Science, Irving K. Barber Faculty of (Okanagan)
Biology, Department of (Okanagan)
DSpace
UBCO
Lindhout, Ivan Andrew
Master of Science - MSc
University of British Columbia
Microglia provide immune defense in the central nervous system (CNS) through functions that include cytokine and cytotoxin secretion and phagocytosis. However, in several CNS disorders such as Alzheimer’s disease, aberrant activation of microglia causes decreased phagocytic activity and increased neurotoxicity due to the release of cytotoxic reactive oxygen (ROS) and nitrogen (RNS) species and pro-inflammatory cytokines. The growth factor interleukin (IL)-34 and neurotrophins (NTs) brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), as well as the proneurotrophin proBDNF are important for trophic signaling and regulation of survival and apoptosis in neurons. The effects of these compounds are mediated through the IL-34 receptor, colony-stimulating factor-1 receptor, and the NT receptors (NTRs), tropomyosin-receptor kinase (Trk)A, TrkB, and p75NTR. These growth factors and proBDNF also have profound CNS effects by binding to their receptors on other cell types, including microglia. IL-34, BDNF, and NGF have been shown to have therapeutic potential by reducing microglial immune activation. However, the large size of BDNF and NGF prevents them from penetrating the blood-brain barrier easily. Therefore, small molecules were developed to circumvent these issues while retaining the ability to activate NTR signaling pathways, including the TrkA ligand GK-2, TrkB ligand GSB-106, and p75NTR ligand LM11A-31. In my thesis work, I addressed knowledge gaps surrounding the effects of these three ligands as well as proBDNF and IL-34 on microglial neuroinflammatory functions. Chapter three demonstrates that IL-34 increased phagocytic activity of unstimulated and stimulated microglia, decreased ROS release by primed microglia-like cells, reduced monocyte chemoattractant protein (MCP)-1 secretion by stimulated microglia-like cells, and raised RNS production by unstimulated microglia. In chapter four, I show that LM11A-31 and proBDNF decreased the release of ROS by microglia-like cells and proBDNF increased the phagocytic activity of microglia. Chapter five shows that GK-2 decreased the release of ROS by primed microglia-like cells and the phagocytic activity of stimulated microglia. GSB-106 lowered the production of ROS in unprimed and primed microglia-like cells and the secretion of MCP-1 in stimulated microglia-like cells. Therefore, my research showed that IL-34, LM11A-31, proBDNF, GK-2, and GSB-106 regulate select immune processes of microglia and microglia-like cells.
NEUROTROPHIN RECEPTOR LIGANDS LM11A-31, PRO-BRAIN-DERIVED NEUROTROPHIC FACTOR, GK-2, AND GSB-106, AND GROWTH FACTOR INTERLEUKIN-34 MODULATE SELECT IMMUNE FUNCTIONS OF MICROGLIA by Ivan Andrew Lindhout B.Sc., The University of the Fraser Valley, 2017 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) June 2024 © Ivan Andrew Lindhout, 2024 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: NEUROTROPHIN RECEPTOR LIGANDS, LM11A-31, PRO-BRAIN-DERIVED NEUROTROPHIC FACTOR, GK-2, AND GSB-106, AND GROWTH FACTOR INTERLEUKIN-34 MODULATE SELECT IMMUNE FUNCTIONS OF MICROGLIA submitted by Ivan Andrew Lindhout in partial fulfillment of the requirements of the degree of Master of Science. Dr. Andis Klegeris, Department of Biology Supervisor Dr. Julien Gibon, Department of Biology Supervisory Committee Member Dr. Richard Plunkett, Department of Biology Supervisory Committee Member Dr. Conor Pranckevicius, Department of Chemistry University Examiner iii Microglia provide immune defense in the central nervous system (CNS) through functions that include cytokine and cytotoxin secretion and phagocytosis. However, in several CNS disorders such as Alzheimer’s disease, aberrant activation of microglia causes decreased phagocytic activity and increased neurotoxicity due to the release of cytotoxic reactive oxygen (ROS) and nitrogen (RNS) species and pro-inflammatory cytokines. The growth factor interleukin (IL)-34 and neurotrophins (NTs) brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), as well as the proneurotrophin proBDNF are important for trophic signaling and regulation of survival and apoptosis in neurons. The effects of these compounds are mediated through the IL-34 receptor, colony-stimulating factor-1 receptor, and the NT receptors (NTRs), tropomyosin-receptor kinase (Trk)A, TrkB, and p75NTR. These growth factors and proBDNF also have profound CNS effects by binding to their receptors on other cell types, including microglia. IL-34, BDNF, and NGF have been shown to have therapeutic potential by reducing microglial immune activation. However, the large size of BDNF and NGF prevents them from penetrating the blood-brain barrier easily. Therefore, small molecules were developed to circumvent these issues while retaining the ability to activate NTR signaling pathways, including the TrkA ligand GK-2, TrkB ligand GSB-106, and p75NTR ligand LM11A-31. In my thesis work, I addressed knowledge gaps surrounding the effects of these three ligands as well as proBDNF and IL-34 on microglial neuroinflammatory functions. Chapter three demonstrates that IL-34 increased phagocytic activity of unstimulated and stimulated microglia, decreased ROS release by primed microglia-like cells, reduced monocyte chemoattractant protein (MCP)-1 secretion by stimulated microglia-like cells, and raised RNS production by unstimulated microglia. In chapter four, I show that LM11A-31 and proBDNF decreased the release of ROS by microglia-like cells and proBDNF increased the phagocytic activity of microglia. Chapter five shows that GK-2 decreased the release of ROS by primed microglia-like cells and the phagocytic activity of stimulated microglia. GSB-106 lowered the production of ROS in unprimed and primed microglia-like cells and the secretion of MCP-1 in stimulated microglia-like cells. Therefore, my research showed that IL-34, LM11A-31, proBDNF, GK-2, and GSB-106 regulate select immune processes of microglia and microglia-like cells. Abstract iv Alzheimer’s disease and other dementias rank as the seventh leading cause of death worldwide. The discovery of novel therapeutics is imperative as current treatments remain ineffective at curing these neurodegenerative diseases or reducing the disability and dependency of affected individuals. These diseases are characterized by nerve cell death, inflammation, and abnormal protein aggregation in the central nervous system (CNS). Microglia, the main immune cells of the CNS, are over-activated in neurodegenerative diseases, resulting in neuron damage and death. Microglial phagocytosis is a process that engulfs and removes accumulated proteins or cellular debris. I determined that certain molecules involved in growth signaling pathways such as interleukin-34, pro-brain-derived neurotrophic factor, and neurotrophin receptor ligands LM11A-31, GK-2 and GSB-106 modulate select microglial immune functions including neurotoxin production and phagocytic activity. These compounds may be promising therapeutic candidates if they attenuate such functions in neurodegenerative disease cases. Lay summary v During this project, I was responsible for knowledge gap identification, experimental design and completion, data analysis, and manuscript preparation for all research conducted in this thesis. All experiments described herein except the phagocytosis assay were conducted in the Dr. Andis Klegeris Laboratory of Cellular and Molecular Pharmacology at the University of British Columbia Okanagan (UBCO) campus. The phagocytosis assay was completed in the Barker Lab at the UBCO campus. The blinded image analysis of the phagocytosis assay experiments was performed by Seamus McRae and Christy Richards. During my M.Sc. studies, I also authored or co-authored the following publications: Lindhout, I. A., Murray, T. E., Richards, C. M., & Klegeris, A. (2021). Potential neurotoxic activity of diverse molecules released by microglia. Neurochemistry International, 148, Article 105117. https://doi.org/10.1016/J.NEUINT.2021.105117. Murray, T. E., Richards, C. M., Robert-Gostlin, V. N., Bernath, A. K., Lindhout, I. A., & Klegeris, A. (2022). Potential neurotoxic activity of diverse molecules released by astrocytes. Brain Research Bulletin, 189, 80–101. https://doi.org/10.1016/J.BRAINRESBULL.2022.08.015 Preface vi Abstract .......................................................................................................................................... iii Lay summary .................................................................................................................................. iv Preface ............................................................................................................................................. v Table of contents ............................................................................................................................ vi List of tables ................................................................................................................................... ix List of figures ................................................................................................................................... x List of abbreviations ..................................................................................................................... xii Acknowledgements ...................................................................................................................... xiv Dedication ...................................................................................................................................... xv Chapter 1: Introduction .................................................................................................................... 1 1.1 Neurotrophins .................................................................................................................. 1 1.2 NTRs and signaling pathways associated with these receptors ....................................... 2 1.3 Interleukin-34, colony-stimulating factor-1 receptor and signaling pathways ................ 3 1.4 Microglia and neuroinflammation ................................................................................... 5 1.5 Effects of neurotrophins on microglia ............................................................................. 6 1.6 Effects of IL-34 on microglia .......................................................................................... 7 1.7 LM11A-31 ....................................................................................................................... 8 1.8 proBDNF ......................................................................................................................... 9 1.9 GK-2 .............................................................................................................................. 10 1.10 GSB-106 ........................................................................................................................ 10 1.11 Ligands and signaling pathways of TrkA/B, p75NTR and CSF-1R ............................. 12 1.12 Cell lines ........................................................................................................................ 12 1.13 Research aims ................................................................................................................ 14 1.14 Hypothesis ..................................................................................................................... 14 Chapter 2: Materials and Methods ................................................................................................. 15 2.1 Chemicals and reagents ................................................................................................. 15 Table of contents vii 2.2 Equipment and supplies ................................................................................................. 15 2.3 Cell cultures ................................................................................................................... 16 2.4 Cell viability assay ......................................................................................................... 16 2.5 Cell death assay (data not shown) ................................................................................. 17 2.6 THP-1/SH-SY5Y supernatant transfer .......................................................................... 17 2.7 ELISA ............................................................................................................................ 18 2.8 Griess assay ................................................................................................................... 18 2.9 Phagocytosis assay ........................................................................................................ 19 2.10 Respiratory burst assay .................................................................................................. 20 2.11 Statistical analyses ......................................................................................................... 21 Chapter 3: IL-34 ............................................................................................................................ 22 3.1 IL-34 hypothesis ............................................................................................................ 22 3.1.1 Hypothesis #1: IL-34 regulates select neuroinflammatory processes of microglia and microglia-like cells. ........................................................................................................ 22 3.2 IL-34 results ................................................................................................................... 22 3.2.1 Cytotoxicity and chemokine secretion of human THP-1 microglia-like cells ...... 22 3.2.2 BV-2 Griess assay ................................................................................................. 26 3.2.3 BV-2 phagocytic activity ....................................................................................... 27 3.2.4 HL-60 respiratory burst ......................................................................................... 28 3.3 IL-34 discussion ............................................................................................................ 31 Chapter 4: LM11A-31 and proBDNF ........................................................................................... 36 4.1 LM11A-31 and proBDNF hypothesis ........................................................................... 36 4.1.1 Hypothesis #2: p75NTR ligands, LM11A-31 and proBDNF, regulate select neuroinflammatory processes of microglia and microglia-like cells. .................................... 36 4.2 LM11A-31 results .......................................................................................................... 36 4.2.1 Cytotoxicity and chemokine secretion of human THP-1 microglia-like cells ...... 36 4.2.2 BV-2 Griess assay ................................................................................................. 38 4.2.3 BV-2 phagocytic activity ....................................................................................... 40 4.2.4 HL-60 Respiratory Burst ....................................................................................... 41 4.3 proBDNF results ............................................................................................................ 43 4.3.1 Cytotoxicity and chemokine secretion of human THP-1 microglia-like cells ...... 43 viii 4.3.2 BV-2 Griess assay ................................................................................................. 45 4.3.3 BV-2 phagocytic activity ....................................................................................... 