**4. Conclusion**

*Extracellular Vesicles and Their Importance in Human Health*

potential for therapeutics, it is growing rapidly.

*has less variability. Synaptosomal associated protein 25 (SNAP25).*

The nervous system is a uniquely isolated environment with limited connection to the systemic circulation. This characteristic has long impeded therapeutic delivery for brain pathologies. The potential of exosome transport, however, is particularly poignant, as exosomes have been observed selectively targeting neurons and glial cells, successfully crossing the blood brain barrier [113]. Improving our understanding of endocytosis mechanisms involved in these particular cells is essential to therapeutic progression. Clathrinmediated endocytosis is the most commonly observed pathway with exosomal trafficking between neurons and glial cells [114, 115]. However, some neurons also utilize macropinocytosis [114] and specific receptors, such as SNAP25 (a SNARE family protein) [116], to take up exosomes (**Figure 6**). Microglia performs phagocytosis similar to their counterparts in the extra-neuronal environment [117]. Using exosomes from two different sources, Chivet et al., illustrated the specificity of exosome targeting seen elsewhere in the body, is also evident in the nervous system. Exosomes from a neuroblastoma cell line (N2a) were preferentially internalized by astrocytes and oligodendrocytes, whereas exosomes from cortical neurons were primarily taken up by hippocampal neurons [118]. It was also shown that pre-synaptic regions were the primary site of internalization of these exosomes [118]. Endocytosis is an important process in the pre-synaptic membrane to recycle released synaptic vesicles [119], indicating that the exosomes may capitalize on this constitutive process for entrance to the neuron. Whether exosomes primarily utilize the specific clathrin-mediated endocytosis in this region [119] or are simply taken by chance with the constant bulk endocytosis [120] still remains unclear. Exosome uptake is a developing area of neuro-research, but with significant

*Cell-specific internalization of exosomes: neurons. Neurons use similar pathways but receptor/ligand binding* 

**3.6 Neurons and glial cells**

**56**

**Figure 6.**

Exosomes are internalized by a multitude of cell types and play an important role in cellular physiology. Our grasp of the mechanisms of this internalization is growing as we are better able to identify characteristics of the cell and the vesicles that facilitate uptake. Pathologic states, such as cancer, have played an integral role in our understanding of how the cellular-exosomal interaction proceeds. Clarity is still needed to better understand the mechanisms by which exosome internalization is so varied from cell to cell and within the same cell. As we have seen with fibroblasts, the vesicle size can dictate mechanism of uptake [111]. The presence or abundance of specific proteins such as scavenger receptors on macrophages [46–48] and lipid profiles in several types of cells, such as external-facing phosphatidylserine [20, 48, 49, 56] all contribute to the specificity of uptake. As has been discussed, cell type can dictate uptake mechanism, particularly with phagocytic cells and professional antigen presenting cells, but even within these specialized cells, differing mechanisms occur regularly and further evaluation is needed to parse the primary determinants.

Various types of endocytosis have been identified as possible mechanisms of intercellular transport of exosomal contents to include macropinocytosis [19, 56, 114], phagocytosis [20], clathrin-mediated [52, 114], caveolin-dependent [95], lipid raft-dependent [43, 46], and clathrin- /caveolin-independent [61] endocytosis. Though much about these processes is unique, there are some aspects where functional overlap exists between them. Macropinocytosis is a form of endocytosis that consists of membrane ruffles forming intracellular vesicles to internalize large amounts of extracellular fluid [30]. This varies from other forms of endocytosis in its formation of separate and distinct intracellular vesicles (macropinosomes) and the internalization of material that is considered non-specific exosomal has been recorded in microglia [56], human epidermoid carcinomaderived A431 cells stimulated by endothelial growth factor receptor (EGFR) and by the pancreatic cancer MiaPaCa-2 cell line [19]. Macropinocytosis is not selective in which molecules are internalized from the extracellular environment, and so uptake may be dictated simply by proximity to the cells and not targeted by the exosome specifically [121]. However, it has been shown that some exosomes naturally induce macropinocytosis internalization [90] and others, through manipulation of exosomal content, can selectively activate this mechanism in order to increase uptake [122]. Phagocytosis is a much more common method of taking up exosomes, especially with phagocytic cells of the immune system. Feng et al., showed that two leukemia cell lines, K562 and MT4, solely utilized phagocytosis for exosome internalization [20, 121].

