CHARACTERIZATION OF OLIGODENDROCYTE-DERIVED EXTRACELLULAR VESICLES ACROSS CELL DIFFERENTIATION by Connor Johnson BSc, University of Northern British Columbia, 2021 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN BIOCHEMISTRY UNIVERSITY OF NORTHERN BRITISH COLUMBIA August 2024 © Connor Johnson, 2024 Abstract Oligodendrocytes (OLs) are the myelinating glia of the central nervous system (CNS), and their dysfunction is a hallmark of several neurodegenerative disorders such as multiple sclerosis. Intercellular communication pathways between OLs and other cell types in the CNS is critical for the proper formation and maintenance of the myelin sheath. Extracellular vesicles (EVs) are a heterogeneous population of secreted membrane vesicles that differ in biogenesis, cargo, and biological functions they exhibit on target cells. One relatively unexplored avenue is the autocrine regulation of myelination mediated by EVs, and given their variety in cargo, potentially they could be involved in this process. However, very little is known about the molecular cargo of OL-derived EVs and nothing is known about how OL-EV cargo changes throughout cell differentiation. This study aims to determine how the molecular composition and functional effects of EVs change over OL cell development using the CG4-OL cell line. Relative to the EV associated marker TSG101, the constitutive secretion of EVs containing the tetraspanin markers CD44, CD63 and CD81 remained consistent across cell differentiation. On the other hand, the expression of CD9 increased suggesting a different population of exosomes and/or microvesicles are secreted from mature OLs. These EVs also had an increase in the cell signaling protein 14-3-3. Treatment of CG4OL cells with OL-derived EV isolated at different stages resulted in no difference in genes associated with progenitor state (Ki67 and Pdgfra) and of the myelin genes, a significant increase (p<0.05) in relative Mog mRNA expression was observed in samples treated with supernatant collected from D0 and D6. Data presented in this thesis, for the first time, characterizes OL derived EVs isolated across cell differentiation and assessed their ability to act as autocrine factors on OL proliferation and differentiation. ii Acknowledgements I want to acknowledge that my research was conducted on the traditional unceded territory of the Lheidli T’enneh First Nation, part of the Dakelh (Carrier) peoples’ territory. I would like to extend my thanks to my supervisor Dr. Kendra Furber for taking me on as a graduate student and continuously pushing me to be the best version of myself. From her, I have learned much about attention detail, perseverance, and determination. I would like to thank my Graduate Supervisory Committee: Dr. Andrea Gorrell and Dr. Stephen Rader for providing me with valuable feedback to push my project forward. I have known Dr. Andrea Gorrell since my undergraduate studies, and she helped solidify my interest in research by supervising me for my undergraduate thesis. She has always made herself available to help me with questions related to research and has provided me with invaluable advice regarding careers in research. For that I am forever grateful. To my colleagues in the Furber lab, I would like to extend my thanks to Victor Liu and Dr. Andrew Giles for training me in addition helping me troubleshoot experiments. Lastly, I would like to thank my friends, family, and cat Momo for being incredibly supportive during my graduate studies. iii Table of Contents Page Abstract ..................................................................................................................................... ii Acknowledgements .................................................................................................................. iii Table of Contents ..................................................................................................................... iv List of Tables ........................................................................................................................... vi List of Figures ......................................................................................................................... vii List of Supplemental Figures ................................................................................................. viii List of Abbreviations ............................................................................................................... ix Chapter 1. Introduction ............................................................................................................. 1 1.1 Myelination, Demyelination, and remyelination............................................................. 1 1.1.1 Development ............................................................................................................. 1 1.1.2 Aging ........................................................................................................................ 5 1.1.3 Pathology .................................................................................................................. 6 1.2 Molecular mechanisms of oligodendrocyte differentiation ............................................ 6 1.2.1 Extrinsic factors and intracellular signaling pathways ............................................. 7 1.2.2 Epigenetics and transcription factors ........................................................................ 8 1.2.3 miRNAs .................................................................................................................... 9 1.3 Role of Extracellular Vesicles in Intercellular Communication in the CNS................. 10 1.3.1 Oligodendrocyte – Neuronal interactions ............................................................... 13 1.3.2 Oligodendrocyte – Astrocyte interactions .............................................................. 16 1.5.3 Oligodendrocyte – Microglia interactions .............................................................. 17 1.5 Model systems for studying oligodendrocyte differentiation ....................................... 17 1.5.1 Oligodendrocyte cell lines ...................................................................................... 18 1.5.2 Oligodendrocyte primary cell culture ..................................................................... 18 1.5.3 Oligodendrocyte-neuronal cell cultures.................................................................. 20 1.6 Thesis scope and significance ....................................................................................... 20 Chapter 2. Materials and Methods .......................................................................................... 22 2.1 CG4-OL Cell Culture .................................................................................................... 22 2.2 Extracellular Vesicle Isolation ...................................................................................... 22 2.3 Nanoparticle Tracking Analysis .................................................................................... 23 2.4 Western Blotting ........................................................................................................... 25 iv 2.5 Primer Design and Validation ....................................................................................... 28 2.6 RNA Isolation and Reverse Transcription .................................................................... 29 2.7 PCR and Gel Electrophoresis ........................................................................................ 30 2.8 qPCR ............................................................................................................................. 30 2.9 MTT assay ..................................................................................................................... 32 2.10 EV treatments .............................................................................................................. 32 Chapter 3. Results ................................................................................................................... 34 3.1 Determination of protein recovery from EV fraction to visualize markers by Western blot....................................................................................................................................... 34 3.2 EV maker assessment across OL cell differentiation .................................................... 36 3.3 Differential ultracentrifugation optimization ................................................................ 39 3.4 Cell culture optimization to reduce OL cell death over cell differentiation.................. 40 3.6 Functional effects of OL-derived EVs collected across cell differentiation on proliferation and differentiation of CG4-OLs. .................................................................... 46 Chapter 4. Discussion ............................................................................................................. 50 4.1 Assessment of OL-derived EV isolations based on MISEV2018 guidelines ............... 50 4.1.1 Shorter EV pelleting centrifugation step increasing common EV marker expression ......................................................................................................................................... 55 4.1.2 DMEM/F12 increases calnexin exclusion in EV fraction ...................................... 55 4.1.3 Stimulating OL EV release ..................................................................................... 57 4.2 Tetraspanin EV markers – the search for unique subpopulations ................................. 59 4.3 Relative expression of EV cargo 14-3-3 increased over differentiation ....................... 60 4.4 Assessing the role of OL EVs as autocrine factors regulating proliferation and differentiation of CG4-OLs ................................................................................................. 62 Chapter 5: Conclusions and Future Directions ....................................................................... 65 References ............................................................................................................................... 66 Supplemental Figures.............................................................................................................. 80 v List of Tables Table 1. Antibody list and conditions used for antigen visualization ………………………27 Table 2. qPCR primer validation summary …………………………………………………29 Table 3. Summarized NTA data of EVs isolated from CG4-OLs used for functional studies ………………………………………………………………………………………………..47 vi List of Figures Figure 1. Classification of extracellular vesicle types, their site of biogenesis, and their contents ……………………………………………………………………………………...11 Figure 2. Summary of known mechanisms of oligodendrocyte mediated neuronal support. 15 Figure 3. Schematic of extracellular vesicle isolation methods …………………………….24 Figure 4. Protein recovered in EV fraction and protein load required to visualize EV markers by Western blot ……………………………………………………………………………...35 Figure 5. Nanoparticle tracking analysis data of isolated EVs across OL differentiation ….36 Figure 6. Extracellular vesicle marker assessment across oligodendrocyte cell differentiation………………………………………………………………………………...38 Figure 7. Comparison of EV markers between 3h and 2h 100K EV pelleting centrifugation step…………………………………………………………………………………………...40 Figure 8. Effects of basal media on calnexin expression found in 100K pellet across oligodendrocyte differentiation………………………………………………………………42 Figure 9. Effect of L-glutamate + D-serine on CG4-OL cell viability across differentiation.43 Figure 10. Comparison of calnexin expression found in 100K pellets between stimulated and unstimulated CG4-OL cells.…………………………………………………………………45 Figure 11. Autocrine effects of OL-EVs on proliferation and myelin gene expression…….49 Figure 12. Ionotropic glutamate receptors mRNA expression in oligodendrocytes across differentiation………………………………………………………………………………...52 vii List of Supplemental Figures Figure S1. Protein required to visualize EV markers by Western blot………………………80 Figure S2a. N1 gels, membranes, and Western blots for Calnexin and 14-3-3……………..81 Figure S3. N1 gels, membranes, and Western blots for lamin A + C……………………….82 Figure S4. N1 gels, membranes, and Western blots for CD63 and CD9……………………83 Figure S5. N1 gels, membranes, and Western blots for CD44, TSG101 and CD81………..84 Figure S6. N2 gels, membranes, and Western blots for Calnexin and 14-3-3………………86 Figure S7. N2 gels, membranes, and Western blots for lamin A + C……………………….88 Figure S8. N2 gels, membranes, and Western blots for CD63 and CD9……………………89 Figure S9. N2 gels, membranes, and Western blots for CD44, TSG101 and CD81………..91 Figure S10. Resolved gels, membranes, and Western blots for comparison of EV markers between 3h and 2h 100K pelleting centrifugation step………………………………………93 Figure S11. Resolved gels, membranes, and Western blots of calnexin expression found in 100K pellet in four basal media compositions……………………………………………….94 Figure S12. Resolved gels, membranes, and Western blots for unstimulated and glutamate stimulated CG4-OL cells..…………………………………………………………………...95 Figure S13. Resolved gels, membranes, and Western blots for unstimulated and glutamate stimulated CG4-OL cells with NAC.………………………………………………………...96 viii List of Abbreviations AB/AM = Antibiotic antimycotic AMPA = α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ANT = adenine nucleotide translocase BrdU = Bromodeoxyuridine bFGF = basic fibroblast growth factor CM = Conditioned media CNS = Central nervous system CNTF = Ciliary Neurotrophic Factor CXCL1 = chemokine ligand 1 CO2 = Carbon dioxide DCX = Doublecortin DM = Differentiation media DMSO = Dimethyl sulfoxide DRG = Dorsal root ganglia ErbB = Erythroblastic leukemia viral oncogene homologue ECM = Extracellular matrix EGF = Epidermal growth factor ERK = Extracellular signal regulated kinase ET-1 = Endothelin-1 ETV5 = ETS variant transcription factor 5 EV = Extracellular vesicle GM = Growth media HDAC = Histone deacetylase KDAC = Lysine deacetylase HES5 = Hes Family BHLH Transcription Factor 5 IPC = intermediate progenitor cells IRAK1 = interleukin-1 receptor associated kinase-1 ISEV = International Society of Extracellular Vesicles ix LINGO-1 = Leucine-rich repeat neuronal protein 1 MAPK = Mitogen activated protein kinase MBP = myelin basic protein MCT = Monocarboxylate transporter MEK = Mitogen-activated protein kinase kinase MISEV = Minimal information for studies of extracellular vesicles MOG = Myelin-oligodendrocyte glycoprotein MRI = Magnetic resonance imaging MS = Multiple sclerosis mTOR = mammalian target of rapamycin MTT = Methyl tetrazole thiol MWI = Myelin water imaging MYRF = myelin regulatory factor NAC = N-acetyl-cysteine NGS = Next generation sequencing NKX2.2 = NK2 homeobox 2.2 NMDA = N-methyl-D-aspartate NRG1 = Neuronal growth factor neuregulin-1 NRT = No reverse transcriptase NSC = neural stem cell NTA = Nanoparticle tracking analysis OL = Oligodendrocyte OLIG2 = oligodendrocyte transcription factor 2 OPC = Oligodendrocyte progenitor cell PBS = Phosphate buffered saline PDGFA = platelet derived growth factor alpha PDGFRA = platelet derived growth factor receptor alpha PI3K = Phosphoinositide 3-kinase PKB = Protein kinase B PLP = Myelin proteolipid protein x PMSF = Phenylmethylsulfonyl fluoride PVDF = Polyvinylidene Fluoride RNE = Relative normalized expression SDS = Sodium dodecyl sulfate SEM = Standard error of the mean sEVs = small extracellular vesicles SIRT2 = Sirtuin 2 SOD-1 = superoxide dismutase-1 SOX6 = Sry-box transcription factor 6 SOX 10 = Sry-box transcription factor 10 STAT3 = signal transducer and activator of transcription 3 TBST = trisaminomethane buffered saline and 0.1% Tween20 TCF7L2 = transcription factor 7-like 2 TEM = Tetraspanin-enriched microdomains TGF = transforming growth factor Tris = trisaminomethane ZFP24 = zinc finger protein 24 xi Chapter 1. Introduction Oligodendrocytes (OLs) are a type of glial cell responsible for myelinating neurons in the central nervous system (CNS) permitting fast transmission of axon potentials (Reiter and Bongarzone 2020). Myelin is a specialized structure generated through the extension and subsequent wrapping of OL plasma membrane around neuronal axons (Stadelmann et al. 2019). The tight packing of the myelin membrane provides the insulating properties crucial for rapid saltatory conduction in addition to providing trophic support for long-term axonal integrity. The dysregulation of myelin maintenance observed in several neurodegenerative pathologies results in decreased neurotransmission rates, among more severe effects such as axon degeneration (Dutta et al. 2006, Kuhn et al. 2019). Demyelinating disorders can be the result of intrinsic factors such as genetic abnormalities and/or extrinsic factors such as growth factor depravation, impaired intercellular communication, environmental toxins, and immune cell dysfunction (Messing et al. 2012, Asai et al. 2015, Prinz and Priller 2017). It is likely that intrinsic and extrinsic factors have synergistic effects with varying contribution levels depending on the disorder. Understanding the mechanisms regulating myelination is crucial for the development of more effective intervention strategies for demyelinating disorders such as multiple sclerosis (MS). 