Exploring G-Protein-Coupled Receptors Regulation, Specificity and Controllability of Exosomes Release in the Neuronal Cell Line SH-SY5Y
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Authors Sadideen, Doraid
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EXPLORING G-PROTEIN-COUPLED RECEPTORS REGULATION,
SPECIFICITY AND CONTROLLABILITY OF EXOSOMES RELEASE IN THE NEURONAL
CELL LINE SH-SY5Y
By
Doraid Tarek Sadideen
______
A Thesis Submitted to the Faculty of the
GRADUATE INTERDISCIPLINARY PROGRAM
IN PHYSIOLOGICAL SCIENCES
In Partial Fulfillment of the Requirements
For the Degree of
MASTER OF SCIENCE
In the Graduate College
THE UNIVERSITY OF ARIZONA
2016
1
STATEMENT BY AUTHOR
This thesis has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.
Brief quotations from this thesis are allowable without special permission, provided that an accurate acknowledgement of the source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.
SIGNED: Doraid Tarek Sadideen
APPROVAL BY THESIS DIRECTOR
This thesis has been approved on the date shown below:
Torsten Falk Date Assistant Professor of Neurology August 5th 2016
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ACKNOWLEDGMENT
This thesis was possible with the support of: Torsten Falk, Brian S. McKay, Scott J.
Sherman, my parents (Tarek Sadideen and Suhair Amer), and siblings (Zeyad, Rana, Yousra and
Ayah Sadideen). As well as a handful of people at the University of Arizona’s Physiological
Sciences Graduate Interdisciplinary Program (PSGIDP).
3
TABLE OF CONTENTS Chapter Page No. List of Figures ...... 6 List of Tables ...... 8 List of Abbreviations ...... 9 Abstract ...... 10 Introduction ...... 11 General introduction ...... 11 Parkinson’s disease ...... 11 Alpha-Synuclein ...... 16 Exosomes ...... 18 SH-SY5Y ...... 19 CHO-K1 ...... 20 MCF-7 ...... 21 Aims of project ...... 22 Materials and Methods ...... 24 Materials ...... 24 Cell lines ...... 24 Antibodies ...... 24 General lab reagents ...... 24 Plasmid ...... 25 Viral α-SYN and GFP ...... 25 Methods ...... 25 Cell culture ...... 25 Plasmid preparation ...... 26 Agarose gel electrophoresis of DNA ...... 28 Transient transfection of DNA ...... 28 Medium collection and cell harvest ...... 29 Bradford protein assay ...... 30 Exosome-depleted serum ...... 30 Cell differentiation ...... 30 Exosome isolation ...... 31 SDS-Polyacrylamide gel electrophoresis ...... 31 Western blotting ...... 32 Silver stain ...... 33 Exosome labeling ...... 33 Exosomes specificity and uptake ...... 34
4 Biotinylation of exosomes ...... 36 Viral α-SYN and GFP transduction ...... 38 Results ...... 39 Transient transfection of DNA ...... 39 Cell differentiation ...... 41 Silver stain ...... 43 Exosomes specificity and uptake ...... 44 Biotinylation of exosomes ...... 47 Discussion ...... 48 References ...... 52
5 List of Figures
Figure 1: Cross section of post-mortem midbrain from a human patient with PD (P) and a control (C) (37), reprinted with permission from BCMJ2001...... 13 Figure 2: Lewy bodies (arrows) in the SNpc, visualized through a hematoxylin-eosin stain (32), reprinted with permission from John Wiley and Sons 2014...... 13 Figure 3: Schematic representation of α-SYN. (A) The regions of α-SYN and most common point-mutations (28, 29), reprinted with permission from Cambridge University Press 2014. (B) Representation of α-SYN in its micelle-bound form (28, 30), reprinted with permission from Nature Publishing Group 2012...... 16 Figure 4: Phase-contrast photomicrograph of SH-SY5Y cells at medium density, scale bar 10 µm, 20X objective...... 20 Figure 5: Phase-contrast photomicrograph of CHO-K1 cells at high density, scale bar 10 µm, 20X objective...... 21 Figure 6: Phase-contrast photomicrograph of MCF-7 cells at medium density, scale bar 10 µm, 20X objective...... 22 Figure 7: Sequence alignment of human GPR143 and human dopamine receptors 1-5 shows ligand contact sites are highly conserved between the receptors (Brian McKay, unpublished)...... 23 Figure 8: A-F photomicrographs of transfected CHO-K1 cells. A-C phase-contrast and D-F fluorescence microscopy images, GFP, D3-GFP and GPR143 respectively, scale bar 10 µm, 20X objective...... 39 Figure 9: A-F photomicrographs of transfected SH-SY5Y cells, A-C phase-contrast and D-F fluorescence microscopy images, GFP, D3-GFP and GPR143 respectively, scale bar 10 µm, 20X objective...... 40 Figure 10: A-F photomicrographs of transfected MCF-7 cells, A-C phase-contrast and D-F fluorescence microscopy images, GFP, D3-GFP and GPR143 respectively, scale bar 10 µm, 20X objective...... 41 Figure 11: Phase-contrast photomicrographs of undifferentiated and differentiated SH-SY5Y in A and B respectively. Processes are longer and more pronounced in the differentiated SH- SY5Y. Scale bar 10 µm, 20X objective...... 42 Figure 12: A-F photomicrographs of transfected differentiated SH-SY5Y cells, A-C phase- contrast and D-F fluorescence microscopy images, GFP, D3-GFP and GPR143 respectively, scale bar 10 µm, 20X objective ...... 42 Figure 13: Silver stain scan of SDS:PAGE of exosome fraction, exosome depleted medium and cell lysate treated with L-DOPA or dopamine. Exo = Exosomes, CM = Exosome-depleted culture medium, CL = Cell lysate...... 43 Figure 14: Graph illustrates total exosome protein analysis of exosome fraction lane (means ± SEM) from silver stain from three experiments. Black, blue and purple GFP, D3 and GPR143 transfected CHO-K1 (A), MCF-7 (B), differentiated SH-SY5Y (D. SH-SY5Y) (C) and SH-SY5Y (D) cell lines respectively. Integrated optical density (IOD) obtained ImageJ via conventional methods plotted against treatment groups. Con = control, D = dopamine, L = L-DOPA treatments. nonparametric, unpaired-test, no significant difference was found. 44 Figure 15: Fluorescent photomicrographs showing that Exo-Red labeled SH-SY5Y exosomes were internalized by MCF-7 and SH-SY5Y shown in A and B respectively.
