A Soft Mechanical Phenotype of SH-SY5Y Neuroblastoma and Primary Human Neurons Is Resilient to Oligomeric Aβ(1-42) Injury

Abstract

Aggregated amyloid beta (Aβ) is widely reported to cause neuronal dystrophy and toxicity through multiple pathways: oxidative stress, disrupting calcium homeostasis, and cytoskeletal dysregulation. The neuro-cytoskeleton is a dynamic structure that reorganizes to maintain cell homeostasis in response to varying soluble and physical cues presented from the extracellular matrix (ECM). Due this relationship between cell health and the ECM, we hypothesize that amyloid toxicity may be directly influenced by physical changes to the ECM (stiffness and dimensionality) through mechanosensitive pathways, and while previous studies demonstrated that Aβ can distort focal adhesion signaling with pathological consequences, these studies do not address the physical contribution from a physiologically relevant matrix. To test our hypothesis that physical cues can adjust Aβ toxicity, SH-SY5Y human neuroblastoma and primary human cortical neurons were plated on soft and stiff, 2D polyacrylamide matrices or suspended in 3D collagen gels. Each cell culture was exposed to escalating concentrations of oligomeric or fibrillated Aβ(1-42) with MTS viability and lactate dehydrogenase toxicity assessed. Actin restructuring was further monitored in live cells by atomic force microscopy nanoindentation, and our results demonstrate that increasing either matrix stiffness or exposure to oligomeric Aβ promotes F-actin polymerization and cell stiffening, while mature Aβ fibrils yielded no apparent cell stiffening and minor toxicity. Moreover, the rounded, softer mechanical phenotype displayed by cells plated onto a compliant matrix also demonstrated a resilience to oligomeric Aβ as noted by a significant recovery of viability when compared to same-dosed cells plated on traditional tissue culture plastic. This recovery was reproduced pharmacologically through inhibiting actin polymerization with cytochalasin D prior to Aβ exposure. These studies indicate that the cell-ECM interface can modify amyloid toxicity in neurons and the matrix-mediated pathways that promote this protection may offer unique targets in amyloid pathologies like Alzheimer's disease.

Keywords: Alzheimer’s disease; extracellular matrix; mechanical properties.

Figure 1.

AFM images of oligomeric and fibrillated Aβ(1–42) preparations from California Peptide and American Peptide Company. Scale bar for the fibrillated species of CA peptide and APC are 750 and 200 nm, respectively. The scale bar for the oligomeric species is 1 μm for both samples. Height data indicate the fibrillated species of each are 2.8 nm (CA) and 4.7 nm (APC), and the height of the oligomeric species is approximately 12.6 nm.

Figure 2.

MTS viability results for SH-SY5Y cells and primary cortical neurons after exposure to increasing concentrations of oligomeric and fibrillated Aβ species for 48 h. Oligomeric Aβ is significantly more toxic than fibrillated Aβ to both cell types (two-way ANOVA, p < 0.05).

Figure 3.

Cell behavior changes dramatically when exposed to varying environments. (a) Confocal images of human primary cortical neurons display an increase in cell spreading and F-actin polymerization on stiffer substrates. Red arrows point to F-actin stress fibers formed in the cells plated on the glass substrate. (b) AFM nanoindentation measurements of cell stiffness indicate an increase in matrix stiffness results in an increase in cell stiffness, when plated on PA gels. (c) Total actin expression for SH-SY5Y cells and primary neurons plated on each PA matrix. Actin expression increases with higher matrix stiffness. All results reported relative to GAPDH expression (one-way ANOVA, p < 0.05). (d) MTS viability of SH-SY5Y and primary neurons on each substrate, when exposed to 10 μM Aβ(1–42). This data suggests a neuroprotective effect, with decreased matrix stiffness resulting in higher cell viability when exposed to the same treatment (two-way ANOVA, p < 0.05).

Figure 4.

AFM cell stiffness measurements of SH-SY5Y cells and human primary cortical neurons after treatment with 1 μM Aβ(1—42) for 24 h. Treatment with oligomeric species induces cell stiffening, while this was not observed after treatment with the fibrillated species (one-way ANOVA, p < 0.05).

Figure 5.

(a) MTS viability and (b) LDH toxicity studies for SH-SY5Y cells and primary neurons when pretreated with cytochalasin D and followed by exposure to 10 μM oligomeric Aβ(1—42) for 48 h (one-way ANOVA, p < 0.05).

Figure 6.

Relative paxillin expression is matrix dependent for SH-SY5Y cells and human primary cortical neurons on PA and tissue culture plastic substrates (one-way ANOVA, p < 0.05).