The release of histone proteins from cells via extracellular

vesicles

Uma Muthukrishnan

Umeå Centre for Molecular Medicine

Umeå 2018

Responsible publisher under the Swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) Dissertation for Licentiate ISBN: 978-91-7601-888-0 Cover: Negative contrast transmission electron micrograph of extracellular vesicles from OLN-93 cells. Courtesy of Linda Sandblad. Electronic version available at: http://umu.diva-portal.org/ Printed by: UmU Print Service, Umeå University Umeå, Sweden 2018

To God and Gurus

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Table of Contents

Table of contents i

Abstract ii

List of original papers in this thesis iv

Abbreviations v

Enkel sammanfattning på svenska vi

Introduction 1

Histone proteins 1

Non-nuclear histone proteins 6

Protein secretory pathways 11

Pathways for exosome biogenesis and secretion 15

Methods for the study of extracellular vesicles 17

Regulation of microvesicle biogenesis 22

Regulation of exosome biogenesis and secretion 23

Molecular composition of extracellular vesicles 25

Functions of extracellular vesicles 28

Cellular stress responses 31

Extracellular vesicles in stress responses 32

Background to Paper I 34

Aims of thesis 35

Materials and Methods 36

Cell culture 36

Western blotting 38

Proteomics 39

Results 41

Discussion 46

Conclusions 51

Acknowledgement 52

References 54

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Abstract

Histones are chromatin-associated proteins localized to the nucleus. However, extracellular histones are present in biofluids from healthy individuals and become elevated under disease conditions, such as neurodegeneration and cancer. Hence, extracellular histones may have important biological functions in healthy and diseased states, which are not understood. Histones have been reported in the proteomes of extracellular vesicles (EVs), including microvesicles and exosomes. The main aim of this thesis was to determine whether or not extracellular histones are secreted via EVs/exosomes. In an initial study (Paper I), I optimized methods for human embryonic kidney (HEK293) cell culture, transfection and protein detection using western blotting. In the main study (Paper II), I used oligodendrocyte cell lines (rat OLN-93 and mouse Oli-neu) to investigate the localization of histones to EVs. Western blotting of EVs purified from OLN-93 cell-conditioned media confirmed the presence of linker and core histones in them. Immunolocalization and transmission electron microscopy confirmed that histones are localized to EVs, as well as intraluminal vesicles (ILVs) within multivesicular bodies (MVBs). This suggests that histones are secreted via the MVB/exosome pathway. Localization of histones in EVs was investigated by biochemical/proteolytic degradation and purification followed by western blotting. Surprisingly, histones were associated with the membrane but not the luminal fraction. Overexpression of tagged histones in HEK293 cells confirmed their conserved, membrane localization. OLN-93 cell EVs contained both double stranded and single stranded DNA but nuclease and protease digestion showed that the association of histones and DNA with EVs was not interdependent. The abundance of histones in EVs was not affected by differentiation in Oli-neu cells. However, histone release was upregulated as an early response to cellular stress in OLN-93 cells and occurred before the release of markers of stress including heat shock proteins. Interestingly, a notable upregulation in secretion of small diameter (50-100 nm) EVs was observed following heat stress, suggesting that a sub-population of vesicles may be involved specifically in histone secretion in response to stress. Proteomic analyses identified the downregulation of endosomal sorting complex required for transport (ESCRT) as a possible mechanism underlying increased histone secretion.

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In Paper III, I developed methods to quantify extracellular histone proteins in human ascites samples from ovarian cancer patients. In summary, we show for the first time that membrane-associated histones are secreted via the MVB/exosome pathway. We demonstrate a novel pathway for extracellular histone release that may have a role in both health and disease. Keywords: Histone, extracellular vesicles, exosomes, extracellular histones extracellular DNA, cellular stress, proteomics, ESCRT complex, Lrrn1, mid-hindbrain organizer, ovarian cancer, ascites

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List of original papers in this thesis

Paper I Kyoko Tossel, Laura C. Andreae, Chloe Cudmore, Emily A. Lang, Uma Muthukrishnan, Andrew G. Lumsden, Jonathan D. Gilthorpe, Carol Irving. Lrrn1 is required for formation of the midbrain-hindbrain boundary and organiser through regulation of affinity differences between midbrain and hindbrain cells in chick. Dev Biol. 2011 Apr 15; 352(2): 341-52 Paper II Uma Muthukrishnan, Balasubramanian Natarajan, Imre Mäger, Hanna Levén May, Iwan Jones, Giulia Corso, Joel Z. Nordin, Oscar Wiklander, Henrik J. Johansson, Janne Lehtiö, Mattias Hällbrink, Matthew J. Wood, Linda Sandblad, Ivan Nagaev, Vladimir Baranov, Lucia Mincheva-Nilsson, Sophie Rome, Adrian Pini, Samir EL Andaloussi, and Jonathan D. Gilthorpe. The exosome membrane localization of histones is independent of DNA and upregulated in response to stress. 2018 manuscript. Paper III Kelly L. Singel, Kassondra S. Grzankowski, ANM Nazmul H. Khan, Melissa J. Grimm, Anthony C. D’Auria, Kayla Morrell, Kevin H. Eng, Bonnie Hylander, Paul C. Mayor, Tiffany R. Emmons, Nikolett Lénárt, Rebeka Fekete, Zsuzsanna Környei, Uma Muthukrishnan, Jonathan D. Gilthorpe, Constantin F. Urban, Kiyoshi Itagaki, Carl J. Hauser, Cynthia Leifer, Kirsten B. Moysich, Kunle Odunsi, Ádám Dénes, Brahm H. Segal. Mitochondrial DNA in the tumor microenvironment activates neutrophils and is associated with worsened outcomes in patients with advanced epithelial ovarian cancer. 2018 manucript under review in the British Journal of Cancer.

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Abbreviations

Note: Only abbreviations used more than three times are listed here. Otherwise they are defined in the text.

EVs OLN-93 Oli-neu HEK293 ILVs MVBs ESCRT NETs AD PTMs LDs TLR IL-1β ER COPII TGN SNAREs Fgf MVs miRNA Vps HRS TSG101 ALIX PLP CD63 FCS UC TEM Rab HSP70 PrP exoDNA CNS MHB Wnt1 LRR Caps EOC FLRT3

Extracellular vesicles Rat oligodendroglial cell line Murine oligodendrocyte progenitor cell line Human embryonic kidney 293 cell line Intraluminal vesicles Multivesicular bodies Endosomal sorting complex required for transport Neutrophil extracellular traps Alzheimer's disease Post-translational modifications Lipid droplets Toll-like receptors Interleukin-1beta Endoplasmic reticulum Coat protein complex II Trans-Golgi network SNAP (Soluble NSF (N-ethylmaleimide-sensitive factor) Attachment protein) REceptors Fibroblast growth factor Microvesicles MicroRNA Vacuolar protein sorting Hepatocyte growth factor-regulated tyrosine kinase substrate Tumor susceptibility gene 101 Apoptosis-linked gene-2-interacting protein X Proteolipid protein Cluster of differentiation 63 Foetal calf serum Ultracentrifugation Transmission electron microscopy Ras-related guanosine triphosphatase Heat shock protein 70 Prion protein Exosomal DNA Central nervous system Midbrain-hindbrain boundary Wnt family member 1 Leucine-rich repeat Capricious Epithelial ovarian cancer Fibronectin leucine-rich transmembrane 3

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Enkel sammanfattning på svenska Histoner är kromatinassocierade proteiner lokaliserade till kärnan. Extracellulära histoner är emellertid närvarande i biovätskor från friska individer och förhöjda under sjukdomstillstånd, såsom neurodegenerering och cancer. Därför kan extracellulära histoner ha viktiga biologiska funktioner i friska och sjuka tillstånd, vilka än inte kan förstås. Histoner har rapporterats i proteomerna av extracellulära vesiklar (EVs), inklusive mikrovesiklar och exosomer. Huvudsyftet med denna avhandling var att bestämma huruvida extracellulära histoner utsöndras via EVs/ exosomer. I en första studie (Paper I), optimerade jag metoder för human embryonal njurcellkultur (HEK293), transfektion och proteindetektion med hjälp av western blotting. I huvudstudien (Paper II) använde jag oligodendrocytcellinjer (råtta OLN-93 och mus Oli-neu) för att undersöka lokaliseringen av histoner till EVs. Western blotting av EVs renade från OLN-93 cellkonditionerade medier bekräftade närvaron av linker och kärnhistoner i EVs. Immunolokalisering och transmissionselektronmikroskopi bekräftade att histoner är lokaliserade till EVs, såväl som intraluminala vesiklar (ILV) i multiverkulära kroppar (MVB). Detta tyder på att histoner utsöndras via MVB/exosom-vägen. Lokalisering av histon i EVs undersöktes genom biokemisk/proteolytisk nedbrytning och rening följt av western blot. Överraskande var histoner associerade med membranet men inte den luminala fraktionen. Överuttryck av märkta histoner i HEK293-celler bekräftade deras konserverade, membranlokalisering. OLN-93-cell-EVs innehöll både dubbelsträngad och enkelsträngad DNA, men nukleas- och proteas behandling visade att associeringen av histon och DNA med EVs inte var beroende av varandra. Histonernas överflöd i EVs påverkades inte av differentiering i Oli-neu-celler. Histonfrisättningen uppreglerades emellertid som ett tidigt svar på cellulär stress i OLN-93-celler och inträffade före frisättningen av markörer av stress innefattande heat- shock proteiner. Intressant nog observerades en anmärkningsvärd uppreglering i sekretion av EVs med liten diameter (50-100 nm) efter värmespänning, vilket tyder på att en subpopulation av vesiklar kan vara inblandad specifikt vid histonsekretion som svar på stress. Proteomiska analyser identifierade nedreglering av endosomalt sorteringskomplex som krävs för transport (ESCRT) komponenter som en möjlig mekanism som ligger till grund för ökad histonsekretion.

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I Paper III utvecklade jag metoder för att kvantifiera extracellulära histonproteiner i humana ascitesprover från äggstockscancerpatienter. Sammanfattningsvis visar vi för första gången att membranassocierade histoner utsöndras via MVB/exosomvägen. Vi demonstrerar en ny väg för extracellulär histonfrisättning som kan spela en roll i både hälsa och sjukdom.

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Introduction Histones are highly basic nuclear proteins and were the earliest family of proteins to be isolated and studied (Parseghian and Luhrs 2006). They are highly conserved and due to their central role as a component of chromatin, they are fundamental for the organization and replication of the genome and in the regulation of gene expression within Eukaryota. Histones can also be localized extracellularly and extracellular histones are known to function as antimicrobial, pro-inflammatory and toxicity-promoting factors (Allam et al. 2014). Activated and damaged cells release histones in association with DNA during apoptosis, necrosis, or as constituent of neutrophil extracellular traps (NETs). Extracelluar histones are also present in proteomes from apparently healthy cells and individuals and their levels increase in a number of different disease states (Pemberton et al. 2010; Chen et al. 2014). This implies that extracellular histones may have a normal function apart from those that are associated with cellular damage or cell death. However, it is unknown if histones can be secreted via a constitutive pathway from healthy, non-stressed cells. Hence, the main aim of this thesis was to identify the pathway for extracellular histone release and understand how this relates to alterations in cellular stress, which is a potential trigger for the release of extracellular histones. Histone proteins Albrecht Kossel published his discovery of histones in 1884. For his work on the chemistry of proteins, including histones; he was awarded the Nobel Prize for Physiology or Medicine in 1910 (Nobelprize.org 2018, April 26). Histones are highly conserved nuclear protein that exists in almost all eukaryotes (Ouzounis and Kyrpides 1996). In addition to eukaryotes, histones are also present in certain prokaryotic species of the domain Archaea that lack a nucleus, highlighting their ancient evolutionary conservation (Ouzounis and Kyrpides 1996; Pereira and Reeve 1998). The origin of the term histone is debated but may derive from the Greek words ‘histanai’ or ‘histos’, meaning to set in place (Parseghian and Luhrs 2006). As this name depicts, the primary function of histones is to ‘set’ DNA and chromatin in place, although they are now known to also function in the regulation of chromatin structure and gene expression (Sarma and Reinberg 2005; Happel and Doenecke 2009). Histones are classified into five major subtypes; H1/H5, H2A, H2B, H3, and H4. Multiple genes may express each histone protein subtype, resulting in histone variants (discussed in detail below). Based on their structures and functions they can also be categorized into two major groups; namely the linker histones (H1/H5) and the core histones (H2A, H2B, H3 and H4).

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Canonical histones Variants

H1 H1.1, H1.2, H1.3, H1.4, H1.5, H1t, H10, H1x, H1∞, H1T2, H1LS1

H2A H2A.X, H2A.Z.1, H2A.Z.2, mH2A1, mH2A2, H2ABbd

H2B H2B.E, TSH2B, H2BFWT H3 H3.2, H3.3, H3.4, H3.5, H3.X, H3.Y, CENPA H4 No variants identified

Table 1. List of histone proteins and their corresponding variants (Maze et al. 2014).

Linker histones The H1/H5 linker histones are highly enriched in lysine residues and consist of a short N-terminal domain, a central globular domain and an extended C-terminal domain (Hartman et al. 1977; Aviles et al. 1978). The N-terminal domain is extremely basic and the C–terminal domain is enriched with lysine, alanine and proline residues (Bustin and Cole 1970). In aqueous solution, the N- and the C-terminal domains of H1 are unstructured. However, they gain specific secondary structures upon binding to DNA (Vila et al. 2001; Roque et al. 2005). The central globular domain is well conserved with nucleosome recognition sites and is rich in hydrophobic residues that form a winged helix motif (Clark et al. 1993; Ramakrishnan et al. 1993). Linker histones (≈ 200 amino acids (aa) in length) have a predicted molecular mass of ≈21 kDa. The linker H1 family consists of eleven variants in mammals and in humans are referred to as H1.1, H1.2, H1.3, H1.4, H1.5, H1t, H10, H1x, H1∞, H1T2, H1LS1).