Four other general categories of endocytosis focus on specific cellular proteins that facilitate the uptake of particles. Clathrin and caveolin are both cytosolic proteins that form specific pits with which to internalize various substances [25]. The exact reasons why and when a cell uses clathrin, caveolin, or neither, is still incompletely understood but particle size and cell type seem to play a role [43, 115, 121]. Caveolin-dependent endocytosis is important in albumin uptake, cholesterol transport, and intracellular signaling. Due to the small size of the caveolae, its endocytosed material tends to be smaller than 60 nm [25]. Clathrin-dependent mechanisms however can internalize particles up to 120 nm. The size restrictions may indicate, with further investigation into which uptake mechanism is utilized by which cells, a possible functional difference between vesicle sizes within the current exosome size range [121]. The clathrin-dependent process is involved in many different cell types and functions ranging from vesicle recycling in the neuronal synapse to organ development and ion homeostasis [25]. Many of the common, well-known endocytosis receptors utilize clathrin coated pits, such as low-density lipoprotein receptor (LDLR) and transferrin receptor (TfR). One of the most commonly used ways to determine which of these mechanisms is in operation is through inhibitory drugs or knocking down certain key players [121]. Dynamin, a GTPase, facilitates the fission of the intracellular clathrin coated vesicle [25, 123]. Dynasore, an inhibitor of dynamin, has been utilized to effectively block endocytosis of extracellular vesicles and establish clathrin-mediated endocytosis as a mechanism of uptake for these vesicles [21, 52, 56]. Following siRNA downregulation of caveolin-1 (the primary protein involved in caveolae-dependent endocytosis), exosome internalization was significantly reduced in B cells [95, 121]. Inhibitory drugs have also been useful in the determination of a third mechanism, lipid-raft mediated endocytosis. The lipid raft is a small portion of the plasma membrane, rich in sphingolipids and sterols, that facilitates various cellular processes [124]. Use of methyl-β-cyclodextrin (MβCD), which alters the cholesterol content of the membrane and disrupts lipid rafts, has been seen by several groups to impair exosomal internalization [43, 44, 97]. While lipid raft-dependent endocytosis is the primary clathrin- and caveolaeindependent mechanism, other pathways and independent interactions have been described in the internalization of exosomes [61, 124]. Endocytosis is the primary method of exosomal delivery of its contents but research is still needed to understand what determines the specific mechanism whether it is cell type, exosome type, or condition specific [121].

Exosome stability, ubiquitous presence, and influential contents make them ideal candidates for therapeutic modalities in a wide variety of pathologies. The significance of exosomal contribution to the cellular network throughout the body still carries untapped potential for conquering some of the most pressing current health challenges including cancer and neurodegeneration. Understanding how these exosomes interact with and enter the myriad of cells in the body will empower our ability to capitalize on this natural social network.

**59**

**Author details**

and Nathan R. Wall1,3\*

†

School of Medicine, Loma Linda, CA, USA

\*Address all correspondence to: nwall@llu.edu

provided the original work is properly cited.

*Cellular-Defined Microenvironmental Internalization of Exosomes*

*DOI: http://dx.doi.org/10.5772/intechopen.86020*

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Amber Gonda1,2†, Ron Moyron1,3†, Janviere Kabagwira1,3†, Paul A. Vallejos1†

1 Center for Health Disparities & Molecular Medicine, Loma Linda, CA, USA

3 Division of Biochemistry, Department of Basic Sciences, Loma Linda University

† All authors contributed to the design of this review, while AG, RM, PV and JK drafted the manuscript. AG, RM, and NRW revised and finalized the manuscript.

2 Department of Pathology and Anatomy, Loma Linda, CA, USA

*Cellular-Defined Microenvironmental Internalization of Exosomes DOI: http://dx.doi.org/10.5772/intechopen.86020*
