Tau hyperphosphorylation impairs cooperative binding to microtubules and perturbs organelle trafficking in neurons

Reviewer #1 (Public review):

Summary:

This work by Beaudet and colleagues aims at exploring the effect of phosphorylation on the formation of tau envelopes and consequently on axonal transport, both in vitro on reconstituted microtubules and in human excitatory neurons derived from IPSCs.

The authors found that a relatively widely used construct in which 14 serine or threonine residues, often hyperphosphorylated in Alzheimer's disease, are mutated to alanines (phosphodeficient), increases the density of tau envelopes compared to wildtype tau, whereas a phosphomimetic (same residues mutated to glutamic acid) reduces envelope density both in vitro and in human excitatory neurons derived from IPSCs.

By analysing the trafficking of different kinesins (KIF1a and KIF5C), they observed different effects of tau phosphorylation status on the movement of these two motors.

They then analyse transport of lysosomes by employing live imaging of lysotracker in human excitatory neurons derived from IPSCs transfected with wildtype, phosphodeficient or phosphomimetic tau, observing that phosphodeficient tau seems to reduce transport of lysosomes while phosphomimetic increases transport compared to wildtype tau.

Strengths:

(1) The work aims to study a novel and underexplored topic in the tau field, tau envelopes, and investigate their relevance to Alzheimer's disease pathology.

(2) Experiments are well conducted and of high quality.

Weaknesses:

Relying only on in vitro reconstituted microtubules and human neurons derived from IPSCs leaves some doubts about the relevance of these results for Alzheimer's disease, considering the embryonic state of IPSCs-derived neurons.

Reviewer #2 (Public review):

This manuscript examines how disease-associated hyperphosphorylation disrupts tau's role as a cooperative microtubule-binding regulator of intracellular transport. Using in vitro reconstitution assays and live-cell imaging in iPSC-derived neurons, the authors employ phosphomutant tau constructs (E14 to mimic hyperphosphorylation, AP to prevent phosphorylation) at 14 disease-associated residues to isolate phosphorylation effects independent of expression system-dependent PTM heterogeneity. The results show that hyperphosphorylated tau fails to form cooperative envelope-like structures on microtubules, instead binding diffusely and dissociating rapidly. In contrast, wild-type and phospho-resistant tau form cohesive envelopes that regulate motor protein access. At the single-molecule level, hyperphosphorylation reduces KIF5C inhibition while maintaining or enhancing KIF1A inhibition through altered processivity and detachment rates. In live neurons, hyperphosphorylated tau phenocopies tau knockout conditions, weakening tau-mediated inhibition of lysosome transport and increasing processive motility. The authors quantify tau binding using Gaussian mixture model-based image analysis and measure tau kinetics via FRAP, demonstrating that hyperphosphorylation-induced loss of cooperative binding correlates with dysregulated organelle transport. These findings establish a mechanism by which phosphorylation-driven disruption of tau's gatekeeper function on microtubules compromises axonal transport prior to aggregation in tauopathies. The paper provides interesting new knowledge for the field, but there are outstanding concerns that could be further addressed by the authors to strengthen and clarify the current manuscript:

(1) Lack of Phosphatase-Treated Control and Explicit WT Phosphorylation Quantification

Wild-type tau expressed in insect and mammalian cells is known to be phosphorylated by endogenous kinases (eg, GSK3, CDK5, MARK). The manuscript acknowledges this in the Discussion but provides no phosphatase-treated lysate control or quantification of endogenous phosphorylation on WT tau via phospho-specific Western blots. This leaves ambiguity about whether observed differences between WT and E14 reflect purely the introduced mutations or confounding baseline differences in phosphostate content.

(2) Limited Normalization of Motor Effects to Measured Tau Lattice Occupancy

Although kinesin trajectories are classified inside vs. outside tau envelopes (inherently normalizing to local tau density), motor parameters are not systematically reported as functions of tau fluorescence intensity across all constructs. Co-purifying MAPs or microtubule-modifying enzymes in cell lysates is not quantified or excluded, leaving residual uncertainty about tau-specificity of observed motor inhibition. This should be at least acknowledged in the results section.