Linker histones represent the most divergent class of the histone family (Table 1), reflecting their varied functions in the nucleus involving chromatin organization and regulation of gene expression (Happel and Doenecke 2009). During development and cellular differentiation, cells express specific H1 variants, which incorporated into chromatin in a replication- independent manner. Linker histones are extremely dynamic and are exchanged rapidly on chromatin (Misteli et al. 2000). The binding of H1 variants to chromatin is greatly influenced by sequences in their C-terminal domains (Th'ng et al. 2005). Core histones Core histones are rich in lysine and arginine residues and have been extremely well conserved throughout eukaryotic evolution (Thatcher and Gorovsky 1994). Core histones are small in comparison to linker histones, with a molecular mass of ≈15-17 kDa. They share a similar structure with a conserved, central histone fold domain that is flanked on either side by highly basic, unstructured domains generally referred to N- and C- termini, or tails (Arents et al. 1991). The histone fold comprises a large proportion of the total primary amino acid sequence,

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consisting of helix-strand-helix motifs (Arents and Moudrianakis 1995). Apart from canonical core histones, several variants have been identified (Table 1) including H2A.X, H2A.Z.1, H2A.Z.2, macroH2A1 (mH2A1), mH2A2, H2ABarr body-deficient (H2ABbd), H2B.E, testis specific (TS) H2B, H2B type W-T (H2BFWT), H3.2, H3.3, H3.4, H3.5, H3.X, H3.Y, H3 like centromeric protein A (CENPA; (Sarma and Reinberg 2005; Maze et al. 2014), which together participate in the organization of chromatin structure, dynamics, and function (Ausio 2006; Molden et al. 2015). Core histone variants have evolved primarily through changes to their N- or C-terminal domains relative to canonical forms and are termed as either heteromorphous or homomorphous, based on larger or smaller sequence variations, respectively (Ausio 2006). Unlike canonical core histones that are DNA-replication dependent and translated during the S phase of the cell cycle, histone variants are largely replication-independent (Grove and Zweidler 1984). Canonical core histones are constantly replaced by their variants at specific sites of chromatin, which results in distinct effects on nucleosome stability, DNA repair or gene expression (Felsenfeld and Groudine 2003) . Functions of nuclear histones Chromatin organization and the nucleosome core particle Arguably the most important function of histones is the packaging of the extremely long cellular content of genomic DNA (≈2 m in humans) into the nucleus in an orderly fashion (Felsenfeld and Groudine 2003). The nucleosome core particle (NCP) forms the structural and repetitive unit of chromatin and is made of core histone subunits and DNA (Kornberg 1974). Core histone subunits are organized into the histone octamer, which establishes the central core of the NCP (Thomas and Kornberg 1975). Studies of crystal structures of the NCP have provided insight into the interactions that occur within the histone octamer (Andrews and Luger 2011). The histone octamer consists of a tripartite configuration made from two dimers of H2A-H2B and one tetramer of H3-H4 (Arents et al. 1991). Core histone heterodimerization is highly specific; H2A dimerizes only with H2B and H3 dimerizes only with H4. NCPs, together with linker histones, initiate the organization of higher order chromatin (Thoma et al. 1979). In order to begin the organization of chromatin, the surface of the histone octamer is surrounded by 1.65 left-handed superhelical turn of DNA that consists of ≈147 base pairs. This represents the first nucleosome unit, where DNA is compressed 5-10 fold (Kornberg 1974; Luger et al. 1997). Neighbouring nucleosomes connect together to form an 11 nm diameter polynucleosome fibre, also known as a ‘beads on a string’ structure. At the next level of organization, nucleosome fibres fold on each other resulting in a 30 nm diameter chromatin fibre. The folding process repeats in an increasing order of diameter (from 300

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to 700 nm and up until 1400 nm) to generate the highest-order chromatin structures (Felsenfeld and Groudine 2003). Ultimately, ≈2 m of genomic DNA becomes packaged into about 120 µm of chromatin, to fit inside the nucleus. However, this incredible feat cannot be accomplished with only core histones. Linker H1 participation begins early in the process of chromatin compaction and plays a crucial role in maintaining chromatin structure and stability (Vignali and Workman 1998). Chromatin organization by linker histones Linker histones (H1/H5) bind to the linker DNA region connecting two nucleosomes and in so doing condense and stabilize the 30 nm chromatin fibre into a higher order structure (Thoma et al. 1979; Felsenfeld and Groudine 2003; Dorigo et al. 2004). Different models have been proposed for the location of H1 in the nucleosome (Vignali and Workman 1998). In the first model, the globular domain of H1 bound to DNA at a single contact site, away from the dyad axis of symmetry and the C-terminal tail interacted with linker DNA (Hayes et al. 1994). In the second model, H1 was proposed to bind to DNA at the dyad axis of symmetry through a second contact site present in the globular domain (Zhou et al. 1998) . Changes in the C-terminal domain are known to affect the affinity of H1 for chromatin (Hendzel et al. 2004). A deletion study has identified the importance of a C-terminal sub-domain of H1 in controlling DNA conformation, chromatin stability, and condensation (Lu and Hansen 2004). Taken together, suggests the influence of the binding affinity of H1 on DNA potentially determines the integrity of chromatin. Hence, H1 plays a major role in chromatin compaction. Regulation of gene expression by post-translational modifications of core histones Gene expression, which is crucial in determining the phenotype of an organism, is tightly regulated by extensive modifications that occur both at nucleosome and chromatin levels (Bannister and Kouzarides 2011). To gain access to DNA, chromatin-associated proteins/enzymes are recruited in response to specific modifications. Post-translational modification (PTM) of core histones and their role in maintaining chromatin structure, status and in the regulation of gene expression have been well studied (Felsenfeld and Groudine 2003; Bannister and Kouzarides 2011). Core histone modifications mark chromatin that is packaged into inactive (heterochromatin; tightly packed) or active (euchromatin; loosely packed) states in order to regulate gene expression (Felsenfeld and Groudine 2003). Numerous core histone PTMs have been identified including methylation, acetylation, ubiquitination, phosphorylation, citrullination, sumoylation, biotinylation, or ADP-ribosylation (Bannister and Kouzarides 2011). Core histone PTMs tend to be more highly enriched in N-tail compared to C-tail regions (Cheung et al. 2000). Interestingly, modifications

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are specific to certain residues, for example lysine and arginine are methylated, serine residues are phosphorylated and lysine residues are acetylated, methylated or ubiquitinylated. Some modifications have been shown to exhibit cross talk with one another, either within the same histone or between different histone proteins and are conserved in mammals (Kouzarides 2007). For example, ubiquitination of H2BK123 controls the methylation of H3K4 and H3K79 sites in human cells (Kim et al. 2009). Initially, histones were assumed only to repress gene expression, as they function in condensing DNA. However, detailed studies of histone PTMs have defined both positive and negative influences on gene expression (Jenuwein and Allis 2001). For example, tri-methylation of histone H3 (H3K4me3) is involved in either gene activation or repression based on the position of the protein in chromatin (Shi et al. 2006). Core histone acetylation is well known to promote gene activation and the same gene can be repressed by the corresponding removal of an acetyl group (deacetylation). In addition to transcription, some of the aforementioned modifications (e.g. acetylation, methylation, phosphorylation, and ubiquitination) regulate DNA repair, replication and chromatin condensation (Kouzarides 2007). Regulation of gene expression by post-translational modification of linker histones Until recently, the involvement of H1 PTMs in regulating chromatin structure and gene expression had not been well characterized. The most well studied modification of H1 is phosphorylation of either N- or C-terminal sites (Happel and Doenecke 2009). However, other modifications have been identified on linker histone H1 including acetylation, methylation, ubiquitination, and formylation (Wisniewski et al. 2007; Weiss et al. 2010). Unlike core histones, many H1 modifications do not reside in the tails but within the central globular domain, which is the binding site of DNA. The association of H1 methylation by human enhancer of zeste homolog (Ezh2) in transcriptional repression has been identified (Kuzmichev et al. 2004). Moreover, H1 has also been found to recruit transcription factors to specific promoter regions and thereby regulate transcription (Vignali and Workman 1998). However, the role of H1 modifications in this study was not investigated. Although much better understood for the core histones, the presence of an extensive range of H1 PTMs suggests their importance in the function regulation of H1. Many core and/or linker histone modifications are inherited parentally and these may determine a number of phenotypes in the offspring (Portela and Esteller 2010). Aberrant histone modifications may not only alter gene expression patterns but are also linked to a number of diseases including cancer and neurodegenerative disorders (Portela and Esteller 2010).

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Non-nuclear histone proteins It has been generally considered that the functions of histone proteins are restricted to the nucleus. However, a number of surprising roles for non-nuclear histone proteins have been described both within the cell and also outside of the plasma membrane (reviewed in (Parseghian and Luhrs 2006). These findings have begun to transform the long-held view that histones only exert their effects in the nucleus and in association with DNA. Known roles of non-nuclear histones Healthy cells A number of studies have found that histones localize outside of the nucleus and have specific functions in healthy cells. An excess of free histones are toxic to cells due to their high positive charge. If not associated with negatively charged DNA, histones need to be chaperoned in some way to prevent cytotoxicity (Singh et al. 2010). A proteomic study performed on mitochondrial proteins isolated from viable cells showed the presence of all five classes of histones (H1, H2A, H2B, H3, and H4), in association with the outer mitochondrial membrane (OMM). Furthermore, a function for H2AX was determined in the regulation of mitochondrial protein transport (Choi et al. 2011). Hence, the OMM might represent a location where non-nuclear histones are normally located in viable cells. In support of this, H2A has been shown to localize to the mitochondrial membrane before translocation to the nucleus with human extracellular signal-regulated protein kinase 1 (hERK1; (Galli et al., 2009). Linker H1 and core histones have been identified as components of cytoplasmic lipid droplets (LDs) in Drosophila embryos (Cermelli et al. 2006). Lipid droplets are a storage location for maternal histone proteins during the rapid phase of nuclear replication/division that occurs in the Drosophila embryo (Cermelli et al. 2006). Interestingly, histones associated with LDs also have an antibacterial function. Jabba is a histone-binding protein present on LDs. In the Jabba mutant, reduced incorporation of histones H2A, H2Av and H2B in LDs is seen. Jabba mutant embryos are susceptible to bacterial infection and growth, whereas the wild-type embryos are not (Anand et al. 2012). The release of H2B from LDs in response to lipopolysaccharide or lipoteichoic acid was observed in the presence of bacterial cell wall components. Furthermore, mice challenged with a systemic infection showed an increase in histone H1 in LDs in circulation, showing that the function of LD-associated histones as antibacterial factors may be highly conserved (Anand et al. 2012). The presence of individual histones and/or nucleosomes on the surface of numerous viable cells types, including immune cells (Hefeneider et al. 1992), skeletal muscle cells (Henriquez et al. 2002), neuronal cells (Bolton and Perry

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1997; Mishra et al. 2010) and Schwann cells (Mishra et al. 2010) suggest that histones may have a wider antimicrobial function as part of the innate immune system (reviewed in (Parseghian and Luhrs 2006). Histones, and H1 in particular, have been shown to act receptors for thyroglobulin on macrophages (Brix et al. 1998), perlecan on skeletal muscle cells (Henriquez et al. 2002), and polysialic acid (PSA) on cerebral neurons and Schwann cells (Mishra et al. 2010), resulting in ligand internalization and also cell proliferation and growth. These reports imply that extracellular histones have signalling functions in healthy cells. Cellular senescence and ageing Studies have recognized the association of non-nuclear histones with the process of cellular ageing. Cytoplasmic histones have been found in non-dividing, or differentiated, cells (Zlatanova et al. 1990). During cellular senescence, histone H1 is lost from the nucleus (Funayama et al. 2006). During this process, the affinity of the N-terminal domain of H1 for DNA is reduced, presumably due to a post-translational mechanism, and expression of H1 tagged on the N- but not the C-terminus with enhanced green fluorescent protein (eGFP) facilitated premature senescence (Funayama et al. 2006). Hence, this suggests that the N-terminus has a specific effect on senescence inducing activity of H1, which is blocked by an eGFP tag. Antimicrobial peptides (AMPs) In addition to the antimicrobial role of histones in LDs described above, non-nuclear and extracellular histones appear to act as an important defence against microbial pathogens and are perhaps a first line of defence of the innate immune response. Extensive studies carried out both in vitro and in vivo on invertebrate and vertebrate models have determined that histones and histone-derived fragments possess anti-microbial properties (Parseghian and Luhrs 2006; Kawasaki and Iwamuro 2008). The antimicrobial function of histones was first reported in vitro by Miller and colleagues (Miller et al. 1942) and later followed by a detailed study by Hirsch (1958), where fractions containing H3 and H4 at nM concentrations were found to kill numerous bacterial species. A number of studies have identified H1 isoforms, or their fragments, as having anti-microbial activity (Hiemstra et al. 1993; Kawasaki and Iwamuro 2008). The activities of core histones within NETs, which are released during a specific form of cell death from activated neutrophils, has been well documented against several pathogens (Brinkmann et al. 2004; Branzk and Papayannopoulos 2013). Histones exert their antimicrobial action via different mechanisms. In vitro, H3 and H4 exert their bactericidal effects by membrane damage, leading to the formation of bacterial membrane blebs (Morita et al. 2013). In contrast, the

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antibacterial activity of H2B occurs following cleavage of H2B by a bacterial outer membrane protease (OmpT) of gram-negative bacteria, (Tagai et al. 2011).. Membrane penetrating peptides A number of cell-free, in vitro and in vivo studies have identified extracellular histones as cell-penetrating peptides (CPPs), able to traverse biological membranes. Direct translocation of linker and core histones across the plasma membrane has been observed in human cells (Hariton-Gazal et al. 2003). However, core histones were unable to penetrate human erythrocytes and Escherichia coli. Histones have also been shown to translocate across the membrane of unilamellar or multilamellar vesicles and liposomes. H1 acquired more α-helical and β-sheet structures upon binding to liposomes (Rosenbluh et al. 2005), indicating a change in protein conformation, becoming more structured. In an in vitro study, core and linker histones were found to penetrate mitochondrial membranes, resulting in membrane destabilization and release of pro-apoptotic proteins (Cascone et al. 2012). These findings indicate that despite their high degree of charge and hydrophilic nature, histones are able to interact with biological membranes and traverse them. However, it should be noted that many of these reports have been carried out with histone proteins at relatively high concentrations (5-20 µM; (Hariton-Gazal et al. 2003), which may not be the situation in vivo. Apoptosis Several studies have reported that linker and core histones become relocalised to non-nuclear locations following the activation of programmed cell death mechanisms stimulated by DNA damage, irradiation, cytotoxic drugs or cellular activation (reviewed in (Parseghian and Luhrs 2006). In response to DNA damage, histone H1 can translocate from the nucleus to mitochondria, where the H1.2 isoform specifically activates the release of cytochrome C (Konishi et al. 2003; Okamura et al. 2008). Loss of nuclear H1 has also been observed in early apoptotic neural cells, suggesting a possible translocation to mitochondria in mediating the release of cytochrome C as observed for H1.2 (Ohsawa et al. 2008). In addition, histones and/or nucleosomes have also been reported at the surface of apoptotic immune cells (Holers and Kotzin 1985; Klein et al. 2014), suggesting a possible role of apoptotic cell surface histones as an ‘eat me’ signal to make them recognizable for phagocytosis. Apoptosis impairs nuclear/plasma membrane integrity and hence, translocation of histones from the nucleus may be a result of membrane disintegration. Nevertheless, histone relocation from the nucleus to subcellular locations/plasma membrane has been observed in several contexts where cells are healthy, as mentioned earlier in this section.

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Extracellular histones in disease Cellular stress can be considered as a common denominator for a number of acute and chronic conditions resulting in histone release, which may act as damage-associated molecular pattern molecules (DAMPs). Extracellular histones are able to mediate a number of downstream responses following activation of specific signalling pathways; Sepsis Release of H3 and H4 have been shown to mediate endothelial dysfunction, organ failure, and death in mouse and primate models of sepsis (Xu et al. 2009). Following histone administration in vivo, sepsis-related dysfunction was provoked and this was efficiently blocked by anti-histone treatments. In another study, binding of extracellular histones to Toll-like receptors (TLR4 and TLR2) mediated liver toxicity, cytokine production, tissue injury and death in sepsis models (Xu et al. 2011). Peritonitis Necrotic cell-derived histones have been shown to trigger peritoneal inflammation by activating Nod-like receptor protein 3 (NLRP3) and inflammasome-dependent release of cytokines such as interleukin-1beta (IL-1β; (Allam et al. 2013). In vitro, H4-mediated IL-1β release was significantly reduced in the presence of antioxidants. This explained the dependency of histone H4 on oxidative stress in triggering NLRP3 activation. Histone administration triggered NLRP3 dependent inflammation in an in vivo model of peritonitis, whereas anti-H4 antibody treatment prevented lethality (Allam et al. 2013). Pancreatitis Extracellular histones mediated cytotoxic and inflammatory responses in acute pancreatitis through the release of HMGB1 (Huang et al. 2014; Kang et al. 2014). Moreover, reduced levels of HMGB1 in the pancreas triggered the release of histones H3 and H4. Anti-histone H3 or anti-HMGB1 treatment protected mice from pancreatitis. Chronic Obstructive Pulmonary Disease Increased levels of extracellular hyperacetylated histone H3.3 have been reported in inflammatory chronic obstructive pulmonary lung disease (Barrero et al. 2013). Histone H3.3 induced impaired protein degradation, increased calcium influx and mitochondrial damage in lung epithelial cells, whereas H3 antibody treatment greatly reduced lung injury following H3 treatment. Stroke Extracellular histone/nucleosome release has been reported during ischemic stroke/reperfusion injury. Injection of histones in vivo in mouse models amplified stroke symptoms with increased infarct volumes, which could be reduced by anti-histone H4 antibody treatment (De Meyer et al. 2012).