47 4.3.4 HL-60 Respiratory Burst ....................................................................................... 48 4.4 LM11A-31 discussion ................................................................................................... 50 4.5 proBDNF discussion ...................................................................................................... 52 Chapter 5: GK-2 and GSB-106 ..................................................................................................... 55 5.1 GK-2 and GSB-106 hypothesis ..................................................................................... 55 5.1.1 Hypothesis #3: TrkA/B ligands, GK-2 and GSB-106, regulate select neuroinflammatory processes of microglia and microglia-like cells. .................................... 55 5.2 GK-2 results ................................................................................................................... 55 5.2.1 Cytotoxicity and chemokine secretion of human THP-1 microglia-like cells ...... 55 5.2.2 BV-2 Griess assay ................................................................................................. 57 5.2.3 BV-2 phagocytic activity ....................................................................................... 59 5.2.4 HL-60 Respiratory Burst ....................................................................................... 60 5.3 GSB-106 results ............................................................................................................. 62 5.3.1 Cytotoxicity and chemokine secretion of human THP-1 microglia-like cells ...... 62 5.3.2 BV-2 Griess assay ................................................................................................. 64 5.3.3 BV-2 Phagocytic activity ....................................................................................... 66 5.3.4 HL-60 Respiratory Burst ....................................................................................... 67 5.4 GK-2 discussion ............................................................................................................ 69 5.5 GSB-106 discussion ...................................................................................................... 71 Chapter 6: Conclusion ................................................................................................................... 73 6.1 Summary table ............................................................................................................... 73 6.2 Hypotheses addressed .................................................................................................... 74 6.3 Future directions ............................................................................................................ 76 6.4 Study limitations ............................................................................................................ 78 6.5 Research significance .................................................................................................... 79 Bibliography .................................................................................................................................. 82 ix Table 1. The significant effects of IL-34, LM11A-31, proBDNF, GK-2 and GSB-106 on immune functions of microglia and microglia-like cells………………………………………………….74 List of tables x Figure 1. TrkA/B, p75NTR and CSF-1R ligands and intracellular signaling pathways………...12 Figure 2. Effect of IL-34 on the secretion of MCP-1 and cytotoxicity of stimulated human THP-1 microglia-like cells towards human SH-SY5Y neuronal cells…………………………………..23 Figure 3. Effect of IL-34 on nitric oxide secretion by BV-2 murine microglia………………….27 Figure 4. Effect of IL-34 on the phagocytosis of latex beads by unstimulated and LPS-stimulated BV-2 murine microglia…………………………………………………………………………..28 Figure 5. Effect of IL-34 on the respiratory burst and viability of differentiated human HL-60 microglia-like cells……………………………………………………………………………….30 Figure 6. Effect of LM11A-31 on the secretion of MCP-1 and cytotoxicity of stimulated human THP-1 microglia-like cells towards human SH-SY5Y neuronal cells……………………..……37 Figure 7. Effect of LM11A-31 on nitric oxide secretion by stimulated BV-2 murine microglia..39 Figure 8. Effect of LM11A-31 on the phagocytosis of latex beads by unstimulated and lipopolysaccharide-stimulated BV-2 murine microglia..………………………………………...40 Figure 9. Effect of LM11A-31 on the respiratory burst and viability of differentiated human HL-60 microglia-like cells……………………………………………………………………………42 Figure 10. Effect of proBDNF on secretion of MCP-1 and cytotoxicity of stimulated human THP-1 microglia-like cells towards human SH-SY5Y neuronal cells………...………………...44 Figure 11. Effect of proBDNF on nitric oxide secretion by stimulated BV-2 murine microglia..46 Figure 12. Effect of proBDNF on the phagocytosis of latex beads by unstimulated and lipopolysaccharide-stimulated BV-2 murine microglia……….....................................................47 Figure 13. Effect of proBDNF on the respiratory burst and viability of differentiated human HL-60 microglia-like cells……………………………………………………………………………49 Figure 14. Effect of GK-2 on secretion of MCP-1 and cytotoxicity of stimulated human THP-1 microglia-like cells towards human SH-SY5Y neuronal cells…………………………………..56 Figure 15. Effect of GK-2 on nitric oxide secretion by BV-2 murine microglia…………...……58 Figure 16. Effect of GK-2 on the phagocytosis of latex beads by unstimulated and lipopolysaccharide-stimulated BV-2 murine microglia……………………….…………………59 List of figures xi Figure 17. Effect of GK-2 on the respiratory burst of differentiated human HL-60 microglia-like cells………………………………………………………………………………………………61 Figure 18. Effect of GSB-106 on secretion of MCP-1 and cytotoxicity of stimulated human THP-1 microglia-like cells towards human SH-SY5Y neuronal cells………………………………...63 Figure 19. Effect of GSB-106 on nitric oxide secretion by BV-2 murine microglia…………….65 Figure 20. Effect of GSB-106 on the phagocytosis of latex beads by unstimulated and stimulated BV-2 murine microglia…………………………………………………………………………..66 Figure 21. Effect of GSB-106 on the respiratory burst of differentiated human HL-60 microglia-like cells………………………………………………………………………………………….68 xii AD: Alzheimer’s disease AKT: Protein kinase B AMP: Adenosine monophosphate ANOVA: Analysis of variance BDNF: Brain-derived neurotrophic factor CBS: Calf bovine serum CD: Cluster of differentiation CNS: Central nervous system CREB: Cyclic-AMP-response element binding protein CSF: Colony-stimulating factor CSF-1R: Colony-stimulating factor-1 receptor CXCL10: Interferon-γ inducible protein 10 DMEM/F12: Dulbecco’s modified Eagle medium/nutrient mixture F-12/Ham DMSO: Dimethyl sulfoxide ELISA: Enzyme-linked immunosorbent assay ERK: Extracellular regulated kinase FITC: Fluorescein isothiocyanate fMLP: N-formyl-methionine-leucyl-phenylalanine gp: Glycoprotein H2O2: Hydrogen peroxide HIV: Human immunodeficiency virus IDE: Insulin-degrading enzyme iNOS: Inducible nitric oxide synthase JAK: Janus kinase JNK: Jun N-terminal kinase KO: Knockout LOD: Limit of detection MAC1: Macrophage antigen complex-1 MAPK: Mitogen-activated protein kinase List of abbreviations xiii MCP: Monocyte chemoattractant protein mRNA: Messenger ribonucleic acid MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NADPH: Nicotinamide adenine dinucleotide phosphate NF-κB: Nuclear factor-κ B NGF: Nerve growth factor NT: Neurotrophin NTR: NT receptor PBS: Phosphate-buffered saline PD: Parkinson’s disease PI: Propidium iodide PI3K: Phosphatidylinositol 3-kinase PLC-γ1: Phospholipase C-γ1 PRR: Pattern recognition receptors PTP: Protein-tyrosine phosphatase Rac1: RAS-related C3 botulinum toxin substrate 1 RAS: Rat sarcoma RNS: Reactive nitrogen species ROS: Reactive oxygen species SDS: Sodium dodecyl sulfate SH2: Src homology 2 STAT: Signal transducer and activator or transcription TGF: Transforming growth factor TLR: Toll-like receptor xiv I extend my heartfelt gratitude to my supervisor, Dr. Andis Klegeris, whose professional guidance, valuable expertise, voluntary support, and prolonged patience has been instrumental throughout the completion of this project. His professionalism and organizational qualities have enriched my academic growth and allowed me to gain many skills that will be fundamental to success in my future career. His mentorship and encouragement were essential for the completion of my program requirements. I would also like to express thanks to my committee members Dr. Julien Gibon and Dr. Richard Plunkett for their valuable feedback, insight, and direction during the formation of my thesis project, as well as their continued patience during its completion. I am also deeply thankful to my dedicated lab colleagues for their invaluable assistance, insightful discussion, and camaraderie. Their teaching and collaboration increased my understanding of lab research and contributed to my project’s success. A special thanks to Seamus McRae and Christy Richards who completed image analysis for my phagocytosis experiments. Thank you to the members of The Barker Lab for showing me proper microscopy use and techniques. To my parents and family, I express profound appreciation for their love, encouragement, and unwavering belief in my capabilities. Their steadfast support and sacrifices have been paramount in my academic pursuits, and I am grateful for their interests in my project. Last, but certainly not least, I extend heartfelt thanks to my beloved wife Sara. Her enduring love, patience and understanding have been a constant source of motivation throughout this journey. Our physical separation during the completion of my research was made much more bearable by her constant support, encouragement, and positive mentality. I will always be grateful for her strength and sacrifices during this time. Acknowledgements xv To my wife Dedication 1 Neurotrophic factors define a family of growth factors in the central nervous system (CNS) that function to promote the growth, survival and differentiation of neurons. They are also fundamental to normal cognitive function, learning and long-term potentiation [reviewed by (McPhee et al., 2020)]. A subset of neurotrophic factors, called neurotrophins (NTs), have specific growth-promoting effects on neurons such as initiating and regulating neurogenesis [reviewed by (Binder & Scharfman, 2004)](Deister & Schmidt, 2006). The members of the NT family include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, and NT-4 [reviewed by (Binder & Scharfman, 2004)]. In the nervous system, many different cell types synthesize certain NTs. Cytokines released from infiltrating macrophages induce the production of NGF by Schwann cells and fibroblasts. BDNF has been shown to be expressed by several populations of sensory neurons (Huang & Reichardt, 2001). In addition, microglia produce BDNF, NGF, NT-3 and NT-4 (Gomes et al., 2013; Nakajima et al., 2001, 2007; Tiberi et al., 2022; Trang et al., 2011; Tzeng & Huang, 2003). NTs are synthesized as proneurotrophins which require N-terminal cleavage to form a mature protein [reviewed by (Al-Yozbaki et al., 2020)]. The main cell growth, survival and differentiation effects of NTs are achieved by these proteins in their mature form. Intracellular cleavage of proneurotrophins occurs in the endoplasmic reticulum or Golgi at di-basic amino acid residues by enzymes furin or pro-convertase [reviewed by (Chao, 2003; Teng et al., 2010)]. They may also be cleaved extracellularly by several different matrix metalloproteinases and the serine protease plasmin [reviewed by (Al-Yozbaki et al., 2020)](Le & Friedman, 2012; R. Lee et al., 2001). Proneurotrophin precursor forms are important for sorting NTs to constitutive or regulated secretory pathways (R. Lee et al., 2001) and for protein folding through interactions in the amino terminal segment [reviewed by (Chao, 2003)]. However, proneurotrophins can also be released into the extracellular space, where they signal by binding to specific receptors and activate certain cell pathways and processes such as apoptosis (R. Lee et al., 2001). Typically, proneurotrophin signaling occurs to initiate cell death in development or injury. Signaling by proneurotrophins or their mature forms is enabled by the presence of specific NT receptors (NTRs) or receptor complexes (Teng et al., 2010). Chapter 1: Introduction 1.1 Neurotrophins 2 NTs bind to NTRs, which are specific receptor tyrosine kinases that participate in many cell-to-cell communication pathways [reviewed by (F. S. Lee et al., 2001)]. These receptors, known as tropomyosin-receptor kinases or tropomyosin-related kinases (Trk)A, TrkB, and TrkC, function to activate several cell signaling pathways [reviewed by (Binder & Scharfman, 2004; Stoleru et al., 2013)]. Each NT binds one or more of the Trk receptors: NGF activates TrkA; BDNF and NT-4 are ligands of TrkB; and NT-3 preferentially binds TrkC [reviewed by (Al-Yozbaki et al., 2020)]. Upon ligand binding, Trk receptors dimerize, resulting in kinase activation and subsequent receptor autophosphorylation of multiple tyrosine residues. This causes several intracellular target proteins such as Src homology 2 (SH2) domain-containing transforming protein 1 to interact with certain binding sites through their SH2 domains, leading to the activation of intracellular signaling pathways [reviewed by (Al-Yozbaki et al., 2020; Binder & Scharfman, 2004)]. Pathways activated by Trk receptors include rat sarcoma (RAS), phospholipase C-γ1 (PLC-γ1), and phosphatexidylinositol 3-kinase (PI3K), as well as the downstream signaling pathways regulated by these proteins including mitogen activated protein kinases (MAPKs) [reviewed by (Reichardt, 2006; Segal & Greenberg, 1996)]. For example, BDNF binds to TrkB and increases rat sympathetic neuron survival by activating PI3K and MAPK/extracellular signal-regulated kinase (ERK) (Atwal et al., 2000). Meanwhile, NGF improves primary rat neuron survival by activating the RAS/PI3K/protein kinase B (AKT) signal transduction pathway (Kaplan & Miller, 2000). NTs also bind with low affinity to a member of the tumor necrosis factor (TNF) receptor superfamily, p75, known as p75NTR. Therefore, high NT concentrations are required to initiate signaling pathways associated with p75NTR (R. Lee et al., 2001). Binding to this receptor activates nuclear factor-kappa B (NF-κB), leading to expression of pro-survival genes, and Jun N-terminal kinase (JNK), resulting in initiation of apoptotic pathways. Modulation of RAS homology family member A (RhoA) activity also occurs through p75NTR which affects growth cone motility [reviewed by (Al-Yozbaki et al., 2020; Reichardt, 2006)]. Although NTs are low-affinity ligands of p75NTR, proneurotrophins bind to this receptor with higher affinity. For example, proNGF and proBDNF preferentially bind to the apoptosis-inducing p75NTR, while NGF and BDNF are the preferred ligands for TrkA and TrkB, respectively, leading to nerve cell 1.2 NTRs and signaling pathways associated with these receptors 3 growth and survival (R. Lee et al., 2001). This occurs through p75NTR dimerization with sortilin, a type I membrane protein. As a p75NTR coreceptor, sortilin is crucial for proneurotrophin binding and activation of p75NTR [reviewed by (Lewin & Nykjaer, 2014)](Paiardini & Caputo, 2008). In addition to activating signal transduction pathways, p75NTR may dimerize with Trk receptors, which affects ligand binding affinities [reviewed by (Al-Yozbaki et al., 2020; Weiss & Schlessinger, 1998)]. For example, p75NTR interacts with TrkA when co-expressed in Sf9 insect cells through the extracellular and intracellular domains of both receptors and the intracellular domain of p75NTR modulates NGF internalization by TrkA (Gargano et al., 1997). Also, in human A293 cells transfected with rat NTRs, p75NTR has been shown to interact with, and affect the ligand binding affinity of, all three Trk receptors. Co-expression of TrkB