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Generation of Stable Cell Lines of APP695 (K595N, M596L) Gene Over-Expression via Lentiviral Over-Expression System

  • CELL BIOLOGY
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Abstract

There are now various theories on the pathophysiology of Alzheimer’s disease (AD), the β-Amyloid (Aβ) cascade hypothesis has garnered a lot of interest. This text applied lentivirus-encapsulated plasmid to transfect APP695 (K595N, M596L) gene into SH-SY5Y cells and thereby obtain a stable cell line over-expressing Aβ. We used qPCR to monitor the mRNA expression of β-Amyloid Precursor Protein (APP) gene, β-Site APP Cleaving Enzyme 1 (BACE1) gene, we also used Western Blot to detect the APP protein, BACE1 protein, and Aβ1-42, respectively, in addition, the expression of Aβ1-42 in SH-SY5Y cell lysate and cell culture supernatant was measured by Enzyme-Linked Immunosorbent Assay (ELISA). The findings demonstrated APP gene over-expression following APP695 (K595N, M596L) gene transfection, and also indicated the successful establishment of the Swedish mutant cell model.

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REFERENCES

  1. Cheignon, C., Tomas, M., Bonnefont-Rousselot, D., et al., Oxidative stress and the amyloid beta peptide in Alzheimer’s disease, Redox Biol., 2018, vol. 14, pp. 450–464. https://doi.org/10.1016/j.redox.2017.10.014

    Article  CAS  PubMed  Google Scholar 

  2. Cheng, Y., Tian, D.Y., and Wang, Y.J., Peripheral clearance of brain-derived Aβ in Alzheimer’s disease: pathophysiology and therapeutic perspectives, Transl. Neurodegener., 2020, vol. 9, рр. 1–11. https://doi.org/10.1186/s40035-020-00195-1

  3. Conway, K.A., Baxter, E.W., Felsenstein, K.M., et al., Emerging β-amyloid therapies for the treatment of Alzheimer’s disease, Curr. Pharm. Des., 2003, vol. 9, pp. 427–447. https://doi.org/10.2174/1381612033391649

    Article  CAS  PubMed  Google Scholar 

  4. Dehury, B., Tang, N., and Kepp, K.P., Molecular dynamics of C99-bound γ-secretase reveal two binding modes with distinct compactness, stability, and active-site retention: implications for Aβ production, Biochem. J., 2019, vol. 476, pp. 1173–1189. https://doi.org/10.1042/bcj20190023

    Article  CAS  PubMed  Google Scholar 

  5. Haass, C., Kaether, C., Thinakaran, G., et al., Trafficking and proteolytic processing of APP, Cold Spring Harbor Perspect. Med., 2012, vol. 2, p. a006270. https://doi.org/10.1101/cshperspect.a006270

    Article  CAS  Google Scholar 

  6. Hampel, H., Vassar, R., De Strooper, B., et al., The β‑secretase BACE1 in Alzheimer’s disease, Biol. Psychiatry, 2021, vol. 89, pp. 745–756. https://doi.org/10.1016/j.biopsych.2020.02.001

  7. Knopman, D.S., Amieva, H., Petersen, R.C., et al., Alzheimer disease, Nat. Rev. Dis. Primers, 2021, vol. 7, p. 33. https://doi.org/10.1038/s41572-021-00269-y

    Article  PubMed  PubMed Central  Google Scholar 

  8. Kovalevich, J. and Langford, D., Considerations for the use of SH-SY5Y neuroblastoma cells in neurobiology, Methods Mol. Biol., 2013, vol. 1078, pp. 9–21. https://doi.org/10.1007/978-1-62703-640-5_2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Liu, C.C., Kanekiyo, T., Xu, H., et al., Apolipoprotein E and Alzheimer disease: risk, mechanisms and therapy, Nat. Rev. Neurol., 2013, vol. 9, pp. 106–118. https://doi.org/10.1038/nrneurol.2012.263

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Lovestone, S., Fleshing out the amyloid cascade hypothesis: the molecular biology of Alzheimer’s disease, Dialogues Clin. Neurosci., 2022, vol. 2, p. 2000. https://doi.org/10.31887/dcns.2000.2.2/slovestone

    Article  Google Scholar 

  11. Stokin, G.B., Lillo, C., Falzone, T.L., et al., Axonopathy and transport deficits early in the pathogenesis of Alzheimer’s disease, Science, 2005, vol. 307, pp. 1282–1288. https://doi.org/10.1126/science.1105681

    Article  ADS  CAS  PubMed  Google Scholar 

  12. Tao, P., Zhao, Y., Song, X., et al., Preparation of the cells transfected with human APPswe695 gene and the determination of Aβ production, Chin. J. Cell Biol., 2019, vol. 41, pp. 72–79. http://dx.chinadoi.cn/10.11844/cjcb.2019.01.0008