1.1 Myelination, Demyelination, and remyelination 1.1.1 Development Within the CNS, developmental myelination occurs in a caudal to rostral direction where the spinal cord is the first structure myelinated, and the cortex is the last (Stadelmann et al. 2019). Myelination in the brain occurs in a predictable sequence of events, such that it begins in areas responsible for homeostatic functions (caudal) and progresses to regions dedicated for 1 higher cognitive tasks (rostral). However, not all axons are myelinated, and their level of myelination varies considerably. Why OLs preferentially myelinate some axons and not others is still not fully understood, but some recent work has begun to unravel this complex question. Recently, experimental paradigms have been designed to uncouple axonal signals from physical cues using engineered nanofibers (Lee et al. 2012, Bechler et al. 2015, 2018). These studies have led to the concept that axon diameter contributes to regulating which axons are myelinated (Lee et al. 2012, Bechler et al. 2015, 2018, Stadelmann et al. 2019). These findings agree with observations in the developing CNS where larger diameter axons are generally myelinated first and more fully compared to smaller axons (Stadelmann et al. 2019). The observations from isolated model systems are most likely due to decreased membrane curvature requirements associated with larger diameter fibers therefore making myelination relatively “easier” in comparison to smaller diameter fibers (Lee et al. 2012, Bechler et al. 2015, 2018, Stadelmann et al. 2019). Other factors such as neurotransmitter release, neuregulin signaling, and interactions with integrin receptors influence myelination, but have so far have not been demonstrated to be essential for myelination to occur (Benninger et al. 2006, Stadelmann et al. 2019, Kang and Yao 2022, Yamada et al. 2022). Myelination has been shown to be regulated to various extents by interactions with proteins found the extracellular matrix such as laminins and fibronectin. Laminins are cell adhesion glycoproteins found in the extracellular matrix (ECM) that have been demonstrated to regulate OL differentiation and myelination by binding to integrins and/or dystroglycan (Stadelmann et al. 2019, Kang and Yao 2022, Yamada et al. 2022). Laminin-211 deficient mice display a 30% decrease in the number of mature OLs as well as ultrastructural abnormalities such as myelin lamellae splitting (Chun et al. 2003). This effect has been described as mediated 2 by laminin interacting with integrin α6 as primary OLs derived from α6 knockout mice have significantly less mature OLs, have less membrane expansion, and have significantly more TUNEL-positive cells indicating increased apoptosis (Colognato et al. 2002). Fibronectin is another cell adhesion protein in the ECM that is produced by astrocytes, microglia, and endothelial cells that has been shown to negatively regulate myelination (Yamada et al. 2022). Evidence for this effect comes from assessing MS plaques as fibronectin levels are upregulated and conversely fibronectin levels are decreased during remyelination (Stoffels et al. 2013). Neuronal growth factor neuregulin-1 (NRG1) signaling has also been demonstrated to regulate myelination: however, it is still heavily debated on the effects it exerts on myelination in the CNS and under what circumstances (Brinkmann et al. 2008, Lundgaard et al. 2013, Stadelmann et al. 2019). NRG1 is a transmembrane growth factor that has three described isoform types; type I and type II can undergo proteolytic cleavage to be released as soluble proteins from neuronal cell surfaces whereas type III is bound to axonal membranes (Brinkmann et al. 2008). Brinkmann et al. 2008 demonstrated that NRG1/erythroblastic leukemia viral oncogene homologue (ErbB) receptor signaling is not required for CNS myelination as ErbB3 -/ErbB4-/- receptor OL conditional knock out mice had no differences in myelination in the optic nerve and corpus callosum compared to control animals. However, overexpressing NRG1 type I and type III resulted in hypermyelination (Brinkmann et al. 2008). The effects of NRG1 type III on CNS myelination have also been assessed using +/- type III NRG1 mice and reduced myelin lamellae were observed in the forebrain (Taveggia et al. 2008). The reduction in myelin lamellae were not observed in the spinal cord or the optic nerve suggesting that the effects NRG1 type III elicits are region specific within the CNS (Taveggia et al. 2008). Interestingly, previous reports have described NRG1 as indispensable for OL survival and differentiation in vitro which 3 contradicts in vivo reports (Vartanian et al. 1999, Flores et al. 2000, Calaora et al. 2001, Park et al. 2001). Spinal cord explants from NRG1 -/- mice embryos displayed OL cell specification defects as few to no OLs were observed (Vartanian et al. 1999). The authors were able to demonstrate that exogenous treatment with NRG was able to rescue OL development in NRG1 null mice and blocking NRG in WT explants displays same phenotype as NRG1 null mice (Vartanian et al. 1999). OLs express a multitude of neurotransmitter receptors and have been shown to respond to nearby neuronal activity (Mitew et al. 2018, Almeida et al. 2021). Mitew et. al. 2018 demonstrated OLs have a preference to myelinate neurons with higher activity in addition to producing thicker myelin sheaths using a chemogenetic approach. These findings agree with previously published work that increased neuronal activity using an optogenetic system (Gibson et al. 2014). The observed preference for more active neurons is believed to be mediated, to some extent, by the vesicular release of neurotransmitters (Gibson et al. 2014, Mitew et al. 2018, Almeida et al. 2021). However, studies that have manipulated vesicular neurotransmitter release have reported different effects. In zebrafish, axonal vesicular fusion has been shown to stimulate myelin sheath formation and growth along reticulospinal axons (Almeida et al. 2021). Blocking axonal vesicular fusion resulted in decreased myelin sheath length as well as decreased proportion of the reticulospinal axon that is myelinated. However, the authors did not identify the vesicular cargo responsible for these effects (Almeida et al. 2021). Conversely, Etxeberria et al. 2016 showed that monocular depravation and decreased neurotransmission increases the number of mature OLs along the optic nerve with no observed effects on the proportion of myelination along the optic nerve. However, the authors did report an observed decrease the myelin sheath length (Etxeberria et al. 2016). This emphasizes the probability that neuronal activity will induce 4 distinct effects on myelination depending on the CNS region, which is most likely explained either due to inherent variations in OL or neuronal populations (Gibson et al. 2014, Etxeberria et al. 2016, Mitew et al. 2018). 1.1.2 Aging As individuals progress in age a variety of structural changes have been observed in the brain by several imaging techniques such as magnetic resonance imaging (MRI) and c (MWI) (Sowell et al. 2003, Dvorak et al. 2021). Regional changes in myelination within the brain vary between individuals and is described as adaptive myelination, whereby experience and sensory input drives neural circuits to be myelinated in such a way that improves the efficiency of signal transduction (Sowell et al. 2003, Stadelmann et al. 2019). Generally, in humans white matter volume increases and peaks at 43-50 years of age and then begins to decrease (Sowell et al. 2003, Dvorak et al. 2021). This age-related decrease in myelin is most pronounced in late myelinating white matter tracts associated with cortical regions and has been associated with cognitive decline in older adults (Peters 2002, Sowell et al. 2003, Furber et al. 2022). Additionally, with increasing age progressively more myelin sheaths exhibit defects such as lamellae splitting at the major dense line and the formation of “balloons” (Feldman and Peters 1998, Peters 2002). Similar myelin structural defects have been observed in the mouse neurotoxic cuprizone model commonly used to study demyelination (Peters 2002). One explanation for the general loss of white matter in aging is the accumulation of oxidative damage that OLs are particularly vulnerable to (Thorburne and Juurlink 1996, Juurlink et al. 1998, Lassmann and van Horssen 2016). This has been attributed due to several factors such as increased iron accumulation with the brain leading to the formation of hydroxide radicals, relatively low levels of antioxidants found in OLs, and the metabolic demand for maintaining 5 extensive myelin membrane sheets (Thorburne and Juurlink 1996, Juurlink et al. 1998, Lassmann and van Horssen 2016, Furber et al. 2022). 1.1.3 Pathology MS is a CNS specific autoimmune disease in which myelin membranes are targeted and damaged by peripheral immune cells (Compston and Coles 2008, Dobson and Giovannoni 2019). MS has four key pathological features; i) inflammation, ii) demyelination and remyelination, iii) neuronal axonal loss or damage, and iv) formation of astrocytic scars (Compston and Coles 2008). During inflammatory episodes in active MS lesions, the increased reactive oxygen species (ROS) production by immune cells adds additional oxidative stress on OLs contributing to cell death (Compston and Coles 2008, Dobson and Giovannoni 2019). Traditionally, MS is described as a two-stage disease with early inflammatory episodes resulting in the relapse-remitting disease and delayed neurodegeneration causing the non-relapsing progression (Compston and Coles 2008, Dobson and Giovannoni 2019). The transition from relapse-remitting to non-relapsing progression typically occurs 10-15 years after an individual presents with clinical symptoms (Compston and Coles 2008, Dobson and Giovannoni 2019). The brain maintains a pool of oligodendrocyte progenitor cells (OPCs) that can migrate to MS lesions and remyelinate demyelinated axons (Compston and Coles 2008, Dobson and Giovannoni 2019). Age, genetic factors, and repeated demyelination effects how efficiently the remyelination process is with a decreasing trend in efficiency over time (Compston and Coles 2008, Dobson and Giovannoni 2019). 1.2 Molecular mechanisms of oligodendrocyte differentiation It has been demonstrated experimentally that OLs have three distinct differentiation stages; OPCs, pre-myelinating OLs, and mature OLs, where each differentiation state transition 6 is mediated by significant changes in gene expression (Emery and Lu 2015). OPCs are highly proliferative, have a bipolar morphology, and are recognizable by the presence of the ganglioside A2B5 and platelet growth factor receptor alpha (PDGFRA) cell surface markers (Fok‐Seang and Miller 1994). Postnatally, OPCs migrate throughout the cortex eventually interacting with target axons resulting in their transition to pre-myelinating OLs (Kuhn et al. 2019). Pre-myelinating OLs lose their bipolar morphology, start to develop filamentous myelin outgrowths, and express the following membrane antigens O4+, O1+, 2’,3’-Cyclic-nucleotide 3’-phosphodiesterase+ (CNP), and galactosylcerimide+ (GalC) (Sommer and Schachner 1981, Braun et al. 1988). The transition to mature OLs is characterized by the production of myelin and myelin-associated proteins such as myelin basic protein (MBP), myelin proteolipid protein (PLP), and myelinoligodendrocyte glycoprotein (MOG) (Braun et al. 1988, Trapp 1990). The differentiation of OPCs into myelinating OLs is regulated by various molecular cues such as; i) extrinsic factors, ii) intracellular signaling pathways iii) transcriptional factors, iv) epigenetics, and v) miRNAs. 1.2.1 Extrinsic factors and intracellular signaling pathways Current available data shows that during the OPC stage extrinsic factors mainly act on pathways that prevent differentiation and promote cell proliferation (Elbaz and Popko 2019). Endothelin-1 (ET-1) is one extrinsic factor involved in OPC proliferation by activating mitogen activated protein kinase (MAPK) pathways and preventing differentiation by decreasing the expression of S100b (Deloulme et al. 2004, Adams et al. 2021). Other extrinsic factors such as basic fibroblast growth factor (bFGF) and platelet-derived growth factor alpha (PDGFA) also promote OPC proliferation through activation of the MAPK/extracellular signal regulated kinase (ERK) cell signaling pathways (Frost et al. 2009, Adams et al. 2021). Triiodothyronine (T3) is a common extrinsic factor used to promote OL differentiation that results in accelerating myelin 7 gene expression and myelination in vivo (Dugas et al. 2012). Many of the extracellular signals that promote OL differentiation act on the protein kinase B (PKB)/ mammalian target of rapamycin (mTOR) cell signaling pathways (Adams et al. 2021). Only one study has reported autocrine inhibition of OPC proliferation in vitro in which adding back OL-conditioned media to culture resulted in decreased DNA synthesis based on bromodeoxyuridine (BrdU) assay (Louis et al. 1992b). This has not been further investigated and represents a gap in the literature. 1.2.2 Epigenetics and transcription factors Epigenetic regulation is also an important mechanism that controls OL development through transcriptional repression and activation. Epigenetic regulation refers to the accessibility of chromatin, which is mediated primarily through histone post translation modifications. Lysine deacetylases (KDACs), previously referred to as histone deacetylases (HDACs), have been shown to be key players for OL differentiation (Ye et al. 2009, Liu et al. 2016a). KDAC1 and KDAC2 have been shown to be crucial for OL specification and differentiation through inhibiting the complex formation between the transcription factors β-catenin and transcription factor 7-like 2 (TCF7L2) (Ye et al. 2009). It has been proposed that KDAC1/2 compete with βcatenin for TCF7L2 thereby converting it from a transcriptional repressor to an activator of OL differentiation genes (Ye et al. 2009). KDAC3 has been demonstrated to target and activate Olig2 expression while also inhibiting astrocyte specification through antagonizing the transcription factor signal transducer and activator of transcription 3 (STAT3) (Zhang et al. 2016). Previous work has identified several transcription factors that dictate whether OLs are maintained in a proliferative state or are committed to differentiate. The transcription factors oligodendrocyte transcription factor 2 (Olig2), NK2 homeobox 2.2 (NKX2.2), zinc finger protein 8 24 (ZFP24), and myelin regulatory factor (MYRF) have been shown to be crucial for OL differentiation to occur (Zhao et al. 2016, Elbaz and Popko 2019). Olig2 increases the expression of Sry-box transcription factor 10 (Sox10), which interacts with several other proteins to permit full OL differentiation (Liu et al. 2007, Hornig et al. 2013). By binding to the enhancer region, Sox10 induces an upregulation in ZFP24 expression (Zhao et al. 2016, Elbaz and Popko 2019). ZFP24 increases the expression of Sox10 and MYRF by binding to their respective enhancer regions (Zhao et al. 2016, Elbaz and Popko 2019). MYRF then facilitates the transition of premyelinating OLs towards the terminal mature myelinating phenotype (Emery et al. 2009). 