6 Photomicrographs were taken within seconds of adding the Exo-Red labeled exosomes. Scale bar 10 µm, 40X objective...... 45 Figure 16: Fluorescent photomicrograph showing Exo-Red labeled SH-SY5Y exosomes were internalized by CHO-K1 cells. Photomicrograph was taken within seconds of adding the Exo-Red labeled exosomes. Scale bar 10 µm, 40X objective...... 45 Figure 17: Fluorescent photomicrographs of Exo-Red labeled SH-SY5Y exosomes shows exosome uptake in CHO-K1 cells at RT but not at -4ºC shown in A and B respectively. Photomicrographs were taken 5 minutes after the addition of Exo-Red labeled SH-SY5Y exosomes and fixation in 4% PFA. Scale bar 10 µm, 40X objective...... 46 Figure 18: Fluorescent photomicrographs of proteinase K untreated and treated Exo-Red labeled SH-SY5Y exosomes added to CHO-K1 cells in A and B respectively, after fixation in 4% PFA. Scale bar 20 µm, 20X objective...... 46 Figure 19: Fluorescent photomicrographs of versene untreated and treated CHO-K1 cells shows Exo-Red labeled SH-SY5Y exosomes were trapped on the cell surface of the cells at RT shown in A and B respectively. Photomicrographs were taken 5 minutes after the addition of Exo-Red labeled SH-SY5Y exosomes and fixation in 4% PFA. Scale bar 10 µm, 40X objective...... 47 Figure 20: A) Silver stain of biotinylated exosomes. 1 = Biotinylated Exo-Red labeled SH-SY5Y exosomes added to MCF-7, 2 = Biotinylated Exo-Red labeled SH-SY5Y exosomes added to SH-SY5Y, 3 = Exo-Red labeled exosome fraction. B) X-ray film of biotinylation blot of biotinylated exosomes. 1 = Biotinylated Exo-Red labeled SH-SY5Y exosomes added to MCF-7, 2 = Biotinylated Exo-Red labeled SH-SY5Y exosomes added to SH-SY5Y...... 48
7
List of Tables Table 1: Lab-Tek II schematic representation of exosome specificity. Cells were incubated at RT after introducing SH-SY5Y exosomes then fixed with 4% PFA...... 35 Table 2: Lab-Tek II schematic representation of exosome uptake. 1-4 wells cells were incubated RT for 2 minutes before fixation with 4% paraformaldehyde. 5-8 cells were incubated at - 4ºC for 2 minutes before fixation with 4% PFA...... 35 Table 3: Lab-Tek II schematic representation of exosome uptake. 1-3 wells cells were incubated at RT for 2 minutes before fixation with 4% paraformaldehyde. 6-8 cells were incubated at - 4ºC for 2 minutes before fixation with 4% PFA...... 36 Table 4: Samples were loaded in a 7% SDS:PAGE. The samples were loaded according to this table...... 38
8 List of Abbreviations
Alzheimer disease: AD, Parkinson’s disease: PD, Substantia nigra pars compacta: SNpc, 5,6- dihydroxyindole: DHI, 5,6-dihydroxyindole-2-carboxylic acid: DHICA, Cysteinyl-3,4- dihydroxyphenylalanine: Cys-DOPA, Alpha-synuclein: α-SYN, 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine: MPTP, 1-methyl-4-phenylpyridinium: MPP+, Monoamine oxidase b: MAO-
B, Dopamine active transporter: DAT, 5,6-dihydroxyindole: DHI, Synuclein alpha: SNCA,
Synuclein beta: SNCB, Synuclein gamma: SNCG, SNARE: soluble NSF attachment protein receptor, Parkin RBR E3 ubiquitin protein ligase: PARK2, PARK7, PTEN-induced putative kinase 1: PINK1, Leucine-rich repeat kinase 2: LRRK2, Deep brain stimulation: DBS, L-3,4- dihydroxyphenylalanine: L-DOPA, Vesicular monoamine transporter 2: VMAT2, Non-Aβ amyloid: NAC-domain, Serine in the 129th position: Ser129, α-SYN phosphorylated in Ser129: pS129-α-SYN, Heat shock cognate protein 73: hsc73, multi-vesicular bodies: MVB, receptor- mediated endocytosis: RME, Endosomal Sorting Complex Required for Transport: ESCRT, intralumenal vesicles: ILVs, vacuolar protein sorting 4-A: Vsp4-A, Tyrosine hydroxylase: TH, δ-
1-pyrroline-5-carboxylic acid: P5CS, Ornithine, aminotransferase: OAT, Dopamine receptor D3:
D3, G protein-coupled receptor 143: GPR143, Green fluorescent protein: GFP, Tetradecanoyl phorbol acetate: TPA, Cytomegalovirus: CMV, Fetal bovine serum: FBS, PFA: paraformaldehyde, inositol 1,4,5-trisphosphate: IP3, phospholipase C: PLC, adenosine triphosphate: ATP, 3',5'-cyclic AMP: cAMP, human embryonic kidney cells: HEK293, human primary fibroblasts: C-013-5C, single-strand RNA: ssRNA, adenosine diphosphate: ADP.
9 Abstract
Parkinson’s disease is a neurodegenerative disease characterized by the buildup of aggregated and spread of misfolded alpha-synuclein. How the misfolded alpha-synuclein contributing to the toxicity and death of neuronal cells has been the focal point of research. The spread of alpha-synuclein has been attributed to many mechanisms, one of which is via cell- derived vesicles called exosomes.
This project aims to examine the controllability of exosome release. SH-SY5Y, MCF-7 and CHO-K1 cells were transfected with dopamine receptor 3-green fluorescent protein, G- protein receptor 143 or green fluorescent protein and treated with either dopamine or L-DOPA.
Medium was harvested and subjected to ultracentrifugation and a silver stain and western blot were performed. There was no significant difference in the total protein in the exosome fraction lanes between the treatment groups or within them.
Another aim was to test the specificity of exosomes. Exosomes isolated from SH-SY5Y or MCF-7 were labeled with Exo-Red dye and introduced to wells containing SH-SY5Y, MCF-7 and CHO-K1 cells at room temperature and -4°C. At room temperature, exosomes were observed intercellular in all of the cell lines, however, they did not deliver their content. At -4°C exosome uptake was halted and they remained on the surface of the cells. Exo-Red labeled SH-
SY5Y exosomes were treated with proteinase K and were introduced to CHO-K1 cells at -4°C and room temperature. CHO-K1 did not take up exosomes, suggesting exosomes contain one or more necessary proteins needed to interact with the cellular membrane to initiate internalization.
CHO-K1 cells were treated with versene to examine the involvement of integrin proteins. Exo-
Red labeled SH-SY5Y exosomes were trapped on the surface of CHO-K1 after versene treatment. Lastly, Exo-Red labeled SH-SY5Y exosomes were biotinylated and magnetically
10 captured then introduced to SH-SY5Y and MCF-7 cells and a silver stain and a biotinylated blot were performed. MCF-7 bound more Exo-Red labeled SH-SY5Y exosomes.
Introduction
General introduction
Neurodegeneration is a category encompassing diseases resulting from the gradual loss of neurons, whether functional or structural loss (1). This loss is often associated with programmed cell-death due to triggers initiating this cascade (2). Some of these triggers can be reactive oxygen species (ROS), nitrogen species (NOS), and aggregation of misfolded proteins (2). The most common neurodegenerative disease is Alzheimer disease (AD) affecting 15% of people above the age of 65 (3) followed by Parkinson disease (PD) in 2% of people above the age of 65 in the United States (4, 49); combined, 50 million people are affected worldwide (5). While they are different, AD leads to decline in cognitive function and PD is a movement disorder (5), they share some common ground. Both disorders are idiopathic with incidents increasing with age, and in both proteins aggregation causes neuronal death (5). Protein aggregates are referred to as inclusions, and were described by Alois Alzheimer, Oskar Fischer and Friedrich Lewy more than
100 years ago (6, 7).
Parkinson’s disease
James Parkinson first described PD in “Essay on the Shaking Palsy” in 1817 (14). It is the second most common neurodegenerative diseases (49). 1-2% of people above the age of 60 suffer from it (38, 44). In 2013, 103,000 deaths were confirmed to be PD cases worldwide (51).
While gender and race susceptibility remain controversial (48), men are 1.5 times more likely to
11 be diagnosed with PD than women (45, 46), and Caucasians are twice likely to be diagnosed with PD than African-Americans, while the latter has double the risk factor (47); possibly due to access to medical facilities (33). There are two categorical classes of symptoms: cardinal
(motor) and non-cardinal (non-motor) symptoms (35). Bradykinesia (slowness of movement), rigidity, postural instability, and tremors are the classical cardinal symptoms (8, 35-50), while sweating, depression, hallucination are part of the non-cardinal symptoms (15, 31).
Pathologically, PD is characterized by the continual loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) (8, 36, 37). The gross anatomy of SNpc is naturally black, due to the dopaminergic neurons expressing neuromelanin (37) in their perikaryon.
Neuromelanin is a collection of black and brown 30 nm diameter spheres with eumelanin, black, on the surface and pheomelanin, reddish-brown, in the core (40, 42). Eumelanic, forms from the oxidative polymerization of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), and pheomelanic, forms from cysteinyl-3,4-dihydroxyphenylalanine (Cys-
DOPA) isomers (39, 40). One study has shown neuromelanin is synthesized from serotonin, norepinephrine, L-DOPA and dopamine (41), while another showed it is formed from the oxidation of catecholamines and dopamine (39), the exact mechanism, substrates involved and whether it is driven by enzymes or auto-oxidative driven remain the subject of research (43). In
PD patients, the SNpc exhibits reduced pigmentation as the pigmented cells have died, as illustrated in Figure 1.
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Figure 1: Cross section of post-mortem midbrain from a human patient with PD (P) and a control (C) (37),
reprinted with permission from BCMJ2001.
Another pathological marker for PD is the presence of Lewy bodies (27, 28) and this is the post-mortem confirmation of PD diagnoses. Lewy bodies are aggregated cytoplasmic inclusion of mostly misfolded alpha-synuclein (α-SYN), however, ubiquitin and tau proteins have been found there as well (8, 27, 28). Figure 2 shows two Lewy bodies (white arrows) in a
SNpc neuronal cell of a Parkinson’s patient.
Figure 2: Lewy bodies (arrows) in the SNpc, visualized through a hematoxylin-eosin stain (32), reprinted with
permission from John Wiley and Sons 2014.