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Trauma Trauma results in marked cellular stress. The release of extracellular histones, or histone administration, in trauma models leads to an inflammatory response, coagulation and acute lung injury. Blocking extracellular histones prevented histone-mediated toxicity in these animal models (Abrams et al. 2013). Retinal detachment The release of histone H3 from stressed retinal cells in vitro or with retinal detachment (RD) in vivo has been reported (Kawano et al. 2014). Extracellular histones mediated pro-inflammatory activity and retinal cell toxicity by modulating the TLR/mitogen-activated protein kinase (MAPK) pathway. The adverse effect of histones on the retina was blocked by vitreous body (the clear liquid present between the lens and the retina of the eye) treatment, TLR/MAPK pathway inhibitors, or anti-histone treatment. Thrombocytopenia and deep vain thrombosis Several studies have identified functions for extracellular histones in platelet activation and aggregation, resulting in thrombus formation both in vitro and in vivo. Extracellular histones mediate platelet activation via ERK, serine/threonine-specific protein kinase (Akt), mitogen activated protein kinase (p38), and nuclear factor κB (NFκB) pathways, as well as TLR2/4, and in stimulating platelet aggregation via cell surface integrins (Semeraro et al. 2011; Carestia et al. 2013). The ability of histones to induce platelet aggregation leads to thrombocytopenia, a condition where individual platelet counts are significantly reduced (Fuchs et al., 2011). In vivo, histones possibly originating from NETs, have been shown to promote deep vein thrombosis (Fuchs et al. 2010; Fuchs et al. 2011). Anti-histone treatment significantly reduced histone-mediated pro-coagulant or thrombus formation both in vivo and in vitro (Lam et al. 2013; Wildhagen et al. 2014). Neurodegenerative diseases Extracellular histones are associated with a number of neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), prion disease and amyotrophic lateral sclerosis (ALS). In vitro interactions of core and linker histones with alpha-synuclein have been observed in AD and PD. Histone H1 is associated with extracellular amyloid plaques in AD (Duce et al., 2006). In prion disease and AD, non-nuclear histone H1 is up regulated in neurons and astrocytes. The presence of extracellular histones has been observed in the transgenic mouse model of ALS (Zhang et al., 2006). Degenerating brain tissue releases histones, including H1, which was found to be highly toxic to neurons at low nM concentrations and drove a pro-inflammatory response in glial cells, mediating astrocyte activation and microglial activation and survival (Gilthorpe et al., 2013). These studies not only implicate an important role for extracellular

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histones in neurodegeneration but also a possible role for secretory pathway engaged in the release of histones. Extracellular histones in circulation Elevated levels of extracellular histones and nucleosomes have been documented in human/animal circulation in several infectious and non-infectious diseases including sepsis, chronic obstructive pulmonary disease, trauma, stroke, autoimmune diseases and cancers (Chen et al. 2014). An increase in modified histone (citrullinated histone H3) levels in serum during sepsis has also been reported (Li et al. 2011; Li et al. 2014b). In the aforementioned studies, high levels of serum histones/nucleosomes typically correlate with lethality. In addition to this, a basal level of circulating histones has been reported in a number of healthy human and animal serum samples, implicating a possible secretion of histones through a regulated pathway (Pemberton et al. 2010). However, it is not clear how these histones are released from cells in the first place. In summary, histones are externalized from cells in a number of in vitro and in vivo contexts and are up-regulated in many diseases. In cases where they have been tested, anti-histone therapies have had beneficial effects. However, apart from cell death, how and when histones might be secreted is not clear. As histones appear to be present normally and in serum from healthy individuals, it important to determine if and how constitutive histone secretion might occur and if this could be upregulated in response to stress. Protein secretory pathways Protein secretion is an essential cellular process, although it may be specialized in certain cell types to facilitate certain functions, and may change in response to adaptation or stress (Rabouille 2017). In eukaryotes, protein secretion involves multiple pathways, which can be classified broadly into canonical or non-canonical pathways (Nickel and Rabouille 2009; Ding et al. 2012). The secretion process involves trafficking, sorting, and transport of proteins to the plasma membrane, or to the extracellular space. A number of intracellular organelles such as the endoplasmic reticulum (ER), Golgi complex, endosomes and their vesicular intermediates are employed in the secretory process. In addition, protein transport often involves PTM, e.g. glycosylation. Canonical and non-canonical pathways of signal-peptide containing proteins The signal peptide is a short amino acid sequence, usually present at the N-terminus of a protein. A newly synthesized protein that carries a signal peptide sequence is recognized by the ER signal recognition particle receptor present in the ER membrane, through which translating proteins enter into the ER lumen

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followed by the cleavage of the signal peptide sequence (Wilkinson et al. 1997). Signal peptide proteins that are targeted to ER can be secreted either through canonical or non-canonical pathways. Canonical ER- and Golgi-dependent secretion The majority of proteins carrying a signal peptide are processed via the canonical ER-Golgi dependent pathway (Fig 1, a; (Burgess and Kelly 1987). Following the entry of secretory proteins into the ER, small GTPases present in the ER participate in cargo selection and export of newly translated, folded proteins from the budding of the ER membrane in coat protein complex II (COPII) coated vesicles (Lee et al. 2004). The Golgi apparatus consists of a group of flattened membrane compartments that process proteins and facilitate their transport to different destinations, such as early endosomes, late endosomes, lysosomes, and plasma membrane, or to the extracellular matrix. The cis-facing Golgi compartment (cis cisternae) faces the ER and the trans-facing compartment faces away from the ER and together form the boundaries of the trans- Golgi network (TGN). The medial Golgi stack, made up of 3 to 8 such membrane compartments, forms the remainder of the Golgi apparatus. Cis cisternae receive newly translated proteins from ER through COPII coated vesicles. Two partly conflicting models have been proposed to explain the transport of proteins within the Golgi (Glick and Nakano 2009). In the Stable Compartment Model, anterograde transport of secretory cargoes happens in long-lived cisternal membranes through Golgi-derived intermediate COPI vesicles and exit via the TGN, whereas resident Golgi proteins remain within the compartments. The small GTPase ADP ribosylation factor 1 (ARF1), along with the coatomer complex, mediates the release of COPI vesicles through membrane fission (Lee et al. 2004). In the Cisternae Maturation Model, cisternae constantly mature from cis to trans, carrying the secretory cargoes and retrograde transport of Golgi resident proteins through COPI vesicles. In both of these models, the TGN is the final location for the sorting and exit of secretory cargos to their final destinations (De Matteis and Luini 2008). In the Golgi, especially at the TGN, proteins may be subjected to final post-translational modification including glycosylation and proteolytic cleavage (Gleeson 1998; De Matteis and Luini 2008). Following the fission of secretory cargo carrier vesicles from the TGN, proteins are ultimately released upon the fusion of these with the plasma membrane. Membrane fusion of inter-Golgi compartments and their vesicular intermediates are mediated by soluble NSF (N-ethylmaleimide-sensitive factor) accessory protein (SNAP) receptors (SNAREs). The ER-Golgi intermediate complex (ERGIC) is the site of retrograde retrieval of ER-resident proteins, with the help of ARF1 and mediated by COPII coated vesicles, in order to enable the continual process of canonical ER-Golgi transport (De Matteis and Luini 2008).

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Non-canonical ER-dependent and Golgi-independent secretion Unlike the canonical pathway, proteins that carry a signal peptide that exit the ER in COPII coated or uncoated vesicles can fuse directly with the plasma membrane (Fig 1, b). For example, α-integrins are secreted membrane-associated proteins that exit the ER via COPII coated vesicles but bypasses the Golgi during their release (Schotman et al. 2008). Another pathway involves the fusion of COPII coated vesicles with late endosomes or lysosomes, which may then fuse with the plasma membrane to release their protein contents (Fig 1, c). For example, the cystic fibrosis transmembrane conductance regulator (CFTR) is a well documented protein secreted via this ER-late endosome pathway (Wang et al. 2004). Non-canonical pathways of leaderless protein secretion Proteins that are secreted but lack a signal peptide sequence are termed leaderless secretory proteins (LSPs). Four major routes of secretion have been defined for LSPs and are also grouped as non-canonical pathways (Nickel and Rabouille 2009). One such pathway is referred to as direct secretion, whilst the other three are based on vesicle-mediated pathways. Direct secretion Cytosolic leaderless proteins can be secreted directly across the plasma membrane without the participation of the ER-Golgi pathway (Fig 1, d). Direct translocation of fibroblast growth factor 2 (Fgf2) has been reported from the cytoplasm to the extracellular space, in which anchoring of the phospholipid phosphatidylinositol-4, 5-bisphosphate (PIP2) to the cytosolic side of the plasma membrane is essential in recruiting Fgf2 for release (Schafer et al. 2004). Secretory lysosomes The lysosome is an intracellular organelle that helps in the digestion of unwanted proteins. However, secretory lysosomes can also carry cytosolic proteins for their release into the extracellular space (Fig 1, e). For example, caspase 1 and activated IL-1β are targeted to secretory lysosomes for exocytosis (Andrei et al. 2004). Microvesicle secretion Direct outward budding, or shedding, of vesicles from the plasma membrane, may lead to the release of proteins enveloped by a double membrane structure and these extracellular vesicles are referred to as microvesicles (MVs) or ectosomes (Fig 1, f). They range from 100-1000 nm in diameter (Thery et al. 2009). MVs may carry a number of proteins normally localized to the plasma membrane or cytoplasm and also nucleic acids. The release of caspase 1 and mature IL-1β has also been reported to occur via MVs (MacKenzie et al. 2001). MVs were initially thought to be the only source of membrane-derived vesicles

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in the extracellular space, or in the circulation. However, a second, mode of vesicular secretion referred to as the exosome pathway, was first documented in the 1970-80s. Exosome secretion Extracellular vesicles of endocytic origin were first discovered by Gunnar Rönnquist and colleagues when studying the activity of a plasma membrane-dependent adenosine triphosphatase in human prostatic fluid (Ronquist and Hedström 1977; Arvidson et al. 1989). Hence, they were named prostasomes. Subsequently, the same class of vesicles was discovered independently in studies of the trafficking of the transferrin receptor in maturing reticulocytes (Harding et al. 1983; Pan et al. 1985) and Rose Johnstone and colleagues coined the term exosome in 1987 (Johnstone et al. 1987).

Figure 1. Protein secretory pathways a. Secretion of signal peptide containing proteins via the cannonical ER-Golgi pathway. b. Proteins that are translated on the ER but bypass the Golgi for their secretion. c. Incorporation of proteins to MVBs from COPII-coated vesicles. d. Direct secretion of proteins across the plasma membrane. e. Secretion of proteins from lysosomes. f. Release of proteins via outward budding of microvesicles. g. Biogenesis of MVBs and subsequent release of ILVs as exosomes. Abbrevaitions; ER-endoplasmic reticulum, COPII-coat protein coated II vesicle, TGN-Trans-Golgi network, MVB-multivesicular body, ILVs-intraluminal vesicles, ECM-extracellular matrix, PM-plasmamembrane. In contrast to the outward budding of MVs from the plasma membrane, the exosome pathways begin with inward budding leading to the formation of early endosomes (EEs). These mature along the EE-late endosome (LE) pathway and

COPII-coatedvesicle

ER

GolgiCOPI-coatedvesicle

Signal-pep8decontainingproteins

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MVB

ILVs Lysosome

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PMECM

Microvesicleshedding

a

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bud inwardly in LEs to form multivesicular bodies (MVBs), which contain numerous intra luminal vesicles (ILVs) formed within an outer limiting membrane (Fig 1, g). Following the fusion of MVBs with the plasma membrane, ILVs are released to the extracellular space as exosomes with a typical diameter of 30-100 nm (Raposo and Stoorvogel 2013). Exosomes are secreted by perhaps all cell types and carry a wide variety of cytoplasmic, nuclear and membrane-derived proteins, as well as nucleic acids including RNA, micro RNA (miR), DNA and bioactive lipids (Colombo et al. 2014; Thakur et al. 2014). Pathways for exosome biogenesis and secretion The sorting of cargo proteins into ILVs during the biogenesis of MVBs is known to occur via distinct pathways, which can be broadly categorized into endosomal sorting complexes required for transport (ESCRT)-dependent or ESCRT-independent (Colombo et al. 2014). ESCRT-dependent biogenesis The role of the ESCRT complex in the biogenesis of ILVs and the sorting of cargo into them has been well established and is conserved from yeast to humans (Henne et al. 2011). Vacuolar protein sorting (Vps) class E proteins are required for the formation of vesicles at the limiting membrane of MVBs and are organized into five major ESCRT complexes (ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III and Vps4). The hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) and signal transducing adaptor molecule (STAM), components of ESCRT-0 recognize ubiquitinated cargos and are recruited to the surface of endosome membranes to engage and initiate the pathway. ESCRT-0 then recruits ESCRT-I components tumor susceptibility gene 101 (TSG101), Vps28, Vps37 and multivesicular body 12 (Mvb12), followed by ESCRT-II members (Vps38/Snf8 or SWItch/Sucrose Non-Fermentable, Vps22, and Vps25). ESCRT-I and ESCRT-II are responsible for sorting cargo proteins and clustering them, resulting in membrane invagination. ESCRT-II then recruits ESCRT-III (charged multivesicular body proteins or CHMP6, CHMP4, CHMP3, CHMP2), which drives vesicle budding during which cargos are sorted. Cargos are deubiquitinated before the final budding into ILV by ESCRT-recruited deubiquitinated machinery such as associated molecular with the SH3 domain of STAM (AMSH) and ubiquitin isopeptidase Y (UBPY) in mammals (Henne et al. 2011). The ESCRT member Vps4 is recruited to the inwardly budding vesicle to disassemble the ESCRT complex and initiate membrane scission, which results in the formation of ILVs within the limiting membrane of the MVB. A reduction in exosome secretion by lowering levels of the ESCRT-0 protein HRS has shown (Tamai et al. 2010). This is also consistent with the effect of silencing early ESCRT components (STAM1 or TSG101) resulting in reduced exosome secretion, whereas silencing of ESCRT-III and accessory proteins

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(CHMP4C, VPS4B, Vps twenty-associated-1 (VTA1) or apoptosis-linked gene-2-interacting protein X (ALIX) increased it (Colombo et al. 2013). However, decreased exosome secretion has been observed following the knockdown of VPS4B and ALIX in another study (Baietti et al. 2012). Although the importance of ESCRTs in exosome biogenesis has been relatively well studied, these aforementioned studies point to a greater complexity in the mechanisms regulating exosome biogenesis/secretion. ESCRT-independent biogenesis The biogenesis of ILVs/MVBs and secretion of exosomes can also occur in an ESCRT-independent manner. In oligodendrocytes, ESCRT-independent biogenesis of exosomes carrying proteolipid protein (PLP) was found to be dependent on ceramide (Trajkovic et al. 2008). Ceramide formation from the precursor sphingomyelin via the activity of neutral sphingomyelinase (nSMase) was found to induce vesicle budding within the limiting membrane of MVBs. Treatment with GW4869, a compound that inhibits the activity of nSMase, resulted in reduced secretion of PLP-positive exosomes (Trajkovic et al. 2008) and other exosome markers such as CD63 (Cluster of differentiation), CD81, or TSG101 (Chairoungdua et al. 2010; Kosaka et al. 2010; Dreux et al. 2012; Hoshino et al. 2013). Another ESCRT-independent pathway involved in the release of syntenin-positive exosomes had been reported (Ghossoub et al. 2014). In this pathway, phospholipase D2 (PLD2) and the small GTPase ARF6 were involved in the budding of ILVs. In melanocytes, protein ubiquitination and the ESCRT machinery are not required for the sorting of pre-melanosomal protein, PMEL into ILVs (Theos et al. 2006). In a subsequent study, it was shown that CD63 participated directly in the sorting of a luminal fragment of PMEL into ILVs in an ESCRT-independent manner, but the disposal of the remaining transmembrane fragment required ESCRT (Niel et al., 2011). Overexpression of CD91, or CD9, has also been reported to result in increased secretion of β-catenin containing exosomes and may represent another example of an ESCRT-independent pathway (Chairoungdua et al. 2010). Syntenin-dependent exosome release is influenced by certain ESCRT members such as ALIX, but not by others, indicating that the complete ESCRT machinery is not essential for ILV production (Baietti et al. 2012). In support to this, ESCRT loss of function did not completely block MVB biogenesis and exosome release (Theos et al. 2006; Stuffers et al. 2009; Colombo et al. 2013). A small integral membrane protein (SIMPLE) and a mutant form were found to lower the secretion of ALIX- and CD63- carrying exosomes (Zhu et al. 2013). Thus, there are multiple pathways

