Prefoldin 5 is a microtubule-associated protein that suppresses Tau-aggregation and neurotoxicity

Reviewer #1 (Public review):

Summary:

In this manuscript, Bisht et al address the hypothesis that protein folding chaperones may be implicated in aggregopathies and in particular Tau aggregation, as a means to identify novel therapeutic routes for these largely neurodegenerative conditions.

The authors conducted a genetic screen in the Drosophila eye, which facilitates the identification of mutations that either enhance or suppress a visible disturbance in the nearly crystalline organization of the compound eye. They screened by RNA interference all 64 known Drosophila chaperones and revealed that mutations in 20 of them exaggerate the Tau-dependent phenotype, while 15 ameliorated it. The enhancer of the degeneration group included 2 subunits of the typically heterohexameric prefoldin complex and other co-translational chaperones.

The authors characterized in depth one of the prefoldin subunits, Pfdn5, and convincingly demonstrated that this protein functions in the regulation of microtubule organization, likely due to its regulation of proper folding of tubulin monomers. They demonstrate convincingly using both immunohistochemistry in larval motor neurons and microtubule binding assays that Pfdn5 is a bona fide microtubule-associated protein contributing to the stability of the axonal microtubule cytoskeleton, which is significantly disrupted in the mutants.

Similar phenotypes were observed in larvae expressing Frontotemporal dementia with Parkinsonism on chromosome 17-associated mutations of the human Tau gene V377M and R406W. On the strength of the phenotypic evidence and the enhancement of the TauV377M-induced eye degeneration, they demonstrate that loss of Pfdn5 exaggerates the synaptic deficits upon expression of the Tau mutants. Conversely, the overexpression of Pfdn5 or Pfdn6 ameliorates the synaptic phenotypes in the larvae, the vacuolization phenotypes in the adult, and even memory defects upon TauV377M expression.

Strengths:

The phenotypic analyses of the mutant and its interactions with TauV377M at the cell biological, histological, and behavioral levels are precise, extensive, and convincing and achieve the aims of characterization of a novel function of Pfdn5.

Regarding this memory defect upon V377M tau expression. Kosmidis et al (2010) pmid: 20071510, demonstrated that pan-neuronal expression of TauV377M disrupts the organization of the mushroom bodies, the seat of long-term memory in odor/shock and odor/reward conditioning. If the novel memory assay the authors use depends on the adult brain structures, then the memory deficit can be explained in this manner.

If the mushroom bodies are defective upon TauV377M expression does overexpression of Pfdn5 or 6 reverse this deficit? This would argue strongly in favor of the microtubule stabilization explanation.

The discovery that Pfdn5 (and 6 most likely) affect tauV377M toxicity is indeed a novel and important discovery for the Tauopathies field. It is important to determine whether this interaction affects only the FTDP-17-linked mutations, or also WT Tau isoforms, which are linked to the rest of the Tauopathies. Also, insights on the mode(s) that Pfdn5/6 affect Tau toxicity, such as some of the suggestions above are aiming at, will likely be helpful towards therapeutic interventions.

Weaknesses:

What is unclear however is how Pfdn5 loss or even overexpression affects the pathological Tau phenotypes.

Does Pfdn5 (or 6) interact directly with TauV377M? Colocalization within tissues is a start, but immunoprecipitations would provide additional independent evidence that this is so.

Does Pfdn5 loss exacerbate TauV377M phenotypes because it destabilizes microtubules, which are already at least partially destabilized by Tau expression?
Rescue of the phenotypes by overexpression of Pfdn5 agrees with this notion.

However, Cowan et al (2010) pmid: 20617325 demonstrated that wild-type Tau accumulation in larval motor neurons indeed destabilizes microtubules in a Tau phosphorylation-dependent manner.

So, is TauV377M hyperphosphorylated in the larvae?? What happens to TauV377M phosphorylation when Pfdn5 is missing and presumably more Tau is soluble and subject to hyperphosphorylation as predicted by the above?