(3) Insufficient Citation of Prior Neuronal Tau Envelope Evidence

In the Introduction, the authors state, "it was an open question if tau forms envelopes in neurons," but this understates existing evidence. Tan et al. (2019) report tau neuronal staining consistent with envelope formation, while Siahaan et al. (2021) provide more direct evidence in non-neuronal cells. The framing should acknowledge and integrate these prior findings.

(4) Unclear Wording on Expression System-Dependent Phosphorylation

The sentence "The phosphostate of tau is strongly dependent on the expression system" requires rewording. It is ambiguous whether this refers to the final phosphostate achieved after expression or the inherent phosphorylating capacity of each system. Clearer language would strengthen the methodological justification.

(5) Insufficient Quantification of Motor and Lysosome Transport Effect Magnitudes in Results Section

The data on molecular motor motility and lysosome transport are densely described. The magnitude of effects (fold-changes, percentage differences) should be explicitly stated in the Results section when first presenting findings to orient readers to biological significance. For example, effect magnitudes for lysosome run lengths, velocities, and directional bias should be quantified in text, not left to figure inspection.

(6) Incomplete Discussion of Projection Domain Necessity for Envelope Formation

The Discussion states the projection domain is "a critical regulator of both tau-tau and tau-microtubule interactions," but does not engage with prior domain dissection work. Tan et al. (2019) found that the entire projection domain is not necessary for envelope formation in vitro. The authors should discuss which projection domain regions are specifically regulated by phosphorylation vs. required for cooperativity, providing a more nuanced interpretation than implied by their current framing.

Author response:

We thank the reviewers for their thoughtful and constructive feedback. Addressing these points will strengthen the manuscript and improve its clarity.

A primary concern involved the justification for using COS7 cell lysates in reconstitution approaches and iPSC-derived neuronal model systems as models for AD. We will clarify the language throughout the manuscript to more explicitly state the study’s goals, emphasize that these systems were selected as robust, well-controlled platforms to test the mechanisms through which tau hyperphosphorylation affects microtubule interactions and tau’s role in regulating intracellular transport, and the limitations of in vitro and iPSC models.

Reviewers also raised the possibility that background phosphorylation could contribute to the effects observed in the pseudo-phosphorylation model. We cite two recent preprints that provide insight into this question through quantitatively assessing tau phosphorylation across expression systems. In the revised manuscript, we will elaborate on how their assessment of tau phosphorylation fits within the scope of our approach and clarify how our experimental controls effectively minimize uncertainty related to background phosphorylation.

Another point concerned the potential influence of other microtubule-associated proteins in lysates and the impact of tau lattice occupancy on motility outcomes. To further strengthen this aspect, we will include additional analyses correlating tau intensity along microtubules with kinesin intensity and motility behavior, and we will more clearly explain how the AP and WT controls provide confidence in the robustness of the system.

Detailed responses to each reviewer comment are provided below point by point. The planned revisions, which include clearer language, stronger justification of the experimental approaches, and additional supporting analyses, will substantially improve the clarity, rationale, and overall impact of the study.

Public Reviews:

Reviewer #1 (Public review):

Summary:

This work by Beaudet and colleagues aims at exploring the effect of phosphorylation on the formation of tau envelopes and consequently on axonal transport, both in vitro on reconstituted microtubules and in human excitatory neurons derived from IPSCs.

The authors found that a relatively widely used construct in which 14 serine or threonine residues, often hyperphosphorylated in Alzheimer's disease, are mutated to alanines (phosphodeficient), increases the density of tau envelopes compared to wildtype tau, whereas a phosphomimetic (same residues mutated to glutamic acid) reduces envelope density both in vitro and in human excitatory neurons derived from IPSCs.

By analysing the trafficking of different kinesins (KIF1a and KIF5C), they observed different effects of tau phosphorylation status on the movement of these two motors.

They then analyse transport of lysosomes by employing live imaging of lysotracker in human excitatory neurons derived from IPSCs transfected with wildtype, phosphodeficient or phosphomimetic tau, observing that phosphodeficient tau seems to reduce transport of lysosomes while phosphomimetic increases transport compared to wildtype tau.

Strengths:

(1) The work aims to study a novel and underexplored topic in the tau field, tau envelopes, and investigate their relevance to Alzheimer's disease pathology.

(2) Experiments are well conducted and of high quality.