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involved in the biogenesis/secretion of exosomes. Some involve the ESCRTs machinery to various degrees and some do not. Factors regulating the number and size of exosomes In addition to exosome biogenesis, a number of studies have reported a role of ESCRT family members in regulating the size of ILVs/exosomes. Apart from cargo sorting, HRS (ESCRT-0) has also been shown to promote larger ILVs, whereas HRS depletion results in smaller vesicles (<50nm; (Colombo et al. 2013; Edgar et al. 2014). In contrast, silencing of STAM1 (ESCRT-0) increased the proportion of larger vesicles >100 nm. Ubiquitinated cargo loading and ESCRT-dependent mechanisms have been shown to result in the formation of larger ILVs (Edgar et al. 2014). Another study has shown that CD63-dependent formation of small ILVs (<40 nm) are suppressed by the HRS-dependent formation of large ILVs (>40 nm; (Edgar et al. 2014). Alteration in the size of ILVs (Stuffers et al., 2009 and Edger et al., 2014) and exosomes (Colombo et al. 2013) are influenced by ESCRT and non-ESCRT members, which infer the existence of subpopulations of MVBs and exosomes of different sizes (Laulagnier et al. 2005; Bobrie et al. 2012a; Oksvold et al. 2014). In summary, studies have revealed the existence of a range of different ILVs and exosomes (30-200 nm) derived from the endosomal pathway, but it still remains unclear how many types and how these are regulated. Methods for the study of extracellular vesicles Although EV preparations purified by different methods are often referred to as exosomes, the most common methods used to purify them cannot completely separate MVs from other types of EV. This is because they share similar properties, for example overlapping size, density and protein content. A variety of names have been given to EVs; exosomes in particular. However, terms such as dexosomes, exosome-like vesicles, exovesicles, prostasomes and synaptic vesicles have also been used (van der Pol et al. 2016). In this thesis, the terminology EV will be used to refer to any particular type of EV where the specific subtype is not known. The term EVs will be used throughout the text unless specifically referring to exosomes. Different strategies have been employed in order to separate heterogeneous populations of EVs based on their size, shape, density and surface markers (Li et al. 2017). The isolation method chosen typically depends on the starting sample volume, viscosity and the degree of purity required for downstream applications. Careful standardization of the selected protocol is essential in determining the integrity, purity, and yield of the isolated vesicles. Each method has advantages and limitations. Several tools have been developed to aid in validating and analysing EVs.

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Isolating and fractionation of extracellular vesicles Differential Centrifugation Differential centrifugation is one of the most widely used methods for the isolation of EVs from e.g., conditioned cell medium (Thery et al. 2006). Sequential centrifugation steps are performed to separate EVs from other cellular and extracellular material based on their density and size with a successive increase in centrifugal force. The conditions used vary between users (Li et al. 2017). Exosome–free FCS (f0etal calf serum) is typically used to generate cell conditioned media since is devoid of calf serum-derived EVs that would contaminate the final product. A low-speed centrifugation step (e.g. 300 g/10 min.) is typically performed to eliminate cellular debris and large apoptotic bodies. A second higher speed centrifugation at 10,000-20,000 g is applied to remove apoptotic bodies and larger MVs. Finally, EVs are sedimented at ≥110, 000 g in an ultracentrifugation (UC) step. The resulting pellet of EVs is typically suspended and washed in a large volume of sample buffer, such as phosphate buffered saline, depending on the downstream application. Vesicle preparations may be stored at 4°C for several days or snap-frozen and stored at -80°C for long-term storage (Zhou et al. 2006). EVs prepared via differential centrifugation may be subject to additional purification steps to increase sample purity. One limitation is that biological activity may be reduced or lost due to vesicle damage or vesicle fusion under high g force. Density gradient ultracentrifugation In order to eliminate other particles, e.g. protein aggregates, that may co-sediment with EVs at 110,000 g or other vesicles that are of similar size to exosomes, density gradient UC may be employed (Escola et al. 1998). The method involves layering a concentrated sample of EVs onto the top or bottom of a density gradient, e.g. made of sucrose, Nycodenz or Optiprep solutions. In sucrose gradient UC, top loading has been shown to enrich vesicles in fractions corresponding to 1.12–1.19 g/ml, whereas bottom loading led to the separation of low density (LD) and high density (HD) vesicles into two different fractions (Willms et al. 2016). Following UC (e.g. ≥110, 000 g/16 hours), EVs band within discrete zones of the gradient that matches their buoyant densities, which can be collected. Fraction densities may be estimated, e.g. by weighing a known volume. Each gradient fraction containing EVs can be washed and pelleted by UC using a large volume of buffer and sample pellets may be analysed immediately or stored at -80C° for later use. In a two-step gradient approach, EVs that float at the interface zone are collected, washed with a large volume of buffer and stored for analysis (Hedlund et al. 2009). These EVs are highly enriched in exosomes. Sucrose gradients have been widely used for EV purification. However, their high osmotic pressure may damage vesicles and not facilitate the efficient separation of vesicles with similar size and density. On the other hand, Iodixanol/Optiprep gradients have been used to separate similarly

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size and density of exosomes from viral particles and from contaminating apoptotic vesicles (Cantin et al. 2008) and EVs derived from conditioned media (Graner et al. 2009) or biological fluids (Iwai et al. 2016). On sucrose gradients, exosome floatation densities range from 1.08–1.22 g/ml and the floatation density of exosomes on Optiprep gradients has been reported to peak at 1.09 g/ml (Ji et al. 2013). Unlike sucrose, the density of the Optiprep solution can be easily measured by absorbance at 244 nm (Ji et al. 2013). UC-based isolation method yield exosomes of high-purity, but the downside of this approach is that it is both time and labour consuming with a low yield (Momen-Heravi et al. 2012). Several other methods have been developed in an attempt to overcome these shortcomings but may result in a preparation with a lower degree of purity. Ultrafiltration EVs of a specific type can be separated from other contaminating vesicles using ultrafiltration (UF) with pore sizes ranging from 100-200 nm in diameter. For example, nanomembrane concentrators have been optimized for the purification of urinary exosomes isolated from a 0.5 ml sample (Cheruvanky et al. 2007). Sequential filtration steps were employed using filters with different pore sizes to enrich exosomes (Li et al. 2017). An initial filtration step through a 100 nm membrane filter was used to eliminate cellular debris and large particles, leaving vesicles and other contaminating proteins in the flow through. Next, the flow through was passed through a molecular weight cut-off filter (500 kDa), where the vesicles were retained. Finally, filtration using a 100 nm track-etched filter concentrated vesicles such as exosomes. A filtration step may also be coupled with UC-based protocols to remove larger particles/vesicles and improve purity. For example, a 0.2 µm filtration step may be used to remove vesicles larger than 200 nm prior to the UC step in an EV isolation protocol as mentioned above. UF is simple to perform. However, this method alone cannot fully eliminate protein or vesicle contamination. Furthermore, losses can be considerable due to the fact that vesicles may stick to the filter membrane or become trapped in the filter pores, resulting in filter blockage. The force required for filtration may also result in vesicle deformation and hence, vesicles of a larger diameter may pass through a smaller sized pore. Size-exclusion chromatography Another size-based separation method for EVs is size-exclusion chromatography (SEC; (Nordin et al. 2015). A single step separation column made out of sepharose and nylon has shown to efficiently separate EVs of 70 nm from plasma (Boing et al. 2014) and has shown to be more efficient in yielding intact EVs compared to precipitation methods (Gamez-Valero et al. 2016). In another comparative study, efficient separation of EVs, increased yield and

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intact vesicle morphology were observed using UF-size-exclusion liquid chromatography (LC) compared to UC (Nordin et al., 2015). Commercially available iZon SEC columns have been used to isolate EVs from bovine milk (Vaswani et al. 2017) and for many other applications. Antibody-coated beads The occurrence of characteristic protein markers on the surface of EVs enables EVs to be purified based on immunocapture. An antibody against an EV/exosome surface marker (e.g. tetraspanin proteins such as CD9, CD63, CD81 etc.) is immobilized on beads, which may be magnetic to aid in separation of the beads from the supernatant, and used to ‘pull down’ exosomes from the sample (Clayton et al. 2001). Crude EV preparations, e.g. following low-speed differential centrifugation, can be used for immunocapture, where specific exosomes can be purified based on the chosen antibody (Tauro et al. 2012). This method has certain limitations, such as the binding of soluble proteins to the beads, or non-specific binding of vesicles/proteins. If used for functional studies there may be an impairment of EV function following elusion from the beads, and this approach is not appropriate for large samples volumes. Exosome precipitation Several exosome isolation kits are commercially available and work on the principle of precipitating exosomes using water-excluding polymers (Taylor et al. 2011). Precipitated exosomes are collected either by a brief centrifugation or filtration step. This method is simple and the yield may be much higher than following density gradient centrifugation, as shown in a comparative study (Yamada et al. 2012). However, because precipitation is not selective, purity is likely to be much lower. Moreover, several kits on the market lack rigorous validation. Methods to analyse extracellular vesicles A range of methods may be used to analyse and validate EVs at various stages of the isolation process. Vesicle shape and size Due to their small size, negative contrast transmission electron microscopy (TEM) has been widely used to analyse the size and morphology of EVs. This is one of the most useful approaches to validate EVs in a sample (Thery et al. 2006). The appearance of vesicles and sample purity with/without protein contamination are clearly evident through TEM. Moreover, size distribution can be estimated. However, it is limited to vesicles that adhere to the EM grid and this may be by other things present in the sample, such as proteins. Another shortcoming includes labelling of vesicles with heavy metals in order to generate negative contrast, as well as drying of the sample on the grid. These can

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generate artefacts such as the classical cup-shaped morphology of exosomes (Li et al. 2017). TEM can also be combined with immunostaining in immuno-electron microscopy (IEM). Here, immunogold labelling of surface antigens on EVs can be visualized on vesicles, which can also give a measure of sample purity and protein marker localization/distribution. Cryo-EM has enabled the size and morphology of EVs to be studied without drying or the need to use chemical fixation or staining procedures (Yuana et al. 2013). In addition, Cryo-EM allows protein structures to be studied in situ in EVs (Zeev-Ben-Mordehai et al. 2014). Light scattering methods EVs are too small to be resolved by conventional light microscopy. Light scattering methods utilize Brownian motion to determine size. Dynamic light scattering (DLS) or photon correlation spectroscopy is one light scattering method, which measures laser scattering by the sample to determine vesicle size and size distribution (Szatanek et al. 2017). DLS may be used to estimate particle sizes ranging from 1 nm to 6 µm. However, this method cannot be used to determine vesicles number or concentration (Lawrie et al. 2009). Nanoparticle tracking analysis (NTA) is another light scattering method used to determine the size and also quantify particles (Nordin et al. 2015). This is a sensitive, fast and user-friendly method that can analyse samples containing ≈ 2 ×108-109 particles/mL (Soo et al. 2012). NTA can also be used in fluorescence mode to distinguish fluorescently labelled EVs. Proteins Following the electrophoretic separation of proteins based on size, western blotting is the most commonly used method to analyse EV proteins. Western blotting helps in the validation of the isolation/purification strategy in addition to a more detailed analysis of selected protein markers. In order to analyse EV proteomes in detail, mass spectrometry (MS) can be used (Thery et al. 2001). Gene ontology (GO) enrichment analysis can then be performed to validate up/down-regulated proteins between samples statistically, in addition to other essential comparative parameters (Yao et al. 2015). Flow cytometry has been widely used to analyse vesicles based on direct fluorescence-labelled surface markers (van der Pol et al. 2012), or indirect labelling. In the indirect method, beads are labelled with a capture antibody against a specific vesicle surface marker protein. A commercially available polystyrene bead-based MACSPlex Exosome kit has been designed to evaluate exosomes with 37 different surface antigens, fluorescently by flow cytometry (Koliha et al. 2016). Nucleic acids Agarose gel electrophoresis is one of the most widely used electrophoretic methods to separate nucleic acids such as DNA and RNA. Nucleic acids present

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in EV samples such as exosomes can be analysed qualitatively using agarose gel electrophoresis. More sensitive methods are available for the quantification of nucleic acids. One method used in this thesis was a Qubit fluorometer. This is a rapid way of estimating the quantity of nucleic acids based on specific fluorescence-based probes after binding to double-stranded DNA (dsDNA), single-stranded (ssDNA), RNA, microRNA or proteins using only a few microliters of input sample (Li et al. 2014a; Vergauwen et al. 2017). Exosomal DNA (exoDNA) or exosomal RNA (exoRNA) profiling can be performed to analyse nucleic acids present in exosomes by sequencing (Huang et al. 2013; San Lucas et al. 2016). Lipids Lipid components including sphingomyelin, cholesterol, phosphatidylserine (PS), ceramide and their derivatives are enriched in EVs, and in particular, exosomes (Laulagnier et al. 2004; Trajkovic et al. 2008). In order to separate lipid classes, chromatographic techniques such as thin layer chromatography and gas-liquid chromatography were initially used in lipid analysis of EVs. A later study on human B-cell EVs utilized MS to analyse their lipid content (Wubbolts et al. 2003; Skotland et al. 2017). Regulation of microvesicle biogenesis As discussed earlier, MVs bud outwardly from the plasma membrane and several proteins that are involved in MV biogenesis have been identified. For example, the interaction of TSG101 with the retroviral gag protein (Booth et al. 2006), or with the endogenous protein arrestin domain containing protein 1 (ARRDC1; (Nabhan et al. 2012) at the plasma membrane triggers the outward budding of MVs. Several studies have identified that the redistribution of cytoskeletal components following cell stimulation (reviewed in (Hugel et al. 2005), expression of the small GTPase ARF6 (Muralidharan-Chari et al. 2009), or reduced levels of actin-nucleating protein diaphanous-related formin 3 (DRF3/Dia2; (Di Vizio et al. 2009) influence the outward budding of MVs. A role for the Rab family of small (20–29 kDa), monomeric, Ras-related guanosine triphosphatase (GTPase) proteins, regulate multiple steps in the intracellular trafficking of vesicles including vesicle formation, vesicle movement and membrane fusion (Stenmark 2009). A role for Rab22A has been reported in MV budding (Wang et al. 2014) and in astrocytes the activity of acid sphingomyelinase triggers the release of large MVs (Bianco et al. 2009). Hence, the regulation of MV budding is likely to be a complex process. However, the regulation of exosome biogenesis and release, which occurs via the endocytic pathway through multiple steps, is perhaps likely to be even more complex.

