A proteomic approach confirms nanostructure-induced neuritogenesis and alterations of the mechanotransductive processes in PC12 cells, a model system for neuronal differentiation and neurosecretion

Introduction Cells are competent to perceive biophysical signals of their microenvironment, including nanotopography, and to convert them into biochemical responses. In this work we investigate 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 Proteins were identified and quantified by a label-free shotgun proteomic approach based on nano-LC-LTQ Orbitrap Velos MS/MS Results We compared the proteome of PC12 cells grown on neuritogenesis-inducing zirconia nanostructure (nrZr, 15 nm rms) with the one of cells grown on flat zirconia (flZr) and Poly-L-Lysine (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 or showed significant t-test difference (Post hoc Bonferroni test p value = 0.0167). 52 proteins were up-regulated or present only in cells grown on nrZr, while 54 proteins were down-regulated in cells grown on nrZr or were present only in cells grown on flZr. Conclusions Several differentially expressed proteins can be associated with the differentiation processes induced by the nanostructure. In fact, most of the proteins are involved in adhesome and/or cytoskeletal organization and their up- or down regulation is consistent with their functions in neuronal differentiation processes and/or neuritogenesis.