Proteomic profile confirms nanostructure-induced neuritogenesis and reflects alterations of the mechanotransductive processes in PC12 cells

BACKGROUND-AIM Cells are competent to perceive biophysical signals of their microenvironment and to convert them into biochemical responses. These signals comprise the microenvironmental nanotopography and in fact cells can sense surface differences on a nanoscopic level that can have a strong impact on cellular mechanics and on cell’s behavior. In this work we tried to understand how nanoroughness of zirconia surfaces, produced by Supersonic Cluster Beam Deposition using a Pulsed Microplasma Cluster Source (PMCS-SCBD) technique, can guide cellular activities in the context of neuronal differentiation. The potential of nanostructured surfaces in inducing neurogenesis would have enormous clinical relevance regarding cell replacement strategies for neurodegenerative diseases. METHODS We performed a label-free shotgun proteomic analysis to compare the proteome of PC12 cells grown on neuritogenesis-inducing zirconia nanostructure (nrZr) with the one of cells grown on flat zirconia (flZr) and Poly-LLysine (PLL) in the presence of Nerve Growth Factor (NGF). An Anova test (FDR 0.05) was carried out to identify proteins differentially expressed among the different conditions. We focused only on the comparison between cells grown on nrZr 15 nm rms and on flZr in order to better understand the effect of the surface nanotopography. Proteins were considered differentially expressed if they were present only in flZr or nrZr 15 nm rms or showed significant t-test difference (Post hoc Bonferroni test p value = 0.0167). RESULTS 52 proteins were up-regulated or present only in cells grown on nrZr 15 nm rms, while 54 proteins were down-regulated in cells grown on nrZr 15 nm rms or were present only in cells grown on flZr. Among these proteins, several of them reflect the differentiation processes induced by the nanostructure. We discovered that most of the proteins are involved in adhesome and/or cytoskeletal organisation and that their up- or down regulation is in line with their functions in neuronal differentiation processes and/or neuritogenesis. CONCLUSIONS Altogether, our results have unravelled some very interesting candidates for more detailed future analysis of mechanotransductive processes induced by nanostructured surfaces.