1.2.3 miRNAs miRNAs are short non-coding RNAs that regulate gene expression post-transcriptionally and have been shown to be involved in OL differentiation (Zhao et al. 2010, Elbaz and Popko 2019). miR-219 is one miRNA that has been described to promote OPC differentiation by targeting known differentiation inhibitors such as Leucine-rich repeat neuronal protein 1 (LINGO-1), Sry-box transcription factor 6 (Sox6), Hes Family basic helix–loop–helix Transcription Factor 5 (HES5), and ETS variant transcription factor 5 (ETV5) (Zhao et al. 2010, Wang et al. 2017). Treatment with miR-219 antisense RNA resulted in a significant decrease in OL maturation using a mouse in vitro model (Zhao et al. 2010). These experiments were recapitulated using a zebrafish in vivo model and deficits in dorsal spinal cord OPCs migration in addition to decreased OL differentiation were observed (Zhao et al. 2010). Recently, miR-146a has been described to promote OPC differentiation and remyelination in the neurotoxic demyelinating cuprizone model (Zhang et al. 2017). The proposed mechanism of miR-146a ability to promote remyelination is by targeting interleukin-1 receptor associated kinase-1 9 (IRAK1) which has been described to inhibit OPC differentiation (Liu et al. 2017, Zhang et al. 2017). 1.3 Role of Extracellular Vesicles in Intercellular Communication in the CNS Extracellular vesicles (EVs) are a heterogeneous population of secreted membrane vesicles that differ in biogenesis, cargo, and the biological functions they exhibit on recipient cells (Willms et al. 2016). Three main types of EVs (figure 1) have been described in the literature which include apoptotic bodies, microvesicles, and exosomes (Tan et al. 2020). Apoptotic bodies are the largest type of EV, are generated by dying cells, and contain cellular fractions as their cargo. Microvesicles are the next largest type of EV and are produced by pinching of the plasma membrane. Lastly, exosomes are the smallest type of EVs and are produced through the endosomal pathway. Classically, the term exosome has been used to describe EVs pelleted by high-speed ultracentrifugation that are below 200nm in size (Théry et al. 2018). However, due to the size overlap between various types of EVs and the lack of exosome-specific markers additional experiments, such as live imaging techniques, are required to demonstrate that the population of interest are exosomes. As such, the International Society of Extracellular Vesicles (ISEV) has recommended researchers to use biophysical properties to 10 describe isolated vesicles, as it more accurately describes different populations of EVs (Théry et al. 2018). Figure 1. Classification of extracellular vesicle types, their site of biogenesis, and their contents. There are three main classes of EVs that are described in the literature: 1) ectosomes/microvesicles, 2) exosomes, and 3) apoptotic bodies. Apoptotic bodies are the largest EV and are generated by dying cells. Ectosomes are the next largest EV type and are produced by pinching of the plasma membrane. Exosomes are the smallest EV type and are generated from the endosomal system. Figure reproduced with permission from Krämer-Albers EM. 2023 Nat Rev Neurosci. 2023 May 31. 11 EVs have surged in research interest due to their growing list of described roles in physiology and pathology (Kowal et al. 2016, Kugeratski et al. 2021). Based on the current literature it has been established that EVs are involved in intercellular communication, acting both locally and systemically (Kowal et al. 2016, Kugeratski et al. 2021). Two main mechanisms in which EVs elicit changes in recipient cells have been demonstrated: i) transfer of functional cargo such as protein, lipid, and nucleic acids or ii) engaging in membrane receptor-mediated signaling (Muller et al. 2017, Kugeratski et al. 2021). There has also been an increasing interest in the use of EVs for clinical applications due to their ability to cross the blood-brain barrier, low immunoreactivity, and can elicit similar or stronger therapeutic effects compared to cell-based therapies (Tan et al. 2020). Exosomes were originally thought to be a homogeneous population of small EVs (sEVs); however, there has been increasing evidence of subpopulations exhibiting distinct physiologic effects on recipient cells (Willms et al. 2016, Tkach et al. 2017). Willms et al. 2016 identified a high- and low-density subpopulation of small EVs using a sucrose gradient that differed in protein and RNA composition. Treatment with each subpopulation resulted in different alterations in gene expression in recipient cells. A recent study of immuno-isolated sEV subpopulations from serum using a mouse sepsis model demonstrated they carry different miRNA cargo by next-generation sequencing (NGS) and quantitative PCR analysis (Wu et al. 2021). Although this study did not assess the functional effects of the isolated sEV subpopulations, due to their different miRNA cargo it is quite possible they exhibit distinct physiological effects (Wu et al. 2021). Kowal et al. 2016 demonstrated differences in protein content between immuno-isolated tetraspanins CD9, CD63, and CD81 small EV subpopulations 12 by a label-free quantitative proteomic analysis. With observed differences in nucleic acid and protein content of EV subpopulations, it suggests different physiological functions. However, this still requires follow-up studies to assess subpopulation function and is a gap in the current literature. 1.3.1 Oligodendrocyte – Neuronal interactions Due to the reduced axonal access to extracellular metabolites caused by myelin wrapping, it has been speculated that OLs provide trophic support to the axons they ensheathe (Figure 2, Nave 2010). This support involves the transfer small molecule carbon metabolites lactate and pyruvate through monocarboxylate transporters (MCT1 and MCT2) in addition to the transfer of extracellular vesicle cargo (Fünfschilling et al. 2012, Frühbeis et al. 2013, Krämer-Albers and Werner 2023). From electron microscopy imaging, multivesicular endosomes are observed in the adaxonal myelin layer which are then able to fuse with the plasma membrane releasing EVs into the periaxonal space (Frühbeis et al. 2013). OL-derived EVs are transferred to neurons and their secretion is mediated through the activity-dependent release of glutamate. Glutamate binding to N-methyl-D-aspartate (NMDA) receptors located in the myelin membrane results in increased intracellular Ca2+ levels which is responsible for the observed increase in exosome release (Frühbeis et al. 2013). This allows for physiologically relevant molecules to be transferred as exosomal cargo to neuronal axons in an ‘on-demand’ fashion given the activity of the axon they ensheathe (Frühbeis et al. 2013, Krämer-Albers and Werner 2023). Recent studies have established that OL-derived EVs are able to confer neuroprotective effects under oxidative stress and nutrient deprivation conditions (Frühbeis et al. 2013, 2019, Fröhlich et al. 2014). This is supported by observed increased neuronal metabolic activity compared to the controls under stress conditions after EV treatement (Frühbeis et al. 2013). It is 13 well recognized that EVs carry functional cargo such as ribonucleic acids, proteins, and lipids to recipient cells resulting in distinct cellular changes (Willms et al. 2016). In a follow-up study, Fröhlich et al. 2014 were able to show that OL-derived EVs transferred antioxidant enzymes superoxide dismutase-1 (SOD1) and catalase to neurons, which explains the previously observed neuroprotective effects under oxidative stress conditions. Additionally, it was determined that OL-derived EVs alter gene expression, signal transduction pathways, and enhance spontaneous neuronal activity in target neurons (Fröhlich et al. 2014). An interesting finding from this study was that neuronal treatment of EVs derived from the precursor OL cell line Oli-neu resulted in decreased the neuronal migration protein doublecortin (DCX) expression whereas mature OLderived EV treatment resulted in increased DCX expression (Fröhlich et al. 2014). The decrease in DCX expression has been demonstrated to be involved in neuronal maturation and differentiation (Brown et al. 2003, Fröhlich et al. 2014). This finding highlights the differences in EV cargo at distinct OL differentiation states and most likely has important consequences during neurogenesis. In contrast, OPCs have been shown to deliver retinoic acid as EV cargo to neurons after neuronal injury providing a signal for growth and regeneration (Goncalves et al. 2018). Recently, Chamberlain et al. 2021 demonstrated that mature OLs increase ATP production in myelinated axons through the transfer of the NAD dependent deacetylase sirtuin 2 (SIRT2) as EV cargo. The authors delineated that the increased ATP production was a result of the deacetylation of mitochondrial adenine nucleotide translocase 1 and 2 (ANT1/2). Overall, the molecular characterization of EVs produced by OPCs and OLs is still in its infancy and requires further investigation. 14 Figure 2. Summary of known mechanisms of oligodendrocyte mediated neuronal support. Extracellular vesicle (EV) mediated and metabolic support of axons by oligodendrocytes maintains axonal energy production and homeostasis. (1) The release of EVs into the periaxonal space and the expression of GLUT1 is mediated by axonal glutamatergic signalling through NMDA receptors. The increased expression of GLUT1 increases glycolytic activity, which in turn increases the production of lactate and pyruvate. These small carbon metabolites can then be shuttled to axons through monocarboxylate transporters (MCT1 and MCT2). In doing so this decreases the energy requirements of neurons by providing metabolic support. (3) SIRT2 has been identified as EV cargo, which is able to deacetylate adenosine nucleotide translocases (ANT1 and ANT2) situated in the inner mitochondrial membrane. This is thought to increase the rate of ATP-ADP exchange thereby increasing axonal ATP levels. (4) Increased mitochondrial activity in axons is required for axonal transport which in turn results in increased production of reactive oxygen species (ROS). The heavy subunit of ferritin (FTH1) is transferred to axons through oligodendrocyte EVs which provides protection from oxidative damage. (5) Transfer of oligodendrocyte EVs has been shown to support axonal transport under stress and unstressed conditions. However, the underlying molecular mechanism that oligodendrocyte EVs stimulate axonal transport remains to be elucidated. Figure reproduced with permission from Krämer-Albers EM. 2023 Nat Rev Neurosci. 2023 May 31. 15 1.3.2 Oligodendrocyte – Astrocyte interactions Within the CNS, reciprocal communication between OLs, neurons, and other glial cells is achieved through the release of soluble factors and EVs (Basso and Bonetto 2016). Astrocytes are a type of glial cell found within the CNS and have been demonstrated to be involved in a variety of cellular processes such as neuronal metabolic support and myelin regulation (Rothstein et al. 1996, Upadhya et al. 2020, Willis et al. 2020). The influence of astrocytes to promote myelination appears to be dependent on reactivity as activated astrocytes promote in vitro myelination whereas myelination was significantly reduced when cultured with quiescent astrocytes (Nash et al. 2011). Astrocytes and OLs have been shown to be connected by gap junctions allowing the transport of metabolites (Stadelmann et al. 2019). It has been reported that the dysregulation of astrocyte-OL gap junctions results in various forms of leukodystrophies (Lundgaard et al. 2014). Astrocytes produce soluble factors such as ciliary neurotrophic factor (CNTF) and chemokine ligand 1 (CXCL1) which have been implicated in OL differentiation and myelination (Stankoff et al. 2002, Padovanu-Claudio et al. 2006). Astrocytes have also recently been demonstrated to be responsible for providing the bulk of cholesterol to OLs after the first phase of myelination as conditional knockout of sterol sensor cleavage-activating protein results in persistent hypomyelination (Camargo et al. 2017a). Potentially cholesterol is transferred from astrocytes to OLs through EVs as cholesterol is enriched in EVs compared to parental cells (Record et al. 2014). Recently, it has been described that the deletion of DICER in astrocytes prevents OL differentiation and subsequent myelination (Liu et al. 2021). Given that DICER is involved in the production of miRNAs (Vergani-Junior et al. 2021), this raises the possibility that astrocyte EVs may modulate OL differentiation through the transfer of miRNA cargo. It is likely that reciprocal communication occurs between astrocytes and OLs to control the timing of OL differentiation and myelination. 16 1.5.3 Oligodendrocyte – Microglia interactions Microglia are the resident macrophages of the CNS and are crucial for proper immune regulation within the brain and spinal cord (Peferoen et al. 2014). They are also involved in several key processes such as synaptic pruning, apoptotic cell clearance, and the production of growth factors (Wake et al. 2012). Upon activation, microglia secrete pro-inflammatory cytokines which OLs are susceptible to due to their high metabolic demands (Peferoen et al. 2014). This is experimentally supported as several inflammatory cytokines produced by microglia have been detected in demyelinating MS lesions which suggest a possible link between microglia activation state and OL damage (Etty N. 1997). Miron et al. 2013 were able to demonstrate that anti-inflammatory/immunoregulatory M2 microglia promote OL differentiation during remyelination using organotypic cerebellar slice cultures. The authors found that the response is mediated, to some extent, by the production of the transforming growth factor (TGF) β superfamily member activin-A (Miron et al. 2013). Fitzner et al. 2011 was able to demonstrate by microscopy that OL-derived EVs are almost exclusively internalized by microglia. Interestingly the EV uptake did not induce microglial activation. There is a need for further studies to determine the molecular changes that occur in OLs due to microglia-derived secretory factors and EVs. 1.5 Model systems for studying oligodendrocyte differentiation Investigating OL development is crucial for determining the mechanisms that govern myelination. Several in vitro models have been developed which are divided into either the use of i) OL cell lines, ii) primary OL cells or iii) OL-neuronal co-culture systems. Given the complexity of the CNS, in vitro models are advantageous as they provide the ability to investigate individual cell types or interactions between a limited number of cell types. These 17 model systems also allow for the manipulation of the cells and culture conditions to suit the research question. However, the disadvantage of these models is they are artificial and more simplistic compared to the interactions experienced in vivo. As such it is highly recommended that findings are corroborated with in vivo models to ensure the validity of findings. 