Expression of WT human Tau (which is associated with most common Tauopathies other than FTDP-17) as Cowan et al suggest has significant effects on microtubule stability, but such Tau-expressing larvae are largely viable. Will one mutant copy of the Pfdn5 knockout enhance the phenotype of these larvae?? Will it result in lethality? Such data will serve to generalize the effects of Pfdn5 beyond the two FDTP-17 mutations utilized.

Does the loss of Pfdn5 affect TauV377M (and WTTau) levels?? Could the loss of Pfdn5 simply result in increased Tau levels? And conversely, does overexpression of Pfdn5 or 6 reduce Tau levels?? This would explain the enhancement and suppression of TauV377M (and possibly WT Tau) phenotypes. It is an easily addressed, trivial explanation at the observational level, which if true begs for a distinct mechanistic approach.

Finally, the authors argue that TauV377M forms aggregates in the larval brain based on large puncta observed especially upon loss of Pfdn5. This may be so, but protocols are available to validate this molecularly the presence of insoluble Tau aggregates (for example, pmid: 36868851) or soluble Tau oligomers as these apparently differentially affect Tau toxicity. Does Pfdn5 loss exaggerate the toxic oligomers and overexpression promotes the more benign large aggregates??

Reviewer #2 (Public review):

Bisht et al detail a novel interaction between the chaperone, Prefoldin 5, microtubules, and tau-mediated neurodegeneration, with potential relevance for Alzheimer's disease and other tauopathies. Using Drosophila, the study shows that Pfdn5 is a microtubule-associated protein, which regulates tubulin monomer levels and can stabilize microtubule filaments in the axons of peripheral nerves. The work further suggests that Pfdn5/6 may antagonize Tau aggregation and neurotoxicity. While the overall findings may be of interest to those investigating the axonal and synaptic cytoskeleton, the detailed mechanisms for the observed phenotypes remain unresolved and the translational relevance for tauopathy pathogenesis is yet to be established. Further, a number of key controls and important experiments are missing that are needed to fully interpret the findings.

The strength of this study is the data showing that Pfdn5 localizes to axonal microtubules and the loss-of-function phenotypic analysis revealing disrupted synaptic bouton morphology. The major weakness relates to the experiments and claims of interactions with Tau-mediated neurodegeneration. In particular, it is unclear whether knockdown of Pfdn5 may cause eye phenotypes independent of Tau. Further, the GMR>tau phenotype appears to have been incorrectly utilized to examine age-dependent, neurodegeneration.

This manuscript argues that its findings may be relevant to thinking about mechanisms and therapies applicable to tauopathies; however, this is premature given that many questions remain about the interactions from Drosophila, the detailed mechanisms remain unresolved, and absent evidence that tau and Pfdn may similarly interact in the mammalian neuronal context. Therefore, this work would be strongly enhanced by experiments in human or murine neuronal culture or supportive evidence from analyses of human data.

Author response:

Reviewer #1:

Summary:
In this manuscript, Bisht et al address the hypothesis that protein folding chaperones may be implicated in aggregopathies and in particular Tau aggregation, as a means to identify novel therapeutic routes for these largely neurodegenerative conditions.

The authors conducted a genetic screen in the Drosophila eye, which facilitates the identification of mutations that either enhance or suppress a visible disturbance in the nearly crystalline organization of the compound eye. They screened by RNA interference all 64 known Drosophila chaperones and revealed that mutations in 20 of them exaggerate the Tau-dependent phenotype, while 15 ameliorated it. The enhancer of the degeneration group included 2 subunits of the typically heterohexameric prefoldin complex and other co-translational chaperones.

In a previous paper, we identified 95 Drosophila chaperones (Raut et al., 2017). We request that “all 64 known Drosophila chaperones” be replaced with “64 out of 95 known Drosophila chaperones” to make it factually correct.