Weaknesses:

Relying only on in vitro reconstituted microtubules and human neurons derived from IPSCs leaves some doubts about the relevance of these results for Alzheimer's disease, considering the embryonic state of IPSCs-derived neurons.

We agree with the reviewer that iPSC-derived neurons represent an immature state compared with the neurons affected in Alzheimer’s disease. However, iPSC-derived neurons, together with in vitro reconstitution, provide insight into (1) whether tau hyperphosphorylation influences its association with microtubules and its ability to form envelope-like structures thought to regulate transport, (2) how tau hyperphosphorylation affects the motility of kinesin motors that are strongly inhibited by tau, and (3) how transport of endogenous degradative organelles such as lysosomes are impacted by tau hyperphosphorylation. We hope that our studies will help to inform future studies examining how tau-related dysfunction evolves in more mature neurons and contributes to the more severe pathological effects observed at later disease stages.

We will include a paragraph in the Discussion section addressing the limitations of this study to better contextualize our findings within the broader effort to understand tauopathies and Alzheimer’s disease.

Reviewer #2 (Public review):

This manuscript examines how disease-associated hyperphosphorylation disrupts tau's role as a cooperative microtubule-binding regulator of intracellular transport. Using in vitro reconstitution assays and live-cell imaging in iPSC-derived neurons, the authors employ phosphomutant tau constructs (E14 to mimic hyperphosphorylation, AP to prevent phosphorylation) at 14 disease-associated residues to isolate phosphorylation effects independent of expression system-dependent PTM heterogeneity. The results show that hyperphosphorylated tau fails to form cooperative envelope-like structures on microtubules, instead binding diffusely and dissociating rapidly. In contrast, wild-type and phospho-resistant tau form cohesive envelopes that regulate motor protein access. At the single-molecule level, hyperphosphorylation reduces KIF5C inhibition while maintaining or enhancing KIF1A inhibition through altered processivity and detachment rates. In live neurons, hyperphosphorylated tau phenocopies tau knockout conditions, weakening tau-mediated inhibition of lysosome transport and increasing processive motility. The authors quantify tau binding using Gaussian mixture model-based image analysis and measure tau kinetics via FRAP, demonstrating that hyperphosphorylation-induced loss of cooperative binding correlates with dysregulated organelle transport. These findings establish a mechanism by which phosphorylation-driven disruption of tau's gatekeeper function on microtubules compromises axonal transport prior to aggregation in tauopathies. The paper provides interesting new knowledge for the field, but there are outstanding concerns that could be further addressed by the authors to strengthen and clarify the current manuscript:

(1) Lack of Phosphatase-Treated Control and Explicit WT Phosphorylation Quantification

Wild-type tau expressed in insect and mammalian cells is known to be phosphorylated by endogenous kinases (eg, GSK3, CDK5, MARK). The manuscript acknowledges this in the Discussion but provides no phosphatase-treated lysate control or quantification of endogenous phosphorylation on WT tau via phospho-specific Western blots. This leaves ambiguity about whether observed differences between WT and E14 reflect purely the introduced mutations or confounding baseline differences in phosphostate content.

Tau contains ~85 putative phosphorylation sites and is modified by several kinases in cells. Studies by Siahaan et al. (2024) and Fan et al. (2025) provide detailed insight into tau phosphorylation, its role in protecting the microtubule lattice from severing enzymes, and the implications of phosphorylation patterns for aggregate formation. Specifically, Fan et al. (2025) show that HEK-expressed tau is phosphorylated by endogenous kinases at 58 residues, with most phospho-occupancy levels below 15%, indicating substantial heterogeneity among individual tau molecules. In the revised manuscript, we will (1) provide justification for the use of the pseudo-phosphorylation model system as an approach to limit heterogeneity among tau molecules, (2) clarify the importance of the WT and AP controls, (3) discuss that E14, WT, and AP tau likely exhibit similar degrees of background phospho-heterogeneity, with WT tau likely exhibiting some overlap between background phosphorylation and the 14 AD-associated sites examined, and (4) expand the discussion to emphasize that although background phosphorylation is present, our results do not suggest that it contributes significantly to the observations reported in this study.

