The role of Piezo1 in an in-vitro neuron-like system

Cells undergo an enormous amount of mechanical stimuli from extracellular matrix and surrounding cells and tissues. These stimuli have been underestimated for years, but recently, several studies highlighted their importance in physiology and diseases. Indeed, many pathological conditions contribute to create peculiar environments that lead to biomechanical abnormalities, as occurs in tumors or in neurodegenerative diseases. Piezo1 is a mechanosensitive calcium-permeable ion channel. Its expression is involved in the maintenance of cerebral homeostasis, making it a relevant potential pharmacological target in different pathologies. This project aims to outline the role of Piezo1 channel in mouse immortalized mesencephalic neuron-derived cell line (A1) by its overexpression (A1-OV) and pharmacological modulation, in different stiffness culture settings. In-vivo, cell environment displays lower elasticity (0.2-64 kPa) as compared to the common in-vitro culture (1x107kPa). We evaluated how cells change and adapt to a different stiffness substrate, analysing shape, elasticity, gene expression, viability, and functional activity. A collateral part of this work was dedicated to the characterization of the nanoindentation technique through the Chiaro nanoindenter, in order to find a correct methodological approach to measure cellular elasticity. A1 cell viability is stiffness-dependent, directly paralleling substrate stiffness. WT A1 cells plated in softer substrate and A1-OV in plasticware change their shape modifying the expression of cytoskeleton components. Notwithstanding in these experimental conditions cells are characterized by higher basal concentration calcium ions, they also display an increased response to the Piezo1 agonist Yoda1 in terms of Ca2+ entry. Furthermore, we assessed the capacity of Amyloid-beta to affect the mechanical property of A1 cells. The non-aggregated “monomeric” peptide-induced cell stiffening, while aggregated Amyloid-beta caused cell softening. Further experiments will be required to confirm these data and correlate them with cell functional changes. Altogether, the data here reported show that neuron-like cells adapt in different stiffness settings changing their characteristics according to the biomechanical features of the substrate in which they are plated or in response to molecular treatment. These changes could, at least partially, involve Piezo1, making it an interesting pharmacological target.