1.5.1 Oligodendrocyte cell lines Cell lines exhibit a more homogeneous phenotype and are more cost effective to culture compared to primary cells. Louis et al. 1992 described the first OL cell line that was derived from neonatal rat forebrain, named the CG4-OL cell line, and has been used in several recent studies (Fang et al. 2013, 2019, Piao et al. 2013). CG4-OL cells display the normal developmental phenotypes of OLs based on cell morphology and immunocytochemistry (Louis et al. 1992a). However, it is important to note that the cell line has not been immortalized and as such over prolonged passaging some of the characteristics of the original progenitors may be lost. Jung et al. 1995 developed another OL cell line called Oli-neu. Unlike the CG4-OL cell line, the Oli-neu cell line is a mouse-immortalized OL cell line that was achieved using retroviral vectors expressing a t-neu tyrosine kinase. However, the Oli-neu cell line has been reported to have minimal differentiation potential in vitro, as minimal MOG expression is observed after culturing in differentiation conditions (Jung et al. 1995). More recently, Lin et al. 2006 described another mouse OPC line, called mOP cells (mouse OL progenitor), that had a greater capacity to differentiate. After 2 years of culturing in proliferation media, the cells have maintained the OL progenitor phenotype. 1.5.2 Oligodendrocyte primary cell culture For primary cell culture both rat and mouse models are used. Traditionally, rats were used to obtain primary OLs as more OPCs can be obtained per animal. However, with the 18 advances in biotechnology and the growing list of knockout/in mice models, there has been a shift to work with mouse models. The shake-off method is used for primary rat OLs and takes advantage of the difference in adherent properties of OPCs, microglia, and astrocytes (Mccarthy and De Vellis 1980, Chen et al. 2007). First a single cell suspension generate from cortical tissue is plated into a cell culture vessel as a mixed glial culture. Five to ten days after plating the mixed glial culture is comprised of a bed of astrocytes with OPCs and microglia growing on top. Isolation of OPCs from mixed glial culture is performed by two shaking steps on an orbital shaker. The shear force generated by an orbital shaker detaches the microglia from the mixed glial culture on the first shake-off, and the second shake-off detaches the OPCs from the astrocytes which are then plated. Currently, most of the literature obtains mouse primary OPCs using the neurosphere protocol described by Chen et al. 2007. Briefly, this protocol uses embryonic or postnatal mouse cortical tissue to expand out resident neural stem cells (NSCs) generating non-adherent neurospheres. The neurospheres are generated over eight days with an epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) containing media. To fate specify the NSCs to OPCs, the media is gradually replaced with a B104 CM containing media over two weeks generating “oligospheres” (Chen et al. 2007). The “oligospheres” are then dissociated and plated at the desired density. Recently, a new neurosphere based protocol to generate OPCs has been described by Dittmann et al. 2023. After the neurospheres are generated, they are dissociated and plated as an adherent monolayer. The media is changed to a chemically defined media containing platelet derived growth factor alpha (PDGFA) and OPCs are observed within two days after plating (Dittmann et al. 2023). 19 1.5.3 Oligodendrocyte-neuronal cell cultures OL-neuronal cell culture systems are used to study myelination as OL-neuron interactions can be recapitulated while providing the ability to use in vitro cell culture methods. The predominant model used to study OL-neuron interactions utilizes dorsal root ganglia (DRG) (Laursen et al. 2009). Briefly, DRG neurons from E15 rats are isolated, plated on growth factor reduced Matrigel and cultured for 21 days in vitro with nerve growth factor containing media. Contaminating cells are removed by pulsing the culture three times for 48h with 5-fluoro-2’deoxyuridine at 1, 4, and 7 days in vitro after seeding. After 21 days in vitro, the media is changed and OPCs are seeded onto the neurons. The main advantage of this co-culture system is that it allows for the evaluation of myelination as well as intercellular communication between neurons and OL. However, the main disadvantage of this system is the time required for the coculture system to be established. The DRGs must be cultured for 3 weeks before they can survive without nerve growth factor, a factor which has been shown to impede myelination, and an additional 2 weeks before significant myelination can be observed (Barateiro and Fernandes 2014). Another aspect that could be viewed as a disadvantage is that DRGs are not considered CNS neurons and some interactions may differ. 1.6 Thesis scope and significance Myelination is essential for proper neuronal function and demyelination is associated with cognitive/physical deficits depending on the CNS region affected. Understanding the mechanisms regulating myelination is crucial for the development of more effective intervention strategies for demyelinating disorders such as MS. One relatively unexplored avenue is intracellular communication mediated by EVs and given their variety in cargo, potentially they could be involved in this process. However, very little is known about the molecular cargo of 20 OL-derived EVs produced from progenitor OLs and nothing is known about how OL-EV cargo changes throughout cell differentiation. Only two studies have performed a proteomic assessment of OL-EVs derived from mature primary mouse OLs (Krämer-Albers et al. 2007b, Frühbeis et al. 2020). Louis et al. 1992b is the only report of autocrine inhibition of OLs cultured in vitro as the addition of OL-conditioned media to culture resulted in decreased DNA synthesis based on BrdU assay. Potentially this effect could be mediated by EVs, or a subpopulation of EVs, produced by OLs. Given the complexity of developmental myelination, characterizing OLEV cargo across cell differentiation could provide insights into molecules involved in autocrine regulation of OL differentiation and myelination in addition to what stage of cell differentiation OL-EVs mediate these effects. I hypothesize that EVs isolated from progenitors and differentiated OLs will contain different common EV marker expression (CD9, CD63, CD81, etc.) that change throughout cell differentiation. These differences may be indicative of unique functional EV populations. The specific objectives of my thesis are the following: · Objective I: Assess EV marker expression across different stages of OL differentiation by Western blot. · Objective II: Assess autocrine effects of OL-derived EVs on cell proliferation and differentiation gene expression by qPCR. This work will lay the foundation for future studies to identify novel mechanisms by which OL differentiation is regulated by EVs which could serve as new avenues for therapeutic intervention strategies in demyelinating disorders. 21 Chapter 2. Materials and Methods 2.1 CG4-OL Cell Culture The CG4-OL cell line is derived from neonatal rat forebrain and were maintained as previously described (Thangaraj et al. 2017, Fang et al. 2019). Briefly, CG4-OL cells were cultured on polyD-lysine coated tissue culture dishes and maintained as undifferentiated progenitors in growth media containing DMEM/F12 or DMEM high glucose, 50 μg/mL of apotransferrin, 5 μg/mL of insulin (bovine), 9.8 ng/mL of biotin, 50 ng/mL of selenium, 1% (v/v) antibiotic antimycotic solution (AB/AM), 1mM Sodium pyruvate, 2.5mM L-glutamine, 15mM HEPES buffer, and 30% (v/v) B104 conditioned medium. Differentiation of CG4-OL cells was achieved by growth in differentiation media containing 2% fetal bovine serum instead of B104 conditioned media. B104 conditioned media was generated by seeding B104 neuroblastoma cells in growth media containing DMEM/F12/10% FBS. Once the cells reached ~70% confluency they were rinsed with PBS, and defined media was be added which contains DMEM/F12, 1% modified N2 supplement (5mg/mL holo-transferrin), and 1% AB/AM. After 3-4 days the conditioned medium was be removed, phenylmethylsulfonyl fluoride (PMSF) was added to a concentration of 5 µg/mL and was centrifuged at 2,000 x g for 10 min at 4℃ to remove cellular debris. The supernatant was then sterile filtered, aliquoted, and stored at -20℃ until future use. 2.2 Extracellular Vesicle Isolation EV depleted B104 conditioned media was obtained by ultracentrifugation of B104 conditioned at 100,000 x g for 18 hours in a SW 28 rotor in a Beckman XL-40 ultracentrifuge; the supernatant was collected and stored at -20℃. EV depleted fetal bovine serum was obtained by ultracentrifugation at 100,000 x g for 18 hours in a SW 41Ti rotor in a Beckman XL-40 22 ultracentrifuge; the supernatant was collected and stored at -20℃. EVs were isolated from CG4OL conditioned media by differential ultracentrifugation and a schematic of the experimental design is represented in Figure 3. CG4-OL cells were seeded at 500,000 cells/T75 flask and were left overnight to recover in growth media. The following morning the media was removed and replaced with 12mL fresh EV depleted growth media per T75 flask. The undifferentiated CG4OL cells were left to grow for two days at which point the progenitor CG4-OL conditioned media was collected (D0) and used to isolated EVs. Each flask was replaced with fresh 12mL EV depleted differentiation media per T75 flask and cultured for two days to generate conditioned media. Conditioned media was collected and processed again on D2, D4, and D6 (Figure 3) To isolate EVs, conditioned media was centrifuged at 300 x g for 10 minutes at 4℃ to pellet detached cells and cellular debris. The supernatant was transferred to new tubes and centrifuged at 10,000 x g for 40 minutes at 4℃ in a SW 41Ti rotor (10K pellet) to pellet apoptotic bodies and larger microvesicles. The supernatant was transferred to new tubes and centrifuged at 100,000 x g for 180 or 120 minutes at 4℃ in a SW 41Ti rotor (100K pellet). A second isolation was performed the same day; the resultant 100K pellets were pooled, washed with PBS, and recentrifuged at 100,000 x g for 1 hour at 4℃ using a SW 60Ti rotor (Figure 3). The supernatant was discarded and 100K pellet was resuspended with 50uL of 0.2µM filtered PBS or sodium dodecyl sulfate (SDS) sample buffer containing 25 mM trisaminomethane (Tris) pH 8.8, 5% (w/v) SDS, and 150mM NaCl. The resuspended pellets were stored at -80℃. 2.3 Nanoparticle Tracking Analysis Particle and size distribution of EVs isolated from CG4-OL conditioned media was determined by nanoparticle tracking analysis (NTA) using a NanoSight LM10 system (Malvern Technologies, Malvern, UK) configured with a 488 nm laser and a high sensitivity scientific 23 a) Day -3 Progenitor Early Differentiation Mid Differentiation Late Differentiation 0 2 4 6 -2 Differentiation Media Growth Media OPCs Progenitors plated plated b) Change to Change to collection collection media media Collect CM Collect CM for D0 for D0 isolation isolation Collect CM for D2 isolation Collect CM for D4 isolation Conditioned Media Conditioned Media 10 min at 300g 10 min at 300g Supernatant Supernatant 40 min at 10 000g* 40 min at 10 000g* 10K Pellet Supernatant 10K Pellet Supernatant Pool 2 or 3 h at 100 000g* 2 or 3 h at 100 000g* 100K Pellet Discard Supernatant Collect CM for D6 isolation 100K Pellet Discard Supernatant Pool 1 h at 100 000g** 100K Pellet Discard Supernatant Figure 3. Schematic of extracellular vesicle isolation methods. (a) Timeline of CG4-OL cell conditioned media collections for extracellular vesicle isolations. (b) Schematic of differential ultracentrifugation protocol for isolating extracellular vesicles from CG4-OL cell conditioned media. *samples processed using SW41 Ti rotor, ** samples processed using SW60 Ti rotor. 24 CMOS camera. Samples were diluted in 0.2 µm filtered PBS within the concentration recommended by the manufacturer. Samples were analyzed under constant flow conditions (flow rate = 70) at room temperature (23.6℃) and 3 x 60s videos were captured with a camera level of 13. Data was analyzed using NTA 3.2.16 software with a detection threshold of 5. 2.4 Western Blotting CG4-OL cells were lysed with SDS sample buffer, the genomic DNA was sheared with 5 passes of a 22-gauge needle, then centrifuged at 16,000 x g for 10 minutes. The insoluble pellet was discarded, and the solubilized protein supernatant was collected and stored at -80℃. The protein concentration of the cell lysates and the EV pellets was determined using a colloidal Coomassie brilliant blue stain dot blot as previously described (Noaman and Coorssen 2018). Equivalent micrograms of protein were loaded on 12% TGX Stain-Free™ gels (BioRad) and separation of samples was achieved at 100V using a running buffer containing 24.7mM Tris, 191.9 mM glycine, and 0.1% (w/v) SDS. Separation was performed under reducing and nonreducing conditions depending on the antigen being assessed (Table 1). After separation, gels were activated and imaged using the stain-free application on the ChemiDoc Imaging System (BioRad). The Western blot was achieved using a modified Towbin (Tobwin et al. 1989) wet tank transfer using low fluorescent polyvinylidene fluoride (PVDF) membrane at 30 mA overnight (16 hr) at 4℃. The transfer buffer contained 24.7 mM Tris, 191.8 mM glycine, 20% (v/v) methanol, and 0.025% (w/v) SDS. After transfer, the blots were imaged for total protein using the stain-free application on the ChemiDoc Imaging System (BioRad). The blots were subsequently blocked for one hour in 5% (w/v) skim milk in trisaminomethane buffered saline and 0.1% Tween20 (TBST) and were transferred into 1% (w/v) skim milk-TBST containing primary antibody (table 1) overnight at 4℃ on a rocker. The following day the blots were 25 washed with TBST and the appropriate secondary (table 1) was added in 1% (w/v) skim milkTBST for one hour. After secondary antibody incubation the blots were washed with TBST and visualized using the ChemiDoc MP Imaging System (BioRad). For sample sets that were spread across two blots, SDS-PAGE and transfer were run in the same apparatus at the same time. To ensure equal loading of each sample, total protein of each lane was visualized using stain-free signal and total protein was determined from Rf0 to Rf1 using rolling disk background subtraction (disk size 100mm) in the ImageLab software (BioRad). The total protein fluorescent intensity for each sample was then divided by the geomean of the sample set to obtain a normalization factor. The integrated band volumes for markers of interest were obtained using rolling disk background subtraction (disk size 1mm) in the ImageLab software (BioRad). Integrated band volumes were then normalized based on total protein (described above). Integrated band volumes for markers of interest are expressed relative to the normalized integrated band volume from D0. All data are presented as mean ± standard error of the mean (SEM). To determine if the mean relative normalized band volumes of EV collection groups differ from D0, one-way ANOVA was used with Bonferroni’s post-test (SigmaPlot 14.5). Differences between groups were considered significant if P < 0.05. 