(2) Limited Normalization of Motor Effects to Measured Tau Lattice Occupancy

Although kinesin trajectories are classified inside vs. outside tau envelopes (inherently normalizing to local tau density), motor parameters are not systematically reported as functions of tau fluorescence intensity across all constructs. Co-purifying MAPs or microtubule-modifying enzymes in cell lysates is not quantified or excluded, leaving residual uncertainty about tau-specificity of observed motor inhibition. This should be at least acknowledged in the results section.

The reviewer raises a valid point. It is challenging to compare conditions where the occupancy of tau on microtubules is similar across conditions, as phosphorylation strongly effects the interaction between tau and microtubules. We will quantify and report tau intensity in single-molecule motility assays. On the second point, while effects from other MAPs or motor proteins could potentially affect kinesin motility, we would expect that these effects would be similar for all tau phosphomutant constructs, such that the effect of tau phospho-states on kinesin motility can be assessed.

(3) Insufficient Citation of Prior Neuronal Tau Envelope Evidence

In the Introduction, the authors state, "it was an open question if tau forms envelopes in neurons," but this understates existing evidence. Tan et al. (2019) report tau neuronal staining consistent with envelope formation, while Siahaan et al. (2021) provide more direct evidence in non-neuronal cells. The framing should acknowledge and integrate these prior findings.

We agree with the reviewer that evidence from several studies using reconstitution systems, fixed neurons, and live cultured cells provides evidence of tau envelope formation in neurons. Specifically, tau envelopes have been observed along taxol-stabilized or GMPCPP-capped GDP microtubules in vitro (e.g., Dixit et al., 2008; Monroy et al., 2018; Tan et al., 2019; Siahaan et al., 2019), in 4% PFA-fixed and Triton X-100–extracted DIV7 mouse hippocampal neurons (Tan et al., 2019), and in live, non-neuronal U-2 OS cells following taxol treatment (Siahaan et al., 2022) or elevated pH (Siahaan et al., 2024). However, to our knowledge, our study is the first to demonstrate tau envelope formation in live neuronal cells under normal cell culture conditions. We will revise this sentence in the manuscript to more precisely position our findings within the context of prior studies.

(4) Unclear Wording on Expression System-Dependent Phosphorylation

The sentence "The phosphostate of tau is strongly dependent on the expression system" requires rewording. It is ambiguous whether this refers to the final phosphostate achieved after expression or the inherent phosphorylating capacity of each system. Clearer language would strengthen the methodological justification.

We agree that the wording here is ambiguous and requires clarification. In the revised manuscript, we will clarify that tau phosphorylation depends on the expression system used; bacterial systems lack the capacity for many post-translational modifications compared with insect and mammalian systems. We will also emphasize that in insect and mammalian expression systems, tau phosphorylation occurs heterogeneously, as demonstrated in previous studies by Siahaan et al. (2024) and Fan et al. (2025).

(5) Insufficient Quantification of Motor and Lysosome Transport Effect Magnitudes in Results Section

The data on molecular motor motility and lysosome transport are densely described. The magnitude of effects (fold-changes, percentage differences) should be explicitly stated in the Results section when first presenting findings to orient readers to biological significance. For example, effect magnitudes for lysosome run lengths, velocities, and directional bias should be quantified in text, not left to figure inspection.

Our initial justification for omitting quantitative data from the results text was to improve readability; however, in doing so, we may have reduced the accessibility and clarity regarding the significance of the findings. In the revised manuscript, we will incorporate the relevant quantifications and statistical significance for the motility data in the text.

(6) Incomplete Discussion of Projection Domain Necessity for Envelope Formation

The Discussion states the projection domain is "a critical regulator of both tau-tau and tau-microtubule interactions," but does not engage with prior domain dissection work. Tan et al. (2019) found that the entire projection domain is not necessary for envelope formation in vitro. The authors should discuss which projection domain regions are specifically regulated by phosphorylation vs. required for cooperativity, providing a more nuanced interpretation than implied by their current framing.

We agree with the reviewer. Tan et al. (2019) demonstrated that the proline-rich region (residues 198–244) within the projection domain of full-length 2N4R tau is the minimal region required to maintain tau’s ability to form envelopes along microtubules. We will incorporate this work on the dissection of the projection domain and discuss how the phosphorylation sites examined in our study are primarily located within this region. Together, these data highlight the proline-rich region as a potential major regulator of tau–tau cooperativity.