No-go Goes the Distance: Long-range Nogo-A Signaling via Extracellular Vesicles

Abstract

Extracellular vesicles (EVs) are nano-to-micro sized lipid encapsulated particles carrying various cytoplasmic proteins, membrane proteins and nucleic acids. These vesicles, which appear to be released by all cells, were long considered merely as a cellular garbage disposal system. However, in the past decade, EVs have emerged as important mediators of intercellular communication, carrying signaling ligands on their surface and transferring nucleic acids such as micro RNAs (miRNAs) between cells. As such, EVs may be considered as multi-modal signaling vehicles, capable of causing functional effects in surrounding cells through multiple parallel mechanisms. In the central nervous system, EVs released from glial cells interact with neurons, affecting processes such as synaptic plasticity and neuronal regeneration after injury. Moreover, hydrophobic morphogens such as Wnts and ephrins have very recently been described to hitchhike on EVs, and to activate signaling events in recipient cells in an EV-associated manner. Against this backdrop, we began to investigate if Nogo-A, a myelin-associated membrane protein that inhibits neuronal growth and regeneration, could also signal as an EV-associated ligand. In the first introductory chapter (Chapter 1), we discuss the concept of EV-mediated signaling in the CNS. Focusing on recent advances and new concepts in this field, we describe the functional relevance of EVs in the healthy, the injured and the regenerating CNS. In the second introductory chapter (Chapter 2), we discuss Nogo-A signaling extensively with a broad scope, all the way from the receptor components and downstream signaling mediators to the functional and clinical relevance of Nogo-A. In Chapter 3, a brief prelude to EV isolation and characterization, we compare various methods of EV isolation with the goal of establishing a reproducible method that produces both high yield and high purity EVs. In Chapter 4, we address the main hypothesis of Nogo-A as an EV-associated ligand. We find that Nogo-A is present in a population of EVs isolated from cell cultures, and that Nogo-A positive EVs can be recovered from human cerebrospinal fluid (CSF). We further show that Nogo-A is active as a signaling ligand on the surface of EVs, and that EV-associated Nogo-A signals at least partially through the newly identified receptor S1PR2. Taken together, these findings indicate that Nogo-A may signal as a cell contact-independent ligand with a potential to act at a long range. In Chapter 5, we conclude with a discussion on future directions for assessing the in vivo functionality of EV-associated Nogo-A. Overall, this thesis contributes conceptually to the field of EVs in the CNS in general, as several novel concepts are discussed. Additionally, a novel perspective on Nogo-A signaling is presented, taking Nogo-A signaling from the confines of the plasma membrane and into solution. The potential relevance of this long-range Nogo-A signaling mechanism in the context of regeneration is discussed.