with p75NTR leads to increased ligand specificity and affinity of TrkB for BDNF, resulting in TrkB phosphorylation. This p75NTR-TrkB interaction engages both the extracellular and intracellular domains of these two receptors and predominantly involves TrkB with phosphorylated intracellular domains (Bibel et al., 1999). The affinity of TrkA and its responsiveness to low concentrations of NGF is also enhanced by interactions with p75NTR [reviewed by (Yano & Chao, 2000)]. Both NGF and NT-3 are ligands of TrkA, while BDNF, NT-3, and NT-4 activate TrkB; however, in the presence of p75NTR, only NGF and BDNF induce functional responses through their respective Trk receptors [reviewed by (F. S. Lee et al., 2001)](Bibel et al., 1999). The Trk and p75NTR coreceptor system regulates and promotes NT-facilitated neuronal differentiation and survival [reviewed by (Teng et al., 2010)]. Interleukin (IL)-34 is a relatively recently characterized cytokine that belongs to the short-chain hematopoietic cytokine family [reviewed by (Baghdadi et al., 2018)]. IL-34 is synthesized and secreted as a 39 kDa homodimeric glycoprotein consisting of 242 amino acid residues [reviewed by (Ge et al., 2019)]. Many adult human tissues express IL-34 messenger ribonucleic acid (mRNA) including the brain, heart, liver, lung, kidney, spleen, testis, ovaries, thymus, prostate, small intestine, and colon. IL-34 was discovered following activation of the colony-stimulating factor (CSF)-1 receptor (CSF-1R) by a molecule that was identified to be 1.3 Interleukin-34, colony-stimulating factor-1 receptor and signaling pathways 4 different than CSF-1 and later turned out to be IL-34. Binding of biotinylated IL-34 to THP-1 cells is inhibited by IL-34, CSF-1, CSF-1R antibodies and IL-34 antibodies; however, it is not blocked by CSF-1 antibodies. As a result, IL-34 was established as a ligand for CSF-1R (Lin et al., 2008), a type III receptor tyrosine kinase [reviewed by (Bourette et al., 2000)]. IL-34 and CSF-1 differ in their amino acid sequence and overall structure. They bind to different domains on CSF-1R, resulting in distinctive biological activities and cell signaling patterns (Chihara et al., 2010; Segaliny et al., 2015). CSF-1R has a higher affinity for IL-34 than CSF-1, due to relative thermodynamic independence of IL-34, negative cooperativity of CSF-1 and hydrophobic interactions in the IL-34: CSF-1R complex (Liu et al., 2012). In addition to the extracellular domain of CSF-1R, alternative receptors for IL-34 include the chondroitin sulfate chains of receptor‐type protein‐tyrosine phosphatase (PTP)-ζ and syndecan-1. IL-34 has lower affinity for PTP-ζ and syndecan-1, than CSF-1R [reviewed by (Baghdadi et al., 2018)](Jie Zhou et al., 2018). In the CNS, IL-34 is primarily produced by neurons, and CSF-1R is predominantly expressed in microglia. [reviewed by (Hu et al., 2021)](Ma et al., 2012; Mizuno et al., 2011). Neural progenitor cells, several subpopulations of neurons, and capillary endothelial cells of the CNS also express CSF-1R [reviewed by (Bo & Bo, 2022)]. In myeloid cells, IL-34 activates many CSF-1R associated signaling pathways including p38 MAPK, NF-κB, caspase 3/8, ERK1/2, signal transducer and activator of transcription (STAT)3, janus kinase (JAK), JNK, PI3K/AKT, and adenosine monophosphate (AMP)-activated protein kinase-uncoordinated-51-like kinase. This results in modulation of cell motility, adhesion, proliferation, differentiation, migration, polarization, cytokine expression, survival, and autophagy (Boulakirba et al., 2018; Eda et al., 2011; Liu et al., 2012; Wei et al., 2010; Jie Zhou et al., 2018). Clonogenicity, proliferation, and motility result from IL-34 binding PTP-ζ and inducing paxillin and focal adhesion kinase in myeloid cells (Nandi et al., 2013). Syndecan-1 also modulates the activation of intracellular signaling pathways associated with IL-34:CSF-1R binding (Segaliny et al., 2015) and may promote the migration of myeloid cells [reviewed by (Baghdadi et al., 2018; Ge et al., 2019)]. In this way, IL-34 is an important regulator of many cell signaling pathways, especially in cells of the mononuclear phagocytic system including microglia, monocytes, dendritic cells, Langerhans cells, osteoclasts, and Kupffer cells [reviewed by (Baghdadi et al., 2018)]. 5 Microglia are the main immune cells of the CNS. These resident macrophages originate primarily from primitive myeloid progenitors in the embryonic yolk sac (Ginhoux et al., 2010). They are distinct from blood-associated myeloid cells like monocytes, even though both cell types assume many similar functions [reviewed by (Hoeffel & Ginhoux, 2015; Q. Li & Barres, 2018)]. Microglia are separated from the other mononuclear phagocytes by the blood-brain barrier and maintain their populations by self-renewal. Therefore, they function independently of circulating bone marrow-derived progenitor cells (Ajami et al., 2007). Although microglia were once thought as being sedentary bystanders in the healthy CNS, recent findings indicate that they constantly survey and respond to the surrounding tissue microenvironment. Microglia communicate with their environment by extending and retracting processes. Microglial secretory capabilities allow them to maintain healthy brain tissue and its normal function [reviewed by (Q. Li & Barres, 2018)]. They react to changes such as brain tissue injury, neurodegeneration, microbial invasion, and the presence of neurotoxic and inflammatory molecules and debris. Microglia respond to these conditions through their sensome: a cluster of genes that are expressed to produce receptors and proteins that sense changes in the CNS (Hickman et al., 2013). Upon activation, these receptors cause phenotypic changes and microglia enter a reactive state. It was previously recognized that microglia become activated into either an M1 inflammation-inducing or an M2 inflammation-resolving state. However, it is currently acknowledged that this classification system is far too simplistic, and microglia may assume many different reactive states which are determined by transcription-level regulation. It is now accepted that microglia are on a pro-inflammatory-anti-inflammatory spectrum based on their transcriptome [reviewed by (Leng & Edison, 2021)]. Also, disease-associated microglia is now a common term for describing pro-inflammatory microglia with down-regulated homeostatic genes and upregulated genes involved in lysosomal, phagocytic and lipid metabolism pathways (Deczkowska et al., 2018). The microglial pro-inflammatory response is often a result of the activation of pattern recognition receptors (PRRs), which bind to damage- or pathogen-associated molecular patterns [reviewed by (Heneka et al., 2015)]. The pathways associated with these receptors are critical for 1.4 Microglia and neuroinflammation 6 an acute neuroinflammatory response allowing destruction or removal of infectious or neurotoxic agents, which minimizes neuron damage. For example, lipopolysaccharide, a molecule found in the cell membranes of bacteria, binds and activates microglial toll-like receptor (TLR)4, resulting in an inflammatory response [reviewed by (Heneka et al., 2015)]. Microglia then release inflammatory cytokines and cytotoxic molecules that recruit other immune cells and destroy the pathogenic or damaging species. In some instances, however, microglia become chronically activated resulting in continued release of pro-inflammatory and cytotoxic molecules. Large concentrations of these harmful molecules can eventually lead to widespread neuroinflammation and neuron death [reviewed by (Martin-Estebane & Gomez-Nicola, 2020)]. Depending on the stimulus and activation state, microglia may perform pinocytosis, phagocytosis, or endocytosis and release proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF, chemokines such as monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory protein-1, or cytotoxic molecules such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Da Pozzo et al., 2019; K. Zhang et al., 2018)[reviewed by (Franco & Fernández-Suárez, 2015). For example, in Alzheimer’s disease (AD), microglia are chronically activated to release many of these molecules as well as perform phagocytosis of amyloid-β, a protein that accumulates in the CNS of AD patients [reviewed by (Leng & Edison, 2021)]. The NTs BDNF and NGF have numerous effects on immune cells. For example, NGF acts on monocytes and macrophages and affects their differentiation, phagocytic activity, IL-1β production, chemotaxis, survival, reactive oxygen and nitrogen species production, and TNF secretion [reviewed by (Vega et al., 2003)]. Importantly, BDNF and NGF also have several notable effects on microglia. For example, NGF- and BDNF-treated BV-2 murine microglia display increased proliferative capacity and survival, which is suppressed by a tyrosine kinase inhibitor (J. Zhang et al., 2003). In models of damage or disease, NTs typically steer microglia towards an anti-inflammatory phenotype. BDNF treatment reduces the neurotoxic effect of lipopolysaccharide-treated BV-2 microglia by inhibiting critical inflammatory regulating kinases, such as p38 MAPK and JNK, and reducing the secretion of inflammatory cytokines TNF and IL-6. Down-regulation of TrkB prevents this inhibitory action of BDNF (S. Y. Wu et al., 2020). Another study shows that BDNF upregulates primary murine microglia phagocytic 1.5 Effects of neurotrophins on microglia 7 activity and the expression of anti-inflammatory cytokine IL-10, but decreases the expression of pro-inflammatory TNF by binding to TrkB and p75NTR (Jiang et al., 2011). By engaging the TrkA, AKT, and Jun signaling pathways, NGF promotes the internalization of soluble amyloid-β oligomers by macropinocytosis in primary mice microglia and increases the expression of neuroprotective genes (Rizzi et al., 2018). Also, agonists of TrkA provide protection from apoptosis and increase the expression of anti-apoptotic B-cell lymphoma-2 (Bcl-2) family members in monocytes (la Sala et al., 2000). Lastly, NGF delivered to ischemic regions in mice results in a polarization shift in microglia from a pro-inflammatory to an anti-inflammatory phenotype (J. Yang et al., 2020). These effects show that NTs BDNF and NGF modulate the microglial inflammatory response; however, their large size and widespread side effects make it difficult to use these NTs therapeutically. Shortly after its discovery in 2008, IL-34 was identified as a very important signaling molecule for the development, proliferation, and maintenance of microglia [reviewed by (Martin-Estebane & Gomez-Nicola, 2020)]. As mentioned earlier, in the CNS, IL-34 is primarily produced by neurons, and CSF-1R is predominantly expressed in microglia. [reviewed by (Hu et al., 2021)](Ma et al., 2012; Mizuno et al., 2011). CSF-1R has protein kinase C activity and its production is increased in locations of CNS injury or pathology (Murphy et al., 2000; Raivich et al., 1998; Sawada et al., 1990). IL-34 and CSF-1R are also important because of their function as part of the microglial sensome which involves detecting changes in the environment and allowing a reaction to many physiological and pathological conditions (Hickman & El Khoury, 2019). Overall, there are much conflicting data surrounding CSF-1R expression and the effects of IL-34 in microglia. For example, CSF-1R deficiency and abundance cause microglial activation (J. Luo et al., 2013; Murphy et al., 2000). Walker et al., (2017) find that IL-34 causes a shift in microglia towards a pro-inflammatory phenotype. In this study, 18 of 31 pro-inflammatory genes are upregulated, 8 are unchanged, and 5 are downregulated by IL-34 as opposed to anti-inflammatory genes of which 10 of 33 are upregulated, 5 are unchanged, and 13 are downregulated. However, the consensus is that IL-34 causes neuroprotective and anti-inflammatory effects in microglia. IL-34-treated lipopolysaccharide-stimulated primary mouse microglia have reduced pro-inflammatory cytokine secretion as the levels of IL-6, IL-1β, and 1.6 Effects of IL-34 on microglia 8 TNF are reduced. Additionally, these cells have increased anti-inflammatory activity as there is an increase in heme oxygenase-1 and arginase-1 mRNA expression (P. W. Lee et al., 2020). IL-34 increases the microglial production of transforming growth factor-β which is reported to reduce superoxide radical production by heme oxygenase-1 and increase amyloid-β degradation and clearance by the insulin-degrading enzyme (IDE) (Ma et al., 2012; Mizuno et al., 2011). These effects describe a neuroprotective phenotype in IL-34-treated microglia and warrant studies on other microglial immune functions under these conditions, which is an objective of my thesis work. LM11A-31 is a water-soluble isoleucine derivative that activates the p75NTR and is a competitive inhibitor of NGF. Similar to NGF, LM11A-31 increases neuronal survival and prevents cell death. These effects are suppressed by p75NTR inhibitors and blocking antibodies (Knowles et al., 2013; Massa et al., 2006). In the presence of damaging stimuli or neurotoxins, LM11A-31 causes the activation of survival-promoting pathways in neurons involving AKT, NF-κB, and cyclic-AMP (cAMP)-response element binding protein (CREB). It also blocks the activation of harmful signaling pathways involving glycogen synthase kinase-3β, cyclin-dependent kinase 5, and JNK (Massa et al., 2006; T. Yang et al., 2008a; G. N. Yin et al., 2021). Many studies report that LM11A-31 reduces the activation of microglia in animal models of human diseases including human immunodeficiency virus (HIV)-infection, Huntington’s disease, AD, cavernous nerve injury, traumatic brain injury, and other tauopathies (James et al., 2017; Nguyen et al., 2014; Shi et al., 2013; Simmons et al., 2016, 2018; Xie et al., 2020, 2021; J. Yang et al., 2020; G. N. Yin et al., 2021). In several of these models, the LM11A-31-induced decrease in microglial activation may lead to improved motor and cognitive performance, reduced neuroinflammation, decreased neuron death or damage, and a restoration of normal neuron physiology (Meeker et al., 2016; Simmons et al., 2018; Xie et al., 2020). In addition, the decline in microglial activation following LM11A-31 treatment may also lead to lower levels of pro-inflammatory cytokines IL-6, TNF, interferon-γ inducible protein 10 (CXCL10), IL-2, IL-1β, and IL-5 (Simmons et al., 2018, 2021). As a result, LM11A-31 contributes to neuroprotection due to its action on microglia (Meeker et al., 2012). However, the specific effects of LM11A-31 1.7 LM11A-31 9 on the inflammatory processes of microglia have not been studied; therefore, my thesis work addresses these knowledge gaps. BDNF can produce contrasting results in the CNS compared to its immature precursor proBDNF (Mizoguchi et al., 2021). Whereas BDNF binds to TrkB on neurons and promotes neuron survival, proBDNF activates p75NTR and downstream apoptotic pathways [reviewed by (Fitting et al., 2020; Mizoguchi & Monji, 2017b)]. This occurs through p75NTR interacting with the mature domain of proBDNF and its coreceptor sortilin binding to the pro-domain (Chen et al., 2005). Microglia are one of the main proBDNF-producing cells in the CNS and its expression is altered by their activation (Duan et al., 2013; Mohammadi et al., 2020; Y. Wang et al., 2017). Microglia also express p75NTR which is upregulated upon infection-induced neuroinflammation. p75NTR alters the activation of immune cells in the brain and an increased expression of surface marker major histocompatibility complex II is observed in microglia of p75NTR knockout (KO) mice. However, microglial phagocytic activity and expression of inducible nitric oxide synthase (iNOS) is unchanged in p75NTR KO mice (Düsedau et al., 2019). Microglia also respond to proBDNF. proBDNF binds to the p75NTR/sortilin complex and activates RAS-related C3 botulinum toxin substrate 1 (Rac1), causing increased intracellular calcium