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Regulation of exosome biogenesis and secretion A number of proteins that are engaged in the process of exosome secretion have been identified. Rab GTPases Rab GTPases act as molecular switches that cycle between GTP-bound (active) and GDP-bound (inactive) states. More than 60 different mammalian Rab proteins have been identified and distinct Rabs are thought to play specific roles in the process of intracellular vesicle transport. Interestingly, roles for several Rab proteins in MVB biogenesis and exosome secretion have been identified (Colombo et al. 2014). Inhibiting the function or expression of Rab11 inhibits the release of exosomes (Savina et al. 2002). Loss of function of Rab2b, Rab5a, Rab9a, Rab27a, or Rab27b inhibits exosome secretion and a role for Rab27a and Rab27b in the docking of late endosomes/multivesicular bodies (MVBs) at the plasma membrane has been described (Ostrowski et al. 2010). In a murine oligodendroglial cell line, as well as in primary oligodendrocytes, Rab35 regulates the release of major myelin PLP-bearing exosomes and in the docking of MVBs at the plasma membrane (Hsu et al. 2010). According to Rab depletion studies carried out in different cell types, Rabs have been shown to regulate the release of exosomal proteins. Although the release of wingless- (wg) or evenness interrupted (Evi) -bearing exosomes is regulated by Rab11 as mentioned above, reduction of Rab27/Rab35 has no effect on this (Koles et al. 2012; Beckett et al. 2013). Rab11 or Rab35 regulate the secretion of flotillin and anthrax toxin via exosomes, but secretion of these proteins was not affected by Rab27a /Rab27b (Abrami et al. 2013). Rab27a depletion reduced the release of small vesicles in several tumor cell lines (Bobrie et al. 2012b; Peinado et al. 2012). In a mouse carcinoma line, Rab27a silencing affected the release of CD63, Alix, and TSG101 in exosomes but did not modulate the release of CD9 or the phosphatidyl serine-bound Mfge8 (Bobrie et al. 2012a). Moreover, an overall reduction in exosome secretion was observed followed by Rab27a and Rab27b silencing (Zheng et al. 2013). Rab7 has been shown to influence the secretion of Alix and syntenin positive exosomes in a tumor cell line (Baietti et al. 2012), whereas Ostrowski et al., found no effect of Rab7 on exosome secretion (Ostrowski et al. 2010). Thus different Rabs appear to be engaged in distinct subsets of late endosomes or MVBs and regulate the secretion of specific subtypes of exosomes and their associated protein cargoes. SNAREs SNAREs facilitate the fusion of membranes/vesicles. However, the role of SNARE proteins in the membrane fusion process involved in exosome secretion has been less well studied. Vesicle-associated membrane protein 7 (VAMP7), a member of the SNARE family, promoted the release of acetylcholine esterase bearing EVs in human leukaemia cells, whereas the release of heat shock

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protein 70 (Hsp70) containing EVs was unaffected following the depletion of VAMP7 in other cells types (Fader et al. 2009). YKT6 v-SNARE homolog (Ykt6), another SNARE member, has been reported in Drosophila late endosomes and is involved in the secretion of wg, syntaxin 1a and Evi positive exosomes (Koles et al. 2012). The Ral GTPases RAL-1 is present on MVB membranes, where it has a role in MVB formation and exosome secretion in C. elegans and in mammalian tumor lines (Hyenne et al. 2015). In the same study, the SNARE protein SYX-5 colocalised with active RAL-1 and had a role in MVB docking at the plasma membrane. Like the Rabs, different SNAREs seem to regulate exosome generation/secretion by acting at different points of membrane fusion in the late endosomal pathway. Other factors regulating exosome secretion Alterations in intracellular calcium levels have been shown to modulate exosome secretion in several studies. The calcium ionophore ionomycin increases exosome secretion by elevating the intracellular calcium concentration in several cell types including a leukaemia cell line (Savina et al. 2002), dendritic cells (Montecalvo et al. 2012), mast cells (Raposo et al. 1996), oligodendrocytes (Kramer-Albers et al. 2007) and cortical neurons (Lachenal et al. 2011). In another study, an influx of calcium following depolarization of rat cortical neurons increased the secretion of exosomes (Faure et al. 2006). In addition to this, neurotransmitters triggered the release of exosomes from neurons and oligodendrocytes (Lachenal et al. 2011; Fruhbeis et al. 2013a). Other intracellular factors, such as increased levels of tumor suppressor protein p53 and its downstream target tumor suppressor activated pathway-6 (TSAP6) induced cell senescence and resulted in increased exosome secretion (Honegger et al. 2013). Intercellular interaction has been shown to regulate exosome secretion. For example, the interaction of murine dendritic cells with T-lymphocytes increased the release of dendritic cells exosomes (Buschow et al. 2009). In addition to the aforementioned studies, several other factors have been shown to modulate exosome secretion, such as high levels of diacylglycerol kinase α that reduced exosome secretion in T-cells (Alonso et al. 2007). Citron kinase (a RhoA effector) promoted the secretion of vesicles bearing exosome markers in HIV-1 viral infected human cells line (Loomis et al. 2006). V0 subunit of V-ATPase induced docking of MVBs at the plasma membrane in C. elegans (Liegeois et al. 2006) and exogenous addition of IL-1β increased exosome secretion in astrocytes (Willis et al. 2017). Furthermore, ARF6 induced phospholipase D2 (Ghossoub et al. 2014) or overexpression of phospholipase D2 (Laulagnier et al. 2004) increased exosome secretion.

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Molecular composition of EVs EVs are composed of a double-lipid membrane and contain both proteins and nucleic acids. Their specific cargos are believed to confer the function of specific vesicle populations. The composition of all proteins, lipids and nucleic acids that have been identified in EVs isolated via different procedures and from numerous sources, including cultured cell types and biological fluids from different organisms, are organized collectively in the databases Exocarta (Mathivanan et al. 2012), EVpedia (Kim et al. 2015a) and Vesiclepedia (Kalra et al. 2012). Proteins Proteomic analysis is one of the most commonly used methods to determine the composition of EVs. MS analysis of peptides derived from proteins carried in EVs has led to the identification of numerous proteins that are enriched in EVs (Fruhbeis et al. 2012). Proteins engaged in the biogenesis of MVBs/exosomes are typically enriched in exosome proteomes and include several members of the tetraspanin family of proteins (CD9, CD63, CD81, and CD82), Rab GTPases, heat shock proteins, ESCRT members, ALIX, flotillin-1 etc. Many of these proteins are considered to be exosome markers and antibodies against these proteins are used routinely to identify them (Thery et al. 2009; van Niel et al. 2011). Other proteins that are found in EV proteomes are representative of the parental cell type (Table 2). As an example, exosomes derived from neural cell types carry neural cell-specific proteins such as neural cell adhesion molecule L1 (L1CAM), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits or prion protein (PrP; (Faure et al. 2006; Lachenal et al. 2011). Oligodendroglial cell-derived exosomes carry myelin proteins such as PLP, 2′, 3′cyclic nucleotide 3′-phosphodiesterase (CNP), myelin-associated glycoprotein (MAG) and myelin-oligodendrocyte glycoprotein (MOG) (Kramer-Albers et al. 2007). Astrocyte exosomes carry glial acidic fibrillary protein (GFAP; Willis et al., 2017) and microglial cell-derived EVs carry surface-bound aminopeptidase N (CD13) and the monocarboxylate transporter 1 (MCT1; (Potolicchio et al. 2005).

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Cell type EVs markers References Neuron L1CAM, AMPA receptor subunits, PrP Faure et al.,

2006; Lachenal et al., 2011

Oligodendrocyte PLP, CNP, MAG, MOG Krämer Albers et al., 2007

Astrocyte GFAP Willis et al., 2017

Microglia CD13, MCT1 Potolicchio et al., 2005

Table 2.Cell-type specific proteins present in EVs

Other proteins better reflect the status if the parental cell, such as a pathological condition. For example, elevated expression of pro-inflammatory cytokines, oncogenic, immunosuppressive and angiogenic factors have been documented in aggressive glioblastoma multiforme tumor-derived exosomes (Graner et al. 2009). For neurodegenerative diseases, several disease-associated proteins such as α-synuclein, amyloid β peptide, tau, PrP or superoxide dismutase-1 have been found in exosomes, either as monomers or as disease-associated misfolded or aggregated forms (Bellingham et al. 2012). Lipids Studies on the lipid composition of EVs have highlighted the enrichment of cell membrane lipid components including sphingomyelin, cholesterol, PS, ceramide and their derivatives (Trajkovic et al. 2008). A study in nematodes has shown enrichment of different lipids in the membrane of EVs with a low content of sphingomyelin and cholesterol, but a high content of plasmalogens, implicating a shift in the enrighment of certain lipids in EVs, perhaps for a specific function (Simbari et al. 2016). EVs are often enriched in surface exposed PS, which is usually present on the inner leaflet of the plasma membrane. This becomes surface exposed in apoptotic cells and can be detected by binding to a fluorescently labeled Annexin V. This method has also been applied in the analysis of exosomes (Fitzner et al. 2011). Exosomes possibly lack the flippase enzyme that is essential in sustaining PS on the inner membrane leaflet (Hugel et al. 2005). A comparative study of the lipid content of EVs found that different EVs contain specific classes of lipids, depending the parental cells of origin (Haraszti et al. 2016). For example, EVs derived from human bone marrow-derived mesenchymal stem cells and hepatocellular carcinoma cells were enriched with cardiolipins but those from the glioblastoma cell line U87 were not. A specific set of lipids enriched in one EV population versus another helped in identifying EV subpopulations in another study (Laulagnier et al. 2005). Changes in cellular

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conditions can alter the lipid composition of EVs. For example, during reticulocyte maturation, a change in lipid composition of EVs has been observed (Carayon et al. 2011), or when cells are cultured under certain pH conditions (Parolini et al. 2009). These studies implicate the altered sorting of lipids into EVs in relation to parental cellular conditions. Nucleic acids Studies on EVs have detected the presence of nucleic acids including RNA and DNA (Valadi et al. 2007; Balaj et al. 2011). Microarray analysis of mast cell exosomes led to the discovery of mRNA and microRNA (miRNA) (Valadi et al. 2007). Subsequent studies have focused on the nucleic acid content of EVs and identified several classes of RNAs. For example, presence of ribosomal RNA (rRNA) subunits such as 28S and 18S detected in EVs derived from human breast cancer cell lines (Jenjaroenpun et al. 2013). In a study carried out on EVs derived from three different colorectal cancer cell lines, RNAs including natural antisense RNA has been reported (Chiba et al. 2012). Interestingly, the RNA profile of exosomal mRNA and miRNA are different from that of their parental cell line, suggesting that incorporation of specific RNAs occurs in exosomes (Creemers et al. 2012). Several studies have identified the transfer RNAs via EVs into recipient cells. For example, transfer of mRNA from human and mouse mast cell exosomes has been shown (Valadi et al. 2007). Exosomal mRNA, miRNA and natural antisense RNA can transfer from human colorectal cancer cell lines into human hepatoma cell lines (Chiba et al. 2012). Transfer of exosomal miRNA from T cells to (Creemers et al. 2012) antigen-presenting cells was also documented (Mittelbrunn et al. 2011). Several studies have investigated the function of exosomal RNAs in intercellular communication (Ramachandran and Palanisamy 2012). Transferred exosomal mRNAs are translatable (Valadi et al. 2007) and can modulate the gene expression profile of recipient cells (Maida et al. 2016). Tumor-specific small RNAs have been found to circulate in cancer patients (Eldh et al. 2014; Nedaeinia et al. 2017) and several patient plasma/serum derived exosomal miRNAs are recognised as prognostic cancer biomarkers (Munagala et al. 2016). After the discovery of RNAs in EVs, both dsDNA and ssDNA have been identified in tumor cell-derived exosomes, where ds exoDNA has been found both intra- and extra-vesicularly (Balaj et al. 2011; Kahlert et al. 2014; Thakur et al. 2014). In addition to this, EVs of astrocytes, glioblastoma cells (Guescini et al. 2010), cancer-associated fibroblasts and EVs derived from cancer patient plasma (Sansone et al. 2017), have been shown to contain mtDNA. Circulating exoDNA derived from pancreatic cancer patients identified exoDNA as the potential source of circulating K-RAS (a proto-oncogene) mutants and tumor

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protein gene TP53 DNA (Allenson et al. 2017). The functional significance of exoDNA is not yet clearly defined. However, like exosomal RNAs, circulating exosomal DNAs are used for non-invasive cancer diagnosis (reviewed in (Kalluri and LeBleu 2016). Functions of EVs The first discovery of exosome-like vesicles was ‘prostasomes’ present in human prostatic fluid (Ronquist and Hedström 1977; Arvidson et al. 1989). Exosomes were discovered while examining how the transferrin receptor was removed from maturing reticulocytes (Harding et al. 1983; Pan et al. 1985; Johnstone et al. 1987). Perhaps for this reason, for a long period exosomes were thought to function as a vehicle for the removal of unwanted proteins from cells. Subsequent studies, particularly with the discovery of proteins that have a signalling function in exosomes (reviewed in (Thery et al. 2009), revealed their potential role in intercellular communication (Thery et al. 2002). Identification of circulating exosomes in biological fluids indicated that signalling might be effected over a long range in vivo (Caby et al. 2005; Ludwig and Giebel 2012). EVs can signal through several mechanisms including the direct interaction of exosome membrane ligands, or their cleavage products, with a receptor present on the surface of target cells (Thery et al. 2009). Alternatively, they may transfer their contents to target cells by means of direct fusion with the plasma membrane, or the following endocytosis by recipient cells (Thery et al. 2009; Mathivanan et al. 2010). For example, in one of the initial studies, dendritic cell exosomes were shown to be endocytosed, processed inside DCs and then secreted as exosomes carrying surface complex of major histocompatibility class II loaded with an alloantigen. This resulted in signalling to CD4+ T-cells, both in vitro and in vivo (Morelli et al. 2004). Subsequent studies on immune cells have identified the specific interaction of exosome surface molecules on recipient cells, either in eliciting or suppressing immune responses (Thery et al. 2009). As an example, mature DC exosomes bearing surface receptor inter cellular adhesion molecule 1 (ICAM1) have been shown to interact with CD8+ DCs cell surface ligand lymphocyte function-associated antigen 1 (lFA1) in order to present antigens (Segura et al. 2007). EVs are relatively well recognized in the field of cancer because of their potential to transform cells to a cancerous state. In one such example, tumor cell-derived exosomes were able to transform recipient cells efficiently to a cancerous state and thereby promote cancer metastasis (Nakano et al. 2015). The signalling properties of EVs also depend on their nucleic acid content. RNAs such as mRNA within of exosomes may be translated and result in changes in gene expression of in recipient cells (Valadi et al. 2007; Skog et al. 2008). Exosomes carrying functional non-coding RNAs such as miRNA

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transfer signals between cancer and stromal cells in the human endometrium (Maida et al. 2016). Numerous studies on exosomal miRNAs have identified their roles in tumor cell migration, metastasis and modulating immune responses (reviewed in (Bell and Taylor 2017). EVs in the nervous system The central nervous system (CNS) contains 4 main cell types including neurons as well as supporting glial cell types; astrocytes, oligodendrocytes, and microglia. As with other organ or tissue systems, effective communication between cells of the nervous system is important for brain homeostasis. The role of EVs in neuron-neuron, glia-glia, or neuron-glia communication under both physiological and pathological conditions have begun to emerge with the main evidence coming from in vitro culture studies (Fruhbeis et al. 2012; Budnik et al. 2016). Main functions of exosomes in the CNS that have been identified so far are highlighted below. Neurons Neurons derived from primary culture or neuron-like cells differentiated from tumor cell lines have been shown to secrete exosomes (Faure et al. 2006; Lachenal et al. 2011; Chivet et al. 2014). The release of exosomes by primary cortical neurons is induced by depolarization (Faure et al. 2006). The presence of L1CAM, PrP and glutamate receptors in these exosomes led to a role being suggested for them in transcellular communication within the brain (Faure et al. 2006). Mature cortical neurons have been shown to release exosomes that are regulated by glutamatergic synaptic signals (Lachenal et al. 2011). In a follow-up study, neuron-neuron communication via exosomes was examined (Chivet et al. 2014), where binding of neuroblastoma-derived exosomes to hippocampal neurons was observed. However, these exosomes were endocytosed specifically by glial cells but not by hippocampal neurons (Chivet et al. 2014). Hence, this shows a selective transfer of exosomes between cell types, which could possibly involve specific mechanisms in their uptake. In the peripheral nervous system, regeneration of the sciatic nerve following injury in vitro or in vivo occurred following internalization of exosomes secreted by dedifferentiated Schwann cells and carried the neurotrophin receptor p75 (Lopez-Verrilli et al. 2013; Escudero et al. 2014). Exosomes have also been reported to be involved in the spread of neurodegeneration, brain tumours and neuroinflammation (Budnik et al. 2016). In relation to neurodegenerative diseases, exosomes have been recognised as a possible mediators in the propagation of disease by carrying disease-specific proteins such as α-synuclein, β-amyloid, β-amyloid precursor protein (APP), tau, scrapie prion PrPSc and misfolded SOD1 (Bellingham et al. 2012). Thus, exosome cargos appear to determine their function and exosomes are potential

