.
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Abstract
Tau protein, the primary component in neurofibrillary tangles characteristic of Alzheimer’s Disease and related dementia disorders, normally regulates microtubule growth and stability. While tau dysfunction contributes to the progression of tauopathies, the role of microtubules in disease has remained unclear. Through forward genetic screening in Caenorhabditis elegans tauopathy models, we found multiple tubulin gene mutations that rescue tau-mediated neurodegeneration. Whole animal behavioral and in vitro biochemical assays were employed to characterize mutation-driven effects on neuron function, neurodegeneration, and effects on tubulin and tau proteins as well as microtubule function. Mutant tubulin genes were found to confer different levels of suppression correlating with the level of mutant gene expression. Mutant tubulins did not drastically alter total tau protein levels, tau phosphorylation or aggregation, however tau-induced neurodegeneration was rescued. The suppression of tau toxicity by tubulin gene mutations cannot be explained by changes in tau or tubulin expression, tau phosphorylation, or tau aggregation state. Rather the tubulin mutations appear to act by influencing global microtubule properties. In vitro experiments using C. elegans tubulin in semi-isolated and isolated contexts have indicated changes to microtubule properties without observable changes to tau-tubulin affinity. This work suggests that manipulation of microtubules can rescue tauopathy even when pathological tau species persist, supporting the importance of understanding microtubule contributions to disease progression and investigation into microtubule targeted gene therapy or small molecule approaches for tauopathy intervention.
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eLife Assessment
Using a genetic screen in C. elegans, Benbow et al., identify mutations in alpha-tubulin genes that suppress Tau-induced neurodegenerative phenotypes. The results solidly support the authors' claim that the tubulin mutants protect against neurodegeneration without altering tau aggregation and hyperphosphorylation. While precise mechanisms of protection by tubulin mutants remain to be established, the results are valuable for understanding the underlying cellular mechanisms of Tauopathies and for the development of therapeutic interventions.
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Reviewer #1 (Public review):
Summary:
This study identifies mutations in alpha-tubulin that suppress Tau-induced neurodegeneration using the C. elegans model of Tauopathy, suggesting a potentially interesting role for microtubule properties in modulating Tau toxicity. These missense mutations cluster in the C-terminal Tau-interacting helix 12 region of alpha-tubulin genes (tba-1, tba-2, and mec-12). Further analysis, particularly using the strongest suppressor tba-2, shows that it rescues Tau-induced behavioral deficits and neuronal loss without significantly altering bulk tau-phosphorylation, aggregation, or binding to soluble tubulin. The authors suggest that altered microtubule properties underlie the neuroprotective effects, and manipulating microtubule properties may have therapeutic potential.
Strengths:
The study is conceptually …
Reviewer #1 (Public review):
Summary:
This study identifies mutations in alpha-tubulin that suppress Tau-induced neurodegeneration using the C. elegans model of Tauopathy, suggesting a potentially interesting role for microtubule properties in modulating Tau toxicity. These missense mutations cluster in the C-terminal Tau-interacting helix 12 region of alpha-tubulin genes (tba-1, tba-2, and mec-12). Further analysis, particularly using the strongest suppressor tba-2, shows that it rescues Tau-induced behavioral deficits and neuronal loss without significantly altering bulk tau-phosphorylation, aggregation, or binding to soluble tubulin. The authors suggest that altered microtubule properties underlie the neuroprotective effects, and manipulating microtubule properties may have therapeutic potential.
Strengths:
The study is conceptually interesting as it shows that Tau-induced neurotoxicity can, in this model, be partially uncoupled from canonical pathological hallmarks such as Tau-hyperphosphorylation and aggregation. The identification of multiple independent mutations in the same structural region of three alpha-tubulin genes provides support for the functional relevance of helix 12 in modulating Tau-induced toxicity. The authors demonstrate significant rescue of behavioral deficits (using motility and manual thrashing assays) and neuronal loss in both WT-tau and FTLD-associated TauV337M in combination with mutant alpha-tubulins, suggesting a general mechanism for tubulin-regulated modulation of Tau-toxicity. Moreover, the correlation between mutant tubulin expression levels and the extent of rescue supports a causal relationship.