levels and p75NTR expression in murine 6-3 and primary microglial cells. This study also reports no changes in the expression of pro-inflammatory phenotype markers TNF, IL-1β, IL-6, and leukocyte common antigen cluster of differentiation (CD)45 or anti-inflammatory markers IL-10, transforming growth factor (TGF)-β, and arginase following treatment with 20 ng/ml proBDNF for 12 h. However, further research with varying concentrations of proBDNF and their effects on other microglial immune responses is warranted. Another study shows that in human peripheral monocytes, exogenous proBDNF causes an upregulation of pro-inflammatory cytokines IL-1β, IL-6, and TNF and induces the infiltration of additional inflammatory cells (W. Shen et al., 2020). proBDNF is decreased in early AD and correlates with the loss of cognitive function. This may contribute to the activation of microglia and resulting neuron death (Peng et al., 2005). Considering these effects of proBDNF on microglia, monocytes, and in neuroinflammatory diseases, further research into its modulation of other microglial immune responses is needed. Since BDNF has numerous effects on microglia and because proBDNF 1.8 proBDNF 10 tends to produce opposite results to those of BDNF, understanding the response of microglia to proBDNF would advance our knowledge of p75NTR signaling as well. Therefore, I studied whether proBDNF modulates microglial neurotoxicity and several other immune functions such as phagocytosis and the production of MCP-1, RNS and ROS. Bis(N-succinyl-L-glutamyl-L-lysine) hexamethylenediamide (GK-2) is a dipeptide mimetic of the TrkA interacting domain of NGF, loop 4. GK-2 has a low molecular weight of 830 Da allowing it to cross the blood-brain barrier and be resistant to peptidases (Antipova et al., 2011). Similar to NGF, GK-2 activates TrkA and the PI3K/AKT pathway associated with neuroprotection; however, it does not trigger the MAPK cascade which contributes to hyperalgesia in vivo (Gudasheva et al., 2019). Although there are no data on the direct effect of GK-2 on microglia, there are many studies investigating its effect in animal models of neurotoxicity and CNS pathology. GK-2 treatment causes neuroprotection and increases the survival of neurons following zinc ion-, hydrogen peroxide (H2O2)-, glutamate-, or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity (Antipova et al., 2011; Seredenin & Gudasheva, 2015; Stelmashook et al., 2015, 2020). Similarly, GK-2 treatment reduces neurodegeneration, neuron damage and neurological deficit in AD, Parkinson’s disease (PD), traumatic brain injury, middle cerebral artery occlusion, cardiac arrest, haemorrhagic stroke, and cerebral ischemia animal models. In these models, GK-2 also promotes neurogenesis, synaptogenesis and likely also gliogenesis (Genrikhs et al., 2018; Gudasheva et al., 2019; Kraineva et al., 2013; Povarnina et al., 2013, 2017; Seredenin & Gudasheva, 2015; Zarzhetskii et al., 2015). Although GK-2 has neuroprotective effects in in vivo models of several neuropathologies, its in vitro effects on microglial neuroprotective activity or microglia-driven immune processes are not known. These knowledge gaps are addressed in my thesis work. To avoid the poor pharmacokinetic properties and undesirable side effects of BDNF, a dipeptide mimetic of the β-turn of BDNF loop 4, hexamethylenediamide bis-(N-monosuccinyl-L-seryl-L-lysine) (GSB-106), was constructed. GSB-106 activates the TrkB receptor and associated signaling pathways including MAPK/ERK, PI3K/AKT and PLC-γ1 (Gudasheva et al., 2021; Zainullina et al., 2019). Although the effects of GSB-106 on microglia are unknown, this 1.9 GK-2 1.10 GSB-106 11 BDNF mimetic has numerous effects on neurons. In vitro, GSB-106 increases the survival of human and murine neuronal cells following toxic insult, induction of apoptosis, or oxidative stress by 6-hydroxidopamine, glutamate, serum-deprivation, or H2O2 (Gudasheva et al., 2013; Logvinov et al., 2013). This neuroprotective effect may be due to the activation of TrkB and subsequent inactivation of the pro-apoptotic B-cell lymphoma (Bcl)-2 associated death promoter (BAD) protein and suppression of caspases 9 and 3/7 (Zainullina et al., 2021). In in vivo murine models of CNS pathologies such as middle cerebral artery occlusion-induced ischemic stroke and major depression, treatment with GSB-106 results in neurogenesis, synaptogenesis, gliogenesis, and anti-depressant effects (Gudasheva et al., 2017, 2018). For example, in rat stroke models, GSB-106 causes a 66% or 24% reduction in brain damage volume, depending on time between middle cerebral artery occlusion and treatment administration (Gudasheva et al., 2016, 2021). Also, in models of mice with major depression, GSB-106 restores reduced locomotor activity and eliminates anhedonia by restoring synaptophysin and CREB levels in the hippocampus by activation of TrkB and PLC-γ1 (Gudasheva et al., 2021). As a result, GSB-106 has many BDNF-like properties such as promoting the survival of neuronal cells, supporting neurogenesis, and its anti-depressant effects. These previous reports had not investigated whether microglia have a role in mediating the actions of this drug on the survival of neurons, so I studied the effects of GSB-106 on microglia. 12 Figure 1. TrkA/B, p75NTR and CSF-1R ligands and intracellular signaling pathways The study of neurobiology and neuroinflammation in vivo is difficult due to cost and ethical issues surrounding these experiments. As a result, many in vitro models have been discovered and are utilized. Typically, primary glial cells and neurons are the first choice; however, a plethora of concerns surround cell extraction and culturing such as the difficult preparation process and slow proliferation rates [reviewed by (Stansley et al., 2012)]. In addition, culturing and isolation processes may affect the function of the cells. For example, primary microglia that are isolated from extracted mixed glial cells often phagocytose neuronal debris, affecting their function and activation state (Henn et al., 2009). Immortalized cell lines are a more efficient model for studying the CNS functions due to faster proliferation rates, more homogenous cell populations, and lower culturing costs. BV-2 murine microglia are a good alternative to primary microglia for studying cell-to-cell interaction and neuroinflammation. They can be induced to display diverse phenotypes. BV-2 microglia were created by infecting primary microglia with a v-raf/v-myc retrovirus to increase proliferation, and are similar to primary cells in some aspects such as their phagocytic activity, 1.11 Ligands and signaling pathways of TrkA/B, p75NTR and CSF-1R 1.12 Cell lines 13 activation states, expressed antigens, and certain secreted factors (Blasi et al., 1990; Henn et al., 2009)[reviewed by (Stansley et al., 2012)]. For example, BV-2 cells are activated by lipopolysaccharide binding to TLR4, leading to the production of pro-inflammatory cytokines and chemokines. Although they differ in the secretion of TNF, IL-6, IL-1β, and MCP-1, they model primary microglia well in migration, ionized calcium binding adaptor molecule 1expression and nitric oxide release (Horvath, Nutile-Mcmenemy, et al., 2008). In addition, like primary microglia, BV-2 cells can phagocytose apoptotic and necrotic cells as well as debris (Hirt & Leist, 2003). The phagocytic activity of BV-2 microglia can be measured experimentally by the uptake of latex microspheres. Kopec and Carroll (1998) show that BV-2 microglia upregulate phagocytosis of latex beads 200% following activation by free amyloid-β42. Surface receptor expression is also similar in BV-2 cells compared to primary microglia. This includes the expression of TLR4 (Dai et al., 2015), TrkA, TrkB (J. Zhang et al., 2003; Jingbin Zhou et al., 2020), p75NTR (Rizzi et al., 2018), sortilin (Du et al., 2022), and CSF-1R (Butovsky et al., 2014; Delaney et al., 2021; Hagan et al., 2020). However, since BV-2 cells do lack some characteristics of primary microglia, other microglia-like cell types may be necessary to study certain functional aspects of this cell type. The human THP-1 monocytic cell line were spontaneously immortalized from peripheral blood monocytes of a child with acute monocytic leukemia [reviewed by (Bosshart & Heinzelmann, 2016). The use of THP-1 cells avoids species differences and incompatibilities and these cells model human microglial immune responses well, such as in the release of pro-inflammatory and neurotoxic molecules (Balon & Wiatrak, 2021; Combs et al., 2000). For example, they produce human MCP-1, a known secreted factor in the microglial immune response (Kolitz et al., 2015). THP-1 cells express Trk receptors, p75NTR (Datta-Mitra et al., 2015; Z. Li et al., 2009; Sniderhan et al., 2009), sortilin (Lv et al., 2019) and CSF-1R (Y. Li & Chen, 1997). The human leukocyte (HL)-60 cell line is a promyelocytic cell line derived from the blood of a patient with acute myeloid leukemia. Dimethyl sulfoxide (DMSO) differentiation causes HL-60 cells to acquire a granulocyte neutrophil-like phenotype (Birnie, 1988; Levy et al., 1990; J. Wu et al., 1990). These cells produce a large respiratory burst following stimulation with chemotactic factors such as N-formyl peptides. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in DMSO-differentiated HL-60 cells, similar to 14 microglia, results in the production of superoxide, which is released during a respiratory burst (Tardif et al., 1998). HL-60 cells also express TrkA (Kaebisch et al., 1996), TrkB (Sun et al., 2014; Takeda et al., 2016), p75NTR (Rende et al., 2006), sortilin (Dao et al., 2020), and CSF-1R (Cellier et al., 1997; Follows et al., 2003; Hsu et al., 1993). SH-SY5Y cells were subcloned from the originally derived neuroblastoma parental line SK-H-SH obtained from a human metastatic bone cancer biopsy. This cell line is commonly used as a neuronal model in studies involving neurotoxicity and neurodegeneration (Pezzini et al., 2017). Undifferentiated SH-SY5Y cells closely resemble immature catecholaminergic neurons (Kovalevich & Langford, 2013). They also lack some receptors of primary neurons such as Trk receptors (Goldie et al., 2014; Kaplan et al., 1993). The use of these cell lines in the experimental assays I employed was beneficial since this approach generated informative data; however, there are several limitations to these models. Further research using other human cell types would be appropriate to confirm my results. Furthermore, the use of 2D and 3D mixed cultures or brain organoids would allow observation of the effects of IL-34, LM11A-31, proBDNF, GK-2 and GSB-106 on microglia in environments similar to the CNS. The goal of this research is to contribute to the expanding knowledge of microglial immune functions and specific molecules that affect those processes. Identifying compounds that alter specific inflammatory states of microglia may also increase the understanding of microglial regulatory mechanisms. Finally, discovering if IL-34, LM11A-31, proBDNF, GK-2 and GSB-106 reduce the pro-inflammatory activation of microglia and microglia-like cells may lead to future therapeutic advancements. IL-34, p75NTR ligands, and TrkA/B ligands regulate select neuroinflammatory processes of microglia and microglia-like cells 1.13 Research aims 1.14 Hypothesis 15 Calf bovine serum (CBS), Dulbecco’s modified Eagle medium/nutrient mixture F-12/Ham (DMEM/F-12), phenol red-free DMEM/F-12, penicillin (10000 U/ml)-streptomycin (10 mg/ml) stock solutions, 0.05% and 0.25% trypsin with ethylenediaminetetraacetic acid, N,N-dimethylformamide, sodium dodecyl sulfate (SDS), Tween 20, and one-step slow 3,3’,5,5’-tetramethylbenzidine-enzyme-linked immunosorbent assay (ELISA) substrate solution, ortho-phosphoric acid, and 0.4% Trypan Blue solution were supplied by Thermo Fisher Scientific (Ottawa, ON, Canada). Lipopolysaccharide, phosphate-buffered saline (PBS) tablets, propidium iodide (PI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), N-formyl-methionine-leucyl-phenylalanine (fMLP), luminol sodium salt, bisbenzimide (Hoechst 33258), Triton™ X-100, sodium nitrite, sulfanilamide, N-(1-933 naphthyl)ethylenediamine dihydrochloride, and DMSO were obtained from Sigma Aldrich (Oakville, ON, Canada). PeproTech (Embrun, ON, Canada) provided human recombinant interferon-γ and IL-34 as well as ELISA development kits for MCP-1. Cedarlane Laboratories (Burlington, ON, Canada) supplied recombinant mouse IL-34. Externally-labelled fluorescein isothiocyanate (FITC) one μm fluorescent polystyrene latex beads were purchased from Bangs Laboratories (Fishers, IN, USA). GK-2 and GSB-106 were acquired as a gift from T.A. Gudasheva, Zakusov Research Institute of Pharmacology, Moscow, Russian Federation. Recombinant mouse proBDNF protein was obtained from Alomone Labs (Jerusalem, Israel). LM11A-31 was synthesized at McGill University (Montreal, QC, Canada) by Dr. Alexey Kostikov. Sorvall RT1 and Legend Micro 17 centrifuges supplied by ThermoFisher Scientific were used to centrifuge cell suspensions. Sterile procedures were performed in a class II type A2 biosafety cabinet from Labconco Corp. (Kansas City, MO, USA). Sterile 24- and 96-well cell culture plates were supplied by Corning (Corning, NY, USA) and used for seeding of cell cultures and spectrophotometric assays. Sterile four-well CELLview culture dishes used for phagocytosis assays were supplied by Greiner Bio-one (Frickenhausen, Germany). T-25 and T-75 cell culture flasks were purchased from VWR International (Mississauga, ON, Canada). Chapter 2: Materials and Methods 2.1 Chemicals and reagents 2.2 Equipment and supplies 16 ThermoFisher Scientific provided sterile 10 cm cell culture dishes and the Model #370 Steri-Cycle High-Efficiency Particulate Air Class 100 CO2 incubator used for ensuring optimal cell growth conditions. Cell counting was performed on a hemocytometer purchased from ChangeBioscience (Castro Valley, CA, USA). A FLUOstar Omega microplate reader supplied by BMG Labtech (Napean, ON, Canada) was used for spectrophotometric, fluorescent and chemiluminescent measurements. Emitted fluorescence of cells was visualized using the Axio Observer.Z1 widefield epifluorescence microscope supplied by Zeiss (Oberkochen, Germany) and imaged using Zen image acquisition software (version 2.0). Fluorescence intensity measurements were completed using ImageJ (version 1.53a) provided by the National Institute of Health (Bethesda, Maryland, USA). The human SH-SY5Y neuroblastoma cell line was donated by Dr. R. Ross (Department of Biological Sciences, Fordham University, Bronx, NY, USA). Dr. G. Garden (Department of Neurology, University of Washington, Seattle, WA, USA) donated the murine BV-2 microglia cell line. The human THP-1 monocytic and HL-60 myelomonocytic cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All immortalized cell lines were maintained in DMEM/F-12 supplemented with 10% CBS (F10), penicillin (100 U/ml) and streptomycin (100 μg/ml) and incubated at 37 ºC in humidified 5% CO2 and 95% air atmosphere in T-75 flasks. Adherent SH-SY5Y and BV-2 cells were detached from the flasks using 0.05% and 0.25% trypsin-ethylenediaminetetraacetic acid solutions, respectively. Long-term cell line storage occurred in liquid nitrogen using DMEM/F-12 media containing 20% CBS, 10% DMSO, penicillin (100 U/ml) and streptomycin (100 μg/ml). All cell culturing procedures were performed in a sterile class II type A2 biosafety cabinet. Unless otherwise stated, experiments were performed by using DMEM/F-12 supplemented with 5% CBS, penicillin (100 U/ml), and streptomycin (100 μg/ml) (F5) and cell cultures were incubated at 37 ºC in humidified 5% CO2 and 95% air atmosphere. Cell viability assessment was based on the ability of metabolically active cells to reduce the water-soluble tetrazolium dye, MTT, to an insoluble purple formazan product, through NADPH coenzyme and dehydrogenases (Stockert et al., 2018). Increased production of purple 2.3 Cell cultures 2.4 Cell viability assay 17 formazan indicated higher metabolic activity or increased number of viable cells per sample. To perform this assay MTT (0.5 mg/ml) was added to culture wells prior to incubation for one h at 37 ºC and 5% CO2. The insoluble formazan product was then dissolved for 3 h with a volume of SDS (20% w/v)/N,N-dimethylformamide (50% v/v) equal to the volume of the media in the culture well. A sample of the well solution was then added to a 96-well plate and optical densities were measured at 570 nm using the Vmax kinetic microplate reader (Molecular Devices, San Jose, CA, USA). Measurements were normalized to the optical density of wells containing untreated cells incubated in the presence of growth medium alone and data are presented as % cell viability using the following formula: % 𝐶𝑒𝑙𝑙 𝑉𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 = 𝑂𝐷!"