Failure of DNA double-strand break repair by tau mediates Alzheimer's disease pathology in vitro

Electron microscopy. The cultures were fixed with 4% paraformaldehyde in

phosphate buffer saline (PBS) for 8 h., washed thrice with PBS, and permeabilized

with 0.1% Triton-X100 and 1% bovine serum albumin in PBS for 10 mins. After

rinsing thrice with PBS, the cultures were incubated in 1% bovine serum albumin

in PBS for 30 mins and in primary antibody solution (anti-Tau-1 ab, 1:2000, 1%

bovine serum albumin in PBS) for 8 h After rinsing thrice with PBS again, the

cultures were incubated in secondary antibody solution (1:100, Nanogold,

Nanoprobes Inc, NY, USA, with 1% bovine serum albumin in PBS) for 8 h., rinsed

with PBS and enhanced with silver acetate solution for 12 mins67. Following this,

the cultures were rinsed thrice with DW, immersed in 0.05% sodium acetate for

1 min, rinsed thrice with DW again, immersed in 0.05% gold chloride solution for

2 mins, and rinsed with DW once more. The cultures were postfixed with 0.1%

osmium tetroxide in PBS for 30 mins and rinsed with DW three times. After

dehydration of the cultures with graded ethanol and embedding in Epon 812

(TAAB Laboratories, UK), the culture dish was removed, and ultrathin sections

(70 nm) were cut parallelly to the cell layers using an ultra-microtome (ReichertJung). Sections on 150 square-mesh grids covered with formvar were stained with

uranyl acetate, followed by lead citrate, and examined under an electron microscope (JEOL, Japan) at 80 kV.

2.

shRNA knockdown of mouse tau. We used MISSION Lentiviral Transduction

Particles to knockdown mouse tau according to the target sequences defined in

Sigma-Aldrich (SHCLNV, NM_010838, Clone ID TRCN0000091300) and MISSION shRNA non-target shRNA control transduction particles (SHC016V-1EA).

Lentiviral particles were transduced 10 μl of 1.4 × 107 VP/mL on day 0 of primary

mouse cortical neuron, which was cultured for seven days.

Comet assay. Primary neurons treated with etoposide were performed according

to Trivigen’s instructions (Trevigen CometAssay, #4250-050-K). Cell susupensions

(1 × 105 cells / ml) in PBS mixed at at a1:10 ratio with Comet LMA agarose

(Trevigen) were pitetted onto CometSlidesTM (Trevigen) and placed at 4 °C in the

dark in the dark. Slides were immersed overnight in Lysis Solution (Trivegen) at

4 °C in the dark and then in Alkaline Unwinding solution (200 mM NaOH, 1 mM

EDTA, pH13) for 1 hr at 4 °C. Slides were electrophoresed in Alkaline Electrophoresis solution (300 mM NaOH, 1 mM EDTA, pH13) for 40 mins at 1 V/ cm,

300 mA, at 4 °C. Slides were incubated in 70% EtOH for 5 mins, dry at 37 °C and

were stained with 1 × SYBR Gold (invitrogen) at room temperature for 30 mins in

the dark. Comets were visualized on BIOREVO BZ-9000 (KEYENCE) and scored

using OpenComet (imageJ).

Statistics and reproducibility. For most analyses, data is shown as the mean ±

standard error (SEM) from 2 to 4 independent experiments. Results were analyzed

using one-way or two-way ANOVA, Tukey-Kramer HSD test, and Student’s t test. The

statistical analyses were performed using JMP® 16 (SAS Institute Inc., Cary, NC, USA).

The level of significance was set at P < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, and

ns = P ≥ 0.05). The making graph were used by GraphPad Prism (GraphPad software

9.0 (https://www.graphpad.com/scientific-software/prism) and JMP® 16 (SAS Institute

Inc., Cary, NC, USA).

Ethics approval and consent to participate. The human samples were taken for

the project ‘Pathological and biochemical studies of neurodegenerative diseases

using human autopsy brain and spinal cord’ (No. R1038) by the Kyoto University

Ethics Committee. Informed and written consents were obtained from all individuals or their guardians, before the autopsy analysis according to the Declaration of

Helsinki. All animal experiments were approved by the Animal Care and Use

Committee of Shiga University of Medical Science (2020-5-8) and Kyoto University of Medical Science (Med Kyo 20017).

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Reporting summary. Further information on research design is available in the Nature

Research Reporting Summary linked to this article.

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Data availability

27.

All data in this study is available at Mendeley Data with https://doi.org/10.17632/

cy5y2wncbz.1. Uncut Western blot images are provided in Supplementary Figures. All

relevant data including the numerical and statistical source data that underlie the graphs

in figures are provided as Supplementary Data 1(as an Excel file). Imformation of human

brain samples is provided in Suppl. Tabel 1.

28.

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Received: 6 June 2021; Accepted: 24 March 2022;

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Acknowledgements

We thank Prof. S. Takeda (Kyoto University) for DSB induction method, K. Asamoto

(Kyoto University) for immunohistochemistry and K. Tanigawa (Kyoto University) for

creating graphs. We thank the Central Research Laboratory, Shiga University of Medical

Science (CRL), and the Medical Research Support Center, Kyoto University Graduate

School of Medicine, for technical support. This work was supported by the intramural

research grant of Shiga University of Medical Science and by a research fund from Kim’s

Korean Ginseng CO. LTD.

Author contributions

M.A.-U. and M.U. gratefully contributed to the study design. M.A.-U. performed

experiments, analyzed data, and wrote a first draft of the manuscript. M.T.U., S.M., R.H.,

T.M., R.T., and A.K. gave critical advice throughout the experiments. T.A. contributed to

human immunohistochemistry. A.S. contributed to primary mouse cortical neuron

culture. T.U. conducted serial section transmission electron microscopy analysis. M.U.

and U.K. contributed to study concept and edited the manuscript. All authors read and

approved the final manuscript.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information The online version contains supplementary material

available at https://doi.org/10.1038/s42003-022-03312-0.

Correspondence and requests for materials should be addressed to Makoto Urushitani.

Peer review information Communications Biology thanks Mohammad Moshahid Khan,

Luca Colnaghi and the other, anonymous, reviewer(s) for their contribution to the peer

review of this work. Primary Handling Editors: Krishnananda Chattopadhyay and

Manuel Breuer. Peer reviewer reports are available.

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