Establishment of an In-Vitro Alzheimer’s Disease Model Through Amyloid-β(25–35) Neurotoxicity in SH-SY5Y Cells

Saviya Kashif; Abdul Hameed; Rehan Imad; Mati ur Rehman

Authors

Saviya Kashif Ziauddin University, Karachi, Pakistan https://orcid.org/0009-0008-9538-1331
Abdul Hameed Ziauddin University, Karachi, Pakistan https://orcid.org/0000-0002-5907-4325
Rehan Imad Ziauddin University, Karachi, Pakistan
Mati ur Rehman Agha Khan University, Karachi, Pakistan

Keywords:

Alzheimer Disease, Amyloid beta-Peptides, SH-SY5Y Cells, Neurotoxicity Models, Apoptosis, Cell Survival, Neuroprotection

Abstract

Background: Alzheimer’s disease (AD) is a progressively neurodegenerative condition marked by amyloid-β (Aβ) accumulation and neuronal death. Despite substantial research, effective disease-modifying medications remain unavailable, underlining the need for dependable preclinical models to test novel therapeutic techniques. In order to enable future screening of possible anti-Alzheimer's drugs, the current work set out to create an in vitro Alzheimer's disease model with an ideal dose of Aβ (25–35) that causes repeatable neuronal cytotoxicity in SH-SY5Y cells.

Methods: The experiment was conducted at Ziauddin University, Karachi. SH-SY5Y neuroblastoma cells were treated to Aβ (25–35) at doses ranging from 0–80 μM during 24 hours. Cell viability was assessed using the MTT test, and the concentration that reduced survival by 50–60% was determined as the best cytotoxic dose for model development. To confirm cell death and observe membrane integrity and nuclear morphology, cells had been stained with propidium iodide (PI) and DAPI, then studied by fluorescence microscopy.

Results: Aβ (25-35) treatment produced a clear, concentration-dependent reduction in viability. At 40 μM, cell survival decreased to ~54%, meeting the target threshold of 50–60% cytotoxicity. Fluorescence microscopy confirmed thesefindings, with PI staining highlighting membrane-compromised cells and DAPI revealing nuclear condensation and fragmentation. Together, these outcomes validate 40 μM Aβ (25–35) as a reliable concentration for establishing a standardized in vitro model of AD.

Conclusion: This study created an in vitro Alzheimer's disease model utilizing SH-SY5Y cells exposing them to Aβ(25-35), with 40 μM causing ~50-60% cytotoxicity and neurotoxicity. This model may serve as a core basis for future in vitro AD research.

DOI: https://doi.org/10.59564/amrj/03.04/013

Author Biographies

Saviya Kashif, Ziauddin University, Karachi, Pakistan

MPhil Student, College of Molecular Medicine

Abdul Hameed, Ziauddin University, Karachi, Pakistan

Associate Professor, College of Molecular Medicine

Rehan Imad, Ziauddin University, Karachi, Pakistan

Assistant Professor, College of Molecular Medicine

Mati ur Rehman, Agha Khan University, Karachi, Pakistan

Assistant Professor, Department of Biological & Biomedical Sciences, Medical College

References

Safiri S, Ghaffari Jolfayi A, Fazlollahi A, Morsali S, Sarkesh A, Daei Sorkhabi A, et al. Alzheimer's disease: a comprehensive review of epidemiology, risk factors, symptoms diagnosis, management, caregiving, advanced treatments and associated challenges. Front Med (Lausanne). 2024 Dec 16;11:1474043.

DOI: https://doi.org/10.3389/fmed.2024.1474043.

Plascencia-Villa G, Perry G. Roles of oxidative stress in synaptic dysfunction and neuronal cell death in Alzheimer’s disease. Antioxidants. 2023;12:1628.

DOI: https://doi.org/10.3390/antiox12081628.

Dias D, Socodato R. Beyond amyloid and tau: the critical role of microglia in Alzheimer's disease therapeutics. Biomedicines. 2025 Jan 23;13(2):279.

DOI: https://doi.org/10.3390/biomedicines13020279.

Viegas J, Sarmento B. Bridging the gap between testing and clinics exploring alternative pre-clinical models in melanoma research. Adv Drug Deliv Rev. 2024 May;208:115295.

DOI: https://doi.org/10.1016/j.addr.2024.115295.

Makhijani P. Investigating the impact of distinct intercellular communication pathways in chronic inflammation [doctoral dissertation]. University of Toronto (Canada).

Yu Y, Yu S, Battaglia G, Tian X. Amyloid‐β in Alzheimer's disease: structure, toxicity, distribution, treatment, and prospects. iBrain. 2024 Sep;10(3):266–89.

Busch L, Eggert S, Endres K, Bufe B. The hidden role of non-canonical amyloid β isoforms in Alzheimer's disease. Cells. 2022;11:3421.

Jurcău MC, Andronie-Cioara FL, Jurcău A, Marcu F, Ţiț DM, Pașcalău N, et al. The link between oxidative stress, mitochondrial dysfunction and neuroinflammation in the pathophysiology of Alzheimer’s disease. Antioxidants. 2022;11(11):2167.

DOI: https://doi.org/10.3390/antiox11112167.