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vehicles for intercellular signalling in various contexts, including during development and in disease. Astrocytes Astrocytes help maintain ionic balance and provide trophic and metabolic support to neurons in the CNS (Nedergaard et al. 2003). Activated astrocytes are known to be involved in neurodegeneration (Barres 2008; Liddelow and Barres 2017) and EVs maybe an important part of their involvement. PrP-bearing exosomes from astrocytes have been shown to protect murine cerebellar neurons against various cellular stresses such as oxidative stress, hypoxia, ischemia and hypoglycemia (Guitart et al. 2016). Treatment of astrocytes with AD-associated β-amyloid resulted in the secretion of exosomes bearing pro-apoptotic proteins. Internalization of these EVs by astrocytes led to cell death and implicates a pathogenic role for exosomes in AD (Wang et al. 2012). Transfer of exosomes bearing mutant SOD1, a protein associated with the pathogenesis of ALS, from astrocytes to motor neurons led to neuronal cell death (Basso et al. 2013). Thus, several studies identified a broader role of astrocyte-derived exosomes in neurodegenerative disorders. Oligodendrocytes Oligodendrocytes myelinate axons in the CNS and increase the conduction velocity of nerve impulses, as well as providing trophic support for axons and facilitating myelin repair (Baumann and Pham-Dinh 2001). Amongst the cell types of the CNS, the mechanisms underlying the biogenesis of oligodendrocyte exosomes has been relatively well studied. The presence of oligodendrocyte MVBs in the periaxonal space supports a role for exosomes in glia-neuron communication (Fruhbeis et al. 2013b). Neuronal signalling through the neurotransmitter glutamate enhanced the secretion of oligodendrocyte exosomes with an increase in intracellular calcium levels (Fruhbeis et al. 2013a). Neurons selectively internalized and retained the function of oligodendrocyte exosomes cargo (Fruhbeis et al. 2013a). Moreover, the uptake of exosomes augmented neuronal metabolic activity and protected them against cellular stress conditions (Fruhbeis et al. 2013a). In addition to this, oligodendrocyte exosomes had an auto-inhibitory effect on myelin sheath formation and this was shown to be regulated by neuronal factors (Bakhti et al. 2011). The regulation of exosome secretion by Rab35 and identification of an ESCRT- independent, ceramide-dependent pathway for exosome secretion was first reported in oligodendrocytes (Trajkovic et al. 2008; Hsu et al. 2010). Exosomes from primary murine oligodendrocytes are enriched in myelin proteins including PLP and protective proteins against oxidative stress (Kramer-Albers et al. 2007). Further studies have identified functions for oligodendrocyte exosomes in pathological contexts. Exosomes derived from Oli-neu cells, a

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murine oligodendrocyte line, were macropinocytosed selectively by a subset of MHC class II-negative microglia in an immunologically silent manner under normal conditions, whereas the uptake of exosomes decreased in interferon gamma- (IFNγ) stimulated microglia. This data suggest that there could be and accumulation of oligodendrocyte exosomes in the extracellular space under neurodegenerative conditions due to reduced clearance by microglia (Fitzner et al. 2011). Microglia Microglia are resident immune cells of the brain that can release pro- or anti-inflammatory signals and also clear extracellular debris. The protein profile of microglia-derived exosomes is similar to that of exosomes derived from several immune cell types (Potolicchio et al. 2005). Primary murine microglia secreted exosomes carry aminopeptidase, important in neuropeptide breakdown, and the lactate transporter MCT-1 (Potolicchio et al. 2005). Treatment with the wingless/int1 3A (Wnt3A) protein induced and altered the secretion of primary microglial exosomes, in which Wnt3A was endocytosed and incorporated into exosomes (Hooper et al. 2012). In addition, serotonin-stimulated microglia triggered the release of insulin-degrading enzyme carrying exosomes, which degrades one of the constituent proteins of extracellular AD plaques; β- amyloid (Glebov et al. 2015). Moreover, inhibition of microglial exosome secretion by nSMase2 silencing prevented the spread of tau protein in an AD model (Asai et al. 2015). These studies have highlighted the neuron-targeted functions for microglial-derived exosomes in ameliorating AD pathology.

In summary, numerous independent lines of evidence provide support for neuron-neuron, glial-glial and neuron-glial communication via exosomes. These studies have shown possible ways through which exosome secretion are regulated and their effective participation in transmitting messages between cells of the nervous system in healthy as well as in disease conditions. Cellular stress responses Cellular stress is evident in almost all pathological situations. Cells respond to a wide range of stresses including heat stress, unfolded/misfolded proteins, oxidative stress, DNA damage, ageing etc. Cellular responses can be protective or destructive, depending on the intensity, duration of the insult and also on the inherent ability of the cell to withstand/overcome such stresses. Depending on the type of stress, cells initiate stress-specific responses such as the heat shock response (HSR), unfolded protein response (UPR) or oxidative stress response (OSR) etc. (Fulda et al. 2010). Based on the load and type of stress, these specific pathways coordinate together to produce a profound effect of either ‘do or die’ in the battle for cell survival. Stress responses modulate secretion, and secretomes are enriched with stress responsive proteins that help to overcome

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the insult and also alert neighbouring cells, as part of a protective response. However, depending on the mode and severity of stress incurred, cells may also activate a destructive response, transferring messages associated with pathology. Understanding proteins that are differentially secreted upon stress and their route of secretion would pave the way to understand and perhaps control the release of disease-associated proteins, which would aid in development of diagnostic and therapeutic approaches against different diseases. The HSR (Richter et al. 2010) stimulates the expression and activity of a wide range of heat shock proteins including Hsp27 and Hsp70. These are inducible molecular chaperones that bind to unfolded proteins/segments that have been denatured by the exposure to elevated temperature. Hsp90 is expressed constitutively and helps prevent premature protein folding under normal condition and is also bound to an inactive monomeric heat shock factor 1 (HSF1) (Shamovsky and Nudler 2008). Under stress conditions, accumulation of unfolded proteins competes with HSF1 for binding to Hsp90. Release of HSF1 induces the expression of Hsp27 and Hsp70 to activate survival pathways and inhibit cell death mechanisms such as apoptosis (Fulda et al. 2010). Hence, the HSR is a general cellular protection mechanism mediated by HSPs against heat stress, but also in other stress conditions including oxidative stress, heavy metals stress etc. The UPR is another well-studied cellular stress response, which is stimulated upon encountering an elevated burden of unfolded/misfolded proteins in the ER. Upon induction of ER stress, three immediate pathways are stimulated by ER stress receptors such as PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1(Ron and Walter 2007). They further stimulate downstream signalling pathways to induce the expression of UPR genes, which perform their function in inhibiting protein translation, enhancing chaperone production etc. to overcome the accumulation of unfolded proteins and help maintain cell survival. Stressed cells have been shown to release signals that lead to an adaptive response in neighbouring cells (Ghazi and Lamitina 2015). This adaptive response could be transmitted via EVs and several studies have identified the secretion of elevated levels of stress-related proteins through EVs, which are presented below. EVs in stress responses The first study to investigate EV secretion in response to stress was reported in B-lymphocytes (Clayton et al. 2005). Heat stress resulted in alterations in the protein content of exosomes and increased exosome release. Secretion of Hsp27, Hsp70, and Hsp90 was identified within the lumen of exosomes. However,

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immature dendritic cells failed to mature (which is essential in triggering immune responses) following treatment with stressed B-cell exosomes (Clayton et al. 2005). In another study, elevated levels of HSP70 in exosomes from peripheral blood mononuclear cells following heat stress has been documented (Lancaster and Febbraio 2005). Exposure to hypoxic conditions was another stress paradigm that was found to increase the release of Hsp60 from adult cardiac myocytes. However, in this case Hsp60 was found to be associated with the exosome membrane rather than the lumen (Gupta and Knowlton 2007). Alterations in both the protein and RNA content of exosomes were found in endothelial cells following a variety of cellular stresses (hypoxia, tumor necrosis alpha-treatment, hyperglycaemia) conditions (de Jong et al. 2012). In vivo responses in exosome release have also been investigated. For example, one study in rat has identified the modulated release of Hsp72 and miR-142-5P in plasma-derived exosomes following an insult from acute stressors such as inescapable tail shock (Beninson et al. 2014). Hence, a number of studies, both in vitro from different cell types and in vivo, have found altered expressions of proteins and RNAs in exosomes in response to stress. Other studies have investigated the potential function of stress-responsive exosomes. Messenger RNAs in murine mast cell exosomes were modulated following oxidative stress. Stress-induced mRNAs in exosomes were found to have a protective role against a later oxidative stress insult, which was abolished when exosomes were exposed to UV light (Eldh et al. 2010). Heat-stressed tumor cell-derived exosomes have been shown to act as chemoattractants for dendritic and T-cells and lead to their activation, which facilitated potent anti-tumor activity and protected mice from tumor growth (Chen et al. 2011). In addition to this, oxidative and heat stress of malignant T- and B-cell lines led to increased secretion of exosomes bearing the activating NK cell receptor natural killer group 2, member D (NKG2D; (Hedlund et al. 2011). Oxidative stress led to increased release of exosomes from retinal cells and these exosomes were found to have a role in promoting angiogenesis (Atienzar-Aroca et al. 2016). Irradiated MCF7 breast epithelial cancer cell-derived exosomes impart genomic instability in non-irradiated cells (bystander cells) and they can intern pass on this effect to their progeny (Al-Mayah et al. 2012; Al-Mayah et al. 2015). In another study, heat-stress EVs induced DNA damage and apoptosis in recipient unstressed bystander cells. However, these cells showed greater resistance to a later heat stress (Bewicke-Copley et al. 2017) suggesting that adaptation had occurred. Hence, the aforementioned studies highlight the potential role of stressed cell exosomes in transferring messages to help defend against a future stress, which could be important in resistance or susceptibility to various pathological scenarios.

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Background to Paper I The midbrain-hindbrain boundary The midbrain-hindbrain boundary (MHB) is an important local organizing centre that forms during embryonic development (Chi. et al. 2003). It patterns the adjoining tectum (midbrain) and cerebellum (hindbrain) through the localized secretion of signalling factors (morphogens) including Fgf8 and Wnt1. Local organizers typically form at a boundary between different compartments. In order for a compartment boundary to form, it is important that cell mixing is restricted between the two adjoining compartments. At the MHB, early expression of the homeobox transcription factors Otx2 and Gbx2 mark the cells that are destined to form the midbrain and hindbrain, respectively (Millet. et al. 1996; Wassarman. et al. 1997 ). During boundary formation, expression of Fgf8 becomes restricted to the posterior side of the MHB (hindbrain) and reinforces a stable boundary (Shamim and Mason 1999). Likewise, the anterior side of the midbrain boundary is occupied with Wnt1 expression. Activation of the Notch signalling pathway is essential for cell lineage restriction and the segregation of cells at the MHB (Wu. and Rao. 1999). However, it is not understood how this occurs and what other extracellular factors are involved in boundary formation. The Drosophila leucine-rich repeat (LRR) proteins capricious (caps) and tartan (trn) play important roles in the compartment boundary that forms in the developing wing disc imaginal disc (Milan. et al. 2001). The LRR Neuronal family (Lrrn1-3) are the closest vertebrate relatives of caps and trn based on sequence similarity and domain organization (Taguchi. et al. 1996). However, whether they are also functionally important for boundary formation is not understood. In chick, Lrrn1 is expressed in a pattern that suggests it may have a role in boundary formation (Christiansen. et al. 2001). Lrrn1 expression is downregulated at forebrain and hindbrain boundaries, as well as at the MHB at the same time that boundary-restricted expression of morphogens are upregulated suggesting a potential role for Lrrn1 in regulating MHB formation (Andreae et al. 2007). In Paper I (Tossell. et al. 2011), both in vivo experiments in the chick embryo in vitro experiments in cell culture were used to help understand the function of Lrrn1 in the formation of MHB.

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Aims of thesis

The overall aim of this thesis was to investigate how extracellular proteins regulate cellular behaviour (Paper I) and how cellular stress or disease state affects protein secretion (Paper II and III). The main aim of Paper II was to understand whether histone proteins are released from cells in a regulated manner and whether this occurs via the exosome pathway. In Paper III I developed methods to translate findings gained in cell culture to patient by quantifying extracellular histones in biofluids from cancer patients. Paper I Investigate the role of the Lrrn1 protein in midbrain-hindbrain boundary (MHB) formation. To optimize cell culture, transfection and western blotting methods to be used in subsequent studies. Paper II To investigate whether or not extracellular histones are secreted via EVs/exosomes. To determine where extracellular histones are located and how their release is regulated. Paper III To develop the methods to quantify extracellular histones in biofluids, using ascites samples from epithelial ovarian cancer (EOC) patients as a model.

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Materials and Methods

Cell culture The idea of culturing cells from a dead organism was first developed by Claude Bernard in 1878 (reviewed in (Oyeleye et al. 2016). In 1885, Roux described a method to culture embryonic chick cells in a saline medium. Subsequently, animal cell culture has become widely used for the culture of specific types of cells in vitro. Cell culture is a useful approach to experimental research that uses cells that have been isolated and adapted to culture ex vivo/in vitro under defined conditions. Cell culture enables cell line to be preserved, maintained and expanded. These can then be used to isolate biological material, depending on the purpose of the study. One advantage is that relatively large amounts of material can be derived from a pure cellular source. It is straightforward to isolate the cells themselves as well as any secreted products from the culture medium. Hence, this approach has been very useful for protein and antibody production, or in the study of cellular characteristics and activities that are involved in protein secretion, including EVs.

The culture of newly isolated cells, e.g. from a tissue sample, is referred to as primary cultural. This typically leads to cell cultures that are heterogeneous, but can be purified to homogeneity via passage or sub-culturing, termed secondary culture. In adherent cell culture, cells typically grow as a single layer or monolayer on a plastic surface and their growth is generally restricted by contact inhibition. However, many transformed or oncogenic cell lines do not retain contact inhibition. Therefore, they can be grown to high density. We utilized such a cell line; human embryonic kidney 293T (HEK293T) cells, for cell aggregation assays in Paper I. Cells immortalisation is important for the continuous growth of cells in culture. The expression of tumor antigens (e.g. T antigens or T-Ags) has been used to generate immortalised cell lines, for example, cancer cells that are transformed by the expression of viral oncoproteins (e.g. SV40 T-Ag). Cells can also acquire spontaneous mutations that enable them to replicate continuously, such as the oligodendroglial cell line, OLN-93 (Richter-Landsberg. and Heinrich. 1996) used in Paper II. Rodent cells can be readily immortalized because they also express the telomerase enzyme. This enables their chromosomes to replicate continuously without undergoing replicative senescence. On the other hand, human cells require telomerase expression in order to escape senescence. Cell culture requires a defined growth medium, temperature and other conditions to mimic the physiological situation as closely as possible (Nema and Khare 2012). The growth medium provides an artificial environment in order

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for cells to satisfy their requirements for bioenergetic and biosynthetic substrates and maintain the cell cycle (Yao and Asayama 2017). Different cell types may require different supplements in order to maintain growth. Serum is typically added to the media as it contains numerous growth-promoting factors. Serum containing media, usually supplemented with foetal calf serum (FCS), as well as serum-free media is available depending on the requirements for optimal growth of cells. Most cells are maintained in a buffered medium at normal extracellular pH (pH 7.4). The pH is usually balanced by the buffering action of CO2 (5-10%) levels in the cell culture incubator and sodium bicarbonate provided in the growth medium. An incubator is also used to maintain the temperature at e.g. 37°C in a humidified atmosphere, optimal for the growth for most cell types. Cells undergo heat stress (heat shock) if the temperature is raised significantly above 37°C and this can be utilized in order to study cellular activity and behaviour under heat stress conditions.