Strengths:

Regarding this memory defect upon V377M tau expression. Kosmidis et al (2010) pmid: 20071510, demonstrated that pan-neuronal expression of TauV377M disrupts the organization of the mushroom bodies, the seat of long-term memory in odor/shock and odor/reward conditioning. If the novel memory assay the authors use depends on the adult brain structures, then the memory deficit can be explained in this manner.

If the mushroom bodies are defective upon TauV377M expression does overexpression of Pfdn5 or 6 reverse this deficit? This would argue strongly in favor of the microtubule stabilization explanation.

We agree that the disruptive organization of the mushroom body may cause memory deficits upon hTauV337M expression and that expression of Pfdn5 or Pfdn6 could reverse the deficits. One possible mechanism by which overexpression of Pfdn5/6 could rescue the Tau-induced memory deficits may be due to the stabilization of microtubules in the mushroom bodies.

Proposed revision: We will assess if Tau-induced mushroom body disruption can be rescued with the overexpression of Pfdn5 or Pfdn6.

Weakness:

What is unclear however is how Pfdn5 loss or even overexpression affects the pathological Tau phenotypes. Does Pfdn5 (or 6) interact directly with TauV377M? Colocalization within tissues is a start, but immunoprecipitations would provide additional independent evidence that this is so.

Our data suggests that Pfdn5 stabilizes neuronal microtubules by directly associating with it, and loss of Pfdn5 exacerbates Tau-phenotypes by destabilizing microtubules. However, as the reviewer notes, analysis of direct interaction between Pfdn5 and hTau^V337M might provide further insights into the mechanism of Pfdn5 and Tau-aggregation.

Proposed revision: We will perform colocalization analysis and coimmunoprecipitation to ask if Pfdn5 colocalizes and directly interacts with Tau.

Does Pfdn5 loss exacerbate TauV377M phenotypes because it destabilizes microtubules, which are already at least partially destabilized by Tau expression? Rescue of the phenotypes by overexpression of Pfdn5 agrees with this notion.

However, Cowan et al (2010) pmid: 20617325 demonstrated that wild-type Tau accumulation in larval motor neurons indeed destabilizes microtubules in a Tau phosphorylation-dependent manner. So, is TauV377M hyperphosphorylated in the larvae?? What happens to TauV377M phosphorylation when Pfdn5 is missing and presumably more Tau is soluble and subject to hyperphosphorylation as predicted by the above?

Proposed revisions: We will overexpress Pfdn5 or Pfdn6 with hTau^V337M and ask if microtubule disruption caused by hTau^V337M is rescued. Further, we will analyze the phospho-Tau levels in controls and Pfdn5 mutant background.

Expression of WT human Tau (which is associated with most common Tauopathies other than FTDP-17) as Cowan et al suggest has significant effects on microtubule stability, but such Tau-expressing larvae are largely viable. Will one mutant copy of the Pfdn5 knockout enhance the phenotype of these larvae?? Will it result in lethality? Such data will serve to generalize the effects of Pfdn5 beyond the two FDTP-17 mutations utilized.

Proposed revision: We will incorporate data about the effect of heterozygous mutation of Pfdn5 on the lethality and synaptic phenotypes associated with the hTau^WT and hTau^V337M in the revised manuscript.

Does the loss of Pfdn5 affect TauV377M (and WTTau) levels?? Could the loss of Pfdn5 simply result in increased Tau levels? And conversely, does overexpression of Pfdn5 or 6 reduce Tau levels?? This would explain the enhancement and suppression of TauV377M (and possibly WT Tau) phenotypes. It is an easily addressed, trivial explanation at the observational level, which if true begs for a distinct mechanistic approach.

We thank the reviewer for suggesting an alternate model for the Pfdn5 function. We will perform the Western blot analysis to assess Tau^WT and Tau^V337M levels in the absence of Pfdn5 or animals coexpressing Tau and Pfdn5. We will incorporate these data and conclusions in the revised manuscript.