Weaknesses:
One of the major claims of this manuscript is that altered microtubule properties suppress Tau toxicity. The only supporting evidence in this context provided by the authors is reduced taxol-stabilized microtubule mass, which does not fully explain neuronal loss or the rescue of behavioral deficits. What remains unclear is whether these mutations alter microtubule dynamics, catastrophe, lattice stability, or axonal transport.
The authors show that mutant tba-2 reduces total tau levels by ~45%. This level of reduction is likely significant but underexplored in the manuscript. Why are the Tau levels reduced? How is Tau getting cleared- is there enhanced autophagy or ubiquitin-proteasome pathway getting upregulated in tba-2 + Tau animals? Or one or more of the Tau species not detectable by the antibodies used in this study? The observation that the mec-12 mutant rescues Tau-induced phenotypes without altering Tau levels suggests that suppression can occur through Tau-independent mechanisms. This raises an important unresolved question regarding the extent to which suppression is Tau-dependent vs Tau-independent across different mutant alpha-tubulin genes, complicating the interpretation of the rescue phenotypes.
Given that Tau primarily associates with the microtubule lattice in vivo, measuring interactions with soluble tubulin may not fully capture biologically relevant binding dynamics and therefore does not exclude the possibility that these mutations alter tau-microtubule interactions at the lattice level or may affect the binding of other MAPs/regulators, thereby altering stability or trafficking.
A large body of conclusions is drawn from behavioral rescue and biochemical assays. This limits the understanding of how molecular changes in tubulin might affect cellular mechanisms of neuroprotection. Are there changes in the neuronal microtubule organization, Tau localization, or its redistribution in the mutant alpha-tubulin background? Are there differences in soluble vs oligomeric vs insoluble Tau in mutant tba-2 and mec-12 animals?
The suppression of behavior in the co-pathology model is interesting but mechanistically insufficient, mainly because the underlying basis of suppression is not examined in these models. Moreover, it remains unclear whether tubulin-Tau genetically interacts with Aβ or TDP-43, and what cellular mechanisms account for the partial rescue observed in these co-pathology models.
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Reviewer #2 (Public review):
Summary:
The manuscript by Benbow et al. identifies, through a genetic screen, key tubulin mutants that, with high confidence, rescue tau-mediated ND phenotypes. This manuscript is well written, and the experimental results strongly support the authors' claims that these tubulin mutants can rescue ND-linked phenotypes in C. elegans while having little to no direct effect on Tau aggregation.
Strengths:
Benbow et al. use a relatively unbiased forward genetic screen to identify mutations associated with phenotypes that suppress tauopathy-related defects. The authors then logically focus on the various α-tubulin missense mutations identified in H12, which are known to localize to the external face of microtubules. The authors also carefully compare their established tauopathy-associated phenotypes in the WT TauH …
Reviewer #2 (Public review):
Summary:
The manuscript by Benbow et al. identifies, through a genetic screen, key tubulin mutants that, with high confidence, rescue tau-mediated ND phenotypes. This manuscript is well written, and the experimental results strongly support the authors' claims that these tubulin mutants can rescue ND-linked phenotypes in C. elegans while having little to no direct effect on Tau aggregation.
Strengths:
Benbow et al. use a relatively unbiased forward genetic screen to identify mutations associated with phenotypes that suppress tauopathy-related defects. The authors then logically focus on the various α-tubulin missense mutations identified in H12, which are known to localize to the external face of microtubules. The authors also carefully compare their established tauopathy-associated phenotypes in the WT TauH model, with and without specific α-tubulin mutations, using appropriate controls throughout. Lastly, the authors provide partial mechanistic insight into the α-tubulin mutant-mediated rescue, showing that these effects are independent of tau aggregation and tau phosphorylation, and instead suggest that the α-tubulin mutations may confer altered microtubule assembly properties based on the sedimentation assays.