#$%& − 𝑂𝐷%'!(!𝑂𝐷%()& − 𝑂𝐷%'!(! × 100 where ODsample = optical density of cell samples with respective treatment, ODlysis = the optical density of unviable cells, and ODlive = the optical density of untreated cells. PI was used to assess cell death through its fluorescence when bound to DNA. PI is not capable of crossing intact cell membranes but could give misleading measurements due to free DNA in sample solutions. PI (20 μg/ml) was added to culture wells and incubated for 15 min at room temperature. Fluorescence intensity was measured using 485/625nm excitation/emission filters by the FLUOstar Omega microplate reader. Values were normalized to readings from culture wells with no viable cells achieved by the addition of 1:100 Triton™ X-100. Data are presented as percent cell death. THP-1 monocytic cells were used as a model of microglia and stimulated to release cytotoxic factors. SH-SY5Y neuroblastoma cells were used as a model of human neurons to detect the neurotoxicity of THP-1 monocytic cell supernatant. One ml of THP-1 cell suspension at a concentration of 5x105 cells/ml was plated in 24-well plates. THP-1 cells were incubated with growth factors for 24 h prior to stimulation with lipopolysaccharide (200 ng/ml) and interferon-γ (150 U/ml) for 48 h. SH-SY5Y cells were plated in 24-well plates at a volume of 400 μl and concentration of 4x105 cells/ml. Following 24 h incubation to allow adherence, the supernatant was removed by aspiration and 400 μl of unstimulated/stimulated and 2.5 Cell death assay (data not shown) 2.6 THP-1/SH-SY5Y supernatant transfer 18 untreated/treated THP-1 supernatant were added to the SH-SY5Y culture wells. THP-1 cell viability and death (data not shown) were measured at the time of transfer and, following a 72 h incubation period, the viability of SH-SY5Y cells was also assessed. Viability values were normalized to those obtained from the samples that received growth medium alone for THP-1 cells and to samples that received supernatant from unstimulated THP-1 cells for SH-SY5Y plates. MCP-1 is an inflammatory cytokine commonly secreted by activated monocytes and microglia. The secretion of MCP-1 by THP-1 monocytic cells was used as a model of chemokine secretion by microglia. THP-1 cells were plated at a concentration of 5x105 cells/ml in 24-well plates at a volume of one ml. Cells were incubated with growth factors for 24 h and then stimulated with lipopolysaccharide (200 ng/ml) and interferon-γ (150 U/ml). Following a 48 h incubation period, cell-free supernatant was removed for use in an ELISA. THP-1 cell supernatants were analyzed for the presence of MCP-1 using PeproTech ELISA development kits according to the manufacturer’s specifications. Concentrations of MCP-1 in supernatant were calculated by comparing optical densities to a standard curve for MCP-1 using the following formula: [𝐶𝑦𝑡𝑜𝑘𝑖𝑛𝑒] = 𝑂𝐷 − 𝑏𝑚 where b and m represent the intercept and slope of the trend line, respectively. The limit of detection (LOD) of the ELISA was calculating using the following equation and the mean OD (ODmean) of the wells containing 0 μM of MCP-1 in F5: 𝐿𝑂𝐷 = [𝑂𝐷#&"* + (2 × 𝑆𝑇𝐷𝐸𝑉 𝑂𝐷#&"*)] − 𝑏𝑚 where STDEV = standard deviation. BV-2 murine microglia were used to analyse the secretion of nitric oxide by microglia. Nitric oxide produced by microglia is quickly broken down into nitrite in cell cultures, which is quantified by the Griess assay. A 500 μl volume of BV-2 cell cultures were plated in 24-well plates at 2x105 cells/ml and incubated for 24 h to allow cells to adhere. Supernatant was then 2.7 ELISA 2.8 Griess assay 19 removed from the wells and replaced with an equal volume of fresh F5. One h later, cells were treated and incubated for 24 h with growth factors and then stimulated with 200 ng/ml lipopolysaccharide. 24 h later, nitrite concentrations were measured by transferring 50 μl cell supernatant to a 96-well plate (Corning) and adding an equal volume of Griess reagent containing 2% sulfanilamide and 5% ortho-phosphoric acid (compound A), and 0.2% N-1-naphthylethylenediamine dihydrochloride (compound B). The remaining supernatant was then used in cell viability and cell death assays. Optical density was measured at 550 nm using the microplate reader and compared to a standard curve (0-50 μM) for nitrite concentrations using the following equation: [𝑛𝑖𝑡𝑟𝑖𝑡𝑒] = 𝑂𝐷 − 𝑏𝑚 where b represents the y-intercept and m the slope of the trend line. The LOD of each Griess assay was calculated using the mean OD (ODmean) of the wells containing 0 μM sodium nitrite in F5 and the following equation: 𝐿𝑂𝐷 = [𝑂𝐷#&"* + (2 × 𝑆𝑇𝐷𝐸𝑉 𝑂𝐷#&"*)] − 𝑏𝑚 where STDEV = standard deviation. The phagocytic activity of BV-2 murine microglia is well documented. This activity can be quantified by the emission of light from cells that have internalized fluorescent latex beads. BV-2 cells were plated at 5x104 cells/ml in glass-bottom 4-well cell dishes at a volume of 500 μl. 24 h later cells were incubated for 15 min with growth factors prior to being stimulated for 24 h with 400 ng/ml lipopolysaccharide. One μm diameter FITC-labelled latex beads were added at a ratio of 10:1 bead:cell and incubated for one h. Fluorescence of free beads was quenched with trypan blue at a final culture well concentration of 0.05%. Cells were washed with 0.5 ml PBS and fixed for 5 min with cold 70% ethanol. Cells were again washed with 0.5 ml PBS and nuclei were stained with 2 μg/ml bisBenzimide in 0.5 ml PBS. Viable cells, as determined by trypan blue exclusion, were visualized with Zeiss AxioObserver.Z1 widefield epifluorescence microscope with excitation/emission of 350/470 nm for bisbenzimide and 470/520 nm for the fluorescent beads and were imaged blindly by colleagues from our laboratory using Zen image acquisition software (version 2.0). Approximately 10 images were captured from each 2.9 Phagocytosis assay 20 experimental sample and the mean fluorescence intensity of 40 live cells (determined by trypan blue exclusion) was quantified in a blinded manner and normalized to the background fluorescence using ImageJ (version 2.0.0) software. Some of the main cytotoxins secreted by microglia are ROS, which contribute to neuron death in many diseases characterized by chronic microglia activation. HL-60 myelomonocytic cells differentiated with DMSO acquire a neutrophil-like phenotype. These cells express NADPH oxidase enzyme subunits and acquire ROS production capabilities similar to that of activated microglia. This enabled me to stimulate the release of ROS through a respiratory burst in HL-60 cells to model ROS release in microglia. HL-60 cells were grown in T-25 flasks and 15-20 ml of cells in F5 were plated in a 12 cm culture dish (ThermoFisher Scientific, Ottawa, ON, Canada) at 2x105 cells/ml. These cultures were differentiated with 1.3% DMSO for 6 days prior to plating one ml of 1x106 cells/ml in wells of a 24-well plate. Cells were treated with growth factors for 15 min and then primed with 500 ng/ml lipopolysaccharide for 24 h to increase the response of these cells to subsequent stimulation with fMLP. 0.5 ml of cell culture was removed from the wells and microcentrifuged, while the remainder of the cultures were used in cell viability and death assays. Cells were suspended to a concentration of 1x106 cells/ml in phenol red-free DMEM/F-12 containing 2% CBS, penicillin (100 U/ml) and streptomycin (100 ng/ml). 85 μl of each cell culture were then transferred to a 96-well plate for 30 minutes. Chemiluminescence was measured at intervals of 21 s, known as a cycle, for a 60-cycle period. 10 μl luminol (10 μg/μl in PBS) was added at cycle 5, which is oxidized by ROS, causing chemiluminescence. Five μl fMLP (20 μM in PBS) was added at cycle 15 to stimulate a respiratory burst of ROS by HL-60 cells. Mars Analysis Software (BMG Labtech) was used to analyze plotted measurements over the 60-cycle period and data was recorded as chemiluminescence intensity using the following equation: 𝐶𝐻𝐿 = 𝐶𝐻𝐿+,-)&. − 𝐶𝐻𝐿/"!&%(*& where CHL(curve2) represents the area under the second curve following cycle 15 (fMLP addition) and CHL(baseline) represents the area under the first curve observed between cycle 5 (luminol addition) and cycle 15 (curve2). This allows for the correction of baseline levels of ROS present in cell supernatants. 2.10 Respiratory burst assay 21 Statistical analyses of all data were conducted using IBM SPSS Statistics (Version 24.0, IBM Corp., Armonk, NY, USA). To determine differences across treatment groups, all data were subjected to a one-way analysis of variance (ANOVA). If the one-way ANOVA showed significant differences between treatment groups, a Dunnett’s post-hoc test was performed to determine which individual treatment groups differed from the control in all data except for the phagocytosis assay data. The phagocytosis assay data were subjected to a Tukey’s post hoc test to compare all individual treatment groups and determine any significant differences between them. Data are presented as means ± SEM and significance was declared when P < 0.05. 2.11 Statistical analyses 22 Neuroinflammatory processes including the overall neurotoxicity, MCP-1 secretion, ROS production, RNS release, and phagocytic activity of unstimulated and stimulated microglia and microglia-like cells was assessed following treatment with IL-34. I assessed whether IL-34 affects the neurotoxic effect and secretion of MCP-1 by unstimulated and immune-activated THP-1 human monocytic cells. Cultured THP-1 cells were stimulated with lipopolysaccharide, interferon-γ, or lipopolysaccharide plus interferon-γ in the absence or presence of IL-34 (10-100 ng/ml). Supernatant from THP-1 cells was then transferred to SH-SY5Y human neuronal cells. When measured at the time of transfer, IL-34, at the highest concentration (100 ng/ml), increased the viability of unstimulated, lipopolysaccharide-, interferon-γ- and lipopolysaccharide plus interferon-γ-stimulated THP-1 cells (Fig.2A-D). Figures 2E-H demonstrate that lipopolysaccharide and interferon-γ alone caused negligible changes to the cytotoxicity of THP-1 cells towards SH-SY5Y neuronal cells; however, a combination of both stimuli caused a greater than 50% decrease in SH-SY5Y viability following the addition of THP-1 supernatant. IL-34 (10-100 ng/ml) did not affect the cytotoxicity of either unstimulated (Fig.2E) or immune-stimulated (Fig.2F-H) THP-1 cells towards SH-SY5Y cells. Figure 2I illustrates that unstimulated THP-1 cells did not secrete detectable levels of MCP-1. Lipopolysaccharide and interferon-γ induced the secretion of this chemokine by THP-1 cells, especially in combination (Fig.2J-L). IL-34 (100 ng/ml) significantly decreased the secretion of MCP-1 by THP-1 cells stimulated with lipopolysaccharide plus interferon-γ (Fig.2L), and a trend towards decreased MCP-1 secretion was observed in THP-1 cells stimulated with inteferon-γ alone (Fig.2K). Chapter 3: IL-34 3.1 IL-34 hypothesis 3.1.1 Hypothesis #1: IL-34 regulates select neuroinflammatory processes of microglia and microglia-like cells. 3.2 IL-34 results 3.2.1 Cytotoxicity and chemokine secretion of human THP-1 microglia-like cells 23 0 ng/mL10 ng/mL50 ng/mL100 ng/mL050100150% THP-1 ViabilityLPS (200 ng/ml) - - - - IFN-γ (150 U/ml) - - - -IL-34 (ng/ml) 0 10 50 100N=5F=10.8P=0.001**A0 ng/mL10 ng/mL50 ng/mL100 ng/mL050100150% THP-1 ViabilityLPS (200 ng/ml) + + + +IFN-γ (150 U/ml) - - - -IL-34 (ng/ml) 0 10 5 100N=5F=17.2P<0.001***B0 ng/mL10 ng/mL50 ng/mL100 ng/mL050100150% THP-1 ViabilityLPS (200 ng/ml) - - - - IFN-γ (150 U/ml) + + + +IL-34 (ng/ml) 0 10 50 100N=5F=38.5P<0.001***C0 ng/mL10 ng/mL50 ng/mL100 ng/mL050100150% THP-1 ViabilityLPS (200 ng/ml) + + + +IFN-γ (150 U/ml) + + + +IL-34 (ng/ml) 0 10 5 100N=5F=49.5P<0.001***D 24 0 ng/mL10 ng/mL50 ng/mL100 ng/mL050100150% SH-SY5Y ViabilityLPS (200 ng/ml) - - - - IFN-γ (150 U/ml) - - - -IL-34 (ng/ml) 0 10 50 100N=6F=1.6P=0.2E0 ng/mL10 ng/mL50 ng/mL100 ng/mL050100150% SH-SY5Y ViabilityLPS (200 ng/ml) + + + +IFN-γ (150 U/ml) - - - -IL-34 (ng/ml) 0 10 5 100N=6F=1.1P=0.4F0 ng/mL10 ng/mL50 ng/mL100 ng/mL050100150% SH-SY5Y ViabilityLPS (200 ng/ml) - - - - IFN-γ (150 U/ml) + + + +IL-34 (ng/ml) 0 10 50 100N=6F=1.8P=0.2G0 ng/mL10 ng/mL50 ng/mL100 ng/mL050100150% SH-SY5Y ViabilityLPS (200 ng/ml) + + + +IFN-γ (150 U/ml) + + + +IL-34 (ng/ml) 0 10 50 100N=6F=0.9P=0.5H 25 Figure 2. Effect of IL-34 on the secretion of MCP-1 and cytotoxicity of stimulated human THP-1 microglia-like cells towards human SH-SY5Y neuronal cells. THP-1 cell-free supernatant was collected from cultures incubated with IL-34 (10-100 ng/ml) or its vehicle solution for 24 h prior to the addition of lipopolysaccharide (200 ng/ml), interferon-γ (150 U/ml), lipopolysaccharide plus interferon-γ, or their vehicle solutions, for 48 h. The viability of THP-1 monocytic cells was assessed with the MTT (A-D) assay and MCP-1 secretion was measured with ELISA (I-L). SH-SY5Y neuronal viability was assessed by the MTT (E-H) assay following 0 ng/mL10 ng/mL50 ng/mL100 ng/mL050010001500MCP-1 (pg/ml)LPS (200 ng/ml) - - - - IFN-γ (150 U/ml) - - - -IL-34 (ng/ml) 0 10 50 100N=3-4F=1.1P=0.4I0 ng/mL10 ng/mL50 ng/mL100 ng/mL050010001500MCP-1 (pg/ml)LPS (200 ng/ml) + + + +IFN-γ (150 U/ml) - - - -IL-34 (ng/ml) 0 10 50 100N=4F=0.3P=0.8J0 ng/mL10 ng/mL50 ng/mL100 ng/mL050010001500MCP-1 (pg/ml)LPS (200 ng/ml) - - - - IFN-γ (150 U/ml) + + + +IL-34 (ng/ml) 0 10 50 100N=4F=2.1P=0.2K0 ng/mL10 ng/mL50 ng/mL100 ng/mL050010001500MCP-1 (pg/ml)LPS (200 ng/ml) + + + +IFN-γ (150 U/ml) + + + +IL-34 (ng/ml) 0 10 5 100N=4F=5.2P=0.024*L 26 a 72 h incubation with THP-1 cell-free supernatant. Data (means ± SEM) are from 3-6 separate experiments performed on different days. The dotted lines represent the LOD of the ELISAs. P and F values obtained from one-way randomized blocks ANOVA are displayed. *P<0.05, ** P<0.01, ***P<0.001 according to Dunnett’s post-hoc test. LPS: lipopolysaccharide. IFN: interferon. I investigated whether IL-34 affects the secretion of nitric oxide by microglia. Using BV-2 murine microglia as models, I quantified the concentration of nitrite, the major breakdown product of NO, in the cell culture supernatants. In unstimulated BV-2 cells, IL-34 (100 ng/ml) caused a significant increase in nitric oxide secretion (Fig.3A). Lipopolysaccharide (200 and 400 ng/ml) increased the secretion of nitric oxide and exposure of BV-2 cells to IL-34 (10-100 ng/ml) had no effect on the nitrite levels in lipopolysaccharide-stimulated cell supernatants (Fig.3B, C). The viability of BV-2 cells was also analyzed and, at 50 and 100 ng/ml, IL-34 significantly increased the viability of unstimulated microglia (Fig.3D). In BV-2 microglia stimulated with 200 and 400 ng/ml lipopolysaccharide, an increase in viability occurred following the addition of 100 ng/ml IL-34 only (Fig.3E, F). 