PD is a complex disease and its etiology remains unknown (8, 34). The complexity is, in part, due to it having genetic and environmental aspects; however, pure onset of genetic or environmental PD is extremely rare (8, 34).
13 Pure environmental onset has been linked to brain injuries, prolonged exposure to heavy metals, pesticides, constant stress, and toxins such as 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) (8-13). MPTP is a neurotoxin, which crosses the blood-brain barrier
(13, 14). MPTP is converted to 1-methyl-4-phenylpyridinium (MPP+) by monoamine oxidase b
(MAO-B). MPP+ utilizes the dopamine active transporter (DAT) on the membrane of dopaminergic neurons (13), once MPP+ is inside the mitochondria, it causes cell death by inhibition of the mitochondrial respiratory metabolism.
Pure genetic onset of PD has been linked to mutations in at least 16 genes including
Synuclein alpha (SNCA), Parkin RBR E3 ubiquitin protein ligase 2 (PARK2), PARK7, PTEN- induced putative kinase 1 (PINK1) and Leucine-rich repeat kinase 2 (LRRK2) genes (8-11).
Familial linked accounts for 15% of the diagnosed cases, and inheritance has been shown to be in the autosomal dominant SNCA and LRRK2 genes (9). Other genes, including the ones listed above are autosomal recessive genes.
There is no disease modifying treatment for PD, however, there are multiple ways to manage the disease. Deep brain stimulation (DBS) is a way to manage the disease, however it involves major surgery; neurostimulator implantation sends electrical impulses at high frequency into subthalamic nucleus, globus pallidus internus, and ventralis intermedius nucleus (17).
Common treatment management is the use of L-3,4-dihydroxyphenylalanine (L-DOPA), also known as levodopa, and it has been the gold-standard treatment for the past 40 years (16).
Prolonged treatment causes L-DOPA-induced dyskinesia, which are involuntary movements, becoming more prominent in a large portion of the patients (16). L-DOPA is the precursor to dopamine capable of crossing the blood-brain barrier (16). L-DOPA can be synthesized directly or indirectly from L-tyrosine or L-phenylalanine respectively (18). Hydroxylation of L-
14 phenylalanine yields L-tyrosine, which is then hydroxylated to L-DOPA (18). Lastly, decarboxylation of L-DOPA yields dopamine (18), which is either packaged into vesicles by vesicular monoamine transporter 2 (VMAT2) (20), broken down by monoamine oxidase (MAO)
(19, 20) or converted to catecholamines (19).
15 Alpha-Synuclein
Α-SYN has emerged as one of the main proteins linked to familial PD. It is an acidic protein of 140 amino acids and a size of 14.5kD (21). A-SYN is one member of the synuclein family: alpha (α), beta (β) and gamma (γ), which are encoded by their respective genes SNCA,
SNCB, SNCG (23). A-SYN is expressed mostly at the presynaptic nerve terminals in several regions in the brain (22, 24); β-SYN co-localizes with α-SYN at most, but not all, the presynaptic nerve terminals, while γ-SYN can be found in glia cells, dopamine neurons and the retina (8, 24)
The function of α-SYN remains unknown (8, 21, 22, 24, 28), however, α-SYN has been linked to involvement in neurotransmitter release, plasticity, interaction with soluble NSF attachment protein receptor (SNARE) proteins, and vesicular recycling (21, 28). α-SYN is comprised of three domains (Figure 3): the positively charged N-terminal region from amino acid residues 1–60; the hydrophobic non-Aβ amyloid component (NAC) from residues 61–95; and the negatively charged C-terminal region from residues 96–140 (8, 21, 22, 24, 28-30).
Figure 3: Schematic representation of α-SYN. (A) The regions of α-SYN and most common point-mutations
(28, 29), reprinted with permission from Cambridge University Press 2014. (B) Representation of α-SYN in
its micelle-bound form (28, 30), reprinted with permission from Nature Publishing Group 2012.
16 Three point mutations associated with early onset autosomal dominant PD are located within the N-terminal domain. A substitution of alanine to threonine at the 53th (A53T) residue was the first mutation identified followed by the discovery of the substitution of alanine to proline at the 30th (A30P) residue and glutamic acid to lysine at the 46th (E46K) residue (8, 21,
24, 28-30). The A53T mutation increases the hydrophobicity of the N-terminal augmenting its fibrillization (29, 30), and the A30P mutation decreases α-SYN’s membrane binding ability (8,
24, 28, 30). The E46K mutation alters the interaction between α-SYN and phospholipids enhancing its toxicity and increasing its binding ability to the presynaptic membrane by conformational change from coil to a cylindrical b-like sheet (8, 28).
The most common modification to α-SYN is its phosphorylation at serine in the 129th position (Ser129) (24, 28). Indeed, 90% of the a-SYN in Lewy bodies are phosphorylated at
Ser129 (pS129-α-SYN). In contrast, only 4% of α-SYN is normally found phosphorylated (8,
28); possible explanations are either the turnover of the protein or the turnover of phosphorylation. When α-SYN aggregates, the access to that site could be blocked and therefore dephosphorylation cannot occur or the turnover the protein is abnormal. In vitro experiments pointed to the phosphorylation of α-SYN at Ser129 by LRRK2, casein kinase I and II, polo-like kinases and G-protein coupled receptor kinases (8), suggesting the involvement of more than one kinase in the phosphorylation of α-SYN. While phosphorylation of Ser129 has been reported to inhibit fibrillization, which is the organized formation of filaments from monomers, it does not inhibit the aggregation of α-SYN monomers. One study hypothesized kinetic stabilization is critical to the aggregation and increase toxicity of pS129-α-SYN (8), however how an increase in aggregation of pS129-α-SYN plays a role in toxicity remains to be answered. There is evidence of α-SYN being transmitted between cells via exocytosis, where one cell deposits α-SYN into
17 the extracellular space and then it is taken up by another neighboring cell (28). Vesicular transport via exosomes is another way α-SYN makes it into cells, and it is hypothesized to be a way for the cells to remove excess amounts of proteins, in this case α-SYN (28). Cell death is another way α-SYN makes its way out of the cell. It is hypothesized that upon cell death, the reservoir of α-SYN is released to the extracellular space (28).
Exosomes
Cells communicate with one another in multiple ways, one of the ways involves the secretion of small vesicles, and one type of these vesicles is an exosome (52, 57). These vesicles range in size between 30 to 140 nm in diameter size (56), and carry cargo including proteins, mRNA and miRNA (57). Proteomic analysis of dendritic-derived exosomes showed the presence of transmembrane proteins such as heat shock protein 73 (hsc73), lactadherin, γ-Actin, gag polyprotein (58, 59) and cytosolic proteins such as tubulin, cofilin, actin and profilin I (58), as well as intracellular transport proteins, signal transduction proteins, and proteins involved with apoptosis such as histones H2A (58). Research suggests exosomes share a subset of proteins from the plasma membrane, Golgi and mitochondria (52), and are formed inside multivesicular bodies (MVBs) (26).
Exosomes formation starts at the cell surface via a mechanism called receptor-mediated endocytosis (RME) or pinocytosis. RME occurs when a ligand binds its receptor and an invagination of the plasma membrane occurs placing the receptor inside creating a vesicle called an endosome (53, 54). The internalization of a portion of cellular membrane is called pinocytosis. (53). Once the invagination occurs, the sorting of membrane proteins into MVBs is manipulated by the addition of a small protein called ubiquitin to lysine residue of the proteins
18 (54). The endosomal-sorting complex required for transport (ESCRT) recognizes the ubiquitin tags and starts sorting them into intralumenal vesicles (ILVs).
ESCRT is made up of four complexes. ESCRT-0 with clathrin-coats creates a network of proteins on endosomal membranes, which in turn, catches the ubiquitin-tagged protein inside the vesicles to be directed to the MVB pathway (54). ESCRT-I binds the resulting complex, ESCRT-
0 and ubiquitinated proteins (54). ESCRT-II binds with ESCRT-1, which is needed for ESCRT-
II’s recruitment of ESCRT-III and assembly. One of the subunits in ESCRT-III is associated with trapping the cargo, membrane distortion and vesicle detachment (54). An ATPase, vacuolar protein sorting 4-A (Vps4-A) complex is necessary for the formation of MVB vesicles. ESCRT-
III recruits deubiquitinases to recycle ubiquitin before the final formation of the vesicle (55). At the formation of the vesicle, Vps4-A detaches from ESCRT-III recycling each of the four complexes to restart the mechanism again (54, 55). After the formation, MVB can fuse with a lysosome to degrade its new exosomes or with the plasma membrane to release them (52).
