Neurotoxicity of Alzheimer’s Disease pathological proteins is differentiation agent-specific

Introduction: Investigation into pressing neurological conditions including Alzheimer’s Disease (AD) hinges on neuronal cultures capable of replicating in vivo responses. The popularly selected SH-SY5Y neuroblastoma cell line is readily available, straightforward to culture, and of human origin (Kovalevich et al., 2021). This and other neuronal-like lines require cells to be differentiated into one or more signaling phenotypes (Fezoui et al., 2000). The involvement of several molecular agonists in facilitating differentiation in vivo is not fully understood. To combat this knowledge gap, most models of neuropathological significance apply one or more of these chemical agents in series. However, the choice of combination and order can govern cell phenotype and, thus, response to stimulants and toxins.
Amyloid-β (Aβ) plaques and inflammatory cytokines are two key hallmarks of AD, which is credited with upwards of 16 new clinical diagnoses per 1000 persons each year (Waugh et al., 2023; Tahami Monfared et al., 2022). In this study, Aβ aggregates and inflammatory cytokines are investigated for pathological effects on SH-SY5Y differentiated into dopaminergic or noradrenergic, neuronal-like phenotypes via distinct protocols.

Materials and

Methods: Human neuroblastoma cell line SH-SY5Y was maintained (37°C, 5% CO2) in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, and 10% (v/v) fetal bovine serum (FBS). Prior to neurotoxicity experiments, SH-SY5Y were seeded in 96-well plates at density 50,000 cells/mL. Cells were differentiated into a dopaminergic phenotype (Shipley et al., 2016) via exposure to 10 μM retinoic acid (RA) (4 days) followed by 80 nM phorbol 12-myristate 13-acetate (PMA) (3 days). Alternatively, cells were differentiated into a noradrenergic phenotype (Krishtal et al., 2019) via exposure to 2 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) (3 days). Differentiation by each type was confirmed via visualization of neurite outgrowth, complete cell adherence to the plate, and increased intercellular spacing as compared to undifferentiated cultures (Feles et al., 2022).
Differentiated SH-SY5Y cells were treated (72 h or 7 days) with Aβ oligomers (2 μM). Cells treated with PBS served as a negative control. Separately, differentiated SH-SY5Y cells were also treated (72 h or 7 days) with supernatants of human THP-1 macrophages, which contained secreted cytokines. In preparation of these supernatants, THP-1 monocytes were differentiated into macrophages by exposure to PMA for 3 days and stimulated with either lipopolysaccharides (LPS) (2 μg/mL) or PBS (negative control). Supernatant samples were diluted 6-fold for SH-SY5Y treatments. Neurotoxicity was assessed via XTT reduction, and cell viability was normalized to the negative control. Significance was analyzed via a paired t-test in GraphPad Prism 9.2.0, where p < 0.05 was considered significant.

Results, Conclusions, and Discussions: SH-SY5Y neuroblastoma cells differentiated by RA and PMA in series (RA/PMA) display hallmarks of a neuronal-like phenotype, depicted by Figure 1. Similarly, cell morphology following dbcAMP differentiation exhibit characteristic neuronal-like properties (Figure 2). Neuronal viability by XTT reduction revealed that cytokines within supernatants generated from stimulated THP-1 macrophages consistently induced a toxic effect on SH-SY5Y cells differentiated by either RA/PMA or dbcAMP (Figure 3). In contrast, neurotoxicity induced by Aβ oligomers was dependent on the differentiation method. Aβ oligomer treatments corresponded to a significant reduction (p < 0.0001) in viability by approximately 20% among dbcAMP-differentiated SH-SY5Y cells, while RA/PMA-differentiated cells treated with Aβ oligomers failed to show a reduction in viability (Figure 3). This observation is validated by existing literature that suggests desensitization of SH-SY5Y to Aβ-induced toxicity is a result of retinoic acid exposure (Krishtal et al., 2019).
In the field of neuronal modeling with SH-SY5Y cells, RA is overwhelmingly the agent of choice for multipurpose differentiation (Figure 1). However, the result herein reinforces the importance of considering alternatives when chemically stimulating neuronal-like cells toward any intended morphology in vitro. Future work will examine the ability of described cell models to examine interactions among neurotoxic agents as well as Aβ oligomerization inhibitors as potential attenuators of human neurotoxicity.

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