3.2.2 BV-2 Griess assay 27 Figure 3. Effect of IL-34 on nitric oxide secretion by BV-2 murine microglia. IL-34 (10-100 ng/ml) or its vehicle solution was added to BV-2 cells 24 h prior to the addition of 200 ng/ml or 400 ng/ml lipopolysaccharide, or its vehicle solution. Following a 24 h incubation, supernatant of BV-2 cells was removed and nitrite concentration measured with the Griess assay (A-C). The viability and death of BV-2 cells were also assessed using the MTT (D-F) assay at the time of supernatant collection. Data (means ± SEM) are presented from 3 independent experiments performed on different days. P and F values were calculated by one-way randomized blocks ANOVA. The dotted lines represent the LOD of the Griess assay. ***P<0.001 according to Dunnett’s post-hoc test. LPS: lipopolysaccharide. Next, I evaluated if IL-34 influences the phagocytic activity of BV-2 murine microglia. I applied IL-34 at the highest concentration tested in other experiments. Cells were treated with IL-34 (100 ng/ml) prior to stimulation with lipopolysaccharide. Figure 4A illustrates that 0 ng/mL10 ng/mL50 ng/mL100 ng/mL010203040Nitrite (µM)LPS (ng/ml) 0 0 0 0 IL-34 (ng/ml) 0 10 50 100N=3F=73.3P<0.001***A0 ng/mL10 ng/mL50 ng/mL100 ng/mL010203040Nitrite (µM)LPS (ng/ml) 200 200 200 200 IL-34 (ng/ml) 0 10 50 100N=3F=2.0P=0.2B0 ng/mL10 ng/mL50 ng/mL100 ng/mL010203040Nitrite (µM)LPS (ng/ml) 400 400 400 400 IL-34 (ng/ml) 0 10 50 100N=3F=1.0P=0.4C0 ng/mL10 ng/mL50 ng/mL100 ng/mL050100150% BV-2 ViabilityLPS (ng/ml) 0 0 0 0 IL-34 (ng/ml) 0 10 50 100N=3F=568.0P<0.001******D0 ng/mL10 ng/mL50 ng/mL100 ng/mL050100150% BV-2 ViabilityLPS (ng/ml) 200 200 200 200 IL-34 (ng/ml) 0 10 50 100N=3F=71.1P<0.001***E0 ng/mL10 ng/mL50 ng/mL100 ng/mL050100150% BV-2 ViabilityLPS (ng/ml) 400 400 400 400 IL-34 (ng/ml) 0 10 50 100N=3F=104.2P<0.001***F3.2.3 BV-2 phagocytic activity 28 stimulation with lipopolysaccharide caused a significant increase in the phagocytosis of fluorescently-labeled latex beads as shown by an increase in the internal fluorescence of BV-2 cells. The same figure also shows that IL-34 (100 ng/ml) significantly upregulated the phagocytic activity of unstimulated and lipopolysaccharide-stimulated BV-2 cells. IL-34 also increased the viability of unstimulated BV-2 microglia but this effect was not significant in lipopolysaccharide-stimulated cells (Fig.4B). Figure 4. Effect of IL-34 on the phagocytosis of latex beads by unstimulated and lipopolysaccharide-stimulated BV-2 murine microglia. BV-2 cells were treated with IL-34 (100 ng/ml) or its vehicle solution for 15 minutes prior to the addition of lipopolysaccharide (400 ng/ml) or its vehicle solution for 24 h. Cells were incubated with fluorescently-labeled latex beads for one h prior to the fluorescence of non-internalized beads being quenched with 0.05% trypan blue. Cells were imaged with a widefield microscope and light emission from viable cells was measured using ImageJ software (A). Cell viability was also analyzed in parallel wells by the MTT (B) assay. Data (means ± SEM) are presented from 4 independent phagocytosis assays performed on different days in which 159 cells total for each of the four experimental conditions were imaged and analyzed. P and F values were calculated by one-way ANOVA. * P<0.05, ** P<0.01, *** P<0.001 according to Tukey’s post-hoc test. LPS: lipopolysaccharide. 0 ng/mL100 ng/mL0 ng/mL100 ng/mL0200004000060000FluorescenceN=159 (4)F=51.1P<0.001******A*LPS (400 ng/ml) - - + +IL-34 (ng/ml) 0 100 0 1000 ng/mL100 ng/mL0 ng/mL100 ng/mL050100% BV-2 ViabilityLPS (400 ng/ml) - - + +IL-34 (ng/ml) 0 100 0 100N=4F=10.3P=0.003**B3.2.4 HL-60 respiratory burst 29 I determined if IL-34 has an effect on the production of ROS by HL-60 human microglia-like cells. Differentiated HL-60 cells were treated with IL-34 (10-100 ng/ml) or its vehicle solution prior to the addition of lipopolysaccharide to prime cells, or its vehicle solution in cells remaining unprimed. fMLP stimulation of unprimed (Fig.5A) and lipopolysaccharide-primed (Fig.5B) HL-60 cells resulted in a large respiratory burst response in the latter case, leading to increased ROS release. The addition of IL-34 (100 ng/ml) to unprimed HL-60 cells showed a trend towards a decrease in ROS release following fMLP stimulation (Fig.5A). IL-34 (100 ng/ml) caused a significant decrease in ROS release by lipopolysaccharide-primed and fMLP-stimulated HL-60 cells (Fig.5B). The viability of unprimed and primed HL-60 cells was unchanged by IL-34 according to the MTT assay (Fig.5C, D). Each MTT viability assay shown in this thesis was also coupled with a PI test that detects cell death. Figures 5E, F are included here as example data obtained by the PI cell death assay for HL-60 cells used in respiratory burst experiments. The percent cell death was not different between the unprimed and primed groups and was not affected by exposure to IL-34 (Fig.5E, F). In other experiments described throughout this thesis, PI assay measurements were performed in addition to the MTT assay. The obtained PI assay data are not shown since they were inconsistent between individual experiments and unreliable compared to the MTT assay data. Significant variation occurred between PI data collected on different days which resulted in large error values. 30 0 ng/mL10 ng/mL50 ng/mL100 ng/mL050000100000ChemiluminescenceLPS (500 ng/ml) - - - - IL-34 (ng/ml) 0 10 50 100N=8F=2.1P=0.1A0 ng/mL10 ng/mL50 ng/mL100 ng/mL050000100000ChemiluminescenceLPS (500 ng/ml) + + + + IL-34 (ng/ml) 0 10 50 100N=8F=8.2P=0.001***B0 ng/mL10 ng/mL50 ng/mL100 ng/mL050100150% HL-60 ViabilityLPS (500 ng/ml) - - - - IL-34 (ng/ml) 0 10 50 100N=5F=0.2P=0.9C0 ng/mL10 ng/mL50 ng/mL100 ng/mL050100150% HL-60 ViabilityLPS (500 ng/ml) + + + + IL-34 (ng/ml) 0 10 50 100N=5F=3.3P=0.056D 31 Figure 5. Effect of IL-34 on the respiratory burst and viability of differentiated human HL-60 microglia-like cells. HL-60 cells were differentiated with DMSO for six days and then treated with IL-34 (10-100 ng/ml) or its vehicle solution for 15 minutes. Cells were primed with lipopolysaccharide (500 ng/ml) for 24 h (B) or kept unprimed by incubating with the vehicle solution (A). A respiratory burst was induced by fMLP and the chemiluminescence caused by ROS reacting with luminol was quantified using a microplate reader (A, B). Cell viability and death were measured in parallel wells using the MTT (C, D) and PI (E, F) assays. Data (means ± SEM) are presented from 8, 5, and 7 independent experiments performed on different days. P and F values were calculated by one-way randomized blocks ANOVA. *** P<0.001 according to Dunnett’s post hoc test. LPS: lipopolysaccharide. Microglia are one of the main cell types in the CNS that express the CSF-1R receptor (Ma et al., 2012; Mizuno et al., 2011). IL-34, binds to its receptor, CSF-1R, and promotes the development, proliferation and maintenance of microglia [reviewed by (Martin-Estebane & Gomez-Nicola, 2020)]. In addition, IL-34 affects the phenotype and activation state of microglia. For example, CSF-1R and IL-34 are part of the microglial sensome, allowing responses to specific conditions and stressors (Hickman & El Khoury, 2019). Conflicting evidence surrounds the response of microglia to IL-34. Walker et al. (2017) report a transition to a pro-inflammatory 0 ng/mL10 ng/mL50 ng/mL100 ng/mL0204060% HL-60 DeathLPS (500 ng/ml) - - - - IL-34 (ng/ml) 0 10 50 100N=7F=0.7P=0.5E0 ng/mL10 ng/mL50 ng/mL100 ng/mL0204060% HL-60 DeathLPS (500 ng/ml) + + + + IL-34 (ng/ml) 0 10 50 100N=7F=0.4P=0.7F3.3 IL-34 discussion 32 microglial phenotype in humans following IL-34 treatment, observed by a larger increase in the expression of pro-inflammatory genes compared to anti-inflammatory genes and downregulated expression of genes involved in amyloid-β phagocytosis. Conversely, others have shown that IL-34 stimulates an anti-inflammatory phenotype in microglia. For example, lipopolysaccharide-stimulated primary mouse microglia show a decrease in the release of pro-inflammatory cytokines IL-6, IL-1β and TNF and an increase in heme oxygenase-1 and arginase-1 mRNA levels following treatment with IL-34 (P. W. Lee et al., 2020). Similarly, an anti-inflammatory effect of IL-34 on mouse microglia is observed as an increase in TGF-β causing an increase in heme oxygenase-1 and IDE production. This leads to decreased superoxide radical production and reduced amyloid-β levels in the surrounding space (Ma et al., 2012; Mizuno et al., 2011). In this study, I showed several notable effects of IL-34 on microglia or microglia-like cells. There is no significant effect of IL-34 on the cytotoxicity of unstimulated or stimulated THP-1 microglia-like cells towards SH-SY5Y neuroblastoma cells. However, I did observe significant effects in the production of individual cytotoxic molecules by microglia and microglia-like cells following exposure to IL-34. Although IL-34 had no effect on the production of nitric oxide in lipopolysaccharide-stimulated BV-2 cells, a large increase in the production of nitric oxide is observed in unstimulated BV-2 cells treated with 100 ng/ml IL-34. This contradicts previous reports that IL-34 pre-treatment enhances the production of nitric oxide by lipopolysaccharide-stimulated BV-2 cells (Kim et al., 2017). This may be due to different concentrations of lipopolysaccharide (1 ng/ml vs 200 ng/ml) used to stimulate BV-2 cells. My results agree with those reported in chicken macrophage (HD11) and fibroblast (OU2) cells where 200 ng/ml IL-34 increases the production of nitric oxide by interacting with CSF-1R (Truong et al., 2018). In addition, I showed that 100 ng/ml IL-34 reduces the production of ROS in lipopolysaccharide-primed, fMLP-stimulated HL-60 microglia-like cells. I did not observe any effect of IL-34 in unprimed HL-60 cells. This differs from previous studies of other cell types where IL-34 induces ROS production in unstimulated THP-1 cells and peripheral blood leukocytes of puffer fish (B. Wang et al., 2017; Yu et al., 2020). However, my results agree with those observed in lipopolysaccharide- or oligomeric amyloid-β42-stimulated primary mouse microglia and BV-2 cells in which IL-34 causes a decrease in superoxide radical production through induction of heme oxygenase-1 expression (P. W. Lee et al., 2020; Mizuno et al., 2011). IL-34 also induces TGF-β production in microglia which is known to reduce superoxide levels 33 produced by lipopolysaccharide-stimulated mouse microglia (Ma et al., 2012; Suzumura et al., 1993). Considering this decreased production of ROS only occurs in lipopolysaccharide-primed HL-60 cells, we conclude that IL-34 must affect a specific signaling pathway associated with lipopolysaccharide activation in HL-60 microglia-like cells. 100 ng/ml IL-34 caused a significant decrease in MCP-1 secretion by lipopolysaccharide plus interferon-γ-stimulated human THP-1 microglia-like cells. A trend towards a decrease in MCP-1 secretion was also observed in microglia-like cells stimulated with interferon-γ alone, but not in those that were stimulated with lipopolysaccharide alone. As a result, it is likely that IL-34 decreases the interferon-γ-stimulated pro-inflammatory activation of microglia. This differs from the results of the respiratory burst assays where IL-34 affects certain signal pathways associated with lipopolysaccharide activation in HL-60 cells. Interferon-γ activates the interferon-γ receptor and JAK/STAT signaling pathways, leading to transcription of interferon-γ-response genes and pro-inflammatory activation of microglia (Rock et al., 2005). In monocytes, activation of p38 MAPK and JAK/STAT signaling pathways is necessary for MCP-1 expression (Burysek et al., 2002). IL-34 also activates p38 MAPK and JAK/STAT pathways, in addition to many other signaling pathways including NF-κB, ERK1/2, JNK, PI3K/AKT and caspase3/8 [reviewed by (Baghdadi et al., 2018)](Truong et al., 2018). The exact pathways activated by IL-34 in microglia are not well characterized. However, it is known that in other cell types CSF-1R and interferon-γ signaling pathways may interact with each other and affect cell activation and gene expression. IL-34-stimulated macrophages are immunosuppressive and interferon-γ causes these cells to revert back to immunostimulatory macrophages (Foucher et al., 2013). Also, IL-34 knockout mice show increased interferon-γ expression in myeloid cells (Hama et al., 2020) and IL-34-treated human peripheral blood mononuclear cells have decreased interferon-γ expression (Zwicker et al., 2016). In osteoclasts, IL-34 and interferon-γ have opposing effects and interferon-γ suppresses CSF-1R expression (Amarasekara et al., 2018; J.-D. Ji et al., 2009). Also, IL-34-treated chicken macrophage and fibroblast cells have increased production of interferon-γ (Truong et al., 2018). In addition, increased expression of CSF-1R following granulocyte macrophage-CSF treatment in human peripheral blood monocytes causes a reduced cellular response to interferon-γ (Finbloom et al., 1993). Previous studies have not measured the effect of IL-34 on MCP-1 production in activated microglia. It is reported that IL-34 causes MCP-1 secretion in unstimulated human macrophages and peripheral blood mononuclear cells (Zwicker 34 et al., 2016). However, IL-34-treated human macrophages do produce less MCP-1 compared to macrophages exposed to the CSF-1R ligand, CSF-1, due to less phosphorylation of STAT family proteins (Chihara et al., 2010). My study shows that unstimulated THP-1 microglia-like cells may behave differently than other macrophages and mononuclear cells, as I did not observe an increase in MCP-1 secretion following treatment with 10-100 ng/ml IL-34. Instead, as mentioned earlier, IL-34 only influenced MCP-1 secretion in lipopolysaccharide plus interferon-γ-stimulated THP-1 microglia-like cells where a greater than 60% decrease is observed. As a result, further research is warranted to determine the mechanism through which IL-34 interacts with interferon-γ and lipopolysaccharide-signaling pathways and whether similar results are observed in microglia activated with other stimulatory molecules, such as amyloid-β. In addition, the effect of IL-34 on MCP-1 secretion by primary murine and human microglia must be studied. Lastly, although Mizuno et al. (2011) describe no changes in the phagocytosis of oligomeric amyloid-β42 by microglia following treatment with IL-34, I was interested in determining if IL-34 had an effect on the phagocytosis of non-immunostimulatory latex beads by unstimulated and lipopolysaccharide-stimulated microglia. Here I report that IL-34 significantly increases the phagocytic activity of unstimulated and lipopolysaccharide-stimulated BV-2 murine microglia. In other studies, CSF-1 causes an increase in the phagocytosis of amyloid-β in microglia through CSF-1R binding (Boissonneault et al., 2009; Smith et al., 2013). Also, in other myeloid cells such as mouse macrophages, reduced CSF-1R signaling results in decreased phagocytic activity, however, this effect was not observed in BV-2 microglia (Delaney et al., 2021). Nevertheless, although BV-2 microglia may not be dependent on CSF-1R signaling for their phagocytic function, it is likely that CSF-1R activation by IL-34 significantly increases the phagocytic activity of unstimulated and lipopolysaccharide-stimulated BV-2 microglia. Therefore, CSF-1R signaling pathways may contribute to the regulation of BV-2 microglial phagocytosis. Although in certain functions, IL-34 and CSF-1 have opposing effects, here I show that IL-34, like CSF-1 in other studies, also increases microglial phagocytic activity. This is supported by another study in which IL-34- and CSF-1-treated human macrophages had comparable levels of Escherichia coli phagocytosis (Chihara et al., 2010). Additionally, reports also indicate varying effects of IL-34 on the phagocytic activity of other myeloid cells such as macrophages and monocytes. IL-34 decreases the internalization of fibrillar and oligomeric amyloid-β42 by mouse macrophages through decreased expression of 35 phagocytic markers CD69 and F4/80 as well as the amyloid-β internalization receptors CD36 and triggering receptor expressed on myeloid cells 2 (Zuroff et al., 2020). Contrarily, recombinant IL-34 in a CSF-1R-dependent manner increases the phagocytic activity of monocytes and macrophages in mudskippers (H.