Weaknesses:
While the claims are largely supported by the experimental outcomes, the authors at times do not provide enough detail in the text for readers to interpret the data sets independently. In addition, some claims appear to be slightly overstated relative to the data or the degree of error associated with those data.
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Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
This study identifies mutations in alpha-tubulin that suppress Tau-induced neurodegeneration using the C. elegans model of Tauopathy, suggesting a potentially interesting role for microtubule properties in modulating Tau toxicity. These missense mutations cluster in the C-terminal Tau-interacting helix 12 region of alpha-tubulin genes (tba-1, tba-2, and mec-12). Further analysis, particularly using the strongest suppressor tba-2, shows that it rescues Tau-induced behavioral deficits and neuronal loss without significantly altering bulk tau-phosphorylation, aggregation, or binding to soluble tubulin. The authors suggest that altered microtubule properties underlie the neuroprotective effects, and manipulating microtubule properties may have therapeutic potential.
Stre…
Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
This study identifies mutations in alpha-tubulin that suppress Tau-induced neurodegeneration using the C. elegans model of Tauopathy, suggesting a potentially interesting role for microtubule properties in modulating Tau toxicity. These missense mutations cluster in the C-terminal Tau-interacting helix 12 region of alpha-tubulin genes (tba-1, tba-2, and mec-12). Further analysis, particularly using the strongest suppressor tba-2, shows that it rescues Tau-induced behavioral deficits and neuronal loss without significantly altering bulk tau-phosphorylation, aggregation, or binding to soluble tubulin. The authors suggest that altered microtubule properties underlie the neuroprotective effects, and manipulating microtubule properties may have therapeutic potential.
Strengths:
The study is conceptually interesting as it shows that Tau-induced neurotoxicity can, in this model, be partially uncoupled from canonical pathological hallmarks such as Tau-hyperphosphorylation and aggregation. The identification of multiple independent mutations in the same structural region of three alpha-tubulin genes provides support for the functional relevance of helix 12 in modulating Tau-induced toxicity. The authors demonstrate significant rescue of behavioral deficits (using motility and manual thrashing assays) and neuronal loss in both WT-tau and FTLD-associated TauV337M in combination with mutant alpha-tubulins, suggesting a general mechanism for tubulin-regulated modulation of Tau-toxicity. Moreover, the correlation between mutant tubulin expression levels and the extent of rescue supports a causal relationship.
Weaknesses:
One of the major claims of this manuscript is that altered microtubule properties suppress Tau toxicity. The only supporting evidence in this context provided by the authors is reduced taxol-stabilized microtubule mass, which does not fully explain neuronal loss or the rescue of behavioral deficits. What remains unclear is whether these mutations alter microtubule dynamics, catastrophe, lattice stability, or axonal transport.
We agree with Reviewer #1’s critique that the evidence presented does not fully explain neuronal loss and requires further investigation. This first manuscript characterized the mutations discovered through forward genetic screening techniques and provided data to support the positive correlation mutant expression and level of suppression. We believe the studies and data presented here help to formulated the next testable hypotheses, and guide the next lines of experimentation. We are encouraged by Reviewer #1’s assessment that exploration of microtubule dynamics, catastrophe, lattice stability and axonal transport will be critical to testing the hypothesis that mutant tubulin drives suppression of tau toxicity through changes to microtubule properties. These suggestions are highly relevant and align with our priorities as we recently submitted an application for a 5-year research award to support these key questions.
To address this specifically, the reviewer recommended “The microtubule-dependent axonal transport should be examined in tubulin mutants and compared with mutant tubulin + Tau conditions. Imaging of mitochondrial or synaptic vesicle markers, along with appropriate quantifications (velocity or run length), may provide a functional readout linking microtubule changes to neuronal survival.”
We agree with the reviewer that these experiments will be highly valuable to further understand the mechanisms underlying suppression, and we have planned to complete these experiments upon receipt of funding that would directly support the completion of these experiments.
