PROTEOMIC ANALYSIS OF NEURONAL CELLS AND TISSUES

Abstract During my three years of PhD I had the opportunity to work with different biological samples analyzing the content and type of proteins through the proteomic approach. In the thesis I describe how a proteomic approach could be useful to analyze different aspect connected to central nervous system. Indeed, the present work is divided in three different parts but the fil rouge is represented by the same analysis technique, a shotgun label-free proteomic approach, for the identification and quantification of expressed proteins, applied to different neuronal cells and tissues: 1) PC12 cells, a wellstudied neuronal cell model due to the ability to easily differentiate into neuron-like cells, 2) Neuro2a cells, another neuronal cellular model very well studied, and 3) the brain of Zebrafish. It is a poikilotherm and eurytherm and therefore it has a wide thermal tolerance, from 6°C to 38°C, temperatures between 24 and 30°C are more suitable for its development, growth and reproduction. Moreover, Danio rerio represents a good animal model for different type of research because it has a generation time of 3-4 months, its maintenance is cheaper than that for rats and mice and required little space. Starting with the first project the proteomic approach is used to dissect at the proteome level similarities and differences between the biochemically and mechanotransductively promoted neuronal differentiation of PC12 cells growth on cluster-assembled zirconia surface with 15nm of roughness, polylysine coated glass in the presence (NGF) or absence (PLL) of NGF. This work lays a substantial cell biological foundation for the intelligent design of substrates for cell culturing based on nanostructured surfaces produced by cluster assembling that mimic more closely physiological 3D extracellular microenvironmental features. Our data suggest that the nanoscale information provided by these surfaces could have a strong potential in favoring neurogenic processes by mechanotransductive processes. The results obtained on zirconia nanostructure can be the fundamental starting point to further characterize the neuronal differentiation process in adequate primary and stem cell systems for regenerative medicine approach. The second part of this work has the aim to understand how the activation of the TrkA pathway is able to trigger biochemical signaling, like ERK1/2, leading to cell differentiation when GM1 oligosaccharide, II3Neu5Ac-Gg4 (OligoGM1), which interacts with NGF receptor TrkA, is administered to cultured murine Neuro2a neuroblastoma cells. The results of our work confirm and reinforce the idea that the molecular mechanisms underlying the GM1 neurotrophic and neuroprotective effects depend on its oligosaccharide chain, suggesting the activation of a positive signaling starting at plasma membrane level. The third part of this thesis regards the determination of the effect of ambient temperature on the molecular mechanism and the behavioural responses in Danio rerio. This project can be framed in a quite interesting area of research, important because global warming occurring in our planet is, especially nowadays, an urgent problem amplified by the anthropic action, the release of CO2 and other greenhouse gases. The huge temperature increase causes climate changes that can deeply alter the habitat of the species, leading to substantial environmental changes possibly impairing the prosecution of the species. We applied for the first time a shotgun proteomic approach to analyze the effect of acclimatization on zebrafish brain proteome and to correlate the results at the protein level with the behavioural tests. As stated above, the shotgun proteomic approach adopted is a powerful analytical method for characterizing the complex proteomes of various types of biological specimens. To characterize the protein component of our sample, we have adopted a quantitative label free shotgun proteomic approach. In recent years, non-gel-based, shotgun proteomic technique has emerged as powerful tool for studying large scale differential protein expression [1]. This method allows to examine the impact of different conditions by achieving the simultaneous identification of thousands of proteins and their quantification in each sample. It does not require a previous purification of the sample, but identifies proteins from tandem mass spectra (MS/MS) of their proteolytic peptides, which are separated by liquid chromatography (LC) [2]. In particular, the identification of the proteins from the MS/MS data was achieved using a database search by MaxQuant which compares acquired mass spectra to a database of known sequences to identify the proteins. 1. Zhu W, Smith JW, Huang CM. Mass spectrometry-based label-free quantitative proteomics. J Biomed Biotechnol. 2010;2010:840518. doi: 10.1155/2010/840518. Epub 2009 Nov 10 2. Washburn MP. Driving biochemical discovery with quantitative proteomics. Trends Biochem Sci. 2011 Mar;36(3):170-7. doi: 10.1016/j.tibs.2010.09.001. Epub 2010 Sep 27