In Paper I, I have used HEK292T cells for cell sorting/aggregation assay as mentioned earlier. In order to study histone secretion in Paper II, I used the rat oligodendrocyte progenitor cell line OLN-93 as a model. This was because of their neural origin and also because they could be grown to high density under serum-free conditions to facilitate EV isolation. Previous work from the group has shown that degenerating neural tissue releases histones (Gilthorpe et al. 2013) and oligodendrocytes are a neural cell type in which linker and core histones have been reported within exosome proteomes (Kramer-Albers et al. 2007; Trajkovic et al. 2008; Hsu et al. 2010) Hence, we focused our efforts on these but also investigated the release of histones via EVs from the murine oligodendrocyte progenitor (Oli-neu) and HEK293T cell lines. There are several important considerations relating to cell culture when working with EVs. In vitro, EVs are isolated from the cell culture media that has been conditioned by the cells that release them. A significant proportion of EVs is typically lost through the various stages of the purification protocol. Hence, in order to isolate a suitable quantity of EVs for downstream applications, a large number of cells are required. For cell monolayers, the volume of conditioned media that can be collected is proportional to the number of growing cells/surface area of the culture. Hence, it is advantageous if cells can be grown to a high cell density. Typically, many culture flasks amounting to a growth area of 2000 cm2 or more need to be used. However, cell media can only be conditioned for a short period (24h-48h) before harvest in order to avoid excessive cell death due to over confluence. Following the collection of conditioned media, cells are not usually reused or maintained due to them becoming over confluent, which may affect their

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subsequent behaviour. An alternative is to grow cells to a high density in smaller volume of media using a bioreactor. In our study, OLN-93 eGFP-CD63 or CD63-RFP stable cell lines were maintained in a bioreactor (CLAD1000, Integra Biosciences AG, Zizers, Switzerland) as previously described (Mitchell et al. 2008). The bioreactor that we used in the thesis consists of two compartments; one for media and another for culturing the cells. Both of the compartments are separated by a 10kD semipermeable membrane, which allows for the efficient exchange of nutrients from the medium compartment and to eliminate waste products from the cell compartment. Using this approach, a high surface area for growth can be achieved using a 3D growth substrate but a minimal volume of medium can be conditioned in the cell compartment to produce a large amount of EVs. In the bioreactor, cells can be maintained for a long period of time with reduced media consumption compared to flasks. It is important to use EV-free media when isolating EVs from cells in order to minimize the amount of contaminating exosomes from FCS present in the media. In this case exosome-free FCS (exo-free FCS) can be generated by UC at 110,000 g for 18 h at 4°C. However, this is not as effective at supporting cell growth as normal serum due to the removal of growth promoting substances from exo-free FCS during the UC process. Hence, cells are typically expanded in normal FCS and then, after washing cells several times in PBS, media containing exo-free FCS is added for the period of EV harvest. Commercially prepared exo-free FCS is also available and does not differ from control FCS in terms of supporting cell proliferation and growth. However, when downstream applications rely on protein analysis, such as western blotting or proteomics, it is particularly important to use serum-free conditions since bovine serum albumin (BSA) becomes a major protein contaminant. Western blotting Western blotting or immunoblotting is a commonly used method for protein detection, size determination and quantification (Mahmood and Yang 2012). This is a convenient method with which to analyse proteins present in EVs. Moreover, guidelines provided by the International Society for Extracellular Vesicles (ISEV) include western blotting as one of the analytical methods that is recommended for use in the analysis and validation of EVs (Lotvall et al. 2014). The development of SDS-PAGE made it possible to separate denatured proteins based on their size, as well as other inherent properties including charge. Proteins in EV samples are denatured at high temperature (95°C) in the presence of SDS, prior to being loaded on an SDS-PAGE gel. Proteins bound by SDS have a net negative charge and move towards the positive electrode. A ladder of proteins of known molecular weights can be used to calibrate the gel. The resolving ability of SDS-PAGE is related to the gel pore size and buffer

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system. During gel electrophoresis, proteins of low molecular weight are retarded to a lesser extent than proteins of higher molecular weight and move faster through the gel. High percentage (e.g., 15-18%) acrylamide gels made with a basic pH (pH 8.8) creates smaller pore sizes for efficient separation of low MW proteins such as histones (10-25 kDa). However, when separated by SDS-PAGE, they run with a greater apparent molecular mass (e.g. H1 runs at ≈ 30 kDa instead of 25 kDa), which possibly arise from their hydrophobic properties (Shirai et al. 2008; Rath et al. 2009). Resolved proteins are then transferred (blotted) onto a membrane, such as supported nitrocellulose or polyvinylidene fluoride (PVDF), using electrophoresis. As mentioned earlier, SDS- bound proteins gain a net negative charge so they move towards the membrane placed towards the positive electrode. After transfer, a specific primary antibody is used to detect the protein and a directly labelled, or enzyme coupled, secondary detection antibody is used to visualize binding of the primary antibody to the protein as a band. The enhanced chemiluminescence (ECL) method is an indirect detection method commonly used with western blotting. Here, the activity of a horseradish peroxidase (HRP) enzyme, conjugated to a secondary antibody, is detected on photographic film or a digital camera by a light-generating reaction. The signal from the bound antibody can be used to determine the position of a protein band on a blot. It may also be used for quantification if suitable standards are available. Although highly sensitive, enzyme-dependent detection is not stable over time. Direct detection methods such as using a near-infrared (NIR) fluorescent dye coupled to a secondary antibodiy (LICOR Odyssey system) are perhaps less sensitive than ECL but have the advantage that more than one primary antibody/protein can be detected on the same blot and quantified easily. Here, the tag on the secondary antibody produces a fluorescent signal that is directly proportional to the amount of bound antibody. LICOR is useful when comparing signal intensities of high and low levels on the same blot. For this reason, it is very useful when detecting/quantifying the same proteins in cell vs EV samples. Proteomics Proteomics refers to the study of the total protein content of a sample from an organism, tissues or cell type or other biological sample (Li et al. 2015). In the 1980s, the idea of identifying proteins on a large scale begun with the help of two-dimensional (2D) gel electrophoresis to separate proteins based on isoelectric point and molecular weight. However, the development of the Edman degradation method of protein sequencing (which utilizes the N-terminus of a protein to sequence individual amino acids) led to a greater potential to identify proteins by their specific sequences. Developments of peptide detection via MS led to further advancement in the identification of specific proteins, as well as

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their post-translational modifications, integrating information from known and predicted proteins from DNA sequencing data. MS-based proteomics is a sensitive method that can be used to identify and quantify proteins present in EVs and the cells that produce them. Proteomic analysis involves liquid chromatographic (LC) separation of proteins coupled with tandem mass spectrometry (MS/MS) in the identification and quantification of proteins (Gaspari and Cuda 2011) . Protein samples are subjected to trypsin digestion in order to convert them to peptides. Several LC principles can be employed to separate peptides based on their charge, affinity and hydrophobicity etc. Ionization sources like an electrospray ionizer (ESI) convert separated peptides to charged ions and the mass/charge ratio of these peptides is subsequently measured in a mass analyser. Specific mass from the dataset is interrogated against a library of known peptides for the protein/organism identification. This helps in distinguishing tryptic peptides from e.g. rat versus cow. Therefore, contaminants from FCS can be detected and distinguished from rat proteins etc.. MS can also be used for protein quantification, where peptide mass intensity is used to calculate protein abundance (Zhang et al. 2010). Unlike western blotting, MS-based proteomics does not require protein specific antibodies to identify and quantify them. Moreover, it also provides a platform for comparing proteins between samples and can be used to assess protein enrichment. In this thesis, the proteomes of cells and the EVs derived from them were compared before and after heat stress in order to investigate changes that occurred between cells and EVs in response to stress.

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Results

Paper I Lrrn1 is required for formation of the midbrain–hindbrain boundary and organizer through regulation of affinity differences between midbrain and hindbrain cells in chick. The MHB is a local organizing centre located in the CNS. It forms at the boundary between the midbrain and hindbrain. Differential cell affinity is one mechanism by which compartment boundaries are established. However, the proteins that may mediate this at the MHB have not been identified. Lrrn1 is one of three proteins that make up the vertebrate LRR neuronal family. It is closely related to the LRR proteins caps and trn in Drosophila that are involved in boundary formation at the wing imaginal disc. Other vertebrate LRR-containing proteins regulate cell affinity, such as fibronectin leucine-rich transmembrane 3 (FLRT3), which can induce the sorting of cells via a mechanisms involving homophilic cell adhesion. In the chick embryo, Lrrn1 showed a dynamic expression pattern at the MHB during its formation. The organizer signalling molecule Fgf8 down regulated Lrrn1. Lrrn1 loss of function led to loss of the morphological constriction that forms at the MHB and loss of Fgf8 expression. Cells overexpressing Lrrn1 were able to cross the MHB and Lrrn1 also regulated the glycosyltransferase lunatic fringe, a modulator of Notch signalling, which is important for boundary formation. Hence, the potential role of Lrrn1 in MHB formation via homophilic cell adhesion was addressed in vitro. Lrrn1 does not induce cell sorting in a heterologous cell system HEK cells were used as a heterologous expression system and were transiently transfected with tagged mouse Lrrn1 expression vectors and also with positive and negative controls. The cells were then mixed with non-expressing cells under conditions where the potential to sort out through homophilic cell adhesion could be assessed. Cells expressing RFP were used as a negative control. LRR constructs were cotransfected with eGFP for visualization and to enable them to be distinguished from the RFP-expressing control cells. Cells were mixed in a sorting assay and shaken overnight in BSA-coated wells, where cells remain unattached to the well but can adhere to each other. We observed that Xenopus laevis (x) FLRT3 transfected cells (positive control) sorted into small clusters, whereas RFP transfected (negative control) cells did not. Cells expressing a mutant xFLRT3 construct that lacked the LRR domain, which is required for homotypic cell adhesion (negative control), did not sort. However,

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no sorting of Lrrn1 transfected cells was observed indicating that Lrrn1 does not regulate boundary formation via a mechanism involving homotypic cell adhesion. Expression of the transfected proteins was confirmed by western blotting. Paper II The exosome membrane localization of histones is independent of DNA and upregulated in response to stress. Extracellular histones contribute to several pathological conditions. However, the release of histones into the extracellular space is generally thought to occur through cell death-related mechanisms, such as apoptosis or necrosis. Since histones circulate in normal human serum and also populate the secretomes of healthy cells, it is possible that they are secreted via a constitutive pathway. Histones lack an ER signal-peptide sequence and are not likely to be secreted via the canonical ER-Golgi pathway. However, EVs are another pathway for protein secretion and the proteomes of exosomes from different cell types often contain histones. Therefore, we investigated the possible secretion of histones via EVs, focussing on exosomes. The rat oligodendrocyte progenitor cell line OLN-93 was used as a model because they could be grown to high density in a bioreactor, or under serum-free conditions, for EV isolation. Oligodendrocyte exosomes contain histones Negative contrast TEM was used to validate the purity and morphology of sucrose cushion isolated EVs. This showed the appearance of membranous vesicles of sizes ranging from 50-150 nm in diameter. Western blotting confirmed the presence of linker and core histones along with the classical markers of exosomes in a single-step sucrose cushion isolated EVs. We developed a discontinuous equilibrium density gradient using OptiPrep to determine the floatation density of the fraction containing histones and found the greatest enrichment in Fraction 6 (1.09 g/ml). Finally, IEM was used to identify histones sucrose cushion purified EVs. Histones are present in MVBs MVs are produced as they bud from the plasma membrane whilst exosomes are generated in the endocytic pathway in MVBs as ILVs. To verify if histones were present on MVs or exosomes, we next extended the analysis of extracellular histones to the intracellular MVB compartment. IEM analysis on ultrathin cryosections of OLN-93 cells confirmed that histones localize to ILVs and to the limiting membrane of MVBs. Interestingly, histone-positive ILVs were of a smaller diameter than histone-negative ILVs, ranging from 30-80 nm. These data confirmed that histones are indeed likely to be present in the endocytic pathway and secreted via exosomes.

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Histones localized to the exosome membrane To determine the localization of histones in EVs, we performed alkaline Na2CO3 (pH 11.5) treatment, which removes peripheral membrane proteins. We found that histones remained associated with the membrane fraction but not the luminal/soluble fraction. To further map the orientation of histones in the membrane, limiting trypsin digestion of intact OLN-93 EVs was used followed by western blotting with N- or C-terminal specific antibodies. We found that the N-termini region of histones was more sensitive to trypsin digestion than the C-terminus. This suggested that the N-termini of histones are relatively exposed at the surface of exosomes. To validate this in a human system, we generated HEK293 cell lines stably expressing histone H2A or H3 with HA and 6xhis tags at the N- or C-termini, respectively. In agreement with the results obtained in OLN-93 EVs, the N-terminal HA tag was more sensitive to proteolysis than the C-terminal 6xhis tag. These observations showed that histones are likely to be exosome membrane associated proteins with relatively exposed N-termini and conserved conformations in rat and human exosomes. DNA is present in EVs but is not dependent on histones for its localization Since histones are strongly associated with DNA in the nucleus and in apoptotic material or in NETs, we also examined the presence of DNA in EVs. This was confirmed using both agarose/ethidium bromide gel electrophoresis and a DNA-specific fluorometric probe (Qubit). Previous studies have reported the presence of ds and ss exoDNA, both inside and outside of exosomes. We found through DNase treatment in combination with Qubit analysis of OLN-93 EVs that the majority of DNA present was ssDNA and also present on the outside of vesicles. Trypsin removal of histones from the vesicle surface did not remove all exoDNA. Moreover, western blotting confirmed the presence of histones in EVs even after the elimination of exoDNA by DNase, suggesting that neither histones nor DNA are dependent on each other for localization to exosomes. Early upregulation of histones in EVs after heat stress The factors that regulate the secretion of histones via exosomes were investigated. The amount of EV-associated histones remained unchanged before and after oligodendrocyte differentiation. However, we found that cellular stress induced histone release via EVs. Heat stress induced a robust response and could be followed by the expression of heat shock proteins, so we chose the heat stress paradigm to study histone secretion at early (0-4 h recovery) and late (4-21 h recovery) time points following a 3 hour period of heat stress at 42°C. Western blotting showed an increased secretion of histones at the early recovery point, which remained high at the late recovery point, where heat shock protein secretion was evident. Live cell staining identified very little cell death (0.8%) at

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the early recovery time. Taken together, these data show that histones are up regulated in exosomes as an early response to cellular stress before the secretion of stress regulated markers such as Hsp70 and loss of cell membrane integrity/cell death in susceptible cells. Stress-induced secretion of small diameter EVs Vesicle size distribution was analysed before and after heat stress. Interestingly, TEM and NTA showed a notable upregulation in the secretion of smaller EVs (50-100 nm diameter) following heat stress, suggesting the possible incorporation of histones into a subpopulation of small diameter vesicles. Downregulation of ESCRTs following heat stress To identify proteins that might regulate histone release via EVs, we performed proteomic analyses on control and heat stressed cells and EVs. This revealed the presence of several distinct linker and core histone proteins in EVs. An overall decrease of histones in cells and a corresponding increase in EVs were observed. Analysis of differentially expressed proteins identified downregulation of ESCRT proteins following stress, which play important roles in MVB biogenesis. Hence, ESCRTs could possibly play a direct or indirect role in histone secretion following heat stress. Paper III Mitochondrial DNA in the tumor microenvironment activates neutrophils and is associated with worsened outcomes in patients with advanced epithelial ovarian cancer. EOC is a prevalent form of cancer. The tumor microenvironment of EOC is characterized by the accumulation of ascites, occurrence of activated immune cells and inflammatory cytokines. Elevated levels of circulating neutrophils have been shown to correlate with a poor outcome in EOC patients. However, the reason for this is unknown. The tumor microenvironment contains mitochondrial DAMPs including mtDNA, which has previously been shown to activate neutrophils and lead to the release of NETs. Therefore, this study was based on the hypothesis that mitochondrial DAMPs could induce NETosis, mediating the formation of tumor-associated thrombosis, platelet activation and thereby increasing the incidence of tumor metastasis. The release of histones/DNA from neutrophils during NETosis is well established and is important for the defence against microbial infection. Moreover, the role of extracellular histones, possibly released in NETs, has previously been reported in platelet activation, aggregation and thrombosis. Therefore, in this study we quantified histones in EOC patient ascites samples and fractionated these to investigate the possible role of ascites-derived histones in platelet activation, thrombosis and cancer progression. My contribution was in developing the method for, as well as quantifying histones in, ascites samples.