SH-SY5Y
The SH-SY5Y cell line is a third subclone from the SK-N-SH cell line (75, 76), which was established from a bone marrow biopsy of a four-year-old female suffering from neuroblastoma (75). They lack polarity, form clusters, have neuroblast morphology (78) and have subtle processes (white arrow in Figure 4). The cells proliferate continuously and have an unsynchronized cell cycle (78), which is one of the limitations of undifferentiated SH-SY5Y cells. Their density must be monitored to ensure less variation in materials collected from them.
The SH-SY5Y cell line is a frequently used cellular model for PD, whether undifferentiated or differentiated, because they are positive for tyrosine hydroxylase (TH) (82). Tyrosine hydroxylase is an enzyme that converts L-tyrosine to L-DOPA in a rate-limiting step, and it is
19 specific to dopaminergic cells because L-DOPA is converted to the neurotransmitter dopamine that is released by dopaminergic cells.
Figure 4: Phase-contrast photomicrograph of SH-SY5Y cells at medium density, scale bar 10 µm, 20X
objective.
CHO-K1
CHO-K1 cells, mesenchymal cells with a fusiform, fibroblastic morphology (64), Figure
5, are a variant derived from the Chinese hamster’s ovary (CHO) (60). CHO-K1 cells are proline auxotroph meaning they are unable to synthesize proline (64). Proline can be synthesized from
L-glutamate or ornithine (65). CHO-K1 lack endogenous δ-1-pyrroline-5-carboxylic acid (P5CS) and ornithine aminotransferase (OAT); the former converts L-glutamate through intermediate steps to proline while the latter converts ornithine to proline (65).
20
Figure 5: Phase-contrast photomicrograph of CHO-K1 cells at high density, scale bar 10 µm, 20X objective.
MCF-7
Mammary gland breast cancer tissue, Michigan Cancer Foundation-7 (MCF-7) Figure 6 , was isolated from a 69-year-old Caucasian female with mammary gland breast cancer (66).
Morphologically MCF-7 cells are squamous epithelium. MFC-7 cells have been shown to release exosomes (67).
21
Figure 6: Phase-contrast photomicrograph of MCF-7 cells at medium density, scale bar 10 µm, 20X objective.
Aims of project
The aims of this thesis are to investigate the specificity and controllability of exosome uptake and release by and from different cells lines. The first aim is to examine whether L-
DOPA or dopamine will decrease or increase exosome release. Experiments have been conducted using SH-SY5Y, CHO-K1 and MCF-7 cell lines. The available literature suggests
CHO-K1 may not release exosomes making it good for negative control, while SH-SY5Y and
MCF-7 have been shown to release exosomes (67, 68, 80). All cell lines do not express dopamine receptor D3 (D3), G-protein receptor 143 (GPR143), formally known as ocular albinism 1 (OA1), or green fluorescent protein (GFP) (61-63, 79). Prof. Brian S. McKay at
University of Arizona analyzed the sequences of the five human dopamine receptors along with
GPR143 (data unpublished, Figure 7). The highlighted highly conserved areas, green, depict the points of contact the ligand makes with each of the dopamine and GPR143 receptors. This data is based on the crystal chromatography structures of dopamine and L-DOPA. Even though,
GPR143 is not traditionally thought to be dopamine receptor, the sequence alignment suggests it
22 is analogous. GPR143 utilizes intracellular calcium stores through the G-protein Gαq (70). There is an increase of pigment epithelium-derived factor secretion when L-DOPA binds GPR143 (70); furthermore, dopamine competitively binds GPR143, however there is no effect on intercellular calcium (70). Dopamine binds D3 and decreases cAMP by inhibiting adenylyl cyclase (61). A study by Kuzhikandathil showed activation of D3 inhibited voltage-dependent calcium channels and the influx of calcium (69).
Figure 7: Sequence alignment of human GPR143 and human dopamine receptors 1-5 shows ligand contact
sites are highly conserved between the receptors (Brian McKay, unpublished).
23 The second aim is to examine whether exosomes uptake are cell-cell or species-species specific. Exosomes isolated from either SH-SY5Y or MCF-7 will be labeled by Exo-Red dye then introduced to SH-SY5Y, MCF-7 and CHO-K1 at room temperature and -4ºC. Exo-Red labeled SH-SY5Y exosomes will be subjected to either biotinylation or proteinase K treatment and then introduced to SH-SY5Y, MCF-7 and CHO-K1 cells.
Materials and Methods
Materials
Cell lines
SH-SY5Y cells were obtained from ATCC (ATCC® CRL-2266™, US). Prof. Brian S.
McKay provided CHO-K1 (ATCC® CCL-61™, US) and MCF-7 (ATCC® HTB-22™, US) cells.
Antibodies
Rabbit monoclonal to alpha synuclein antibody [MJFR1] (ab138501) and donkey anti- rabbit IgG antibody, horseradish peroxidase-conjugate secondary antibody (AP182P) were purchased from Abcam (Cambridge, MA) and EMD Millipore (Cambridge, MA) respectively.
General lab reagents
All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.
24 Plasmid
D3, WT, CMV promoter, pcDNA 3.1+, was purchased from cDNA Resource Center at the University of Missouri-Rolla (Rolla, MO). The GPR143 1.36 was obtained from Prof. Brian
S. McKay. The GPR143 plasmid is expressed from the pEGFP N-1 vector, and has a GFP protein conjugated to the C-terminal via a linker (70).
Viral α-SYN and GFP
Viral α-SYN, AAV5-CBA-Alpha-Synuclein 1x1013 vg/ml, and viral GFP, AAV5-CBA- eGFP 9.5x1012 vg/ml were obtained from Prof. Julie Miller at the University of Arizona.
Methods
Cell culture
SH-SY5Y, CHO-K1 and MCF-7 cells were cultured as described before with modification (71), medium of choice was RPMI 1640 for SH-SY5Y and DMEM for CHO-K1 and MCF-7 with high glucose, 23.8 mM sodium bicarbonate, penicillin (100 ID/ml), streptomycin (100 µg/ml), 5% heat-inactivated fetal bovine serum (FBS) and DMEM was supplemented with 0.6 mM L-proline in incubator (model VMR 2300) at 37°C with humidified atmosphere and 5% CO2 (71, 72); medium was changed every two to three days. Once the T-25 flask was 65-70% confluent the medium was decanted and 2 mL of versene added for 3 minutes.
Then versene was replaced with 2.5 mL 0.25% trypsin-EDTA and cells were placed in the incubator for 5 minute; 2.5 mL of medium was added to inactivate the trypsin-EDTA and then the cells were pelleted down using 400X g for 5 minutes; cells were either reseeded or set up for an experiment.
25 Plasmid preparation
The D3, GPR143 and GFP plasmids were prepared first by transforming Escherichia coli according to Invitrogen’s (One Shot® TOP10 Chemically Competent E. coli, C4040-06) protocol. Cell transformation, is the process of changing a cell by introducing exogenous DNA into it (74). Four agar plates were prepared with 1% ampicillin; three receiving D3, GPR14 or
GFP while the fourth did not receive plasmids. While competent E. coli vials were thawed on ice; 5 µL of each plasmid was mixed with 50 µL of 0.1X Tris-EDTA (T.E.) buffer. The latter mixture added to corresponding vials and then incubated on ice for 30 minutes. The vials were heat shocked at 37°C in water bath for 30 seconds then placed back on ice for 2 minutes. 250 µL of Super Optimal Broth (S.O.C.) medium added to each vial aseptically and placed in Innov™
4300 incubator shaker for 1 hour at 230 RPM. The incubator shaker helps the cells recover and express the antibiotic resistant gene. The content of the vials was emptied in their respective agar plates and spread evenly. Plates were placed upside-down in an incubator (Imperial III incubator) overnight.
The following day, the agar plates were inspected for bacterial colonies. If bacteria covered the plate then the ampicillin was not effective in killing colonies not expressing the plasmid, and if there were no colonies then the bacteria did not incorporate the plasmid or the ampicillin concentration was high. The plate contained 7-10 colonies; to grow the transformed E. coli each colony was touched by a pipette tip and discarded into round-bottom polypropylene tubes (Falcon™ REF 352059) containing 2.5 mL of Terrific Broth (T.B.) and 3 µL of 50 mg/mL ampicillin. The round-bottom tubes were covered by the cap and not closed shut, and placed in incubator shaker at 37°C and 200 RPM for 18 hours.