The authors show that mutant tba-2 reduces total tau levels by ~45%. This level of reduction is likely significant but underexplored in the manuscript. Why are the Tau levels reduced? How is Tau getting cleared- is there enhanced autophagy or ubiquitin-proteasome pathway getting upregulated in tba-2 + Tau animals? Or one or more of the Tau species not detectable by the antibodies used in this study? The observation that the mec-12 mutant rescues Tau-induced phenotypes without altering Tau levels suggests that suppression can occur through Tau-independent mechanisms. This raises an important unresolved question regarding the extent to which suppression is Tau-dependent vs Tau-independent across different mutant alpha-tubulin genes, complicating the interpretation of the rescue phenotypes.
We think the reviewer has addressed an important point that there may be both tau-dependent and tau-independent mechanisms at work here, and we will add greater nuance to this in our discussion. Additionally, we agree these two potential mechanistic pathways merit further exploration. To address this, we have planned to conduct experiments using reporter C. elegans lines crossed with our mutant tubulin/tau-transgenic lines to detect potential upregulation of these pathways as mechanisms for tau clearance.
Given that Tau primarily associates with the microtubule lattice in vivo, measuring interactions with soluble tubulin may not fully capture biologically relevant binding dynamics and therefore does not exclude the possibility that these mutations alter tau-microtubule interactions at the lattice level or may affect the binding of other MAPs/regulators, thereby altering stability or trafficking.
In the discussion we acknowledge the limitation of only examining the binding affinity between soluble tubulin and tau and intend to complete further studies with polymerized microtubules containing mutant α-tubulin. We will expand discussion of this in the text. Similar to reviewer 1, we have also concluded that the next line of experimentation will focus on mutant alpha-tubulin effects on the microtubule polymer such as changes to MAP interactions, stability and trafficking. We have applied for and hope to receive funding to address these questions in the near future.
To address this concern specifically, we plan to conduct these experiments using C. elegans extracts to polymerize microtubules and subsequently test the binding of recombinant human tau. These co-sedimentation experiments are expected to be included in the revised manuscript.
A large body of conclusions is drawn from behavioral rescue and biochemical assays. This limits the understanding of how molecular changes in tubulin might affect cellular mechanisms of neuroprotection. Are there changes in the neuronal microtubule organization, Tau localization, or its redistribution in the mutant alpha-tubulin background? Are there differences in soluble vs oligomeric vs insoluble Tau in mutant tba-2 and mec-12 animals?
The reviewer raises relevant questions regarding elucidation of the mechanisms underlying mutant tubulin-mediated suppression at the cellular level. To address this concern we will analyze the cellular distribution of tau in neurons from mutant and non-mutant C. elegans.
Ultimately, our goals are to identify and connect the underlying biochemical mechanisms with the observed prevention of cell death as Reviewer 1 has identified. Their suggestion to explore cellular-level changes such as mutant tubulin effects on tau distribution is highly relevant. We therefore plan to test this directly by imaging neurons in C. elegans strains expressing fluorescently labeled tau and/or immunohistochemical techniques to stain for tau in C. elegans neurons.
The suppression of behavior in the co-pathology model is interesting but mechanistically insufficient, mainly because the underlying basis of suppression is not examined in these models. Moreover, it remains unclear whether tubulin-Tau genetically interacts with Aβ or TDP-43, and what cellular mechanisms account for the partial rescue observed in these co-pathology models.
In agreement with Reviewer #1’s assessment, we have concluded these data, while interesting, do not substantially expand our understanding apart from the existing data. Without additional information regarding the underlying mechanisms, they do not provide substantial novel insights and we have therefore chosen to remove the co-pathology data sets from the revised version of the manuscript to refine the scope of the data and hypotheses discussed in this work.
Reviewer #2 (Public review):
Summary:
The manuscript by Benbow et al. identifies, through a genetic screen, key tubulin mutants that, with high confidence, rescue tau-mediated ND phenotypes. This manuscript is well written, and the experimental results strongly support the authors' claims that these tubulin mutants can rescue ND-linked phenotypes in C. elegans while having little to no direct effect on Tau aggregation.
