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Genome-wide CRISPR–Cas9 Knockout Screening Reveals a TSPAN3-mediated Endo-lysosome Pathway Regulating the Degradation of α-Synuclein Oligomers

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Abstract

Misfolding and aggregation of α-Synuclein (α-Syn), which are hallmark pathological features of neurodegenerative diseases such as Parkinson’s disease (PD) and dementia with Lewy Bodies, continue to be significant areas of research. Among the diverse forms of α-Syn – monomer, oligomer, and fibril, the oligomer is considered the most toxic. However, the mechanisms governing α-Syn oligomerization are not yet fully understood. In this study, we utilized genome-wide CRISPR/Cas9 loss-of-function screening in human HEK293 cells to identify negative regulators of α-Syn oligomerization. We found that tetraspanin 3 (TSPAN3), a presumptive four-pass transmembrane protein, but not its homolog TSPAN7, significantly modulates α-Syn oligomer levels. TSPAN3 was observed to interact with α-Syn oligomers, regulate the amount of α-Syn oligomers on the cell membrane, and promote their degradation via the clathrin-AP2 mediated endo-lysosome pathway. Our findings highlight TSPAN3 as a potential regulator of α-Syn oligomers, presenting a promising target for future PD prevention and treatment strategies.

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Data Availability

The raw sequencing data have been deposited in the NCBI Sequence Read Archive (SRA: SRR14660095).

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Acknowledgements

We thank Prof. Zhuohua Zhang, School of Life Sciences, Central South University, for the discussion and suggestions and Professor Jiada Li from the School of Life Sciences, Central South University, for his support in the purification of α-synuclein.

We are grateful for resources from the High Performance Computing Center of Central South University.

Funding

This work was supported by the National Key Research and Development Program of China (Grant No. 2021YFA0805200), National Natural Science Foundation of China (Grant No. 82171258), Changsha Science and Technology Foundation (Grant No. kq2004083), the Innovative Team Program 2019RS1010 from the Department of Science & Technology of Hunan Province, the Innovation-Driven Team Project 2020CX016 from Central South University, NHC Key Laboratory of Birth Defect for Research and Prevention, Hunan Provincial Maternal and Child Health Care Hospital, No. KF2021001. and Fundamental Research Funds for the Central Universities of Central South University (2022ZZTS0234, 2023ZZTS0254).

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Authors and Affiliations

  1. Center for Medical Genetics and Hunan Key Laboratory of Medical Genetics, School of Life Science, Central South University, Changsha, 410078, Hunan, China

    JunJian Hu, Xinjie Guan, Miao Zhao, Pengqing Xie & Jieqiong Tan

  2. Department of Central Laboratory, SSL Central Hospital of Dongguan City, Affiliated Dongguan Shilong People’s Hospital of Southern Medical University, Dongguan, China

    JunJian Hu

  3. Mr. & Mrs. Ko Chi-Ming Centre for Parkinson’s Disease Research, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, SAR, China

    Xinjie Guan

  4. Institute for Research and Continuing Education, Hong Kong Baptist University, Shenzhen, China

    Xinjie Guan

  5. Department of Neurology, Xiangya Hospital, Central South University, Changsha, 410008, Hunan, China

    Jifeng Guo

  6. Hunan Key Laboratory of Animal Models for Human Diseases, School of Life Sciences, Central South University, Changsha, Hunan, China

    Jieqiong Tan

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Designed the study: TJQ, HJJ, GJF. Performed experiments: HJJ, GXJ, XPQ. Analyzed the data: HJJ, TJQ. Drafted the manuscript: TJQ, HJJ. All authors critically revised the manuscript and approved the final version.

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Hu, J., Guan, X., Zhao, M. et al. Genome-wide CRISPR–Cas9 Knockout Screening Reveals a TSPAN3-mediated Endo-lysosome Pathway Regulating the Degradation of α-Synuclein Oligomers. Mol Neurobiol 60, 6731–6747 (2023). https://doi.org/10.1007/s12035-023-03495-5

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