26 The next day, the round-bottom tubes were taken out of the incubator shaker and glycerol stocks were made from each round-bottom tube by taking 250 µL of its content and adding 56.25
µL of 80% glycerol into a labeled 1.5 mL microcentrifuge tube and placed in -80°C. The remaining mixture is subjected to isolation of plasmid DNA from E. coli and was done according to the protocol shipped with the miniprep kit by Bio-Rad (Quantum Prep® Plasmid Miniprep
Kit). 2 mL of each round-bottom tube’s content were placed into labeled microcentrifuge test tubes and centrifuged at 14000X g for 1-2 minutes using Eppendorf centrifuge (5417R rotor FA
45-24-11). Then the supernatant was discarded by inverting the microcentrifuge tubes over 10% bleach bucket, and before the inverted microcentrifuge tube was returned to its upright position,
Kimwipe was used to remove left over fluids. 200 µL of resuspension solution was added and the tubes were vortexed until the pellet was suspended in its entirely. 250 µL of lysis solution was added and gently inverted ten times. Then, 250 µL of neutralization solution was added and tubes were gently inverted ten more times. After that, the content of the tubes were pelleted for 5 minutes at 14000X g. During this time, a spin filter was added to each of a corresponding 1.5 mL labeled centrifuge tube and the quantum prep mixture (Q.P.M.) was mixed to ensure a homogenized solution for better DNA binding. The supernatant from the previous spinning step was placed into the microcentrifuge tube containing spin filter and 200 µL of Q.P.M. was added and mixed via pipetting. Then centrifuged for 30 seconds at 14000X g, the flow-through was discarded and 500 µL of washing buffer was added and centrifuged for 2 minutes at 14000X g.
Flow-through was discarded again and a dry spin at 14000X g for 1 minute to ensure all the washing buffer was removed. Lastly, 100 µL of Nanopure water was added to each spin filter and spun down at 14000X g for 1 minute and plasmids were stored at 4°C. Nucleic acid
27 quantitation for each plasmid was preformed using Cytation 3 Cell Imaging Multi-Mode Reader from BioTek (Winooski, VT).
Agarose gel electrophoresis of DNA
The plasmids were run on 0.8% agarose gels. Briefly, 0.8 g of agarose was dissolved in
50 mL running buffer (40 mM Tris, 20 mM Acetate and 1 mM EDTA, pH 8.6) in an Erlenmeyer flask, weighed and placed in a microwave; the mixture was swirled every 15 seconds to dissolve the agarose. The flask was weighed after the agarose was dissolved and the difference in volume was replaced with Nanopure water. After the Erlenmeyer flask cooled to 55°C, 2.5 µL of ethidium bromide (EtBr), which binds DNA and RNA with high affinity (73), was added and the mixture was poured into a cassette with the comb. The mixture solidified after 40 minutes and 10
µL EtBr was added to 250 mL of running buffer and mixed then added to the instrument. 2 µL of
6x loading buffer was added to microtubes containing 5 µL of each plasmid and GeneRuler 100 bp Plus DNA Ladder (SM0321, ThermoFisher, Waltham, MA) was loaded at 0.5 µg concentration. The instrument was run on constant voltage of 110 mV for 45 minutes. Then the gel was photographed with a Bio-Rad ChemiDoc™ XRS+ System.
Transient transfection of DNA
The cells were transfected using FuGENE 6 purchased from Promega (Madison, WI), according to their protocol (FuGENE® 6 Transfection Reagent, CAT. No. E2691). FuGENE 6 requires fewer steps than traditional methods (94). Briefly, transfection complex was prepared by adding 10 µL FuGENE 6 transfection reagent and 7 µg of GPR143, D3 or GFP to 250 µL minimum essential medium (MEM), because medium containing FBS hinders the formation of the transcription complex (94), in glass round-bottom tubes. Since D3 does not have a GFP tag, 3
28 µg of GFP plasmid was added to the tube containing D3; the logic being if GFP made into the cells there is a good possibility D3 would as well (95). The tubes were gently tapped to mix the reagent:DNA complex and left for 15 minutes. The cells were re-suspended in MEM medium and approximately 1x106 cells ~ 1 mL was added to each tube and gently tapped to mix the reagent-DNA:cells every 30 minutes. After 2 hours the content of each tube was emptied into a
T-25 flask containing 4 mL of medium and place in the incubator. After 18 hours, the cells were inspected for expression of the plasmid under fluorescence microscopy (Olympus IX71, program
Picture Frame Ver. 2.3, camera lens 2.3A, camera Microfire_C, microscope objective 10x).
Medium collection and cell harvest
Two days prior to medium collection and cell harvest, flasks were split into three groups: group A: dopamine treated cells, group B: L-DOPA treated cells, and group C: control. SH-
SY5Y, CHO-K1 and MCF-7 treatment groups received RPMI and DMEM, respectively, supplemented with 5% exosome-depleted FBS, 1%, penicillin (100 ID/ml), streptomycin (100
µg/ml), 100 mg/mL of geneticin for SH-SY5Y and 500 mg/mL for CHO-K1 and MCF-7. Group
A received 1 µM dopamine, group B received 1 µM L-DOPA and group C did not received any treatment. Each group comprised of cells transfected with D3, GPR143 and GFP plasmid.
After two days, the medium was collected and subjected to ultracentrifugation for exosome isolation, detailed in the exosome isolation section, and total cellular protein was harvested using 0.1N NaOH (83). Briefly, after the medium was collected, 500 µL of 0.1 N
NaOH was add to each T-25 flask and the cells were scraped and collected into a 1.5 mL microcentrifuge tube. Each tube was sonicated at 3WATTS using the Sonic Dismembrator F60 to sheer any DNA present in the sample and then centrifuged at 30,000X g using an Eppendorf
29 centrifuge (5417R rotor FA 45-24-11) for 30 minutes. The supernatant was then collected and placed into labeled 1.5 mL microcentrifuge tubes and stored at -20°C.
Bradford protein assay
Bradford protein assay uses Coomassie Brilliant Blue G-250 to measure protein levels in a given sample (77) standardized to known concentrations of bovine serum albumin (BSA). This method utilizes the maximum absorbency of G-250 at 595 nm, which binds the protein turns from neutral (green) to unprotonated (blue). The intensity of this color is proportional to the concentration of protein in the sample. Briefly, BSA standards were 1 mg/mL, 0.75, 0.50, 0.375,
0.25, 0.125, 0.06 and 0.03. Standards and samples were loaded at 25 µL in triplicates in a Costar flat bottom 96-well plate (3596) and 225 µL of Coomassie Brilliant Blue G-250 was added to each well. The plate was then read in FlexStation3 and a standard absorbency curve was generated.
Exosome-depleted serum
FBS was subjected to ultracentrifugation to create exosome-depleted FBS (89) via
Beckman Coulter Optima TLX Ultracentrifuge (rotor TLS-55), which then was added to DMEM and RPMI 1640 medium. Briefly, FBS was spun down at 104,000X g for 1 hr and 30 minutes; the supernatant was collected, then spun again at 104,000X g for 1 hr and 30 minutes, removing exosomes from the serum. For all experiments in which we collected cell derived exosomes, the cells were cultured using exosome-depleted FBS.
Cell differentiation
SH-SY5Y were treated with RPMI 1640 containing100 nM of L-DOPA and tetradecanoyl phorbol acetate (TPA) (84-86) every two days to induce terminal differentiation.
30 Exosome isolation
Isolation of exosomes from collected CHO-K1, MCF-7 and differentiated and undifferentiated SH-SY5Y medium was done using differential ultracentrifugation protocol modified from Stamer et al and Thery et al (87-90). Briefly, medium was collected every three days and was spun down at 500X g using Eppendorf centrifuge (5417R rotor FA 45-24-11) for 4 minutes to remove cells without rupturing them. Supernatant then spun down using at 25,000X g to remove any cell debris. The supernatant was spun at 104,000X g for 1 hr and 30 minutes to isolate the exosomes from the medium using Beckman Coulter Optima TLX Ultracentrifuge
(rotor TLS-55). The supernatant was collected as exosome-depleted medium for silver stain and western blot analysis to identify total protein and whether there is specificity between exosomes and α-SYN excreted from the cell. The resulting pellet was re-suspended and washed in cold phosphate-buffered saline (PBS, 0.137 M NaCl, 2.68 mM KCl, 0.01 M Na2HPO4, 1.76 mM
KH2PO4, 0.498 mM MgCl2•H2O, pH 7.4) and pelleted again at 104,000X g for 1 hr and 30 minutes to purify the exosomes. The resulting pellet was emulsified in 25 µL of 4X laemmli buffer for comparison between the different groups (88, 89, 91).