26 Table 1. Antibody list and conditions used for antigen visualization. ANTIGEN CAT # & DISTRIBUTOR Ab22595 (AbCam) SPECIES & CLONALITY Rabbit pAb 1:10,000 REDUCING/NONREDUCING Reducing SECONDARY USED 1:2500 goat antiRabbit Starbright blue 700 (BioRad) CD9 SA35-08 (Novus) Rabbit mAb 1:5000 Non-reducing 1:2500 goat antiRabbit Starbright blue 700 (BioRad) CD44 Ab189524 (AbCam) Rabbit mAb 1:1000 Non-reducing 1:2500 goat antiRabbit Starbright blue 700 (BioRad) CD63 MCA4754 (Bio-Rad) Mouse mAb 1:1000 Non-reducing 1:2500 goat antiMouse Starbright blue 520 (BioRad) CD81 MCA1846 (Bio-Rad) Hamster mAb 1:1000 Non-reducing 1:2500 Goat F(ab')2 antiHamster IgG:Dylight®800 (BioRad) TSG101 Ab125011 (AbCam) Rabbit mAb 1:1000 Non-reducing 1:2500 goat antiRabbit Starbright blue 700 (BioRad) LAMIN A + LAMIN C Ab133256 (AbCam) Rabbit mAb 1:10,000 Reducing 1:5000 goat antiRabbit HRP (BioRad) 14-3-3 Ab14112 (AbCam) Rabbit mAb 1:2000 Reducing 1:2500 goat antiRabbit Starbright blue 700 (BioRad) CALNEXIN DILUTION 27 2.5 Primer Design and Validation Primer pairs for four myelin-related genes (Pdgfra, Cnp, Plp, and Mog) and one proliferation marker (Ki67) were designed and optimized following the Minimal Information for Quantitative Real-Time PCR Experiments (MIQE) guidelines (Bustin et al. 2009). Accession numbers of genes of interest were obtained from the NCBI Nucleotide database and primer sets were designed using the NCBI Primer-Blast software. The following technical parameters were used: (i) PCR product size between 70 to 350 base pairs (bp); (ii) optimal primer melting temperature (Tm) of 62.0°C, ranging between 60.0-64.0°C; (iii) at least one primer must span exon-exon junction. Primer pairs were analyzed with several other in silico tools to ensure minimal primer secondary structure formation (OligoAnalyzer 3.1, IDT; https://eu.idtdna.com/calc/analyzer) and potential amplicon secondary structures (UNAFold; http://www.unafold.org/mfold/applications/dna-folding-form.php). After in silico screening, three sets of primer pairs for each gene of interest were selected for validation as previously described (Smith 2022). Briefly, primer specificity was assessed by amplicon PCR followed by visualization with gel electrophoresis to ensure single product formation at the correct molecular weight. Primer sets that produced a single product at the correct molecular weight were then further assessed for their viability to be used for qPCR experiments as previously described (Smith 2022). Primer pairs that passed validation had efficiencies within a range of 90% to 110% and a standard curve correlation coefficient (r2) ≥ 0.98. The standard curve comprised points up to five orders of magnitude using a 1:5 dilution. Designed primers that were viable for qPCR experiments are summarized in table 2. Primers pairs with acceptable efficiencies were then assessed for temperature dependence via a thermal gradient. 28 Table 2. qPCR primer validation summary Gene Primers Ki67 Fwd: CATCAAACGGAGCGGCGATG R2 71 Efficiency (%) 101.2 128 94.3 0.990 155 92.1 0.995 166 94 0.994 255 94.4 0.988 Length (bp) 0.996 Rev: TACTCCTTCCAAACAGGCAGG PDGFRA Fwd: CTCACTTTTTCCTCCGGGCT Rev: ATGAGGCTCGGCCCTGT Fwd: GCCCAACAGGATGTGGTGAG CNP Rev: GAGGATGAGGGCTTGTCCAG Fwd: ACCACCTGCCAGTCTATTGC PLP Rev: AGGTCATTTGGAACTCGGCT Fwd: TATCGGCGAGGGAAAGGTTG MOG Rev: TCTGCACGGAGTTTTCCTCTC 2.6 RNA Isolation and Reverse Transcription CG4-OL cell media was removed, and the cells were washed with PBS to reduce contaminating proteins. Total RNA was then isolated using the Aurum™ Total RNA mini kit (#7326820, Bio-Rad, Hercules, WA, USA) following the manufacturers protocols. RNA was eluted in nuclease-free water, RNA quantity and purity were measured with an ND-1000 Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Samples were stored at -80˚C until further processing. Reverse transcription (RT) cDNA was obtained with the iScript™ gDNA Clear cDNA Synthesis Kit (#1725035, Bio-Rad) according to the manufacturer’s protocol. In each 20uL reaction 1ug of RNA template was used including the no reverse transcriptase (NRT) control. The cDNA was then diluted to a final concentration of 10ng/uL. 29 2.7 PCR and Gel Electrophoresis Oligonucleotide primers were purchased from Integrated DNA Technologies (IDT; IA, USA). PCR reactions were performed in a total reaction volume of 25μL using GoTaq® G2 Master Mix (#M7823, Promega, Madison, WI, USA), 250nM of forward and reverse primers, and 20ng cDNA template. The thermocycler conditions used were the following: 3 minutes of polymerase activation at 95°C, followed by 37 cycles of (i) denaturation at 95°C for 30 seconds, (ii) annealing at 58°C for 30 seconds and (iii) extension at 72°C for 30 seconds, and then final extension step for 5 minutes at 72°C. PCR products were separated on a 3% agarose gel and visualized with GelRed nucleic acid stain (#SCT123 Millipore Sigma, Darmstadt, Germany) using Bio-Rad ChemiDoc MP imaging system (Bio-Rad Laboratories, Hercules, CA, USA). Assessment of CG4-OL differentiation was done using the following genes and primer pairs: Pdgfra (Fwd: CTCACTTTTTCCTCCGGGCT, Rev: ATGAGGCTCGGCCCTGT), Cnp (Fwd: GCCCAACAGGATGTGGTGAG, Rev: GAGGATGAGGGCTTGTCCAG), Plp (Fwd: ACCACCTGCCAGTCTATTGC, Rev: AGGTCATTTGGAACTCGGCT), and Mog (Fwd: TATCGGCGAGGGAAAGGTTG, Rev: TCTGCACGGAGTTTTCCTCTC). 2.8 qPCR qPCR reactions were performed in a total volume of 15uL per well, consisting of SSO Advanced™ universal SYBR® Green Supermix (#1725271; Bio-Rad), 250nM of each genespecific forward and reverse primer pairs and 20ng cDNA template. Reactions were performed on a Bio-Rad CFX384 real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA) with the following parameters: initial denaturation and enzyme activation at 95°C for 90 seconds, followed by 40 cycles of (i) denaturation at 95°C for 15 seconds and (ii) combined annealing/extension step at 58℃ for 30 seconds. A melt curve from 65.0°C to 95.0°C in 0.5°C 30 increments for 5 seconds was performed at the end of every plate run to confirm single product formation. All reactions were run in triplicate. The following genes were analyzed by qPCR assays: ki67 (Fwd: CATCAAACGGAGCGGCGATG, Rev: TACTCCTTCCAAACAGGCAGG), Pdgfra (Fwd: CTCACTTTTTCCTCCGGGCT, Rev: ATGAGGCTCGGCCCTGT), Plp (Fwd: ACCACCTGCCAGTCTATTGC, Rev: AGGTCATTTGGAACTCGGCT), Mbp (Fwd: GGGTGTACGAGGTGTCAA, Rev: AACTACCCACTACGGCTCCC), and Mog (Fwd: TATCGGCGAGGGAAAGGTTG, Rev: TCTGCACGGAGTTTTCCTCTC). Data were analyzed using CFX Maestro software using automated baseline and threshold settings. The relative quantity of each gene is represented relative to untreated control and was normalized to three reference genes – Hprt1 (Fwd: CTCATGGACTGATTATGGACAGGAC, Rev: GCAGGTCAGCAAAGAACTTATAGCC), Sdha (Fwd: CACCGTGAAAGGCTCTGACT, Rev: CATACCGCAGAGATCGTCCA), and Tbp (Fwd: TGGGATTGTACCACAGCTCCA, Rev: CTCATGATGACTGCAGCAAACC). Relative normalized expression values >0 indicate an increase in gene expression relative to untreated control, and conversely values <0 indicate a decrease in gene expression relative to untreated control. All primer efficiencies are withing the acceptable range outlined in the MIQE guidelines, as such efficiencies were set at 100% for relative normalized expression (RNE) calculations. All data presented as mean ± standard error of the mean (SEM). To determine if the RNE values differed from the untreated control, oneway ANOVA was used compared to control with Bonferroni’s post-test (SigmaPlot 14.5). Differences between groups were considered significant if P < 0.05. 31 2.9 MTT assay Methyl tetrazole thiol (MTT, Cat# M5655, Sigma) cell viability assays were performed in 96 well plates using the BioTek Synergy Neo2 Hybrid Multimode Reader. Briefly, 100 µL of 1mg/mL MTT solution was added to each well and was incubated for three hours at 37℃/5% CO2. After three hours had passed, the media was carefully removed from each well, 100 µL of dimethyl sulfoxide (DMSO) was added to each well and was incubated for five minutes at 37℃ in a cell culture incubator. The 96 well plate was then loaded into the Synergy Neo2, shaken for 2 minutes before scanning at 570nm. Samples were run in triplicate and are expressed as the mean relative to the control ± standard deviation. One-way ANOVA was used compared to control with Bonferroni’s post-test (SigmaPlot 14.5). Differences between groups were considered significant if P < 0.05. 2.10 EV treatments EVs were isolated from CG4-OL cell culture conditioned media at D0, D2, D4, and D6 collected from six T75 flasks as described in section 2.2 with one exception; after the final 1h 100,000 x g spin the resultant pellet was resuspended in PBS, brought up to a volume of 450uL, and 50uL aliquots were made and stored at -80℃ until use. After the first 120min 100,000 x g spin, 200uL aliquots were made from the supernatant and stored at -80℃ until use. CG4-OL cells were seeded at 10,000 cells/cm2 as OL progenitors in 6-well tissue culture plates and were left to recover for two days. On day two, the growth media was removed and replaced with differentiation media containing one aliquot of either EVs or 100K supernatant. Half volume media changes were performed daily containing one aliquot of either EVs or 100K supernatant for four days. On day four in differentiation media, the media was removed, the cells were rinsed 32 with PBS, total RNA was isolated and cDNA conversion was performed as described in section 2.5. 33 Chapter 3. Results 3.1 Determination of protein recovery from EV fraction to visualize markers by Western blot EVs (n=5, pooled from 12 T75 flasks) were isolated from CG4-OL cell conditioned media at D0, D2, D4, and D6 were assessed for total protein. Total protein recovered was consistently lowest at D0 and increased up to D4 before decreasing at D6 (figure 4a). EVs isolated from D4 were used to perform protein titers to determine the optimal amount of protein to load for visualization EV markers (CD9, CD44, CD63 CD81, and TSG101) by Western blot as this sample consistently had the most material. All EV markers could be visualized across the entire protein titer range (20ug-5ug). To conserve on material to allow assessment of multiple targets per replicate, 5ug was loaded to visualize EV markers (figure 4b). Samples were also evaluated by nanoparticle tracking analysis to ensure isolated EVs are within the expected size range (figure 5). All isolation time points are within the size range (40-200 nm) reported in the literature (Théry et al. 2018). 34 a) 250 150 100 D0 b) 20 135 ± 30 71 ± 14 0 41 ± 11 50 CL 15 10 D2 5 94 ± 18 Total protein (ug) 200 D4 20 D6 EVs 15 10 5 ug 75 CD44 50 TSG101 20 CD81 75 50 CD63 37 20 CD9 Figure 4. Protein recovered in EV fraction and protein load required to visualize EV markers by Western blot. (a) Bar graph of total protein recovered by differential ultracentrifugation in 100K pellet corresponding to EV enriched fraction across oligodendrocyte cell differentiation (n=5). (b) Western blot protein titer for extracellular vesicle markers CD44, TSG101, CD81, CD63, and CD9 from D4 cell lysate (CL) and EV fraction (EVs). 35 a) b) Mean: 160.2 nm Mode: 116.5 nm StDev: 59.6 nm 6.95x1010±1.015x109 particles c) d) Mean: 157.5 nm Mode: 120.0 nm StDev: 50.8 nm 7.95x1010±1.70x108 particles Mean: 172.7 nm Mode: 133.3 nm StDev: 59.8 nm 6.4x1010±1.07x109 particles Mean: 158.7 nm Mode: 132.3 nm StDev: 53.3 nm 5.3x1010±1.55x109 particles Figure 5. Nanoparticle tracking analysis data of isolated EVs across OL differentiation. Data from (a) D0, (b) D2, (c) D4, and (d) D6 isolated EV samples from CG4-OLs. Mean and mode size is within expected range reported in the literature. 3.2 EV maker assessment across OL cell differentiation To confirm that the CG4-OL cell cultures were differentiating, PCR and agarose gel electrophoresis was performed on four differentiation genes – Pdgfra, Cnp, Plp, and Mog. As expected Plp and Mog expression increases over the six days in differentiation media compared to D0 (figure 6a). EVs were isolated from CG4-OL cell conditioned media (n=2) at D0, D2, D4, and D6 were assessed for five EV markers of interest (CD9, CD44, CD63, CD81, and TSG101), one cargo marker (14-3-3), and two exclusion markers (calnexin and lamin A + C). Both exclusionary markers, the ER marker calnexin and the nuclear envelop marker lamin A + C, that 36 are used to assess EV purity were found to increase in the 100K pellet over the collection period indicating increased cell death (figure 6b-c). TSG101 has previously been described as a marker specific to tetraspanin-enriched EVs and thus could potentially used to assess purity (Kowal et al. 2016). As such a linear regression analysis was performed between TSG101 expression and percent calnexin exclusion which indicates a correlation (figure 6c, r2=0.97). The percent calnexin exclusion was calculated in the following way: ቀ1 − ଵ଴଴୏ ୤୰ୟୡ୲୧୭୬ ୠୟ୬ୢ ୢୣ୬ୱ୧୲୭୫ୣ୲୰୷ େୣ୪୪ ୪୷ୱୟ୲ୣ ୠୟ୬ୢ ୢୣ୬ୱ୧୲୭୫ୣ୲୰୷ ቁ ∗ 100 = percent calnexin exclusion. The band densitometry was then reprocessed, where after total protein normalization each band was normalized to the integrated band volume of TSG101 and data are expressed relative to D0. The EV markers CD44, CD63, and CD81 relative expression remain consistent across cell differentiation (figure 6d). On the other hand, the relative expression of CD9 and 14-3-3 increased (figure 6d). 37 Pdgfra Cnp Plp Mog 10K 100K CL 10K 100K CL 75 50 75 30 75 Lamin A + C 37 CD63 20 75 20 CD9 CD44 CD81 TSG101 Calnexin 14-3-3 100 1.5 80 1.0 0.5 Calnexin Lamin A+C 60 40 20 0.0 90 % Calnexin Exclusion 2.0 % Exclusion Relative to D0 D6 50 TSG101 c) D4 10K D6 CL D4 D2 10K D2 CL D0 D0 100K b) 100K a) 2 80 r =0.97 70 60 50 40 0 D0 D0 D2 D4 D6 D2 D4 D6 0.4 0.6 0.8 1.0 TSG101 expression relative to D0 Normalization factor 1 Lane total protein Geomean of sample set total protein Normalization factor 2 Target antigen integrated band volume TSG101 integrated band volume CD63 CD81 3 2 1 0 3 2 1 0 D0 D2 D4 D6 * 2 0 Relative to D0 Realtive to D0 4 14-3-3 10 * 8 8 6 4 * * * 2 0 D0 D2 D4 D6 3 2 1 0 D0 D2 D4 D6 CD9 10 6 4 Relative to D0 Relative to D0 d) CD44 4 Relative to D0 4 Express relative to D0 target antigen D0 D2 D4 D6 38 D0 D2 D4 D6 Figure 6. Extracellular vesicle marker assessment across oligodendrocyte cell differentiation. (a) PCR analysis of OL differentiation markers Pdgfra, Cnp, Plp, and Mog across cell differentiation. (b) Western blot analysis of lamin A+C, calnexin, 14-3-3, CD63, CD9, CD44, and CD81 in cell lysates (CL), 100K fraction (EVs) and 10K fraction (Apoptotic bodies and microvesicles) expressed relative to D0. (c) Linear regression analysis of the expression of the relative expression of TSG101 and percent calnexin exclusion in EV fraction is correlated (r2=0.97). This suggests that the relative decrease in TSG101 in EV fraction results from the accumulated cell death over cell differentiation. (d) Bar graphs of relative expression of CD63, CD81, CD44, CD9, and 14-3-3 in EVs across OL cell differentiation to TSG101. Individual points represent n=2 isolation sets while the bar represents the mean ± SEM; one-way ANOVA with Bonferroni’s post-hoc test compared to control (D0), *p<0.05 3.3 Differential ultracentrifugation optimization The time that conditioned media is centrifuged to pellet EVs varies considerably throughout the literature, with the 100,000 x g spin ranging from 1-6h. Initially, increasing the 100,000 x g spin from 2h to 3h resulted in a higher protein yield; however with increased centrifugation time the risk of pelleting unwanted material may also increase. To investigate this further, a direct comparison was performed to assess if a 2h 100,000 x g spin performed better than 3h for three EV markers by Western blot analysis (figure 7). After total protein normalization across the two replicates, TSG101 and CD81 expression were higher in the 2h centrifugation time compared to 3h whereas the opposite trend was observed for CD44 (Figure 7). Given that CD81 and TSG101 are classically used as EV markers, the 2h 100,000 x g centrifugation was carrier through to future experiments. 39 CD44 N2 Adj. Total Band Vol. (Int) 2h N1 N2 3h N1 3e+8 CD44 TSG101 CD81 3e+8 2e+8 2e+8 1e+8 5e+7 0 3h CD81 7e+7 7e+8 6e+7 6e+8 Adj. Total Band Vol. (Int) Adj. Total Band Vol. (Int) TSG101 2h 5e+7 4e+7 3e+7 2e+7 1e+7 0 5e+8 4e+8 3e+8 2e+8 1e+8 0 3h 2h 3h 2h Figure 7. Comparison of EV markers between 3h and 2h 100K EV pelleting centrifugation step. Western blot analysis of CD44, TSG101, and CD81 in 100K pellets centrifuged for 3 hours and 2 hours. Bar graphs represent integrated total band volume for CD44, TSG101, and CD81 found in 100K pellet pellets centrifuged for 3 hours and 2 hours. 