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  1. Author Response

    Reviewer 1

    Employing in vitro and Drosophila model, the authors interrogate which domain of Hsp27 binds to which region on Tau, and how these interactions facilitate the proteinaceous aggregation. They utilized various biochemical, biophysical, cellular, and genetic tools to dissect the association, and identified the structural basis for the specific recognition of Hsp27 to pathogenic p-Tau. Conceivably, Hsp27 may play some role in preventing Tau abnormal aggregation and p-Tau pathology in AD. Overall, the data support the main claim, especially, the biophysical data are very impressive. Nevertheless, the manuscript could be strengthened by complementary cellular or biochemical methods for validation. For example, the authors can use a stably transfected Tau cell line to interrogate Hsp27's role in its cellular aggregation or proteinaceous inclusions by immunoblotting. Immunofluorescent and immunohistochemical staining and IB with different antibodies may be conducted to validate the observations.

    REPLY: We sincerely thank the reviewer for the positive assessment of our work, and for providing very insightful suggestions. We appreciate the reviewer for considering our biophysical data to be impressive. We totally agree with the reviewer that the work could be strengthened by complementary cellular methods for validation. In our work, we used the Drosophila tauopathy model, where expression of human TauR406W in the Drosophila nervous system leads to age-dependent neurodegeneration recapitulating some of the salient features of tauopathy in FTDP-171,2, to interrogate the role of Hsp27 in aggregation and proteinaceous inclusions of pTau.

    In our Drosophila Tau model study, three different antibodies including a total Tau antibody 5A63, a pTauSer262 specific antibody4, and a hyper-phosphorylated Tau antibody AT8 that recognizes hyper-phosphorylation of Tau at Ser202 and Thr205 sites5 were used in western blot analysis to explore the role of Hsp27. As shown in Figure R1-1A and 1B, overexpression of Hsp27 significantly reduced the level of both pTauSer262 and hyper-phosphorylated Tau at both 2 and 10 days after eclosion (DAE). In addition, we further examined the morphology of the fly brain as well as the accumulation of hyper-phosphorylated Tau by immunofluorescence staining. Consistent with previous findings, brains with neuronal expression of TauR406W exhibited an accumulation of filamentous pTau and a reduction of brain neuropil size indicative of neurodegeneration (Figure R11C-F). Importantly, overexpression of Hsp27 restored the size of brain neuropil and suppressed the accumulation of filamentous pTau (Figure R1-1C-F), suggesting that Hsp27 protects against mutant TauR406W - induced neurodegeneration. Taken together, our Drosophila results show that Hsp27 protects against synaptic dysfunction in a Drosophila tauopathy model by reducing pTau aggregation, which well supports our biophysical data.

    Figure R1-1 Hsp27 reduces pTau level and protects against pTau-induced synaptopathy in Drosophila. (This figure represents Fig. 2A-F in the revised manuscript) (A) Brain lysates of 2 and 10 days after eclosion (DAE) wild-type (WT) flies (lanes 1 and 6), flies expressing human Tau with GFP (lanes 4 and 9), or human Tau with Hsp27 (lanes 5 and 10) in the nervous system were probed with antibodies for disease-associated phospho-tau epitopes S262, Ser202/Thr205 (AT8), and total Tau (5A6). Actin was probed as a loading control. Brain lysates of flies carrying only UAS elements were loaded for control (lanes 2, 3, 7, and 8). (B) Quantification of protein fold changes in (A). The levels of Tau species were normalized to actin. Fold changes were normalized to the Tau+GFP group at 2 DAE. n = 3. (C) Brains of WT flies or flies expressing Tau+GFP or Tau+Hsp27 in the nervous system at 2 DAE were probed for AT8 (heatmap) and Hsp27 (green), and stained with DAPI (blue). Scale bar, 30 μm. (D-F) Quantification of the Hsp27 intensity (D, data normalized to WT), brain optic lobe size (E), and AT8 intensity (F, data normalized to the Tau+GFP group). n = 4.

    Reviewer 2

    Abnormal accumulation and aggregation of amyloid-β protein are one of the main pathological hallmarks of Alzheimer's disease. It is well known that molecular chaperones play central roles in regulating tau function and amyloid assembly in disease. In this manuscript, Zhang, Zhu, Lu, Liu, et al., have investigated that Hsp27, a member of the small heat shock protein, specifically binds to phosphorylated Tau, which prevents pTau fibrillation in vitro and in a Drosophila tauopathy model. Using NMR spectroscopy and cross-linking mass spectrometry, the authors found that the N-terminal domain of Hsp27 directly binds to phosphorylation sites of pTau. Overall, the study is important and provides the demonstration of interactions between Hsp27 and pTau.

    REPLY: We sincerely thank the reviewer for the positive remarks of this work, and appreciate that the reviewer summarizes the major conclusions of our manuscript, and evaluates our work is important in the area of fundamental biology of the interaction between chaperones and clients, and its implications in AD pathology.

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  2. Evaluation Summary:

    The authors found elevated Hsp 27 levels in brains from Alzheimer disease patients. Hsp27 co-localized with p-Tau, efficiently prevented pTau fibrillation in vitro, and mitigated neuropathology of pTau aggregation in a Drosophila tauopathy model. A series of biochemical assays is presented to supporrt the claim that Hsp27 prevents abnormal Tau aggregation and p-Tau pathology. Overall, the study is well designed and presented, and the data convincingly support this major conclusion, which is relevant to colleagues studying neurodegeneration.

    (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. Reviewer #1 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    Employing in vitro and Drosophila model, the authors interrogate which domain of Hsp27 binds to which region on Tau, and how these interactions facilitate the proteinaceous aggregation. They utilized various biochemical, biophysical, cellular, and genetic tools to dissect the association, and identified the structural basis for the specific recognition of Hsp27 to pathogenic p-Tau. Conceivably, Hsp27 may play some role in preventing Tau abnormal aggregation and p-Tau pathology in AD. Overall, the data support the main claim, especially, the biophysical data are very impressive. Nevertheless, the manuscript could be strengthened by complementary cellular or biochemical methods for validation. For example, the authors can use a stably transfected Tau cell line to interrogate Hsp27's role in its cellular aggregation or proteinaceous inclusions by immunoblotting. Immunofluorescent and immunohistochemical staining and IB with different antibodies may be conducted to validate the observations.

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  4. Reviewer #2 (Public Review):

    Abnormal accumulation and aggregation of amyloid-β protein are one of the main pathological hallmarks of Alzheimer's disease. It is well known that molecular chaperones play central roles in regulating tau function and amyloid assembly in disease. In this manuscript, Zhang, Zhu, Lu, Liu, et al., have investigated that Hsp27, a member of the small heat shock protein, specifically binds to phosphorylated Tau, which prevents pTau fibrillation in vitro and in a Drosophila tauopathy model. Using NMR spectroscopy and cross-linking mass spectrometry, the authors found that the N-terminal domain of Hsp27 directly binds to phosphorylation sites of pTau.

    Overall, the study is important and provides the demonstration of interactions between Hsp27 and pTau.

    Read the original source
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