Hoffmann LF, Martins A, Majolo F, Contini V, Laufer S, Goettert MI. Neural regeneration research model to be explored: SH-SY5Y human neuroblastoma cells. Neural Regen Res. 2023 Jun;18(6):1265–6.

DOI: https://doi.org/10.4103/1673-5374.358621.

Ioghen OC, Ceafalan LC, Popescu BO. SH-SY5Y cell line in vitro models for Parkinson disease research—old practice for new trends. J Integr Neurosci. 2023 Jan 16;22(1):20.

Kaya ZB, Santiago-Padilla V, Lim M, Boschen SL, Atilla P, McLean PJ. Optimizing SH-SY5Y cell culture: exploring the beneficial effects of an alternative media supplement on cell proliferation and viability. Sci Rep. 2024 Feb 27;14(1):4775.

Mohammed I, Noor Aishah, Min J, Vidyadaran S, Sulaiman M, Bharatham H. The effect of DMEM and DMEM:F12 culture media on the growth of SH-SY5Y cells. Life Sci Med Biomed. 2018;2.

DOI: https://doi.org/10.28916/lsmb.2.3.2018.23.

Yu X, Li Y, Mu X. Effect of quercetin on PC12 Alzheimer's disease cell model induced by Aβ25–35 and its mechanism based on Sirtuin1/Nrf2/HO-1 pathway. Biomed Res Int. 2020;2020:8210578.

DOI: https://doi.org/10.1155/2020/8210578.

Zhang H, Wei W, Zhao M, Ma L, Jiang X, Pei H, et al. Interaction between Aβ and tau in the pathogenesis of Alzheimer's disease. Int J Biol Sci. 2021;17(9):2181–92.

DOI: https://doi.org/10.7150/ijbs.57078.

Zhang Y, Chen H, Li R, et al. Amyloid β-based therapy for Alzheimer’s disease: challenges, successes and future. Sig Transduct Target Ther. 2023;8:248.

DOI: https://doi.org/10.1038/s41392-023-01484-7.

Azargoonjahromi A. The duality of amyloid-β: its role in normal and Alzheimer’s disease states. Mol Brain. 2024;17:44.

DOI: https://doi.org/10.1186/s13041-024-01118-1.

Kozina A, Herbert-Alonso G, Díaz A, Flores G, Guevara J. Effect of the aggregation state of amyloid-beta (25–35) on brain oxidative stress in vivo. PLoS One. 2024;19(10):e0310258.

DOI: https://doi.org/10.1371/journal.pone.0310258.

Han J, Qu Q, Qiao J, Zhang J. Vincamine alleviates amyloid-β 25–35 peptides-induced cytotoxicity in PC12 cells. Pharmacogn Mag. 2017;13(49):123–8. DOI: https://doi.org/10.4103/0973-1296.196309.

You F, Li Q, Jin G, et al. Genistein protects against Aβ25–35 induced apoptosis of PC12 cells through JNK signaling and modulation of Bcl-2 family messengers. BMC Neurosci. 2017; 18:12.

DOI: https://doi.org/10.1186/s12868-016-0329-9.

Alanazi MM, Albaker AB, Alzaagi LA, Alsabhan JF, Alasmari F, Almutairi MM, et al. Oxytocin protects PC12 cells against β-amyloid-induced cell injury. Pharmaceuticals. 2025;18:390.

DOI: https://doi.org/10.3390/ph18030390.

Li LX, Liu MY, Jiang X, Xia ZH, Wang YX, An D, et al. Metformin inhibits Aβ25–35-induced apoptotic cell death in SH-SY5Y cells. Basic Clin Pharmacol Toxicol. 2019;125(5):439–49.

DOI: https://doi.org/10.1111/bcpt.13279.

Liu Y, Jiang L, Li X. κ-carrageenan-derived pentasaccharide attenuates Aβ25–35-induced apoptosis in SH-SY5Y cells via suppression of the JNK signaling pathway. Mol Med Rep. 2017;15(1):285–90.

Henríquez G, Mendez L, Varela-Ramirez A, Guerrero E, Narayan M. Neuroprotective effect of Brazilin on amyloid β (25–35)-induced pathology in a human neuroblastoma model. ACS Omega. 2020;5(23):13785–92.

DOI: https://doi.org/10.1021/acsomega.0c00396.

Jarero-Basulto JJ, Gasca-Martínez Y, Rivera-Cervantes MC, Gasca-Martínez D, Carrillo-González NJ, Beas-Zárate C, et al. Cytotoxic effect of amyloid-β1–42 oligomers on ER and Golgi arrangement in SH-SY5Y cells. NeuroSci. 2024;5:141–57.

DOI: https://doi.org/10.3390/neurosci5020010.

Do BK, Jang JH, Park GH. Effects of corticosterone on beta-amyloid-induced cell death in SH-SY5Y cells. Biomol Ther. 2024;32(1):77–83.

DOI: https://doi.org/10.4062/biomolther.2023.133.

Liu Q, Song S, Liu L, Hong W. In vivo seeding of amyloid-β protein and implications in modeling Alzheimer’s disease pathology. Biomolecules. 2025;15:571.

DOI: https://doi.org/10.3390/biom15040571