Strengths:
Benbow et al. use a relatively unbiased forward genetic screen to identify mutations associated with phenotypes that suppress tauopathy-related defects. The authors then logically focus on the various α-tubulin missense mutations identified in H12, which are known to localize to the external face of microtubules. The authors also carefully compare their established tauopathy-associated phenotypes in the WT TauH model, with and without specific α-tubulin mutations, using appropriate controls throughout. Lastly, the authors provide partial mechanistic insight into the α-tubulin mutant-mediated rescue, showing that these effects are independent of tau aggregation and tau phosphorylation, and instead suggest that the α-tubulin mutations may confer altered microtubule assembly properties based on the sedimentation assays.
Weaknesses:
While the claims are largely supported by the experimental outcomes, the authors at times do not provide enough detail in the text for readers to interpret the data sets independently. In addition, some claims appear to be slightly overstated relative to the data or the degree of error associated with those data.
We appreciate the feedback regarding the need for additional clarity for independent analysis of the datasets. We will revise the figures and text to increase clarity for the readers. We will review statements and edit language in accordance with their degrees of error as appropriate.
The authors measure tau binding affinities using soluble tubulin but do not assess tau binding to assembled microtubules. This is an important limitation, as the physiologically relevant interaction involves α/β-tubulin heterodimers, either free or incorporated into the microtubule lattice. Furthermore, the binding analysis appears to focus only on the D429N α-tubulin mutant, which further limits physiological relevance, as β-tubulin, which is also required for normal tau binding, is not explicitly considered.
We acknowledge that the limited conclusions may be drawn from soluble tubulin interactions with tau and additional analysis with polymerized microtubules will be useful in understanding tau-microtubule binding affinity. The analysis was completed with isolated pools of tubulin from C. elegans, not recombinant mutant tubulin, so this is a heterogenous mixture of tubulin composed of α/β heterodimer subunits, and a mixture of the mutant isotype within the larger pool of wild type isotypes. While this further complicating the analysis, and is the likely source of variability, it incorporates the normal heterodimer subunit biochemistry.
Given that tau prominently binds the microtubule lattice we agree with the reviewers that the assessment that experiments with polymerized microtubules containing mutant tubulin would offer a greater understanding of the effects of mutant alpha-tubulin on microtubule properties and potential mechanisms of toxic tau suppression. To test this directly we intend to complete co-sedimentation experiments using C. elegans extracts from wild type and mutant tubulin expressing C. elegans incubated with recombinant human tau.
In conclusion, the thoughtful commentary and suggestions from reviewers will help improve the manuscript. We plan to complete the following experiments to address their concerns.
(1) Assess tau localization in mutant tba-2 and mec-12 C. elegans as compared to tau-transgenic C. elegans without tubulin mutations. We plan to use immunohistochemical techniques and/or imaging of Dendra2-labeled tau to assess the sub-compartmental distribution of tau in C. elegans neurons. This addresses Reviewer #1’s question of whether the mutant tubulin changes tau localization in neurons.
(2) Assess changes mutant-tubulin driven changes to tau affinity for polymerized microtubules. To address both reviewers concerns regarding the limitations of biding experiments with tau and soluble tubulin, We plan to use C. elegans extracts to tests whether microtubule polymers containing mutant alpha-tubulin alter tau-microtubule co-sedimentation.
(3) Using C. elegans reporter lines we plan to assess whether tau clearance occurs in tba-2 mutant tubulin C. elegans through the upregulation of autophagy or ubiquitin degradation pathways.
(4) Evaluate the neuroprotective effects of mutant alpha-tubulin in cholinergic neurons using a C. elegans strain expressing a fluorescent label specifically in cholinergic neurons.
We plan to make textual revisions to increase clarity, aid in independent analysis of the presented datasets, and better address the possibility of both tau-dependent and tau-independent mechanisms. We appreciate the Reviewers attentive reading and thoughtful feedback for the improvement of this manuscript.
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