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6-OHDA Induces Oxidation of F-box Protein Fbw7β by Chaperone-Mediated Autophagy in Parkinson’s Model

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Abstract

Parkinson’s disease (PD) is the most common movement disorder disease, and its pathological feature is the degenerative loss of dopaminergic neurons in the substantia nigra compacta (SNc). In this study, we investigated whether distinct stress conditions target F-box protein Fbw7β via converging mechanisms. Our results showed that the 6-hyroxydopamine (6-OHDA), which causes PD in animals’ models, led to decreased stability of Fbw7β in DA neuronal SN4741 cells. Further experiments suggested that oxidized Fbw7β bound to heat-shock cognate protein 70 kDa, the key regulator for chaperone-mediated autophagy (CMA), at a higher affinity. Oxidative stress also increased the level of lysosomal-associated membrane protein 2A (LAMP2A), the rate-limiting receptor for CMA substrate flux, and stimulated CMA activity. These changes resulted in accelerated degradation of Fbw7β. 6-OHDA induced Fbw7β oxidation and increased LAMP2A in the SNc region of the mouse models. Consistently, the levels of oxidized Fbw7β were higher in postmortem PD brains compared with the controls. These findings for the first time revealed the specific mechanism of ubiquitin ligases, oxidative stress, and CMA-mediated protein degradation, to provide a new theoretical basis for further clarifying the mechanism of PD.

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Acknowledgements

The authors thank Ms. Lin Zhang for her English proof reading. This project was supported by grant from the National Natural Science Foundation of China (81471377).

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Authors and Affiliations

  1. Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan, Hubei, 430000, China

    Xiufeng Wang, Heng Zhai & Fang Wang

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  1. Xiufeng Wang
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  2. Heng Zhai
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  3. Fang Wang
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Contributions

Xiufeng Wang, acquisition of data, literature research, and manuscript preparation; Heng Zhai, acquisition of data; Fang Wang, study concept and design, critical revision of the manuscript for important intellectual content, and study supervision.

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Correspondence to Fang Wang.

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Electronic Supplementary Material

Supplementary Fig. S1

The overexpression of Fbw7β was confirmed by western blot. SN4741 cells were inoculated into six-well plate to culture till 80% fusion and the pGCSIL vector encoding the mouse Fbw-7β and control was transfected into SN4741 cells (JPEG 62 kb)

High-resolution image (TIFF 2374 kb)

Supplementary Fig. S2

Motor activity examination after 6-OHDA lesioning. Amphetamine (2.5 mg/kg)-induced rotations were counted after 6-OHDA lesioning, as described in the supplementary methods. a Time course of rotational behavior. b Total number of rotations in 60 min. Values shown are means ± SD of three experiments (total nine mice in each group). * P < 0.05 (JPEG 181 kb)

High-resolution image (TIFF 2189 kb)

Supplementary Fig. S3

Levels of Fbw7β in the SNc region of 6-OHDA-treated mice and PD patients. a Lysates prepared from 6-OHDA-treated mice brains were analyzed for Fbw7β by western blot (* P < 0.05). b Fbw7β levels in the brain of human PD patients analyzed by western blot (JPEG 216 kb)

High-resolution image (TIFF 7000 kb)

Supplementary Fig. S4

The oxidization level of HMGB1 in PD patients and control. The lysates were prepared from the striata of postmortem PD patients and controls (JPEG 56 kb)

High-resolution image (TIFF 3661 kb)

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Wang, X., Zhai, H. & Wang, F. 6-OHDA Induces Oxidation of F-box Protein Fbw7β by Chaperone-Mediated Autophagy in Parkinson’s Model. Mol Neurobiol 55, 4825–4833 (2018). https://doi.org/10.1007/s12035-017-0686-0

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  • DOI: https://doi.org/10.1007/s12035-017-0686-0

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