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Presence of elevated HI levels in ovarian cancer ascites Histones were analysed in unfractionated ascites by western blotting using standards containing known amounts of histones. Based upon the understanding of histones as a component of in EVs (Paper II), centrifugation at 10,000 g was first used to pellet histones in large MW complexes including apoptotic bodies. The 10,000 g supernatant samples were analysed by western blotting. Interestingly, 10,000 g centrifugation did not deplete histones from the supernatant. H1 was detected in almost all ascites supernatants samples but only a few ascites supernatants showed the presence of core histone H3. In addition to this, H1 concentrations were 10-fold higher than those of H3 in ascites supernatants. No correlation was found between histone content of ascites and the ability of the samples to induce platelet activation. This indicates that the majority of histones in ascites are linker histone H1 and, due to the absence of core histones in the samples and the absence of ascites-induced platelet activation, they are not likely to be derived from NETs. This raises the interesting possibility that histones in ascites are associated with exosomes.

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Discussion

Paper I Lrrn1 does not promote homophilic cell adhesion Proteins that harbour LRRs sequences are though to have a role in cell adhesion through protein-ligand interaction. Homophilic cell adhesion occurs through the binding of the same cell surface proteins to each other. Both caps and FLRT3 promote cell adhesion via homophilic binding (Karaulanov et al. 2006; Shinza-Kameda et al. 2006). Hence, we used xFLRT3 as a positive control in an aggregation assay to induce cell sorting of xFLRT3-expressing cells. Lrrn1 did not induce cell sorting in this assay and is unlikely to bind homophilically. Mouse Lrrn1 constructs were used here because they were readily available (Haines et al. 2005) and were tagged with epitopes that enabled their detection via western blotting, but this is not thought to the reason for the lack of sorting observed, since xFLRT3 was functional in HEK cells. There is also a high degree of sequence conservation between mouse and chicken Lrrn1 (Andreae et al. 2007). Other possible reasons for a lack of sorting could be due to the lack of a necessary co-receptor in HEK cells, or a possible change in confirmation of the FLAG-tagged Lrrn1 proteins used in this assay. Paper II Histones in exosomes are not a contaminant from apoptotic vesicles The presence of histones in exosomes proteomes is defined in certain publications as a contaminant, mainly because of the presence of histones in apoptotic vesicles (Thery et al. 2001). Apoptotic vesicles (50-500 nm in diameter) are released from apoptotic cells (Thery et al. 2009). Apoptotic bodies are larger in size and are typically removed by 10,000 g spin/0.2 μm filtration steps (Lobb et al. 2015). Apoptotic vesicles contain histones with a sucrose floatation density of 1.24-1.28 g/ml (Thery et al. 2001). Density gradient fractionation of EVs did not show the presence of histones in high-density fractions (1.23 g/ml) and contamination of exosome-containing fractions (1.08-1.11 g/ml) with apoptotic or aggregated material was not observed via TEM. Although we can’t exclude categorically that histones are part of a complex that copurifies with EVs, all evidence from Paper II suggests that histones are a component of exosomes. We have attempted to confirm this using on double IEM and also immunocapture using an anti-histone or an anti-EV marker antibody. This has not been successful so far, largely for technical reasons relating to anti-histone antibody specificity or sensitivity (data not shown). However, these would be good ways to verify the presence of specific histones on the surface of exosomes.

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Membrane localization of histones in exosomes The identification of histones in exosome proteomes has led to the assumption that they are present in the lumen, together with other cytoplasmic or nuclear proteins (Colombo et al. 2014). In our study, we found that linker and core histones are localized to the membrane of exosomes. A previous study on mitochondrial proteins has documented the presence of all five histone types on the OMM (Choi et al. 2011). Studies on ovarian and breast cancer exosomes have identified the presence of H2A on the exosome surface (Peng et al. 2011; Nangami et al. 2014). Moreover, histones and their variants were identified in detailed proteomics carried out on exosome proteins cleaved from the outer membrane (Cvjetkovic et al. 2016). In addition, fetuin-A, a serum glycoprotein, has been shown to regulate the transfer of nuclear histones to exosome membranes (Nangami et al. 2014). We found the localization of histones to the limiting membrane of MVBs and ILVs also supporting the fact that histones are localized to the exosome membrane. Moreover, quantitative proteomic analysis showed a lack of stoichiometry in core histones, which does not support the idea that they are present as a nucleosomes-like complex with DNA. Nevertheless, we cannot exclude the fact that that binding of soluble histones from an extracellular source could occur to the exosome surface. The N-terminal region of histones appears to be relatively exposed to proteolysis on the exosome surface. A similar N-terminal exposed orientation was observed for histone H2A and H2B in the OMM (Choi et al. 2011). The N-terminal region of histones is highly enriched with PTMs, which are important for histone function in chromatin organisation and gene expression. One study has reported the possible role of histones on the exosome surface in cell adhesion and uptake (Nangami et al. 2014). Hence, histones could play roles both in exosome uptake by target cells and in exosome signalling. In our study, the specific subset of histones identified by proteomics have also been found in other studies from other cell types, suggesting a broader role for histone that are incorporated into the exosome membrane (Willms et al. 2016). In future it would be interesting to investigate PTMs present on histones from unstressed and stressed cells in order to identify possible targeting modifications and also better understand changes in histone PTMs in relation to stress. These could be useful to define molecular changes in healthy vs disease states. Enrichment of histones in small diameter ILVs Studies on the biogenesis and secretion of exosomes have identified the existence of subpopulations of exosomes that vary in size, biophysical properties and composition (Simons and Raposo 2009; Willms et al. 2016). Our study showed that EVs released by OLN-93 cells varied in diameter ranging from 50-150 nm. Interestingly, we observed histone labelling on small diameter ILVs and heat stress led to an increase in the secretion of exosomes of small diameter,

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suggesting a link between incorporation of histones into small diameter vesicles for secretion. However, IEM on exosomes labelled with anti-histone antibodies resulted in the labelling of large diameter exosomes. Therefore, we cannot exclude the fact that histones are also present in large diameter EVs. Our heat stress and oxidative stress data suggest that the pathway regulating histone sorting into small ILVs is activated in response to stress and thereby found increased secretion of small diameter vesicles following heat stress. Lack of association of DNA and histones in EVs We found DNA both inside and outside of EVs. Although the localization of histones within exosomes has not been reported, the general assumption is that histones are luminal proteins and bound to DNA, e.g. (Colombo et al. 2014). We found that the majority of exoDNA is extravesicular and the striking abundance of ss, compared to ds exoDNA agrees with previous findings (Balaj et al. 2011). However, we found that the association of histones with the membrane is not DNA dependent. This suggests that exosomal histones are not associated with DNA in the same way as they are in nucleosomes. The presence of circulating histone-DNA complexes found in various pathological (Xu et al. 2011; Gould et al. 2015; Kim et al. 2015b), as well as non-pathological (Holdenrieder et al. 2001) states, suggests perhaps that these are not membrane-associated and are not part of EVs. However, we cannot exclude that the increased levels of circulating histones and DNA in several pathological situations arises from exosomes, at least in part. Detailed studies are warranted in order to understand the exact membrane topology of histones and DNA within exosomes, as well at the types of extracellular histone- or DNA-containing complexes present in the circulation. Role of ESCRTs in histone release ESCRT members are important in sorting proteins during exosome biogenesis and in addition can influence vesicle size and release. We found a significant downregulation of ESCRTs in heat stressed exosomes, where histones release were increased, as well as an increased release of small diameter vesicles. Hence, downregulation of ESCRTs may have a direct or indirect role in the secretion of histones, perhaps via an ESCRT-independent pathway. Several pathways of ESCRT-independent exosome secretion have been described including ceramide- (Trajkovic et al. 2008), CD63- (van Niel et al. 2011) and PLD2-dependent (Ghossoub et al. 2014) ones. These studies showed the existence of exosome subpopulations and infer a great degree of heterogenity. Since histone secretion increased in conjunction with a significant decrease in ESCRT secretion in EVs, it suggests the involvement of a novel ESCRT-independent pathway in relation to increased histone secretion and smaller diameter exosomes.

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Paper III Human ovarian cancer ascites contain histones. A 10,000g spin failed to deplete histones from EOC ascites samples. This could be because histones present in ascites are not associated with large complexes or microparticles. Association of histones with EVs or in a soluble form is a likely reason that they remained in the 10,000 g ascites supernatant. Tumor cells release more exosomes than normal cells and tumor exosomes carry tumor antigens (Cho et al. 2005). The interaction between tumor and non-tumor cells via exosomes is thought to promote tumor progression (Li and Wang 2017). EOC cells release exosomes and the presence of H2A on the surface of EOC exosomes has been documented in one study (Peng et al. 2011). Moreover, EOC exosomes function in enhanced cell migration, tumor progression and angiogenesis (Li and Wang 2017). Hence, although the limited material available in this study did not enable us to study EOC exosome-derived histones directly, taken together the available evidence suggests that some of the histones detected in ascites could have been EV-derived. The abundance of H1 in EOC ascites implicates a unique role of this histone in EOC prognosis. In future, investigations are warranted to identify the types and levels of circulating histones in EOC patient ascites in relation to the outcome. Final discussion and future directions Although the existence of extracellular histones has been well documented in human biofluids and they are known to be elevated under pathological conditions, little is known about their source. This is mainly because histones are well recognized as nuclear proteins and although highly abundant, they are typically restricted to the nucleus and enclosed by a double membrane. Hence, it is often believed that the only route for histones to be released from cells is via cell death. Hence, the release of histones via a regulated secretory pathway has not been addressed. Our work adds a new dimension to the understanding of extracellular histones. The binding of histones to the exosome membrane, independently of DNA, highlights a specific association to the membrane that has not been appreciated before. The rapid release of histones in response to stress, possibly via small diameter EVs, supports a role for histones as stress messengers in communication to neighbouring or distant cells. Proteomic data suggest not only a possible link in downregulation of ESCRT in the secretion of histones following heat stress, but also the existence of a novel pathway for exosome biogenesis. How and why histone release is triggered following cellular stresses need to be addressed. This will expand our understanding of elevated extracellular histones in several disease conditions. The abundance of histones in small diameter EVs is intriguing, as these perhaps have the potential to travel long distances more easily and spread messages between cells at a greater distance. Detailed

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investigations on exosomal histones in relation to cell signalling may help explain numerous related occurrences of extracellular histones in diseases. The loading of histones into MVBs/exosomes could help reduce the toxicity of highly basic, soluble histones, which could easily induce cytotoxic effects. The intracellular storage of histones in MVBs appears to be similar to their presence in LDs. Hence, a role for exosomal histones in microbial defence needs to be investigated. Our heat stress models help in generating high levels of histone in exosomes for these studies.

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Conclusions

1. Core and linker histones are localised to exosomes.

2. Presence within small ILVs within MVBs suggests localisation to exosomes but whether they are also present in MVs is unclear.

3. Histones are membrane-associated. 4. N-termini remain sensitive to proteolysis and may be exposed on the vesicle

surface. 5. Membrane localization of histones does not depend on exo-DNA. 6. Exo-DNA does not depend on histones for EV localization. 7. Heat stress-induced immediate upregulation of histones in exosomes along

with the increased release of small diameter EVs. 8. Proteomic analyses identified a specific subset of histones in exosomes and

suggested the possibility that ESCRT-downregulation is involved in the increased secretion of histones.

9. Core and linker histones are present in human ovarian cancer ascites

samples and found 10-fold enrichment with linker histone H1 in ascites 10,000g supernatants.

10. The retention of H1 following centrifugation at 10,000g suggests H1 is not

associated with high MW complexes/large vesicles in ovarian cancer ascites. 11. The levels of nucleosomes derived from acid extracted ascites were

significantly higher only in few epithelial ovarian cancer (EOC) ascites compared to donor serum.

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Acknowledgement

Jonathan Gilthorpe, thank you very much for giving me the opportunity to learn and explore in the JG lab from the very beginning. I sincerely admired your ever-flowing ideas and passion for work. I enjoyed working in the lab and for all the histone discussions! Thanks for guiding me all these years. I am grateful to all the collaborators, without them the thesis wouldn’t have taken shape. Lucia Mincheva-Nilsson, Sophie Rome and Linda Sandblad, thanks for your great effort in providing beautiful EM images. Samir EL Andaloussi, Henrik J Johansson, Imre Mäger and coworkers; thank you all for your immense support and continuous input especially during the final stage of the EV project. Many thanks to all the departments at the 6M building, especially ultracentrifuge and LICOR facilities for their extreme support to the EV project. My sincere thanks to all cell lines involved in the projects and patient ascites samples and my special thanks to OLN-93 cells for bearing me all these years whether I culture or contaminated them. Åsa Gilthorpe, you have been very supportive and special thanks for helping me out with the Swedish abstract. Gerhard Gröbner, I had an opportunity to learn and work with circular dichroism because of you and I thank you very much for that. Gilthorpe lab members past and present; Elin, Manuela, Bala, Ann, Ash, Iwan, Hanna and Harsha you have all helped me at various stages through the project and always been supportive. Ann and Manu, I admired your friendly nature with everyone and thank you very much for your tremendous support during my maternity days. Harsha, many thanks for your support in the project. ALS group members, I thank you all for accepting me into your research community. Stefan Marklund, Peter Andersen and Thomas Brännström, great team and inspiration for others and thanks for arranging the memorable ALS meeting in Istanbul and for accepting me as a part of it. Isil, Anna Brive and Eva Bern, special thanks for all the care and support. Denise and Lelle, thanks for sharing the office space and for all the support. Lelle, I always felt it was easy to talk with you and thanks for all the fun we had in exchanging little Swedish/Tamil words and for training me to bake something edible for others ;). UCMM lab groups and members, thank you very much for accepting me and for letting me to use the UCMM/lab facility. Helena Edlund, Simon Tuck, Lena Gunhaga, Jonas Von Hofsten, Ulf Ahlgren, Leif Carlsson, Sara Wilson, Lars Nilsson and Gautam Kao, your suggestions and scientific discussions during the seminars were valuable. Vijay, thanks for your endless support during my stay in UCMM. Ági Regős,

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thank you very much for your friendship and help at all times. Tanushree and Hanna Nord, thanks for all the discussions and fun during the journal club. Per Arne Lundström, thank you very much for taking care of my orders and delivering them on time. Lina Sollen, thank you very much for your extensive support for all these years. I also thank Birgitta and Tina for their administrative support. Ravi, Vidya, Anubhav, Aaron, Shani, Kamaraj, Ajay, Dhana, Raghu, Munendhar, Sharvani, Dinesh, Srinivas, Philge, Nisha, Reshma, Shyam, Sajna, Sinosh, Edwin and Maria, I thank you all for get-togethers and for providing homely Indian food in Umeå with love and care. Artist, Krishna Kumar, and the poorna prajna faculty improvement head, Avanthi mam, thank you very much for pouring positive energy and love since the day I met you. Many thanks to My Parents for always believing in me whatever I do, my lovable brother and his family, Srikanth, Aarthi and Kamalee and all other family members/friends for their continuous support and encouraging words at all times. Father-in-law, my indebted thanks for your immense love and blessing. I thank my mother-in-law for her support throughout these years. Bala, thanks for showing me the place like Umeå, otherwise, I wouldn’t probably get to experience the outside culture and beauty. I am always grateful for that. I am glad we left so many good memories of this place. Thank you very much for your love and support throughout these years to make this possible. Navira, my love and light, thanks for being a part of my life.

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