SDS-Polyacrylamide gel electrophoresis
SDS-PAGE is a technique used to resolve proteins based on their mass-to-charge ratio
(91). The 10% separating gels were made by mixing 7.5 mL of 1.5 M Tris, pH 8.8, 300 µL 10%
SDS, 300 µL 50% glycerol, 40 µL TEMED, 10 mL of 30% acrylamide-bis (29:1), and bringing the volume up to 30 mL using Nanopure water, then 200 uL of 10% APS was added, swirled and poured into two casts and Nanopure water was added on top of the acrylamide-bis solution. After polymerization, the water on top of the separating gel is decanted and stacking gel solution was made by mixing 2.5 mL 0.5M Tris pH 6.8, 300 µL 10% SDS, 300 µL 50% glycerol, 42 µL
31 TEMED, 1.8 mL 30% acrylamide-bis and bring the volume to 10 mL with Nanopure water then
220 uL 10% APS was added to initiate polymerization and poured on top of the separating gel and a 15 well comb was placed. After the polymerization process is over, the comb is taken out carefully and the wells cleaned by Nanopure water then the gel is placed in the electrophoresis apparatus and 1X running buffer is added into the wells, the samples with laemmli buffer and
Precision Plus Protein Prestained Protein Standards ladder (1610373, Hercules, CA) are boiled for 5 minutes at 105°C then loaded into the wells and the electrophoresis was run at a constant
60 mA for 1 hr and 30 minutes.
Western blotting
This technique detects proteins of interests from homogenated tissue or cellular lysate
(92), once the proteins are separated using gel electrophoresis they are transferred to one of two membranes, nitrocellulose or PVDF and then probed with a primary, which binds to a specific antigen and secondary anti-bodies which binds the primary antibody (92, 93) then enhanced chemiluminescence (ECL) solution is added and through a electrochemical reaction it emits lights while it decays which is captured on an x-ray film. Briefly, samples loaded on a 10% SDS-
PAGE, transferred to nitrocellulose, blocked with Tris-buffered saline and 0.2% Tween 20
(TBS-T) with 10% nonfat dry milk. Membranes were incubated overnight in rabbit monoclonal
α-SYN diluted 1:500 at 4°C with slow agitation. Washed three times in TBS-T for 10 minutes, then incubated for 1 hr with a secondary anti-rabbit horseradish peroxidase-conjugate diluted in milk at 1:5000 dilution. Membranes then washed three times for 10 minutes in TBS-T and X- rayed on Carestream Biomax MR film and ECL was used as a method of detection of the protein-antibody complex.
32 Silver stain
Silver stain is a method of protein detection (97). This detection method is 100 times more sensitive than Coomassie blue stain (97, 98). It was carried out according to Bio-Rad’s
(Silver Stain Plus™, 161-0449) protocol. Once a gel electrophoresis was complete, the gel was removed from the cassette and placed in a pan filled with Nanopure water; the plate was subjected to constant gentle agitation. The rinsing protocol subjects the gel to be bathed in 300 mL Nanopure water three times at 10 minutes each. During the rinsing phase-contrast, 500 mL of fixative solution was prepared (50% Reagent-Grade Methanol, 10% Reagent-Grade Acetic
Acid and 10% Fixative Enhancer Concentrate and 30% Nanopure water), replacing the water for
30 minutes with constant agitation. After the fixative phase, the gel was rinsed and developer acceleration (5% Na2CO3) and the staining solution (5 mL Silver Complex, 5 mL Reduction
Moderator Solution and 5 mL Image Development Reagent in 35 mL Nanopure water) were prepared.
The developer accelerator and staining solution were mixed right before adding them to the gel after the rinsing protocol, and the pan placed on the agitator again and monitored because once the reaction occurs the intensity amplifies rapidly. When the desired intensity was reached, the developer accelerator and staining solution are decanted and stopping solution made of 5% acetic acid in Nanopure water was added for 15 minutes. After this period has passed, the gel was rinsed then scanned via Epson Perfection V550 at 600 dpi.
Exosome labeling
The exosome labeling kit was purchased from System Biosciences (Palo Alto, CA), and the procedure was carried out according to the manufacturer’s protocol (Exo-GLOW™ Exosome
Labeling Kits, EXOC300A-1) with some modifications. Briefly, medium was collected from
33 SH-SY5Y and MCF-7 cells, spun down according to the exosome isolation protocol (87-90) and after resuspension in 500 µL of PBS, 25 µL of 10x Exo-Red was added to the exosomes. The solution was mixed by inverting the tube ten times and incubated at 37°C for 10 minutes then 50
µL of the ExoQuick-TC, to stop the labeling reaction, and placed on ice for 30 minutes. The tube was centrifuged at 25,000X g for 3 minutes, supernatant was decanted and the pellet, composed of labeled exosomes, was suspended in 100 µL of PBS.
Exosomes specificity and uptake
To test specificity, exosomes from SH-SY5Y cells were collected and labeled with Exo-
Red and then were introduced to CHO-K1, MCF-7 and SH-SY5Y cells. Briefly, Lab-Tek II 8 well glass slide chambers were coated with poly-D-lysine and washed with PBS. Cells were passaged and seeded in triplicates and placed in the incubator in 500 µL of medium. The following day, the medium was decanted and the chambers were washed with PBS, 10 µL of
Exo-Red labeled SH-SY5Y exosomes were added to each chamber and left for 2 minutes at RT, then washed with PBS. To fix the cells, 500 µL 4% paraformaldehyde (PFA) was added to each chamber, Table 1, after washing with PBS and placed on ice for 5 minutes. PFA was washed, the chambers were removed from the slide and two drops of 80% glycerol in PBS mounting solution was added to each section and a coverslip was added. The slides were imaged using
Olympus IX71 fluorescence microscopy.
34 CHO-K1 receiving CHO-K1 receiving CHO-K1 receiving SH-SY5Y receiving SH-SY5Y red- SH-SY5Y red- SH-SY5Y red- SH-SY5Y red- labeled exosomes labeled exosomes labeled exosomes labeled exosomes
SH-SY5Y receiving SH-SY5Y receiving MCF-7 receiving SH- MCF-7 receiving SH- SH-SY5Y red- SH-SY5Y red- SY5Y red-labeled SY5Y red-labeled labeled exosomes labeled exosomes exosomes exosomes
Table 1: Lab-Tek II schematic representation of exosome specificity. Cells were incubated at RT after
introducing SH-SY5Y exosomes then fixed with 4% PFA.
To examine if exosome uptake is an active process, preparation procedure was as mentioned above except exosomes were added to CHO-K1 cells at RT or -4ºC.
To study if there are protein on the surface of exosomes that help with the uptake of exosomes, preparation procedure was as mentioned above except exosomes were added to 25 µL of MCF-7 exosomes were used and received 1 µL of proteinase K to digest proteins on the surface of exosomes and 10 µL was added to cells in Lab-Tek II 8 well glass slide chamber; schematic of wells as in Table 2. Slides were imaged using fluorescence microscopy.
1) Control CHO-K1 3) CHO-K1 + 5) Control CHO-K1 7) CHO-K1 + + SH-SY5Y red- + SH-SY5Y red- SH-SY5Y red- labeled exo treated SH-SY5Y red- labeled exo treated labeled exo not with proteinase K at labeled exo not with proteinase K at - treated with RT treated with 4ºC proteinase K at RT proteinase K at -4ºC
2) Control MCF-7 + 4) MCF-7 + 6) Control MCF-7 + 8) MCF-7 + SH-SHY5Y red- SH-SHY5Y red- SH-SHY5Y red- SH-SHY5Y red- labeled exosomes not labeled exosomes labeled exosomes not labeled exosomes treated with treated with treated with treated with proteinase K at RT proteinase K at RT proteinase K at -4ºC proteinase K at -4ºC
Table 2: Lab-Tek II schematic representation of exosome uptake. 1-4 wells cells were incubated RT for 2
minutes before fixation with 4% paraformaldehyde. 5-8 cells were incubated at -4ºC for 2 minutes before
fixation with 4% PFA.
To examine if integrin proteins are involved in exosome uptake, the preparation procedure was as mentioned above except cells were subjected to versene for 2 minutes, then 10
35 µL of Exo-Red labeled SH-SY5Y exosomes to the labeled chambers in Table 3 and images were taken using fluorescence microscopy.