3.4 Cell culture optimization to reduce OL cell death over cell differentiation To increase the purity of the isolated EVs, several different media compositions were assessed to reduce OL cell death over the course of six days in differentiation media. Initially, four different basal media compositions were assessed for total protein recovered in 100K pellet at D6 (n=6) and percent calnexin exclusion (n=3). The conditions assessed were the following: DMEM High glucose (Cond 1), DMEM High glucose + 15mM HEPES (Cond 2), DMEM High 40 glucose + 15mM HEPES + 2mM L-glutamine (Cond 3), and DMEM/F12 + 15mM HEPES + 2mM L-glutamine (Cond 4). The recovered protein was relatively consistent across the four conditions (Figure 8a); however, condition 4 had the highest percent exclusion of calnexin and as such was used for future experiments (Figure 8b and c). Another way to mitigate contamination due to cell death is stimulate cells to release EVs over a shorter collection period. Previous studies have used glutamate stimulation to obtain OL EVs over a shorter collection period, but this was only performed against MBP+ OLs and not OPCs. To assess the effects of glutamate on OPCs, glutamate stimulation was performed with the co-agonist D-serine on undifferentiated OLs at three different concentrations (50µM, 100µM, and 200µM) for three exposure durations (figure 9a). Two days after L-glutamate + D-serine stimulation cell viability was assessed using an MTT assay and no decrease in cell viability was observed (figure 9a). Given that glutamate is released as a neurotransmitter by neurons in vivo cell viability was assessed to determine whether constitutively adding glutamate to the media formulation had any adverse effect compared to a short 2h stimulation (Figure 9b). Cell viability was assessed using an MTT assay and no decrease in cell viability was observed across all conditions tested. However, due to concerns with potentially activating intracellular Ca 2+ dependent proteases a short 2h 100µM glutamate + D-serine stimulation was chosen to use for future experiments. 41 a) 7 5 4 0 4.3 ± 0.37 1 4.1 ± 0.53 2 3.4 ± 0.26 3 4.1 ± 0.27 Total protein (ug) 6 Cond 1 Cond 2 Cond 3 Cond 4 100K Cond 4 CL 100K CL Cond 3 100K Cond 2 CL CL 100K Cond 1 b) N1 N2 N3 50 c) * % Exclusion 40 30 20 10 0 Cond 1 Cond 2 Cond 3 Cond 4 Figure 8. Effects of basal media on calnexin expression found in EV fraction across oligodendrocyte differentiation. Four basal media compositions were compared: DMEM High glucose (cond 1), DMEM High glucose + 15mM HEPES (cond 2), DMEM High glucose + 15mM HEPES + 2mM L-glutamine (cond 3), and DMEM/F12 + 15mM HEPES + 2mM L-glutamine (cond 4). (a) Bar graphs of total protein recovered in 100K pellet across oligodendrocyte cell differentiation between different basal media formulations (n=6). (b) Western blot analysis of calnexin expression found in 100K pellet and cell lysate in four different basal media compositions. (c) Bar graphs of percent calnexin exclusion between four different basal media compositions across oligodendrocyte cell differentiation. Individual points represent n=3 while the bar represents the mean ± SEM; one-way ANOVA with Bonferroni’s post-hoc test compared to control (D0), *p<0.05 42 200 a) Control 50uM 100uM 200uM 180 % Cell Viability 160 140 120 100 80 60 40 20 0 0.5h 1h 2h Treatment time b) 120 % Cell Viability 100 80 60 40 20 0 l M M tro 0u 0u n 0 0 o 1 C r1 h 2 uM 10 1u M M M M 1u 1u 1u . 0 0 0 0 0. 0. Figure 9. Effect of L-glutamate + D-serine on CG4-OL cell viability across differentiation. (a) MTT assay performed on undifferentiated (D0) CG4-OL cells after treatment of 50µM, 100µM, and 200µM L-glutamate + D-serine. MTT assay was performed two days post treatment in GM, bars represent the mean ± standard deviation (n=3); One-way ANOVA with Bonferroni’s post-hoc test compared to control. (b) MTT assay performed on differentiated CG4-OL cells after short (2h) stimulation of L-glutamate + D-serine in GM at D0 measured after 6 days of differentiation and continuous supplementation in DM for 6 days. MTT assay was performed after six days in differentiation media, bars represent the mean ± standard deviation (n=3) relative to untreated control cells; One-way ANOVA with Bonferroni’s post-hoc test compared to control. 43 The EV fraction isolated on D6 consistently had the lowest percent calnexin exclusion (figure 6b-c), so the effect of glutamate stimulation was assessed at this collection point. CG4OL cells were stimulated at D0 for 2h before changing to differentiation media and for 2h after collecting conditioned media at D6 to generate constitutive and stimulated released EVs. There was less protein in the 100K pellet from the 2h glutamate stimulated conditioned media compared to conditioned media generated from two days of cell culture (Figure 10a-c). Interestingly, the percent calnexin exclusion was nearly identical between the stimulated and unstimulated 100K pellets (figure 10a-c). Additionally, the percent calnexin exclusion was higher than previously observed in earlier experiments (figure 6b-c, figure 8b-c). Given that OLs are exceptionally prone to oxidative stress, the addition of 10 ng/mL N-acetyl-cysteine (NAC) to the media was assessed to reduce cell death and therefore increase the percent calnexin exclusion (Figure 10d-f). EVs were isolated from conditioned media collected with and without glutamate stimulation at D0 and D6 as described above. Similar results were obtained compared to cells grown without NAC. However, given that NAC is included in primary OL cell culture media formulations it was included for future experiments. 44 N3 Treated Unstimulated Total Protein (ug) 60 0 Untreated Stimulated 15 10 5 Untreated Stimulated 100K Glu 10K Glu 100K 10K CL N2 20 0 Treated Unstimulated e) N1 25 f) 100 % Calnexin exclusion % Exclusion 20 57 ± 5.6 40 62 ± 8.5 % Exclusion 80 30 80 4.7 ± 0.81 d) Treated Unstimulated 60 40 20 0 68± 11 c) 100 25± 2.3 Untreated Stimulated N2 64 ± 12 0 N1 3.0 ± 1.5 5 100K CL 15 10 10K 20 10K Glu 25 100K Glu b) 15 ± 4.7 Total Protein (ug) a) 30 Untreated Unstimulated Treated Stimulated Figure 10. Comparison of calnexin expression found in the EV fraction between stimulated and unstimulated CG4-OL cells. Cells were stimulated at D0 for 2h and at D6 for 2h with 100µM L-glutamate + Dserine. Media was collected from D4-D6 (unstimulated) and after short (2h) stimulation at D6 supplemented without (a-c) and with (d-f) 10ng/mL NAC (a) Total protein recovered in 100K pellet between 100 µM Lglutamate + D-serine stimulated and unstimulated CG4-OL cells at D6 (n=3). (b) Western blot analysis of calnexin expression found in 10K, 100K pellet, and cell lysate of stimulated and unstimulated CG4-OL cells at D6. Glutamate + D-serine stimulation was done for 2h, and conditioned media was collected whereas untreated collection was performed two days after a full volume media change (n=3) (c) Bar graphs of percent calnexin exclusion between stimulated and unstimulated CG4-OL cells at D6 (n=3). (d) Total protein recovered in 100K pellet between glutamate stimulated and untreated CG4-OL cells at D6 grown (n=2). (e) Western blot analysis of calnexin expression found in 10K, 100K pellet, and cell lysate of stimulated and unstimulated CG4-OL cells at D6 grown with 10ng/mL NAC. (n=2) (f) Bar graphs of percent calnexin exclusion between stimulated and unstimulated CG4-OL cells at D6 (n=2). 45 3.6 Functional effects of OL-derived EVs collected across cell differentiation on proliferation and differentiation of CG4-OLs. After optimizing the cell culture conditions, the functional effects of isolated OL EVs on proliferation and differentiation were evaluated by qPCR assays. To determine functional effects of OL derived EVs on proliferation and differentiation, EVs were isolated as previously described and added back to culture daily for four days after changing to differentiation media (Figure 11). NTA analysis of all isolated CG4-OL EVs are within the expected size range reported in the literature and are comparable to initial isolations (table 3.) Differentiation genes (Plp, Mbp, Mog) and progenitor genes (Ki67 and Pdgfra) were assessed after treatment with EVs or supernatant at D4. Ki67, Pdgfra, Plp, Mbp, and Mog were detected in untreated, supernatant treated and EV treated CG4-OLs (figure 11). There were no differences in Ki67, Pdgfra, Plp, or Mbp relative mRNA expression compared to the control (figure 11). However, there was a downward trend for Plp and Mbp observed in samples treated with EVs across cell differentiation whereas an upward trend was observed for Mog. Interestingly, a significant increase (p<0.05) in relative Mog mRNA expression was observed in samples treated with supernatant. 46 Table 3. Summarized NTA data of EVs isolated from CG4-OLs used for functional studies. Sample ID Mean size (nm) Mode size (nm) N1_D0 N1_D2 N1_D4 N1_D6 N2_D0 N2_D2 N2_D4 N2_D6 N3_D0 N3_D2 N3_D4 N3_D6 N4_D0 N4_D2 N4_D4 N4_D6 202.5 172 154 152.4 172 181.3 155.5 182.7 176.2 186 152.5 181.4 172 186.4 160.5 167.1 155.0 122.7 113.5 120.4 137.6 137.6 114.2 123.4 122.9 120.1 136.9 133.9 138.1 117.1 124.2 112.5 47 Standard deviation (nm) 74.4 61.8 61.8 62.1 57.7 73.6 58.9 76.4 65 82.6 57.3 77.1 67.3 75.2 57.1 76.8 Concentration (particles/mL) 3.14x1010±3.65x108 7.19x1010±2.37x109 8.23x1010±5.57X109 7.38x1010±7.84x107 5.12x1010±4.88x108 3.78x1010±1.45x109 8.72x1010±2.12x109 8.28x1010±2.53x109 6.39x1010±2.25x109 5.12x1010±2.07x109 1.02x1011±3.73x109 8.41x1010±7.31x109 5.75x1010±1.98x109 3.85x1010±1.82x109 6.94x1010±1.03x109 9.94x1010±3.01x109 1 Supernatant Supernatant 2 Change to DM 3 RNA isolation Daily ½ vol media change Pdgfra 3 2 1 0 -1 -2 -3 Mbp 3 2 1 0 -1 -2 -3 EVs 48 C trl D 0. D 2. D 4. D 6. D 0 D 2 D 4 D 6 Relative Normalized Expression (Log2 Fold Change) 4 C trl D 0. D 2. D 4. D 6. D 0 D 2 D 4 D 6 OPCs plated 0 EVs 2 C trl D 0. D 2. D 4. D 6. D 0 D 2 D 4 D 6 -1 Relative Normalized Expression (Log2 Fold Change) C trl D 0. D 2. D 4. D 6. D 0 D 2 D 4 D 6 Relative Normalized Expression (Log2 Fold Change) Day -2 Relative Normalized Expression (Log2 Fold Change) C t rl D 0. D 2. D 4. D6 . D 0 D 2 D 4 D 6 Relative Normalized Expression (Log2 Fold Change) Daily treatment (Supernatant/EVs) Ki67 3 2 1 0 -1 -2 -3 Supernatant Plp * Supernatant EVs 3 2 1 0 -1 -2 -3 Supernatant EVs Mog 3 * 1 0 -1 -2 -3 EVs Figure 11. Autocrine effects of OL-EVs on proliferation and myelin gene expression. (a) Schematic of cell culture and treatment conditions used to assess autocrine effects of OL-derived EVs. (b-f) Expression patterns of proliferation marker Ki67 and OL genes (Pdgfra, Plp, Mbp, Mog) are represented as Log2 FC normalized to reference gene panel (Hprt1, Sdha, & Tbp) and expressed relative to untreated control (ctrl). Individual points represent the n=4 biological replicates while the bar represents the mean ± SEM; one-way ANOVA with Bonferroni’s post-hoc test compared to control (ctrl), *p<0.05 49 Chapter 4. Discussion In the last decade, research interest in EVs has surged due to their growing number of described roles in health and disease. The release and transfer of EVs between cells is one mechanism of intercellular communication that transports nucleic acid species and proteins. Intercellular communication pathways between OLs and other cell types in the CNS is critical for the proper formation and maintenance of the myelin sheath. The characterization of OL derived EVs has been primarily focused on the effects they elicit on neurons under stress and unstressed conditions. Very little is currently known about the cargo or functional effects of EVs derived from OPCs compared to mature OLs and how these change over the course of cell differentiation. Total protein recovered in EV fraction increases and peaks at D4. Isolated EV fractions had increased relative expression of the EV marker CD9 and the cell signaling protein 14-3-3. Of the progenitor and differentiation genes assessed after four days of treatment with CG4-OL EVs or EV depleted supernatant, relative Mog expression increased in samples treated with supernatant. Data presented in this thesis, for the first time, characterizes OL derived EVs isolated across cell differentiation and assessed their ability to act as autocrine factors on proliferation and differentiation genes. 4.1 Assessment of OL-derived EV isolations based on MISEV2018 guidelines The international society of extracellular vesicles (ISEV) has published the minimal information for studies of extracellular vesicles (MISEV2018) to ensure proper characterization and reporting of EV research (Théry et al. 2018). The MISEV2018 guidelines describe the characterization work involved in assessing isolated EV fractions to meet the following criteria: assess size distribution by NTA or electron microscopy, demonstrate isolated EV fraction is positive for three common EV markers (usually tetraspanin proteins CD9, CD63, and CD81), 50 one transmembrane/lipid-bound protein (such as TSG101) or cytosolic protein, and has reduced/no expression of one exclusionary marker such as calnexin. Current publications on OL EVs do not conform to the MISEV2018 guidelines and do not state protein loads used to visualize EV markers by Western blot (Krämer-Albers et al. 2007a, 2007b, Frühbeis et al. 2013, 2020, Fröhlich et al. 2014). As such, changes in isolated OL-EV fractions were evaluated across cell differentiation using the CG4-OL cell line following the MISEV2018 guidelines. First, the protein yield of EVs isolated across OL cell differentiation was evaluated as this has not been reported in the literature and is essential for planning future experiments. EV fractions isolated at D0 consistently had the lowest total protein, with protein yields increasing at later time points and peaking at D4 (figure 4a). The transition from OPCs towards mature OL phenotype is mediated by significant changes in gene expression, and most likely explains the observed increase up to D4. One possible explanation is the production of ionotropic glutamate receptors which includes the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (subunits: GRIA1, GRIA2, GRIA3, and GRIA4), kainate (subunits: GRIK1, GRIK2, GRIK3, GRIK4, and GRIK5), and NMDA (subunits: GRIN1, GRIN2a, GRIN2b, GRIN2c, GRIN2d, GRIN3a, and GRIN3b) which typically have the highest mRNA expression in OPCs and the lowest in mature OLs based on brainseq data (Figure 12, Zhang et al. 2014). Typically, there is a lag period between changes in mRNA expression and protein expression, the observed decrease in mRNA expression observed in newly formed OLs compared to OPCs could reflect steadystate expression levels to ensure appropriate protein levels are maintained (Liu et al. 2016b). 51 AMPA Receptor Subunits a) 180 OPC Newly formed OL Myelinating OL 160 140 FPKM 120 100 80 60 40 20 0 G IA R b) 1 G R 2 IA G R 3 IA G R 4 IA Kainate Receptor Subunits 60 OPC Newly formed OL Myelinating OL 50 FPKM 40 30 20 10 0 1 IK R G c) R G 3 IK R G 2 IK G 4 IK R G 5 IK R NMDA Receptor Subunits 30 OPC Newly formed OL Myelinating OL 25 FPKM 20 15 10 5 0 G 1 IN R R G 2a IN G 2b IN R G 2c IN R IN R G 2d IN R G 3a 3b IN R G Figure 12. Ionotropic glutamate receptors mRNA expression in oligodendrocytes across differentiation. Expression values of (a) AMPA (b) Kainate, and (c) NMDA receptor subunits found in OPCs, newly formed OLs, and myelinating OLs. Figure generated from open source RNAseq FPKM counts (Zhang et al., 2014). 