-Y. Shen et al., 2020). In haemorrhagic fever with renal syndrome patients, increased blood plasma IL-34 levels may cause an increase in CD14++ and CD16- phagocytic monocytes (Tang et al., 2019). As a result, IL-34 has an important effect on the phagocytic activity of many cell types, as my study illustrates in unstimulated and lipopolysaccharide-stimulated BV-2 microglia where it causes an increase in phagocytosis. This may be important for clearance of debris or pathogenic stimuli such as amyloid-β in neuroinflammation and disease. 36 Neuroinflammatory processes including the overall neurotoxicity, MCP-1 secretion, ROS production, RNS release, and phagocytic activity of unstimulated and stimulated microglia and microglia-like cells was assessed following treatment with p75NTR ligands. I assessed whether LM11A-31 affects the neurotoxic effect and secretion of MCP-1 by unstimulated and immune-activated THP-1 human monocytic cells. Cultured THP-1 cells were stimulated with lipopolysaccharide and interferon-γ in the absence or presence of LM11A-31 (1-1000 nM). Supernatant from THP-1 cells was transferred to SH-SY5Y human neuronal cells. At the time of transfer, a decrease in THP-1 cell viability was observed in lipopolysaccharide plus interferon-γ-stimulated cells (Fig.6A, B). LM11A-31, at the lowest concentration (1 nM), significantly increased the viability of unstimulated THP-1 cells compared to vehicle-treated cells (Fig.6A). Figures 6C, D demonstrate that the viability of SH-SY5Y cells was decreased to approximately 50% following the addition of stimulated THP-1 supernatant compared to unstimulated THP-1 supernatant. The same figure shows that the presence of LM11A-31 (1-1000 nM) had no effect on the cytotoxicity of unstimulated or stimulated THP-1 cells towards SH-SY5Y cells. I also measured the concentration of MCP-1 in THP-1 cell supernatants. Stimulation with lipopolysaccharide and interferon-γ increased the secretion of this chemokine but LM11A-31 (1-1000 nM) had no effect in both unstimulated and stimulated cell cultures (Fig.6E, F). Chapter 4: LM11A-31 and proBDNF 4.1 LM11A-31 and proBDNF hypothesis 4.1.1 Hypothesis #2: p75NTR ligands, LM11A-31 and proBDNF, regulate select neuroinflammatory processes of microglia and microglia-like cells. 4.2 LM11A-31 results 4.2.1 Cytotoxicity and chemokine secretion of human THP-1 microglia-like cells 37 0 nM1 nM10 nM100 nM1 µM050100150% THP-1 ViabilityLPS (200 ng/ml) - - - - - IFN-γ (150 U/ml) - - - - - LM11A-31 (nM) 0 1 10 100 1000N=4F=3.7P=0.035**A0 nM1 nM10 nM100 nM1 µM050100150% THP-1 ViabilityLPS (200 ng/ml) + + + + + IFN-γ (150 U/ml) + + + + + LM11A-31 (nM) 0 1 10 100 1000N=4F=2.8P=0.07B0 nM1 nM10 nM100 nM1 µM050100% SH-SY5Y ViabilityLPS (200 ng/ml) - - - - - IFN-γ (150 U/ml) - - - - - LM11A-31 (nM) 0 1 10 100 1000 N=4F=3.0P=0.06C0 nM1 nM10 nM100 nM1 µM050100% SH-SY5Y ViabilityLPS (200 ng/ml) + + + + + IFN-γ (150 U/ml) + + + + + LM11A-31 (nM) 0 1 10 100 1000 N=4F=2.1P=0.2D 38 Figure 6. Effect of LM11A-31 on the secretion of MCP-1 and cytotoxicity of stimulated human THP-1 microglia-like cells towards human SH-SY5Y neuronal cells. THP-1 cell-free supernatant was collected from cultures that were incubated with LM11A-31 (1-1000 nM) or its vehicle solution for 24 h prior to stimulation with lipopolysaccharide (200 ng/ml) and interferon-γ (150 U/ml) for 48 h. The viability of THP-1 monocytic cells was assessed with the MTT (A, B) assay and MCP-1 secretion was quantified using an ELISA (E, F). SH-SY5Y cell viability was assessed by the MTT (C, D) assay following a 72 h incubation with THP-1 supernatant. Data (means ± SEM) are presented from 4 and 3 independent experiments performed on different days. The dotted line represents the LOD of the ELISA. P and F values were calculated by one-way randomized blocks ANOVA. ** P<0.01 according to Dunnett’s post hoc test. LPS: lipopolysaccharide. IFN: interferon. I evaluated whether LM11A-31 affects the secretion of nitric oxide by microglia. Using BV-2 murine microglia, I quantified the concentration of nitrite in the cell culture supernatants. Nitrite levels were below the LOD for most unstimulated BV-2 treatment groups; however, stimulation with lipopolysaccharide caused a large increase in nitric oxide release (Fig.7A, B). LM11A-31 had no effect on the production of nitrite in unstimulated or stimulated BV-2 cells (Fig.7A, B). Lipopolysaccharide stimulation caused a decrease in BV-2 cell viability (Fig.7C, D). Low concentrations of LM11A-31 (1 nM) caused increases in the viability of both stimulated 0 nM1 nM10 nM100 nM1 µM05001000MCP-1 (pg/ml)LPS (200 ng/ml) - - - - - IFN-γ (150 U/ml) - - - - - LM11A-31 (nM) 0 1 10 100 1000 N=3F=0.7P=0.6E0 nM1 nM10 nM100 nM1 µM05001000MCP-1 (pg/ml)LPS (200 ng/ml) + + + + + IFN-γ (150 U/ml) + + + + + LM11A-31 (nM) 0 1 10 100 1000 N=3F=0.9P=0.5F4.2.2 BV-2 Griess assay 39 and unstimulated cells, whereas high concentrations of LM11A-31 (1000 nM) decreased the viability of stimulated BV-2 microglia (Fig.7C, D). Figure 7. Effect of LM11A-31 on nitric oxide secretion by stimulated BV-2 murine microglia. BV-2 cells were treated with LM11A-31 (1-1000 nM) or its vehicle solution for 24h prior to stimulation with lipopolysaccharide (200 ng/ml) or the addition of its vehicle solution. Following a 24 h incubation, supernatant from BV-2 cells was removed and nitrite concentration was measured using the Griess assay (A, B). The viability of BV-2 cells was also assessed using the MTT (C, D) assay at the time of supernatant collection. Data (means ± SEM) are presented 0 nM1 nM10 nM100 nM1 µM01020304050Nitrite (µM)LPS (200 ng/ml) - - - - - LM11A-31 (nM) 0 1 10 100 1000N=3F=1.0P=0.5A0 nM1 nM10 nM100 nM1 µM01020304050Nitrite (µM)LPS (200 ng/ml) + + + + + LM11A-31 (nM) 0 1 10 100 1000N=3F=1.6P=0.3B0 nM1 nM10 nM100 nM1 µM050100% BV-2 ViabilityLPS (200 ng/ml) - - - - - LM11A-31 (nM) 0 1 10 100 1000N=3F=30.4P<0.001***C0 nM1 nM10 nM100 nM1 µM050100% BV-2 ViabilityLPS (200 ng/ml) + + + + + LM11A-31 (nM) 0 1 10 100 1000N=3F=33.0P<0.001****D 40 from 3 independent experiments performed on different days. P and F values were calculated by one-way randomized blocks ANOVA. The dotted line represents the LOD of the Griess assay. *P<0.05, ***P<0.001 according to Dunnett’s post-hoc test. LPS: lipopolysaccharide. I evaluated if LM11A-31 at the highest concentrations used in other assays influences the phagocytic activity of BV-2 murine microglia. Cells were treated with LM11A-31 (1 μM) prior to stimulation with lipopolysaccharide. Figure 8A illustrates that stimulation with lipopolysaccharide caused a significant increase in the internalization of fluorescently-labeled latex beads through phagocytosis by BV-2 cells. This figure also shows that LM11A-31 (1 μM) did not have an effect on the phagocytic activity of unstimulated and lipopolysaccharide-stimulated BV-2 cells although there may be a trend toward downregulation in the latter case. The viability of BV-2 cells was also assessed, but there was no effect of lipopolysaccharide stimulation or LM11A-31 treatment (Fig.8B). Figure 8. Effect of LM11A-31 on the phagocytosis of latex beads by unstimulated and lipopolysaccharide-stimulated BV-2 murine microglia. BV-2 cells were treated for 15 minutes with LM11A-31 (1 μM) or its vehicle solution prior to the addition of lipopolysaccharide (400 ng/ml) or its vehicle solution for 24 h. Cells were incubated with fluorescently-labeled latex beads for one h. Then the fluorescence of non-internalized beads was quenched with 0.05% trypan blue and the cells were imaged with a widefield microscope. 0 uM1 uM0 uM1 uM01000020000300004000050000FluorescenceLPS (400 ng/ml) - - + + LM11A-31 (µM) 0 1 0 1N=194 (5)F=27.2P<0.001******A0 µM1 µM0 µM1 µM020406080100% Cell ViabilityLPS (400 ng/ml) - - + + LM11A-31 (µM) 0 1 0 1N=5F=1.8P=0.2B4.2.3 BV-2 phagocytic activity 41 Emitted light from viable cells, as determined by trypan blue exclusion, was measured using ImageJ software (A). Cell viability was also analyzed by the MTT (B) assay in parallel wells. Data (means ± SEM) are presented from 5 independent phagocytosis assays performed on different days in which 194 cells total for each of the four experimental conditions were imaged and analyzed. P and F values were calculated by one-way ANOVA. *** P<0.001 according to Tukey’s post-hoc test. LPS: lipopolysaccharide. I determined if LM11A-31 has an effect on the production of ROS by human HL-60 microglia-like cells. Differentiated HL-60 cells were treated with LM11A-31 (10-1000 nM) or its vehicle solution prior to priming the cells with lipopolysaccharide or adding its vehicle solution to unprimed cells. fMLP stimulation of unprimed (Fig.9A) and lipopolysaccharide-primed (Fig.9B) HL-60 cells led to a large respiratory burst response in the latter case, resulting in increased ROS release (Fig.9A, B). LM11A-31 had no significant effect on the release of ROS by unprimed, fMLP-stimulated HL-60 cells, however, there is a trend towards a decrease in ROS release by these cells. The addition of LM11A-31 (1000 nM) caused a significant decrease in ROS release by lipopolysaccharide-primed, fMLP-stimulated HL-60 cells, as shown by decreased chemiluminescence (Fig.9B). Lipopolysaccharide-primed HL-60 cells had a small decrease in viability compared to unprimed cells but LM11A-31 had no additional effect on cell viability (Fig.9C, D). 4.2.4 HL-60 Respiratory Burst 42 Figure 9. Effect of LM11A-31 on the respiratory burst and viability of differentiated human HL-60 microglia-like cells. HL-60 cells were differentiated for six days with DMSO, prior to treatment with LM11A-31 (10-1000 nM) or its vehicle solution. 15 minutes later, HL-60 cells were primed with lipopolysaccharide (500 ng/ml) or kept unprimed by incubating with its vehicle solution. 24 h later, a respiratory burst was stimulated with fMLP, and the chemiluminescence of the luminol-ROS reaction was measured on a microplate reader (A,B). Cell viability was quantified by MTT (C,D) assay in parallel wells. Data (means ± SEM) are 0nM10nM100nM1uM050000100000150000ChemiluminescenceLPS (500 ng/ml) - - - - LM11A-31 (nM) 0 10 100 1000N=6F=3.0P=0.06A0nM10nM100nM1uM050000100000150000ChemiluminescenceLPS (500 ng/ml) + + + + LM11A-31 (nM) 0 10 100 1000N=6F=4.5P=0.019*B0nM10nM100nM1uM050100150% Cell ViabilityLPS (500 ng/ml) - - - - LM11A-31 (nM) 0 10 100 1000N=4F=1.2P=0.4C0nM10nM100nM1uM050100150% Cell ViabilityLPS (500 ng/ml) + + + + LM11A-31 (nM) 0 10 100 1000N=4F=0.4P=0.8D 43 presented from 6 and 4 independent experiments performed on different days. P and F values were calculated by one-way randomized blocks ANOVA. * P<0.05 according to Dunnett’s post hoc test. LPS: lipopolysaccharide. I evaluated whether proBDNF affects the neurotoxic effect and secretion of MCP-1 by unstimulated and immune-activated THP-1 human monocytic cells. Cultured THP-1 cells were stimulated with lipopolysaccharide and interferon-γ in the absence or presence of proBDNF (0.5-500 ng/ml). Supernatant from ΤΗP-1 cells was then transferred to SH-SY5Y human neuroblastoma cells. When measured at the time of transfer, lipopolysaccharide and interferon-γ stimulation seemed to cause a decrease in THP-1 viability and 50 ng/ml proBDNF caused a significant decrease in immune-stimulated THP-1 viability compared to vehicle-treated cells (Fig.10A, B). Figures 10C-D illustrate that SH-SY5Y cell viability was decreased to approximately 40% in cultures receiving lipopolysaccharide plus interferon-γ-stimulated THP-1 supernatant compared to those receiving unstimulated THP-1 supernatant. Transfer of unstimulated proBDNF-treated (0.5 ng/ml) THP-1 supernatant caused a significant increase in SH-SY5Y viability (Fig.10C). Transfer of stimulated proBDNF-treated (500 ng/ml) THP-1 supernatant caused a small but significant decrease in SH-SY5Y cell viability (Fig.10D). Lipopolysaccharide and interferon-γ stimulation of THP-1 cells also caused an increase in MCP-1 secretion but proBDNF (0.5-500 ng/ml) had no effect on the concentration of this chemokine in THP-1 supernatant (Fig. 10E, F). 4.3 proBDNF results 4.3.1 Cytotoxicity and chemokine secretion of human THP-1 microglia-like cells 44 0 ng/mL0.5 ng/mL5 ng/mL50 ng/mL500 ng/mL050100% THP-1 ViabilityLPS (200 ng/ml) - - - - - IFN-γ (150 U/ml) - - - - - proBDNF (ng/ml) 0 0.5 5 50 500N=4F=3.3P=0.050A0 ng/mL0.5 ng/mL5 ng/mL50 ng/mL500 ng/mL050100% THP-1 ViabilityLPS (200 ng/ml) + + + + + IFN-γ (150 U/ml) + + + + + proBDNF (ng/ml) 0 0.5 5 50 500N=4F=4.6P=0.018*B0 ng/mL0.5 ng/mL5 ng/mL50 ng/mL500 ng/mL050100% SH-SY5Y ViabilityN=4F=5.2P=0.011**CLPS (200 ng/ml) - - - - - IFN-γ (150 U/ml) - - - - - proBDNF (ng/ml) 0 0.5 5 50 500 0 ng/mL0.5 ng/mL5 ng/mL50 ng/mL500 ng/mL050100% SH-SY5Y ViabilityN=4F=8.6P=0.002*DLPS (200 ng/ml) + + + + + IFN-γ (150 U/ml) + + + + + proBDNF (ng/ml) 0 0.5 5 50 500 45 Figure 10. Effect of proBDNF on secretion of MCP-1 and cytotoxicity of stimulated human THP-1 microglia-like cells towards human SH-SY5Y neuronal cells. THP-1 cell-free supernatant was collected from cells treated with proBDNF (0.5-500 ng/ml) or its vehicle solution for 24 h prior to 48 h stimulation with lipopolysaccharide (200 ng/ml) and interferon-γ (150 U/ml). The viability of THP-1 cells was assessed with the MTT (A, B) assay and MCP-1 secretion was quantified using an ELISA (E, F). SH-SY5Y cell viability was measured by the MTT (C, D) assay following a 72 h incubation with THP-1 supernatant. Data (means ± SEM) are presented from 3 or 4 different experiments performed on different days, as indicated. The dotted lines represent the LOD of the ELISAs. P and F values obtained from one-way randomized blocks ANOVA are displayed. *P<0.05, ** P<0.01, according to Dunnett’s post-hoc test. LPS: lipopolysaccharide. IFN: interferon I evaluated whether proBDNF influences the secretion of nitric oxide by microglia. The concentration of nitrite was quantified in BV-2 murine microglia supernatant. Stimulation with lipopolysaccharide caused a large increase in nitric oxide release, however, proBDNF (0.5-500 ng/ml) had no effect in unstimulated or stimulated cells (Fig.11A, B). Lipopolysaccharide stimulation decreased the viability of BV-2 cells (Fig.11C, D). In unstimulated cells, low concentrations of proBDNF (0.5-5 ng/ml) caused an increase in BV-2 viability (Fig.11C). A 0 ng/mL0.5 ng/mL5 ng/mL50 ng/mL500 ng/mL050010001500MCP-1 (pg/ml)N=3-4F=0.9P=0.5ELPS (200 ng/ml) - - - - - IFN-γ (150 U/ml) - - - - - proBDNF (ng/ml) 0 0.5 5 50 500 0 ng/mL0.5 ng/mL5 ng/mL50 ng/mL500 ng/mL050010001500MCP-1 (pg/ml)N=4F=1.2P=0.4FLPS (200 ng/ml) + + + + + IFN-γ (150 U/ml) + + + + + proBDNF (ng/ml) 0 0.5 5 50 5004.3.2 BV-2 Griess assay 46 decrease in cell viability was observed in lipopolysaccharide-stimulated BV-2 cells treated with higher concentrations of proBDNF (50-500 ng/ml) (Fig.11D). Figure 11. Effect of proBDNF on nitric oxide secretion by stimulated BV-2 murine microglia. BV-2 cells were treated with lipopolysaccharide (200 ng/ml) or its vehicle solution for 24 h following treatment with proBDNF (0.5-500 ng/ml) or its vehicle solution for 24 h. Supernatant from BV-2 cells was removed and nitrite concentration was measured using the Griess assay (A, B). The viability of BV-2 cells was also measured using the MTT (C, D) assay at the time of supernatant collection. Data (means ± SEM) are presented from 3 independent experiments performed on different days. P and F values were calculated by one-way 0 ng/mL0.5 ng/mL5 ng/mL50 ng/mL500 ng/mL010203040Nitrite (µM)LPS (200 ng/ml) - - - - - proBDNF (ng/ml) 0 0.5 5 50 500N=3F=0.4P=0.8A0 ng/mL0.5 ng/mL5 ng/mL50 ng/mL500 ng/mL010203040Nitrite (µM)LPS (200 ng/ml) + + + + + proBDNF (ng/ml) 0 0.5 5 50 500N=3F=0.2P=0.9B0 ng/mL0.5 ng/mL5 ng/mL50 ng/mL500 ng/mL050100% BV-2 ViabilityN=3F=57.8P<0.001*****CLPS (200 ng/ml) - - - - - proBDNF (ng/ml) 0 0.5 5 50 5000 ng/mL0.5 ng/mL5 ng/mL50 