1) Versene treated 3) CHO-K1 + SH- 7) Versene treated CHO-K1 + SH- SY5Y red-labeled MCF-7 + SH-SY5Y SY5Y red-labeled exo at RT empty red-labeled exo at - exo at RT 4ºC
2) Versene treated 6) Versene treated 8) Versene treated CHO-K1 + SH- MCF-7 + SH-SY5Y CHO-K1 + SH- SY5Y red-labeled empty red-labeled exo RT SY5Y red-labeled exo at -4ºC exo at RT
Table 3: Lab-Tek II schematic representation of exosome uptake. 1-3 wells cells were incubated at RT for 2
minutes before fixation with 4% paraformaldehyde. 6-8 cells were incubated at -4ºC for 2 minutes before
fixation with 4% PFA.
Biotinylation of exosomes
Exosomes isolated from SH-SY5Y were biotinylated then introduced to SH-SY5Y and
MCF-7. Briefly, Exosomes were pulled down by adding 125 µL 40% PEG 8000 to SH-SY5Y exosomes suspended in 500 µL PBS and spun down at 14000 RPM for 10 minutes. Supernatant was decanted and exosomes were washed twice by suspending them in 500 µL of biotinylation buffer (100 mM NaCl, 50 mM NaHCO3, 2 mM CaCl2, pH 8.0) and spun down at 14000 RPM for
10 minutes. 500 µL of 1 mg/mL of biotin in biotinylation buffer was added to the exosomes and incubated on ice for 25 minutes. Exosomes were spun down at 14000 RPM for 10 minutes, supernatant was decanted and another 500 µL of biotin was added and incubated for 25 minutes on ice. T-25 flasks of SH-SY5Y and MCF-7 were decanted of their medium and 2 mL of biotinylation buffer was added and placed in a -4°C ice bath. Hypotonic lysis buffer (10 µM
MnCl2, 10 µM PPO4) was prepared containing 1X protease inhibitor cocktail (P8340). Each flask received 500 µL of hypotonic lysis buffer and cells were scraped and the lysed cells were placed in glass homogenizers and moved the glass pistons ten times. The solution was
36 transferred to a 15 mL falcon tubes and 50 µL of magnetic streptavidin beads in 1 mL of biotinylation buffer were added and spun down at 15000 RPM for 2 minutes. Tube caps were parafilmed and were agitated for an overnight capture in the fridge.
For magnetic antigen capture, hypotonic lysis solution was placed on ice, 1.5 mL
Eppendorf tubes placed in Dynal MPC-E Magnetic Particle Concentrator (product number
120.04). Falcon tubes always kept on ice and using a transfer pipet solution was transferred 1 mL at a time to 1.5 mL microcentrifuge tube. Once the magnet has taken action, the streptavidin beads magnetized to one side of the Eppendorf tube, using transfer pipet fluid was removed from the tube without disturbing the beads, until all 15 mL were subjected to the magnet. 1 mL of hypotonic lysis solution was added to the Eppendorf tubes containing the beads; the tubes were gently tapped and were placed back in the Magnetic Particle Concentrator and fluid was removed from the tube without disturbing the beads. This washing step was done 9 additional times. After the washing step, 25µL of laemmli buffer was added to each Eppendorf tube to emulsify the magnetic streptavidin beads and boiled for 5 minutes at 105ºC and then ran on a 7% SDS:PAGE.
Well contained the following samples, Table 4.
37 Exo-Red Exo-Red Exo-Red Exo-Red Exo-Red
labeled labeled labeled labeled labeled
SH- SH- SH- SH- SH-
SY5Y SY5Y SY5Y SY5Y SY5Y
Ladder Empty exosomes exosomes exosomes Empty Empty exosomes exosomes
added to added to added to added to
SH- MCF-7 SH- MCF-7
SY5Y cells SY5Y cells
cells cells
Table 4: Samples were loaded in a 7% SDS:PAGE. The samples were loaded according to this table.
Viral α-SYN and GFP transduction
SH-SY5Y, CHO-K1 and MCF-7 cells were infected with AAV5-CBA-Alpha-Synuclein or viral AAV5-CBA-eGFP at an MOI of 5. Briefly, when T-25 flasks were 50% confluent, one group of flasks received AAV5-CBA-Alpha-Synuclein while the other received AAV5-CBA- eGFP in 2 mL of medium and placed in the incubator for 2 hrs while gently agitating the flasks every 10 minutes. Then 3 mL of medium was added to each flask and place in the incubator.
After two days, 60% of the cells are expressing the GFP protein when viewed under a fluorescence microscope.
38 Results
Transient transfection of DNA
After 18 hrs post transfection, T-25 flasks were imaged under fluorescence microscopy.
They were treated with geneticin to create pools of stable protein expressing cells. SH-SY5Y received 100 mg/mL, while CHO-K1 and MCF-7 received 500 mg/mL.
Figure 8: A-F photomicrographs of transfected CHO-K1 cells. A-C phase-contrast and D-F fluorescence
microscopy images, GFP, D3-GFP and GPR143 respectively, scale bar 10 µm, 20X objective.
Figure 8 shows the transfected CHO-K1 cells; GPR143 (F) expression was not as prominent due to the presence of 500 µM of L-tyrosine in the media. L-tyrosine binds GPR143, while it does not stimulate signaling (70), causes endocytosis of the receptor.
39
Figure 9: A-F photomicrographs of transfected SH-SY5Y cells, A-C phase-contrast and D-F fluorescence
microscopy images, GFP, D3-GFP and GPR143 respectively, scale bar 10 µm, 20X objective.
SH-SY5Y, in Figure 9, shows the expression of GFP, D3-GFP and GPR143 respectively.
40
Figure 10: A-F photomicrographs of transfected MCF-7 cells, A-C phase-contrast and D-F fluorescence
microscopy images, GFP, D3-GFP and GPR143 respectively, scale bar 10 µm, 20X objective.
Figure 10 shows plasmid expression in MCF-7 of GFP, D3-GFP and GPR143.
Cell differentiation
After pools of cell lines were created in SH-SY5Y, they were treated with medium containing100 nM of L-DOPA and 100 nM of TPA to create terminally differentiated cells,
Figure 11.
41
Figure 11: Phase-contrast photomicrographs of undifferentiated and differentiated SH-SY5Y in A and B respectively. Processes are longer and more pronounced in the differentiated SH-SY5Y. Scale bar 10 µm, 20X
objective.
Figure 12 shows differentiated SH-SY5Y cells expressing GFP, D3-GFP and GPR143
Figure 12: A-F photomicrographs of transfected differentiated SH-SY5Y cells, A-C phase-contrast and D-F
fluorescence microscopy images, GFP, D3-GFP and GPR143 respectively, scale bar 10 µm, 20X objective .
42 Silver stain
Exosome fraction, exosome-depleted medium and cell lysate were ran on 10%
SDS:PAGE and subjected to silver stain as shown in Figure 13.
Figure 13: Silver stain scan of SDS:PAGE of exosome fraction, exosome depleted medium and cell lysate
treated with L-DOPA or dopamine. Exo = Exosomes, CM = Exosome-depleted culture medium, CL = Cell
lysate.
Exosome fraction lanes from silver stains were analyzed using ImageJ for total exosome protein in the lanes between treatment groups for each receptor within a cell line. For n of 3 using nonparametric unpaired t-test analysis, there was no significant difference in the total protein in the exosome fraction lanes between the treatment groups within each selected cell line.
Graphs were generated by plotting integrated optical density (IOD) versus treatment groups,
Figure 14.
43
Figure 14: Graph illustrates total exosome protein analysis of exosome fraction lane (means ± SEM) from silver stain from three experiments. Black, blue and purple GFP, D3 and GPR143 transfected CHO-K1 (A),
MCF-7 (B), differentiated SH-SY5Y (D. SH-SY5Y) (C) and SH-SY5Y (D) cell lines respectively. Integrated
optical density (IOD) obtained ImageJ via conventional methods plotted against treatment groups. Con = control, D = dopamine, L = L-DOPA treatments. nonparametric, unpaired-test, no significant difference was
found.
Exosomes specificity and uptake
SH-SY5Y Exo-Red labeled exosomes showed no specificity as they were internalized by
SH-SY5Y and MCF-7 cells as shown in Figure 15. Photomicrographs were taken within seconds of introducing the exosomes.