52 Given that OLs main biological function is to form myelin sheaths to wrap neuronal axons, increased competency to the neurotransmitter glutamate over cell differentiation aids in proper myelination (Li et al. 2013, Gibson et al. 2014, Mitew et al. 2018, Almeida et al. 2021). It has been shown experimentally that NMDA receptor stimulation increases myelin protein expression and arborization complexity in cultured OLs (Li et al. 2013). Binding of glutamate to these ionotropic receptors results in the influx of Ca2+ raising intracellular levels. Experimentally increasing intracellular calcium levels, typically with the calcium ionophore Ionomycin, has been employed as a strategy to increase the release of EVs over a shorter collection period (KrämerAlbers et al. 2007b, Lachenal et al. 2011). As such, changes in ionotropic glutamate receptor expression throughout OL differentiation, allowing for greater calcium influx, may explain the observed increase in recovered protein in the EV fraction. The NTA data partially supports this as the bulk D4 isolated EV fraction had the highest particle counts, however D6 had the lowest counts whereas D0 and D4 had comparable counts (Figure 5). It is important to note that the particle counts do not follow the same trend observed in total protein found in the isolated EV fraction (Figure 4a, Figure 5) and as such, this difference could potentially be explained by different/increased protein cargo found at later collection points. To maximize our sample usage to probe for several EV markers of interest, a protein titer was performed to determine the amount of sample that is required to visualize targets by Western blot (figure 4b). All the EVs markers (CD44, TSG101, CD81, CD63, and CD9) were able to be visualized across the entire protein range. As such 5ug of input sample was chosen for future experiments to ensure all EV markers of interest can be assessed. To confirm isolated EVs are within the expected size range, isolated samples were assessed using NTA. Bulk isolated EVs from OL cell culture CM are within the expected size range reported in the literature (40-200 53 nm) but have a larger mean size than the reported 95 nm for primary mature OL-derived EVs (Frühbeis et al. 2013, Théry et al. 2018). These size differences most likely are due to differences in producing cells, however they both are within the accepted size range indicating that the centrifugation steps pellet the appropriate sized vesicles. Calnexin as well as lamin A + C as an additional exclusionary marker for EV purity were assessed in isolated EV fractions (Théry et al. 2018). As EV biogenesis occurs from either the plasma membrane for microvesicles or the endosome for exosomes, calnexin is an endoplasmic reticulum protein and lamin A + C is a nuclear protein that are used as exclusionary markers as they are generally not found in isolated EV fractions (Théry et al. 2018). Increased expression levels found in the EV fraction are associated with cell death, either apoptosis or necrosis, as small membrane fragments may form vesicular-like structures that pellet with desired EVs (Théry et al. 2018). Over the course of OL cell differentiation, the percent exclusion of both calnexin and lamin A + C decreased after switching to differentiation media which is indicative of increased cell death (Figure 6b-c). It has been reported in the literature that OLs have increased cell death as they differentiate in culture and in vivo (Mayer and Noble 1994, Thorburne and Juurlink 1996, Juurlink et al. 1998, Lassmann and van Horssen 2016). It is estimated that greater than 50% of newly formed OLs die during developmental myelination, most likely due to competition for trophic factors causing local depletion (Barres et al. 1992). Minimizing cell death is essential for assessing EVs as decreased purity of EV fraction, due to co-isolation of membrane fragments, may skew expression levels depending on the antigen (Théry et al. 2018). 54 4.1.1 Shorter EV pelleting centrifugation step increasing common EV marker expression Differential ultracentrifugation remains to be the most common method used to isolate EVs from cell culture conditioned media primarily due to the low cost and minimal technical expertise required (Li et al. 2017, Aliakbari et al. 2024). There is considerable variety in the duration of the 100,000 -120,000 x g EV pelleting step reported in the literature ranging anywhere form 1-6h. Given that this method relies on the size and density of the EVs in the CM to pellet, increasing centrifugation time can result in pelleting unwanted materials thus reducing sample purity (Li et al. 2017). Previously, our initial optimization of the differential ultracentrifugation protocol was based solely on total protein recovered. To increase the purity of our isolated EV fraction, a direct evaluation was performed between two- and three-hour EV pelleting step on three common EV markers expression by Western blot analysis (Figure 7). Both TSG101 and CD81 expression were highest in the two-hour condition whereas CD44 expression was higher in the three-hour condition (Figure 7). Given this data, there is most likely extra contaminating material that is being pelleted with the longer centrifugation time. Changing to a 2h EV pelleting step will most likely increase calnexin exclusion, however not entirely without eliminating OL cell death as well. 4.1.2 DMEM/F12 increases calnexin exclusion in EV fraction Media composition plays a crucial role in the overall growth and health of cultured cells. Studies that culture either primary OLs or OL cell lines have used DMEM/F12, DMEM, or DMEM high glucose as basal media (Jung et al. 1995, Chen et al. 2007, Thangaraj et al. 2017, Dittmann et al. 2023). To directly assess if the observed increase in cell death across differentiation can be attributed to basal media choice and/or cell culture additives, the effect of basal media on calnexin exclusion was evaluated in the isolated EV fraction at late 55 differentiation (D6) where the lowest cell viability was consistently observed (Figure 5b-c). The four media compositions evaluated had similar protein recovered in the 100K EV fraction; however, DMEM/F12 +15mM HEPES + 2mM L-glutamine had the best exclusion (Figure 8a-c). One important observation is that DMEM high glucose basal media showed significant variability in calnexin exclusion over different experiments (Figure 8c, condition 1, Figure 5b-c) which most likely reflects that the health status of the CG4-OL parent cultures used differed. Given that DMEM/F12 is the most common basal media in the literature for culturing OLs, it is not surprising that this was the best performing condition. Ham’s F12 supplement was originally designed for serum free culture of Chinese Hamster Ovary cells (Ham 1965), and as such has several additional factors that are not found in DMEM formulations that could potentially explain the increased calnexin exclusion. It contains several inorganic salts (copper sulfate-5H20, ferrous sulfate-7H20, and zinc sulfate-7H2O) that contain metals used in key biological processes and as cofactors for antioxidant proteins (Powell 2000, Gaetke and Chow 2003, Imam et al. 2017). Given OLs vulnerability to oxidative stress, increasing the availability of trace elements could potentially increase their capacity to deal with oxidative stress in a cell culture system (Thorburne and Juurlink 1996, Juurlink et al. 1998, Lassmann and van Horssen 2016). However, it is important to note that too much or too little of these trace elements can result in adverse effects (Gaetke and Chow 2003, Imam et al. 2017). The F12 supplement also contains vitamin B12, linoleic acid and DL-α-lipoic acid which have been demonstrated to be important during OL differentiation and myelination. Vitamin B 12 has acts as a cofactor in folate metabolism making it crucial for fatty acid metabolism (Gröber et al. 2013, Wu et al. 2019) and has been shown to increase remyelination in a peripheral rat focal demyelination model (Nishimoto et al. 2015). B12 deficiency is associated with brain atrophy and cognitive 56 impairments in older adults (Vogiatzoglou et al. 2008). During OL differentiation there is increased cell arborization and membrane expansion to produce myelin membrane (Simons and Nave 2016). Given that myelin is a lipid rich structure, during developmental myelination OLs utilize extracellular lipids from dietary sources as well as from astrocytes rather than producing all de novo (Camargo et al. 2017b). As such decreasing metabolic demand on OLs to synthesize lipid molecules de novo through the addition of exogenous lipids could also aid in improving cell viability during differentiation in cell culture. It has also recently been demonstrated that oleic acid and linoleic acid prevent glutamate-induced OL cell death in a primary cell culture system through binding of free fatty acid receptor 1 (Maruyama et al. 2023). 4.1.3 Stimulating OL EV release The main biological role of OLs is to myelinate neuronal axons, and as such express neurotransmitter receptors such as ionotropic glutamate receptors to respond to neuronal activity (Frühbeis et al. 2013). Binding of glutamate to these receptors results in increased intracellular calcium levels which has been shown in other cell types to stimulate EV release. One advantage of stimulating cells to release their EV contents is that collection period can be reduced to a shorter window, which in theory could reduce contaminating vesicular structures in the EV fraction. Frühbeis et al. 2013 demonstrated that increased EV release in OLs can be mediated by glutamate and this effect was demonstrated to be facilitated by NMDA receptors. Specifically in this study, mature primary OLs were stimulated for 5h with 100µM L-glutamate and D-serine which is a NMDA co-agonist. Given the study did not evaluate these effects on OPCs, the effect on cell viability of varying concentrations of L-glutamate and D-serine were evaluated here. Both discrete and continuous supplementation over a broad concentration range (0.0001-200µM) did not affect cell viability (Figure 9). Given that no difference was observed, the effect on 57 calnexin exclusion from the EV fraction with two short L-glutamate and D-serine treatments at early (D0) and late (D6) differentiation were evaluated at D6. There was less protein recovered in the EV fraction from the stimulation period (2h) compared to the EV fraction from constitutive release (48h), most likely due to the time frame for each. Regardless, no difference in the relative calnexin exclusion was observed between both conditions (Figure 10a-c). The same trends were also observed with the addition of the antioxidant NAC (Figure 10d-f). Interestingly, the calnexin exclusion for both conditions were approximately 20% greater than for initial isolations (Figure 5c, Figure 10c and f). Given OLs appear to have a preference to myelinate axons releasing higher levels of glutamate, a high concentration of glutamate for a short period may be more relevant to what is experienced by OPCs and OLs in vivo (Gibson et al. 2014, Mitew et al. 2018, Almeida et al. 2021). In the developing CNS, glutamate released into the synaptic cleft is primarily collected back into the axon terminals to be repackaged into synaptic vesicles but reuptake can also be done by astrocytes. On the other hand, prolonged exposure of OLs to glutamate, commonly referred to as excitotoxicity, results in cell stress and apoptosis (Matute et al. 2001, Belov Kirdajova et al. 2020, Maruyama et al. 2023). These results contradict current literature, and this is mostly likely due to the short exposure duration compared to the prolonged exposure times used investigated excitotoxicity in OLs (Matute et al. 2001, Belov Kirdajova et al. 2020). One limitation is that the use of calnexin exclusion in isolated EV pellets is used to assess purity and acts as an indirect measure of cell death. As such to determine if short glutamate exposure/stimulation does in fact increase cell viability over cell differentiation, an immunocytochemistry approach assessing an apoptotic marker, such as cleaved caspase-3, would most likely provide the most compelling evidence. Initially, the goal was to assess if stimulation 58 could increase calnexin exclusion in the isolated EV pellet from progenitors as well as mature OLs. However, the protein yield from the 2h treatment on progenitors was too low to assess calnexin exclusion. Given the increased calnexin exclusion EV fraction from media collected at D6 after a short (2h) stimulation on progenitor CG4-OLs at D0, this was carried forward for collecting EV fraction for functional study. The optimization of cell culturing conditions to improve cell viability increased calnexin exclusion in EV fraction to 68% (figure 10d). Given the linear relationship relative the EV marker TSG101 and percent calnexin exclusion (r 2=0.97), it is reasonable that decreased expression in TSG101 was due to cell death (figure 6c). After normalizing to TSG101, expression the EV markers CD44, CD63, and CD81 remain consistent across cell differentiation (figure 6). On the other hand, the expression of the EV marker CD9 increased (figure 6). This suggest that there are different subpopulations of EVs being produced over OL differentiation. Relative expression of the cargo cell signaling protein 14-3-3 also increased over cell differentiation which may act in regulating OL differentiation. 4.2 Tetraspanin EV markers – the search for unique subpopulations The tetraspanin protein superfamily have been shown to be heavily enriched in endosomal membranes and as such are routinely used to characterize EVs. Tetraspanins have been shown to influence membrane architecture by clustering and interacting with various transmembrane and signaling proteins forming tetraspanin-enriched microdomains (TEMs). Different tetraspanin proteins have been demonstrated to interact with unique proteins as well as influence protein sorting (Andreu and Yáñez-Mó 2014). These observations have fueled the search for unique EV subpopulations with biological or diagnostic value across several cell types. For example, EV mediated export of β-catenin is achieved by CD82 and CD9 in 59 association with E-cadherin (Chairoungdua et al. 2010). Another tetraspanin, CD81, has been shown to be responsible for Wnt 11 as EV cargo in cancer associated fibroblasts (Luga et al. 2012). Recently, using immuno-isolations a proteomic assessment was performed on CD9+, CD63+, and CD81+ EVs isolated from human dendritic cells (Kowal et al. 2016). Each respective subpopulation had unique identified protein species as well as a larger proportion of shared protein species (Kowal et al. 2016). The observed increase in CD9 expression in our isolated EVs across OL cell differentiation could reflect an increase/change in a subpopulation with distinct biological effects (figure 5d). This is the first example suggesting temporal OL EV subtype changes and presents an avenue for future investigation. Although there was no observed difference in CD44, CD63, or CD81 expression by Western blot analysis (figure 5d) that does not necessarily indicate that there are not functional differences between EVs isolated at each respective time point (D0, D2, D4, D6). Western blotting is a targeted approach to evaluate antigens of interest and as such is unable to assess global changes in protein species across the samples in question. To address this limitation and obtain an overall picture of the temporal changes in all protein species found in isolated EVs across OL differentiation, a proteomics analysis would need to be employed. However, given the cost associated with proteomic analyses, improving the cell culture conditions to reduce cell death thereby increasing EV purity is crucial. 4.3 Relative expression of EV cargo 14-3-3 increased over differentiation 14-3-3 proteins are a family of conserved regulatory proteins that consist of seven isoforms and have been demonstrated to bind to a several functionally different signaling proteins such as kinases, phosphatase, and transmembrane receptors (Fu et al. 2000, Hermeking and Benzinger 2006). Many of these interactions have been shown to be involved in signaling 60 pathways related to cell growth, survival, and differentiation. It has been recently described in OL-EV cargo (Frühbeis et al. 2020) and given the diversity of cellular process it is involve in, this warranted further investigation. The interaction between 14-3-3 and Raf-1 has been heavily characterized as it plays an essential role in the signal transduction pathway elicited by growth factors regulating cell division (Michaud et al. 1995, Muslin et al. 1996, Morrison and Cutler 1997, Hermeking and Benzinger 2006). It has also been shown to interact with the cell cycle regulator protein Cdc25, regulating its cellular localization to either cytoplasm (phosphorylated & 14-3-3 bound) or nuclear (dephosphorylated & 14-3-3 unbound) (Kumagai and Dunphy 1999). 