Finally, the authors argue that TauV377M forms aggregates in the larval brain based on large puncta observed especially upon loss of Pfdn5. This may be so, but protocols are available to validate this molecularly the presence of insoluble Tau aggregates (for example, pmid: 36868851) or soluble Tau oligomers as these apparently differentially affect Tau toxicity. Does Pfdn5 loss exaggerate the toxic oligomers and overexpression promotes the more benign large aggregates??

We will perform the Tau solubility assay in control, in the absence of Pfdn5 or animals coexpressing Tau and Pfdn5. Moreover, we will also ask if the large Tau puncta formed in the absence of Pfdn5 are soluble oligomers or stable aggregates. We have found that the coexpression of Tau and Pfdn5 does not result in the formation of Tau aggregates. We will incorporate these and other relevant data in the revised manuscript.

Reviewer #2 (Public review):

Bisht et al detail a novel interaction between the chaperone, Prefoldin 5, microtubules, and tau-mediated neurodegeneration, with potential relevance for Alzheimer's disease and other tauopathies. Using Drosophila, the study shows that Pfdn5 is a microtubule-associated protein, which regulates tubulin monomer levels and can stabilize microtubule filaments in the axons of peripheral nerves. The work further suggests that Pfdn5/6 may antagonize Tau aggregation and neurotoxicity. While the overall findings may be of interest to those investigating the axonal and synaptic cytoskeleton, the detailed mechanisms for the observed phenotypes remain unresolved and the translational relevance for tauopathy pathogenesis is yet to be established. Further, a number of key controls and important experiments are missing that are needed to fully interpret the findings.The major weakness relates to the experiments and claims of interactions with Tau-mediated neurodegeneration. In particular, it is unclear whether knockdown of Pfdn5 may cause eye phenotypes independent of Tau. Further, the GMR>tau phenotype appears to have been incorrectly utilized to examine age-dependent, neurodegeneration.

We have consistently found the progression of eye degeneration in the population of animals expressing Tau^V337M, measured as the number of fused ommatidia/total number of ommatidia, with age. A few other studies have also shown age-dependent progressive degeneration in Drosophila retinal axons or lamina (Iijima-Ando et al., 2012; Sakakibara et al., 2018). We appreciate other studies that have proposed hTau-induced eye degeneration as a developmental defect (Malmanche et al., 2017; Sakakibara et al., 2023).

Proposed revision: a) We will analyze the age-dependent neurodegeneration in the adult brain to further support our main conclusion that Pfdn5 ameliorates hTauV337M-induced progressive neurodegeneration.

b) We have used three independent Pfdn5 RNAi lines (the RNAi's target different regions of Pfdn5) – all of which enhance the Tau phenotypes. The knockdown of any of these RNAi lines with GMR-Gal4 does not give detectable eye phenotypes. We will include these data in the revised manuscript.

This manuscript argues that its findings may be relevant to thinking about mechanisms and therapies applicable to tauopathies; however, this is premature given that many questions remain about the interactions from Drosophila, the detailed mechanisms remain unresolved, and absent evidence that tau and Pfdn may similarly interact in the mammalian neuronal context. Therefore, this work would be strongly enhanced by experiments in human or murine neuronal culture or supportive evidence from analyses of human data.

Proteome analysis of Alzheimer's brain tissue shows that the Pfdn5 level is reduced in patients (Askenazi et al., 2023; Tao et al., 2020). Moreover, the Pfdn5 expression level was found to be reduced in the blood samples from AD patients (Ji et al., 2022). Another study further validates the age-dependent reduction of Pfdn5 in the tauopathy transgenic murine model (Kadoyama et al., 2019). Together, these reports highlight a potential link between Pfdn5 levels and tauopathies. We will revise the manuscript to reflect these findings in more detail.

References

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