Extracellular Vesicles-mediated communication among brain cells

This thesis results from the research activity conducted during my Ph.D. in Biochemistry and Molecular Biology (BiBiM 2.0) starting in 2019. The Pegaso initiative of the Tuscan Region sponsored the BiBIM 2.0 Ph.D. program, and I conducted most of my work in the Biochemistry lab of Claudia Martini, Full Professor of Biochemistry, at the Department of Pharmacy of the University of Pisa, Italy. In 2021, I spent six months in the lab of Marcal Vilar at the Molecular Basis of Neurodegeneration Unit lab, located in the Institute of Biomedicine of València, Spain. The main topic of my Ph.D. thesis is the role of extracellular vesicles (EVs) as mediators of intercellular communication in the brain. This choice was motivated by a naïve interest in the basic biochemistry underlying these subcellular structures. Furthermore, EVs represent an excellent opportunity to build fully biological protein- or nucleic acid- based drugs in the future. This premise prompted our research group to investigate two aspects of EV function. On the one hand, I set-up and validated one protocol for extracting EVs from two human microglial cell lines. I performed a biophysical characterization on these EVs and investigated their biological effect both on microglia itself and on a line of glioblastoma cells. We started analyzing the effect on EVs from untreated microglia, and we carried on by analyzing EVs from inflamed microglia. Next, I investigated one possible role of neurotrophins as a molecular system by which EVs could gain specificity of action. This was motivated by my previous studies on neurotrophins and their receptors, performed during my master’s degree, and by the possibility of joining the lab of Marcel Vilar, an internationally recognized expert on neurotrophins. The results of these studies have been organized into three chapters, and one appendix. In Chapter 1, I will introduce EVs, presenting a brief historical context and how they are classified. I will focus on their function in cell-to-cell communication and their implication in neuroinflammation, which will be a significant part of this thesis. I will also talk about the present knowledge of their production and secretion from the parental cells and their uptake by the recipient cells. In Chapter 2, I am going to give an introduction concerning microglia and the role of microglia-derived EVs in the communication among brain cells. After that, I will present my experimental work in characterizing the biophysical properties of EVs extracted from two microglia cell lines (C20 and HMC3), investigating their biological effect and ability to propagate inflammation to neighboring cells. Chapter 3 will focus on a possible mechanism to increase EVs’ targeted delivery. The first part will introduce the neurotrophin system, and the second part will integrate all the experimental work I performed on the neurotrophins and their receptors, with a particular focus on the investigation of the NGF-mediated p75-TrkA interaction as a molecular mediator of EVs uptake by recipient cells. In Chapter 4, I will draw the main conclusions from my experimental work and delineate how this could be prosecuted in the following years. Finally, appendix I will describe the work I’ve done during my period abroad in the Institute of Biomedicine of Valencia. This work didn’t involve extracellular vesicles but was dedicated to a better understanding of the neurotrophin system.