Optimization of SH-SY5Y Differentiation to Study Neuronal Mechanosensitivity

Abstract

Neurodegenerative disorders, including Alzheimer’s disease, pose a global social burden strongly linked with aging populations. Despite extensive research efforts, the underlying causes of the neuropathological changes that result in neuronal death are unknown, making the improvement of in vitro models a priority to address. Moreover, these pathological events are accompanied by alterations in brain rigidity, and while it is well established how various cell types can feel these mechanical cues and adapt their function, neuronal mechanosensing remains mostly unexplored. For this reason, in this project we aimed to investigate the mechanobiology of Alzheimer's disease by optimizing the differentiation of SH-SY5Y cells into pseudo-neurons. To facilitate neuronal differentiation, we adopted a novel strategy in which, by introducing a genetic construct in the SH-SY5Y cells, we could control the expression of the neuronal master gene NGN2. We demonstrated that this system, along with an optimized differentiation protocol, led to the production of more mature neuron-like cells. Subsequently, the response of differentiated cells to mechanical stimulation was tested by culturing them in polyacrylamide gels with varying stiffness. Immunostaining and image analysis confirmed the morphological adaptation of neuron-like cells to matrix stiffness, presenting better morphology at a higher stiffness. Finally, we observed detectable levels of the co-transcriptional factor YAP and confirmed that its nuclear presence is mechanoregulated in neurons. In conclusion, by combining differentiation techniques, genetic modification, and mechanical stimulation, our study provides a new model to explore Alzheimer's disease mechanobiology, potentially leading to innovative therapeutic strategies by uncovering complex underlying mechanisms.