    Google Scholar 

  13. Van Gool, B., Storck, S.E., Reekmans, S.M., et al., LRP1 has a predominant role in production over clearance of Aβ in a mouse model of Alzheimer’s disease, Mol. Neurobiol., 2019, vol. 56, pp. 7234–7245. https://doi.org/10.1007/s12035-019-1594-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Vassar, R., Bennett, B.D., Babu-Khan, S., et al., β-Secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE, Science, 1999, vol. 286, pp. 735–741. https://doi.org/10.1126/science.286.5440.735

    Article  CAS  PubMed  Google Scholar 

  15. Wang, H., Xu, X.X., Pan, Y.C., et al., Recognition and removal of Amyloid-β by a heteromultivalent macrocyclic coassembly: A potential strategy for the treatment of Alzheimer’s Disease, Adv. Mater., 2021, vol. 33, p. 2006483. https://doi.org/10.1002/adma.202006483

    Article  ADS  CAS  Google Scholar 

  16. Watamura, N., Sato, K., and Saido, T.C., Mouse models of Alzheimer’s disease for preclinical research, Neurochem. Int., 2022, vol. 158, p. 105361. https://doi.org/10.1538/expanim.22-0164

    Article  CAS  PubMed  Google Scholar 

  17. Wu, T., Lin, D., Cheng, Y., et al., Amyloid cascade hypothesis for the treatment of Alzheimer’s disease: progress and challenges, Aging Dis., 2022, vol. 13, pp. 1745–1758. https://doi.org/10.14336/AD.2022.0412

    Article  PubMed  PubMed Central  Google Scholar 

  18. Yang, Y., Arseni, D, Zhang, W., et al., Cryo-EM structures of amyloid-β42 filaments from human brains, Science, 2022, vol. 375, pp. 167–172. https://doi.org/10.1016/j.cclet.2022.06.046

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  19. Yiannopoulou, K.G. and Papageorgiou, S.G., Current and future treatments in Alzheimer disease: An update, J. Cent. Nerv. Syst. Dis., 2020, vol. 12, pp. 1–12. https://doi.org/10.1177/1179573520907397

    Article  Google Scholar 

  20. Zhao, J., Wang, M., Liu, W., et al., Activation of cannabinoid receptor 2 protects rat hippocampal neurons against Aβ-induced neuronal toxicity, Neurosci. Lett., 2020, vol. 735, p. 135207. https://doi.org/10.1016/j.neulet.2020.135207

    Article  CAS  PubMed  Google Scholar 

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ACKNOWLEDGMENTS

The authors thank the Analytical & Testing Center of Shenzhen Institute of Advanced Technology, CAS.

Funding

This research was supported by the National Natural Science Foundation of China (62074155, 62175252), the Basic and Applied Basic Research Foundation of Guangdong Province (2020A1515110142), the Shenzhen Overseas Innovation Team Project (no. KQTD20180413181834876), the Shenzhen Science and Technology Innovation Commission (KCXFZ202002011008124, JCYJ20210324101405016), and the SIAT Innovation Program for Excellent Young Researchers (E25402), the Natural Science Foundation of Ningxia (2020AAC03151), the Innovation and Entrepreneurship Training Program for College Students of Ningxia in 2020 (S202010752048).

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Authors and Affiliations

  1. School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, China

    Huitao Zhang, Shifan Wang, Huihui Sun & Juan Ding

  2. Bionic Sensing and Intelligence Center, Institute of Biomedical and Health Engineering, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China

    Huitao Zhang, Rui Hao, Zitong Yu, Shi Hu, Jingyi Ren, Yanhang Hong, Pengcheng Zhang & Hui Yang

  3. Research Center for Medical Artificial Intelligence, Institute of Biomedical and Health Engineering, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China

    Huitao Zhang & Yi Zhang

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  1. Huitao Zhang
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  2. Rui Hao
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  3. Zitong Yu
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  4. Shi Hu
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  11. Hui Yang
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Contributions

Conceptualization: J. D., Y. Z. and H. Y.; Methodology: H. Z., R. H., J. D.; Formal analysis and investigation: H. Z., Z. Y., R. H., S. H.; Writing—original draft preparation: H. Z., J. R., S. W., H. S.; Writing—review and editing: J. D., Y. Z., H. Y., Y. H.; Funding acquisition: H. Y., P. Z.; Supervision: J. D., Y. Z. and H. Y.

Corresponding author

Correspondence to Juan Ding.

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Huitao Zhang, Hao, R., Yu, Z. et al. Generation of Stable Cell Lines of APP695 (K595N, M596L) Gene Over-Expression via Lentiviral Over-Expression System. Biol Bull Russ Acad Sci 51, 30–36 (2024). https://doi.org/10.1134/S1062359023602586

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