14-3-3 has also been shown to interact with Bad, a member of the Bcl-2 family, and antagonizes its proapoptotic activity by preventing binding with Bcl-XL and Bcl-2 thus providing control of cell death (Zha et al. 1996). However, given their known interactions with proteins involved in the signal transduction pathways elicited by growth factors and proteins involved in control of cell division, it is possible they influence this process in glial cells. Given the observed increase in 14-3-3 expression in isolated EVs across OL cell differentiation (figure 5d) it is possible that the transfer of 14-3-3 as EV cargo could potentially affect cell state progression such as promoting/preventing differentiation or cell death. Interestingly there appears to be some level of cell type specificity in terms of isoform effects. For example, 14-3-3γ has been shown to be upregulated in vascular smooth muscle cells when growth serum and platelet derived growth factor containing media (Autieri et al. 1996). The expression patterns of 14-3-3 isoforms vary considerably during mouse embryogenesis, neural development, and human neurological disorders suggesting cell type/stage specific effects (Cheah et al. 2012, Toyo-Oka et al. 2014). Recently, 14-3-3ε and 14-3-3ζ have been demonstrated to be crucial for neurogenesis using a loss of function mouse model (Toyo-Oka et 61 al. 2014). Double knockout (dKO) mice displayed increased number of proliferating cells in the subventricular zone, broader distribution of intermediate progenitor cells (IPCs), increased neuronal differentiation, and significantly decreased median survival compared to control mice (Toyo-Oka et al. 2014). These effects have been attributed to altered catenin levels where δcatenin levels increased whereas β-catenin and αN-catenin levels decreased in dKO mice (ToyoOka et al. 2014). Currently there are no studies that have directly investigated the role of 14-3-3 proteins in glial cell development or differentiation. However, several studies have described pathways involved in OL differentiation are also pathways 14-3-3 has been shown to act on such as phosphoinositide 3-kinase (PI3K)/mTOR, PKB, Mitogen-activated protein kinase kinase (MEK)/ERK, and β-catenin (Mitew et al. 2014). The inhibition of PI3K and mTORC1/2 prevents OL differentiation measured by decreased MBP expression (Dai et al. 2014) and increased expression of 14-3-3 in cancer cells has been shown to activate PI3K/PKB signalling (Neal et al. 2012, Wu et al. 2020). The MEK/ERK signalling pathway has been demonstrated to control myelin thickness as well as OL differentiation (Yang et al. 2016, Elbaz and Popko 2019). As previously mentioned, 14-3-3 acts on Raf-1 which is an upstream signaling protein in MEK/ERK signaling (Yang et al. 2016). It is tempting to speculate that the transfer of 14-3-3 as EV cargo could affect OL differentiation, potentially in an isoform specific manner, however this would need to be investigated in future studies. 4.4 Assessing the role of OL EVs as autocrine factors regulating proliferation and differentiation of CG4-OLs The differentiation of OLs during postnatal development is a highly regulated process involving both external factors as well as significant changes in gene expression (Emery and Lu 2015). It is well accepted that the expansion of OPCs is in response to PDGFA produced by type-I astrocytes, and then once a critical density is reached, local depletion of PDGFA results in 62 differentiation towards a mature myelinating phenotype (Elbaz and Popko 2019, Adams et al. 2021). However not all OPCs differentiate, and a pool of progenitors remains in the adult CNS (Foerster et al. 2019, Beiter et al. 2022, Dennis et al. 2024). How these OPCs are retained in a progenitor state throughout lifespan is poorly understood, but this pool is critical for the injury response and repair under pathological conditions (Foerster et al. 2019, Beiter et al. 2022, Dennis et al. 2024). Early studies on CG4-OLs showed that cells secrete substance(s) that decrease proliferation (Louis et al. 1992b), yet this was never explored further and the molecules responsible for this effect were never identified. These molecules could be released as soluble factors into the media or the effect may be mediated by EVs due to their ability to transfer protein, nucleic acid species, and lipids between cells (Takeda and Xu 2015). As such the functional effects OL-derived EVs were evaluated OL gene expression using qPCR assays. There was no change in genes associated with progenitor state such as Pdgfra and Ki67. From the myelin genes associated with differentiation, only Mog expression showed upregulation in response to treatment with EV depleted supernatant collected from D0 and D6 (Figure 11). Additionally, an upward trend for Mog was observed in samples treated with isolated EVs. Mog is one of the last genes to be upregulated during developmental myelination (Smith 2022). RNA was isolated after four days of treatment in DM which could explain why it was the only gene to consistently show increased expression compared to control. Given that Plp and Mbp expression peaks earlier, by evaluating a single time point, it is possible that the untreated control cells caught up with the changes elicited by EV treatments by this time point. To address this, earlier collection time points would need to be assessed. Although the supernatant is “EV depleted” this is not entirely true. The recommendation to make EV depleted FBS is to centrifuge for 18h at 63 100,000 x g and in the case for these experiments, the supernatant used after pelleting EV fraction for 2h at 100,000 x g was referred to as “EV depleted”. As such, potentially the reason an observed significant increase in the Mog expression in the EV depleted supernatant samples is due to a combined effect of a soluble protein and residual EVs. However, it does appear that OLs secrete factors, both soluble and EV, that effect differentiation. 64 Chapter 5: Conclusions and Future Directions Although OL differentiation and myelination has been studied for decades, our understanding is far from complete. This research aimed to assess the role of EVs in this process and if they act as autocrine factors. In support of this, the work presented in this thesis (i) assessed marker expression across different stages of OL differentiation by Western blot and (ii) evaluated the autocrine effects of OL-derived EVs on cell proliferation and differentiation gene expression by qPCR. First, common EV markers outline by the MISEV2018 guidelines were evaluated and found that relative expression of CD9 and cargo protein increased over differentiation. The cargo protein 14-3-3 was also found to increase over differentiation, so further work into assessing the role of 14-3-3 isoforms in OL differentiation is warranted to delineate which are involved in this process. It would also be of interest to determine if CD9+ EVs are a distinct population using global omics-based approaches. While, the functional effects of OL-derived EVs from different cell development stages were limited, more robust investigation using different techniques across multiple time points may provide more insight. Alternatively, OL-EVs may not act as autocrine factors but instead act preferentially on other neural cell types. Currently there are no approved therapeutic interventions targeted towards remyelination or neurodegeneration in progressive MS. Understanding the molecular mechanisms that coordinate myelination will allow for the development of novel treatments for demyelination in aging and disease. This work has begun to investigate how OLs participate in intracellular communication with non-neuronal cells in the CNS to coordinate myelination, potentially leading to novel therapeutic interventions for remyelination. 65 References Adams, K. L., K. D. Dahl, V. Gallo, and W. B. Macklin. 2021. Intrinsic and extrinsic regulators of oligodendrocyte progenitor proliferation and differentiation. Seminars in Cell and Developmental Biology 116:16–24. Aliakbari, F., N. B. Stocek, M. Cole-Andre, J. Gomes, G. Fanchini, S. H. Pasternak, G. Christiansen, D. Morshedi, K. Volkening, and M. J. 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N1 gels, membranes, and Western blots for Calnexin and 14-3-3. Associated with figure 6 main text. (a-b) uncropped TGX Stain-Free SDS-PAGE gels activated and auto exposed imaged using the Stain-Free application on the ChemiDoc imaging system. (c-d) uncropped Post transfer membranes imaged using the Stain-Free application on the ChemiDoc imaging system with identical exposure times. (e-f) uncropped Western blot images of calnexin expression imaged with identical exposure times. (g-h) uncropped Western blot images of 14-3-3 expression imaged with identical exposure times. 81 b) c) d) e) f) 10K X X 100K D2 10K CL 100K X a) Ladder CL D0 10K X X 100K D2 10K CL 100K Ladder CL X D0 Supplemental Figure S3. N1 gels, membranes, and Western blots for lamin A + C. Associated with figure 6 main text. (a-b) uncropped TGX Stain-Free SDS-PAGE gels activated and auto exposed imaged using the Stain-Free application on the ChemiDoc imaging system. (c-d) uncropped Post transfer membranes imaged using the StainFree application on the ChemiDoc imaging system with identical exposure times. (e-f) uncropped Western blot images of lamin A + C expression imaged with identical exposure times. 82 c) d) e) f) g) h) 10K X X 100K D2 10K CL 100K Ladder CL b) X D0 10K X X 100K D2 10K CL 100K Ladder CL a) X D0 Supplemental Figure S4. N1 gels, membranes, and Western blots for CD63 and CD9. Associated with figure 6 main text (a-b) uncropped TGX Stain-Free SDS-PAGE gels activated and auto exposed imaged using the Stain-Free application on the ChemiDoc imaging system. (c-d) uncropped Post transfer membranes imaged using the Stain-Free application on the ChemiDoc imaging system with identical exposure times. (e-f) uncropped Western blot images of CD63 expression imaged with identical exposure times. (g-h) uncropped Western blot images of CD9 expression imaged with identical exposure 83 times. 10K CL 100K a) b) c) d) e) f) g) h) i) j) 84 D6 10K CL 100K 10K X X D4 Ladder CL 100K X 10K X X D2 CL 100K Ladder X D0 Supplemental Figure S5. N1 gels, membranes, and Western blots for CD44, TSG101 and CD81. Associated with figure 6 main text. (a-b) uncropped TGX Stain-Free SDS-PAGE gels activated and auto exposed imaged using the Stain-Free application on the ChemiDoc imaging system. (c-d) uncropped Post transfer membranes imaged using the Stain-Free application on the ChemiDoc imaging system with identical exposure times. (e-f) uncropped Western blot images of CD44 expression imaged with identical exposure times. (g-h) uncropped Western blot images of TSG101 expression imaged with identical exposure times. (i-j) uncropped Western blot images of CD81 expression imaged with identical exposure times. 85 a) 10K X X 100K 10K CL 100K Ladder CL X b) c) d) e) f) g) h) 86 10K X X D4 100K 10K CL D2 100K Ladder CL X D0 D6 Supplemental Figure S6. N2 gels, membranes, and Western blots for Calnexin and 14-3-3. Associated with figure 6 main text. (a-b) uncropped TGX Stain-Free SDS-PAGE gels activated and auto exposed imaged using the Stain-Free application on the ChemiDoc imaging system. (c-d) uncropped Post transfer membranes imaged using the Stain-Free application on the ChemiDoc imaging system with identical exposure times. (e-f) uncropped Western blot images of calnexin expression imaged with identical exposure times. (g-h) uncropped Western blot images of 14-3-3 expression imaged with identical exposure times. 87 f) 10K X X e) 100K d) D6 10K CL c) 100K b) Ladder CL a) X D4 10K X X 100K D2 10K CL 100K Ladder CL X D0 Supplemental Figure S7. N2 gels, membranes, and Western blots for lamin A + C. Associated with figure 6 main text (a-b) uncropped TGX Stain-Free SDS-PAGE gels activated and auto exposed imaged using the StainFree application on the ChemiDoc imaging system. (c-d) uncropped Post transfer membranes imaged using the Stain-Free application on the ChemiDoc imaging system with identical exposure times. (e-f) uncropped Western blot images of lamin A + C expression imaged with identical exposure times. 88 a) 10K X X 100K 10K CL 100K Ladder CL X b) c) d) e) f) g) h) 89 10K X X D0 100K 10K CL D2 100K Ladder CL X D0 D2 Supplemental Figure S8. N2 gels, membranes, and Western blots for CD63 and CD9. Associated with figure 6 main text (a-b) uncropped TGX Stain-Free SDS-PAGE gels activated and auto exposed imaged using the Stain-Free application on the ChemiDoc imaging system. (c-d) uncropped Post transfer membranes imaged using the Stain-Free application on the ChemiDoc imaging system with identical exposure times. (e-f) uncropped Western blot images of CD63 expression imaged with identical exposure times. (g-h) uncropped Western blot images of CD9 expression imaged with identical exposure times. 90 D2 a) b) c) d) e) f) g) h) i) j) 91 Ladder CL 100K 10K CL 100K 10K X X X CL 100K 10K CL 100K 10K X X Ladder X D0 D0 D2 Supplemental Figure S9. N2 gels, membranes, and Western blots for CD44, TSG101 and CD81. Associated with figure 6 main text (a-b) uncropped TGX Stain-Free SDS-PAGE gels activated and auto exposed imaged using the Stain-Free application on the ChemiDoc imaging system. (c-d) uncropped Post transfer membranes imaged using the Stain-Free application on the ChemiDoc imaging system with identical exposure times. (e-f) uncropped Western blot images of CD44 expression imaged with identical exposure times. (g-h) uncropped Western blot images of TSG101 expression imaged with identical exposure times. (i-j) uncropped Western blot images of CD81 expression imaged with identical exposure times. 92 a) b) c) d) e) f) 3h 2h X X X Ladder N1 N2 N1 N2 N1 N2 X X 2h X X X Ladder N1 N2 X X 3h Supplemental Figure S10. Resolved gels, membranes, and Western blots for comparison of EV markers between 3h and 2h 100K pelleting centrifugation step. Associated with figure 7 main text (a) uncropped TGX Stain-Free SDS-PAGE gels activated and auto exposed imaged using the Stain-Free application on the ChemiDoc imaging system. (b) uncropped Post transfer membrane imaged with auto exposure using the Stain-Free application on the ChemiDoc imaging system. (c) uncropped Western blot images of CD44 expression. (d) uncropped Western blot images of TSG101 expression. (e) uncropped Western blot images of CD81 expression 93 X Ladder CL Cond 1 100K CL Cond 2 100K CL Cond 3 100K CL Cond 4 100K X a) b) c) d) e) f) g) h) i) Ladder CL Cond 1 100K CL Cond 2 100K CL Cond 3 100K CL Cond 4 100K N3 N2 Ladder CL Cond 1 100K CL Cond 2 100K CL Cond 3 100K CL Cond 4 100K X N1 Supplemental Figure S11. Resolved gels, membranes, and Western blots of calnexin expression found in 100K pellet in four basal media compositions. Associated with figure 8 main text. Four basal medias compared were the following: DMEM H glucose (cond 1), DMEM H glucose + 15mM HEPES (cond 2), DMEM H glucose + 15mM HEPES + 2mM L-glutamine (cond 3), and DMEM/F12 + 15 mM HEPES + 2mM L-glutamine (cond 4) (a-c) uncropped TGX Stain-Free SDS-PAGE gels activated and auto exposed imaged using the Stain-Free application on the ChemiDoc imaging system. (d-f) uncropped Post transfer membranes imaged using the Stain-Free application on the ChemiDoc imaging system. (g-i) uncropped Western blot images of calnexin expression. 94 N2 N3 X X Ladder CL 10K 100K 10K Glu 100K Glu X X X X Ladder CL 10K 100K 10K Glu 100K Glu X X X X Ladder CL 10K 100K 10K Glu 100K Glu X X N1 a) b) c) d) e) f) g) h) i) Supplemental Figure S12. Resolved gels, membranes, and Western blots for unstimulated and glutamate stimulated CG4-OL cells. Associated with figure 10 main text. (a-c) uncropped TGX Stain-Free SDS-PAGE gels activated and auto exposed imaged using the Stain-Free application on the ChemiDoc imaging system. (d-f) uncropped Post transfer membranes imaged using the Stain-Free application on the ChemiDoc imaging system. (g-i) uncropped Western blot images of calnexin expression. 95 a) b) c) d) e) f) X X 100K Glu 100K 10K Glu CL 10K Ladder X X N2 X X Ladder CL 10K 100K 10K Glu 100K Glu X X N1 Supplemental Figure S12. Resolved gels, membranes, and Western blots for unstimulated and glutamate stimulated CG4-OL cells with NAC. Associated with figure 10 main text. (a-b) uncropped TGX Stain-Free SDSPAGE gels activated and auto exposed imaged using the Stain-Free application on the ChemiDoc imaging system. (c-d) uncropped Post transfer membranes imaged using the Stain-Free application on the ChemiDoc imaging system with identical exposure times. (e-f) uncropped Western blot images of calnexin expression. 96