44
Figure 15: Fluorescent photomicrographs showing that Exo-Red labeled SH-SY5Y exosomes were
internalized by MCF-7 and SH-SY5Y shown in A and B respectively. Photomicrographs were taken within
seconds of adding the Exo-Red labeled exosomes. Scale bar 10 µm, 40X objective.
SH-SY5Y Exo-Red labeled exosomes showed no specificity, as they were internalized by CHO-K1 cells, however, the exosomes did not release their content, Figure 16. This results suggest there is no specificity between species in the selected cell lines. Photomicrographs were taken within seconds of introducing the exosomes.
Figure 16: Fluorescent photomicrograph showing Exo-Red labeled SH-SY5Y exosomes were internalized by
CHO-K1 cells. Photomicrograph was taken within seconds of adding the Exo-Red labeled exosomes. Scale
bar 10 µm, 40X objective.
Uptake of SH-SY5Y Exo-Red labeled exosomes was stopped when exosomes were added to CHO-K1 at -4ºC in comparison to RT, Figure 17.
45
Figure 17: Fluorescent photomicrographs of Exo-Red labeled SH-SY5Y exosomes shows exosome uptake in
CHO-K1 cells at RT but not at -4ºC shown in A and B respectively. Photomicrographs were taken 5 minutes
after the addition of Exo-Red labeled SH-SY5Y exosomes and fixation in 4% PFA. Scale bar 10 µm, 40X
objective.
Proteinase K treated Exo-Red labeled SH-SY5Y exosomes were not internalized by
CHO-K1 at RT, Figure 18.
Figure 18: Fluorescent photomicrographs of proteinase K untreated and treated Exo-Red labeled SH-SY5Y
exosomes added to CHO-K1 cells in A and B respectively, after fixation in 4% PFA. Scale bar 20 µm, 20X
objective.
Exo-Red labeled SH-SY5Y exosomes were not internalized by the CHO-K1 cells after the treatment of versene; they were trapped on the cell surface. Photomicrographs were taken 5 minutes after the cells were fixed in 4% PFA, Figure 19.
46
Figure 19: Fluorescent photomicrographs of versene untreated and treated CHO-K1 cells shows Exo-Red labeled SH-SY5Y exosomes were trapped on the cell surface of the cells at RT shown in A and B respectively.
Photomicrographs were taken 5 minutes after the addition of Exo-Red labeled SH-SY5Y exosomes and
fixation in 4% PFA. Scale bar 10 µm, 40X objective.
Biotinylation of exosomes
Biotinylated SH-SY5Y Exo-Red labeled exosomes alone and after their introduction to
SH-SY5Y and MCF-7 cells were ran on a 7% SDS:PAGE gel. There were 6 loaded wells as shown above in Table 4. After the SDS:PAGE was done, the gel was cut and one part went to be silver stained and the other was used for a biotinylated blot, Figure 20.
47
Figure 20: A) Silver stain of biotinylated exosomes. 1 = Biotinylated Exo-Red labeled SH-SY5Y exosomes
added to MCF-7, 2 = Biotinylated Exo-Red labeled SH-SY5Y exosomes added to SH-SY5Y, 3 = Exo-Red labeled exosome fraction. B) X-ray film of biotinylation blot of biotinylated exosomes. 1 = Biotinylated Exo-
Red labeled SH-SY5Y exosomes added to MCF-7, 2 = Biotinylated Exo-Red labeled SH-SY5Y exosomes
added to SH-SY5Y.
Discussion
SH-SY5Y, MCF-7 and CHO-K1 cells were transfected with D3-GFP, GPR143 and GFP to study the effect of dopamine and L-DOPA on modulating the release of exosomes. As shown in Figure 14, there was no significant difference between the exosome fraction lanes between cells expressing D3-GFP, GP143 or GFP whether treated with dopamine, L-DOPA or left untreated. A study from the McKay lab, showed a significant decrease in the amount of exosomes released from RPE cells in situ when treated with L-DOPA (101). Based on the results of Locke et al, we wanted to investigate if exosome release could be regulated in the selected cell
48 lines after treatment with dopamine or L-DOPA. Cells cultured in flasks yields a specific population of cells that express fibronectin and vitronectin enabling them to adhere to the flask, where cells not expressing these proteins will not adhere and will die. Thus, we are lowering the diversity of cells in comparison to cells in situ. This could be a possible explanation for the difference in results. Another possible difference, studies showed RPE cells express D1, D2 and
D5 receptors (102, 103) in addition to GPR143, while the selected cell lines do not express D3 or
GPR143 naturally. Expression of multiple dopamine receptors in RPE cells could point to an additive or synergic effect once the cells were treated with 100 nM L-DOPA. Furthermore,
GPR143 utilizes Gaq to increase the concentration of cytosolic calcium by modulating the levels of inositol 1,4,5-trisphosphate (IP3) through activation of phospholipase C (PLC) (107). IP3 binds its receptor on the endoplasmic reticulum (ER) causing intercellular calcium to increase (107).
D3, on the other hand, decreases intercellular calcium by stopping the conversion of 3',5'-cyclic
AMP (cAMP) from adenosine triphosphate( ATP) by inhibiting adenylyl cyclase (AC) (108).
Heusermann et al. (2016), showed 80% of exosomes from human embryonic kidney cells
(HEK293) were taken up by human primary fibroblasts (C-013-5C) after 2 hr. CHO-K1, SH-
SY5Y and MCF-7 cells internalized Exo-Red labeled exosomes isolated from SH-SY5Y or
MCF-7 within 20 seconds of introducing them to the cells, Figure 15 and Figure 16, showing lack of cell-cell and species-species specificity in the selected cell lines. However, the contents of exosomes were not readily released in the CHO-K1 cells as shown in Figure 16, because the
Exo-Red dye fluoresces red when it intercalates single-strand RNA (ssRNA) and when it dissociates it fluoresces green, and there was no green hue in the photomicrographs.
The next question to answer was whether exosome uptake was an active process. Exo-
Red labeled SH-SY5Y exosomes were added to two groups of CHO-K1 cells, one at RT and the
49 other at -4ºC. As shown in Figure 17, there was exosome uptake at RT (A) but not at -4ºC (B) indicating this is an active process. Enzymes required to convert ATP to adenosine diphosphate
(ADP) are functioning at RT, however in subzero temperatures, in this case, -4ºC they are not active eliminating the energy from the conversion of ATP to ADP.
We also conducted experiments to determine if exosomes have membrane resident proteins aiding with their internalization. Exo-Red labeled SH-SY5Y exosomes were treated with proteinase K and then added to CHO-K1 cells. Proteinase K cleaves peptides on the surface of cells or exosomes, and that is shown in Figure 18. Proteinase K did not cleave peptides inside the exosomes because there was no green signal when the cells were viewed through fluorescence microscopy. Untreated exosomes were internalized by the CHO-K1cells, while there was no exosome uptake in the proteinase K treated ones, Figure 18, indicating one or more proteins help with their internalization. Wang et al. (2015), showed the involvement of integrins in exosome uptake. To test the possible involvement of integrins, CHO-K1 cells were treated with versene before adding Exo-Red labeled SH-SY5Y exosomes. Integrin proteins are comprised of α and β subunits and they require calcium to function. Versene is a calcium chelator causing the subunits to dissociate. The exosomes were trapped at the cell surface in the versene treated CHO-K1 cells as shown in Figure 19. This result indicates there is a receptor on the cell surface that is binding a protein found on the exosome surface. To investigate this protein, biotinylated Exo-Red labeled SH-SY5Y exosomes were added to SH-SY5Y and MCF-7 cells and using magnetic antigen capture technique the biotinylated exosomes were collected and the samples were ran on a 7% SDS:PAGE. After gel electrophoresis was done the gel was cut and one part was silver stained, while the other was blotted. Figure 20, A shows the total protein found in the SH-SY5Y and MCF-7 cells and B shows the biotinylated proteins from exosomes.
50 Panel A in Figure 20, shows multiple protein bands and a prominent protein band in the region of 70 to 80 kDa on the silver stain that is not present in the biotinylated blot. This signifies that the potential protein helping exosome uptake resides between 70 to 80 kDa. To determine which protein it is in future studies, gel electrophoresis at a higher concentration of acrylamide, 12%, needs to be done to insure more separation between the proteins. The band will be cut out, the protein isolated and identified by sequencing.
This thesis provides a foundation to study exosome communication and regulation of their cellular uptake, a process that currently remains largely unknown. This work lays the groundwork for future studies investigating potential exosome biomarkers to help identify human diseases and possibly early diagnosis of diseases such as PD.
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