Characterization of a paracrine target engagement in an in vitro model of extracellular vesicles-mediated cell-to-cell communication

This thesis summarizes the main findings of the research project conducted from August 2016 until August 2020 at the Laboratory for Biomedical Neurosciences (LBN) of the Ente Ospedaliero Cantonale (EOC) Switzerland, performed under the supervision of PD Dr. Paolo Paganetti (Neurodegeneration Group) and the academic mentorship of Prof. Dr. Anne Spang (Growth & Development Group, Biozentrum, University of Basel, Switzerland).
The project was developed in the context of a broader study aiming to investigate cellular and molecular mechanisms governing cell-to-cell communication in neurodegeneration. Specifically the aim of this project, which is described in detail in Chapter 2, was to further elucidate the cellular mechanisms consenting a paracrine protein target engagement in an in vitro model of extracellular vesicles-mediated cell-to-cell communication.
This thesis is organized as follows. Chapter 1 presents extracellular vesicles in cell-to-cell communication and the controversial link between autophagy and the aggregation of aberrant protein species, which forms the context of the research thesis work. Chapter 1, Section 1. contains a literature review of the evidence supporting the interplay of the endocytic and autophagic pathways in the target engagement between an extracellular vesicle cargo and its cytosolic cellular target. The relevance of this engagement is then discussed in light of the possible implications in the cellular processes driving the aggregation of aberrant protein species. This section is compiled into a manuscript, which has been submitted to a peer-reviewed journal. Chapter 1, Section 2. includes a perspective on the use of chloroquine during the novel coronavirus (i.e. SARS-CoV-2) pandemic and the possible implications on clinical trials of neurodegenerative disorders. This possible correlation is discussed in the context of the cellular mechanisms linking the autophagy-lysosome pathway and cellular mechanisms involved in the pathogenesis of neurodegenerative diseases. The manuscript has been submitted to a peer-reviewed journal. Chapter 2 briefly introduces the problem of identifying a functional delivery of EV-transported luminal proteins within the cytosol of recipient cells. The chapter then presents the research model, the reasons behind why this model was chosen, and outlines the objectives of the study. Chapter 3 contains the main research findings. The manuscript uploaded to bioRxiv preprint server on May 28th 2020 describes a novel cellular mechanism consenting target engagement between an extracellular vesicle transported luminal protein and its cytosolic cellular target. Furthermore, data generated in this study highlights a possible dichotomic role of autophagy in protein aggregation, which is yet to be confirmed. A study that complements the research thesis work is then presented in Chapter 4 in the form of a manuscript. The manuscript describes the split GFP technique that was implemented as part of a study aiming at investigating protein-protein interaction in in vitro models of neurodegeneration. This method was used in the main research study of the thesis to demonstrate target engagement between two proteins that have distinct cell origins. The manuscript was published in Scientific Reports on October 25th, 2017. In these first chapters (1-to-4) only material that was considered sensible for publication in peer-reviewed journals and relevant for the aim of the study is included. Chapter 5 gathers additional preliminary data generated during the doctoral study, which is intended to complement the main research study. Chapter 6 includes a discussion addressing the most significant findings of this research, their implications in EV-mediated cell-to-cell communication, and the possible dichotomic role of autophagy in cellular mechanisms driving the intracellular build-up of aberrant protein species. The Chapter includes a future outlook perspective and outlines additional questions that remain to be explored in these fields. The thesis concludes in Chapter 7 with a brief section dedicated to concluding remarks.