ng/mL500 ng/mL050100% BV-2 ViabilityN=3F=28.0P<0.001*****DLPS (200 ng/ml) + + + + + proBDNF (ng/ml) 0 0.5 5 50 500 47 randomized blocks ANOVA. The dotted line represents the LOD of the Griess assay. **P<0.01, ***P<0.001 according to Dunnett’s post-hoc test. LPS: lipopolysaccharide. Next, I evaluated if proBDNF affects the phagocytic activity of BV-2 murine microglia at the highest concentrations used in other experiments. BV-2 cells were treated with proBDNF (500 ng/ml) prior to stimulation with lipopolysaccharide. Figure 12A illustrates that stimulated cells had significantly increased phagocytic activity compared to unstimulated cells as shown by increased fluorescence of internalized latex beads. The same figure shows that proBDNF (500 ng/ml) caused a significant increase in phagocytic activity of lipopolysaccharide-stimulated BV-2 cells. Lipopolysaccharide stimulation and proBDNF (500 ng/ml) exposure had no affect on the viability of BV-2 cells (Fig.12B). Figure 12. Effect of proBDNF on the phagocytosis of latex beads by unstimulated and lipopolysaccharide-stimulated BV-2 murine microglia. Cells were exposed to lipopolysaccharide (400 ng/ml) or its vehicle solution for 24 h following a 15 minute incubation with proBDNF (500 ng/ml) or its vehicle solution. BV-2 cells were then exposed to fluorescently-labeled latex beads for one h. The fluorescence of non-internalized beads was quenched with 0.05% trypan blue and the cells were imaged with a widefield microscope. Light emitted by viable cells was normalized to the background using ImageJ software (A). Cell viability was also analyzed by the MTT (B) assay in parallel wells. Data (means ± SEM) are 0 ng/mL500 ng/mL0 ng/mL500 ng/mL0200004000060000FluorescenceLPS (400 ng/ml) - - + + proBDNF (ng/ml) 0 500 0 500**N=199 (5)F=39.0P<0.001***A0 ng/mL500 ng/mL0 ng/mL500 ng/mL050100% Cell ViabilityB N=5F=1.1P=0.4LPS (400 ng/ml) - - + + proBDNF (ng/ml) 0 500 0 5004.3.3 BV-2 phagocytic activity 48 presented from 5 independent phagocytosis assays performed on different days in which 199 cells total for each of the four experimental conditions were imaged and analyzed. P and F values were calculated by one-way ANOVA. **P<0.01 *** P<0.001 according to Tukey’s post-hoc test. LPS: lipopolysaccharide. I determined if proBDNF has an effect on the production of ROS by HL-60 human microglia-like cells. Differentiated HL-60 cells were treated with proBDNF (5-500 ng/ml) or its vehicle solution for 15 minutes prior to being primed with lipopolysaccharide (500 ng/ml) for 24 h. fMLP stimulation of unprimed (Fig.13A) and lipopolysaccharide-primed (Fig.13B) HL-60 cells led to a large respiratory burst response in the latter case, leading to increased ROS release (Fig.13A, B). Although increasing concentrations of proBDNF may cause slight decreases in ROS release by unprimed, fMLP-stimulated HL-60 cells, this effect was not significant (Fig.13A). However, proBDNF (500 ng/ml) caused a significant decrease in ROS release by lipopolysaccharide-primed and fMLP-stimulated HL-60 cells as shown by chemiluminescence (Fig.13B). As shown by the MTT assay, proBDNF (500 ng/ml) significantly increased the viability of unprimed HL-60 cells (Fig.13C) but had no effect on lipopolysaccharide-primed cells (Fig.13D). 4.3.4 HL-60 Respiratory Burst 49 Figure 13. Effect of proBDNF on the respiratory burst and viability of differentiated human HL-60 microglia-like cells. Differentiation of HL-60 cells was induced by a six day incubation with 1.3% DMSO, prior to treatment with proBDNF (5-500 ng/ml) or its vehicle solution for 15 minutes. HL-60 cells were then primed with lipopolysaccharide (500 ng/ml) (B) or its vehicle solution was added to unprimed cultures (A). 24 h later, a respiratory burst was stimulated with fMLP and the chemiluminescence of the luminol-ROS reaction was measured on a microplate reader (A,B). Cell viability was assessed by the MTT (C,D) assay in parallel wells. 0 ng/mL5 ng/mL50 ng/mL500 ng/mL050000100000150000ChemiluminescenceLPS (500 ng/ml) - - - - proBDNF (ng/ml) 0 5 50 500N=7F=1.7P=0.2A0 ng/mL5 ng/mL50 ng/mL500 ng/mL050000100000150000ChemiluminescenceLPS (500 ng/ml) + + + + proBDNF (ng/ml) 0 5 50 500N=7F=4.3P=0.019*B0 ng/mL5 ng/mL50 ng/mL500 ng/mL050100150% Cell ViabilityLPS (500 ng/ml) - - - - proBDNF (ng/ml) 0 5 50 500N=3F=15.5P=0.003*C0 ng/mL5 ng/mL50 ng/mL500 ng/mL050100150% Cell ViabilityN=3F=1.0P=0.5DLPS (500 ng/ml) + + + + proBDNF (ng/ml) 0 5 50 500 50 Data (means ± SEM) are presented from 7 and 3 independent experiments performed on different days. P and F values were calculated by one-way randomized blocks ANOVA. * P<0.05 according to Dunnett’s post hoc test. LPS: lipopolysaccharide. LM11A-31 has been extensively shown to reduce microglial activation leading to decreased neuroinflammation and neurotoxicity, as well as improved neuron health and performance in human disease models (James et al., 2017; Meeker et al., 2016; Nguyen et al., 2014; Shi et al., 2013; Simmons et al., 2016; Xie et al., 2020, 2021; T. Yang et al., 2020; G. N. Yin et al., 2021). In addition, LM11A-31 decreases the level of pro-inflammatory cytokines IL-6, TNF, CXCL10, IL-2, IL-1β, and IL-5 in mouse models of neuroinflammatory diseases (Simmons et al., 2018, 2021). I tested whether LM11A-31 influences the neurotoxic effect of lipopolysaccharide plus interferon-γ-stimulated THP-1 microglia-like cells. LM11A-31 had no effect on the overall neurotoxic effect of lipopolysaccharide plus interferon-γ-stimulated THP-1 cells or their secretion of MCP-1. Recent studies in humans showed that 1-300 nM LM11A-31 ameliorates neurotoxic phenotypes in monocyte-derived macrophages stimulated with HIV glycoprotein (gp)120 compared to untreated cultures and decreased the damage to neurons that received medium from these cultures (Killebrew et al., 2022). Although my results in THP-1 microglia-like monocytes do not support these claims, several important differences between these experiments must be noted, in addition to the differences in cell types. I activated THP-1 microglia-like cells with lipopolysaccharide and interferon-γ, molecules which have been shown to cause different inflammatory responses than HIV gp120 in macrophages (Killebrew et al., 2022). I also observed neuron viability following a 72 h incubation with THP-1 supernatant instead of observing changes in neuronal calcium levels for 1 h. Neuronal damage may not be observed in my model as neurons could possibly recover after 72 h or the damage may not be severe enough to affect metabolism and viability. Killebrew et al. (2022), also show that LM11A-31 does not change MCP-1 secretion by HIV gp120-activated macrophages. Although human microglia express more MCP-1 protein than monocyte-derived macrophages, I show that production of MCP-1 by stimulated THP-1 microglia-like cells is also not affected by LM11A-31. Even though MCP-1 production is decreased in the striatum of LM11A-31-treated R6/2 4.4 LM11A-31 discussion 51 Huntington’s disease model mice, this effect may be through decreased expression of this chemokine in other cell types. Killebrew et al. (2022) examined the levels of proteins released by macrophages and microglia treated with LM11A-31 and concluded that the neuroprotective activity of LM11A-31 may be due to increased production of growth factors or decreased production of non-protein neurotoxic molecules, a parameter which they did not assess. I studied the effects of LM11A-31 on the production of ROS and nitric oxide in HL-60 microglia-like cells and BV-2 murine microglia. LM11A-31 had no effect on the production of nitric oxide by lipopolysaccharide-stimulated BV-2 murine microglia. However, these results may be confounded by some viability differences caused by LM11A-31 treatment. Using HL-60 microglia-like cells, I observed that LM11A-31 (1 μM) caused a significant decrease in ROS production by lipopolysaccharide-primed, fMLP-stimulated cells. Therefore, this supports the previous conclusion by Killebrew et al. (2022) that the neuroprotective effect of LM11A-31 may be due to a decrease in non-protein neurotoxic molecules, such as ROS. Increased ROS production is a fundamental characteristic of activated microglia. Other studies show that microglial activation, neuroinflammation and neurodegeneration are decreased by LM11A-31 binding to p75NTR (M. Ji et al., 2018; Simmons et al., 2018; Xie et al., 2021). My results further support these findings, and it is likely that LM11A-31 decreases ROS production by reducing microglial activation, resulting in neuroprotection. In other studies, 6-hydroxydopamine-activated p75NTR-deficient rat pheochromocytoma (PC)12 cells have increased peroxide generation (Mi et al., 2009) and NGF binding to p75NTR in rat neurons causes increased mitochondrial superoxide production (Pehar et al., 2007). Therefore, it is evident that p75NTR is important in regulating the production of ROS. LM11A-31-treated macrophages also acquire an alternative type II activation (M2b)-like phenotype, which is often implicated in active tissue remodeling, phagocytosis and decreased TLR signaling (Akhmetzyanova et al., 2019; Killebrew et al., 2022). I tested whether LM11A-31 alters the phagocytic activity of BV-2 microglia. I report no significant changes for unstimulated or lipopolysaccharide-stimulated microglia; however, there may be a trend towards a decrease in phagocytic activity of LM11A-31-treated, lipopolysaccharide-stimulated microglia. These results may be confounded by the response of BV-2 microglia to lipopolysaccharide in which their phagocytic activity is increased (Majerova et al., 2014), an effect which is not typical of inflammatory microglia. In my experiments, lipopolysaccharide-stimulation and a LM11A-31- 52 induced transition to an “M2b-like” phenotype may cause similar phagocytic activity levels in BV-2 cells. Therefore, studies assessing the effect of LM11A-31 on the phagocytic activity of microglia activated by other immune modulators may be warranted. It is also worth highlighting here that only the single, highest concentration of LM11A-31 (1μM) was used in the phagocytosis assay. Although this concentration was effective at decreasing ROS production in HL-60 microglia-like cells, the lowest concentration (1 nM) was shown to increase the viability of THP-1 microglia-like cells and BV-2 murine microglia. Therefore, it would be worth repeating the phagocytosis assay with lower concentrations of LM11A-31, such as 1 nM, which may be a more effective dose. Although the exact mechanism of LM11A-31 neuroprotection is not fully elucidated, it is hypothesized that it competes with NGF and proNGF for p75NTR binding (Massa et al., 2006). In this manner, LM11A-31 inhibits the effects of NGF and proNGF and their signaling pathways through p75NTR (Friesland et al., 2014; B. Li et al., 2016; T. Yang et al., 2008b). This may also prevent the production of pro-inflammatory cytokines IL-6, IL-1β, IL-8 and TNF in microglia (Minnone, Soligo, et al., 2017). Therefore, I conclude that the LM11A-31-induced decrease in ROS production by microglia-like cells may be due to p75NTR inhibiting cell activation caused by other ligands, similar to the p75NTR antagonist TAT-Pep5 in other studies (Xu et al., 2018). Considering LM11A-31 is currently in a phase IIA clinical trial for AD, further research and monitoring of its effects on activated microglia and regulation of neuroinflammation is warranted (ClinicalTrials.gov Identifier: NCT03069014). The effect of proBDNF on inflammatory cells of the CNS has been studied very little compared to the mature form of this NT, BDNF, which causes lipopolysaccharide-stimulated BV-2 microglia to achieve an anti-inflammatory phenotype by inhibiting p38 and JNK inflammatory kinases (S. Y. Wu et al., 2020). In oxygen and glucose-deprived BV-2 cells, BDNF decreases apoptosis, the expression of inflammatory cytokines TNF, IL-1β and IL-6 as well as the PRR TLR2/4, and the production of ROS (Jingbin Zhou et al., 2020). The phagocytic activity of primary murine microglia is also increased by BDNF (Jiang et al., 2011). proBDNF, through the p75NTR, often produces the opposite effects as BDNF in many cell types [reviewed by (Fitting et al., 2020; Mizoguchi & Monji, 2017a)]. For example, in monocytes, exogenous 4.5 proBDNF discussion 53 proBDNF upregulates pro-inflammatory cytokines (W. Shen et al., 2020). Previous research has shown that the absence of p75NTR in microglia does not affect their phagocytic activity or iNOS expression (Düsedau et al., 2019). Although 20 ng/ml proBDNF activates Rac1 and increases the intracellular concentration of calcium and p75NTR expression in murine 6-3 and primary microglia cells, it does not have an effect on pro-inflammatory markers TNF, IL-1β, IL-6 and CD45 or anti-inflammatory markers IL-10, TGF-β, and arginase (Mizoguchi et al., 2021). However, no research was previously reported on the effect of various concentrations of proBDNF on activated microglia. Therefore, I studied proBDNF-induced changes in neurotoxicity, phagocytic activity and production of MCP-1, ROS and RNS by immune-activated microglia and microglia-like cells. My results show an increase in neurotoxicity of supernatant from lipopolysaccharide and interferon-γ stimulated THP-1 microglia-like cells pretreated with 500 ng/ml proBDNF. Conversely, the supernatant from unstimulated THP-1 cells pretreated with 0.5 ng/ml proBDNF have decreased neurotoxicity. However, there does not seem to be a dose-dependent effect in these viability assays where significant differences are observed. Also, the changes in THP-1 viability suggest that these results may be due to experimental inconsistencies. Nevertheless, more research into the varying effects of proBDNF in different activation states of microglia is warranted. In addition, I show no change in MCP-1 secretion in THP-1 cells pretreated with proBDNF prior to stimulation. Several other studies have also investigated how proBDNF contributes to inflammation and pain in the CNS. proBDNF is concluded to be involved in inflammatory processes and its increased levels often correspond with activated microglia, pro-inflammatory molecule secretion or markers of apoptosis and necroptosis (C. Luo et al., 2016; Mohammadi et al., 2020). Furthermore, proBDNF may be important for the production of certain inflammatory cytokines, although varying results have been recorded (Yu et al., 2020). Similar to my study showing no effect of proBDNF on MCP-1 secretion in activated THP-1 microglia-like cells, Shen et al. (2020) report no effect of proBDNF on the expression of pro-inflammatory cytokines by monocytes stimulated with lipopolysaccharide. However, they do observe that proBDNF upregulates CD40 and the expression of pro-inflammatory cytokines IL-1β, IL-6 and TNF in unstimulated monocytes. In addition, blocking proBDNF expression inhibits inflammatory responses induced by acute aortic dissection (W. Shen et al., 2020). However, Mizoguchi et al. (2021) also report no changes in the expression of proinflammatory markers 54 TNF, IL-1β, IL-6 and CD45 in murine microglia. P75NTR may function in the immune response of microglia as their activation status was changed in p75NTR KO mice. Although myeloid cells from these animals had higher secretion of pro-inflammatory mediators and anti-inflammatory IL-10, these increases were not observed in microglia, which agrees with my data showing no effect of proBDNF on pro-inflammatory MCP-1 secretion (Düsedau et al., 2019). In my study, proBDNF did not change the production of RNS by lipopolysaccharide-stimulated BV-2 murine microglia. However, proBDNF pre-treatment of interferon-γ-stimulated rodent microglia cells does cause a decrease in nitric oxide production (Mizoguchi et al., 2021). Therefore, proBDNF may interfere with interferon-γ signaling pathways but not those interacting with lipopolysaccharide. Further research is required to fully elucidate the effects of proBDNF on microglia and microglial inflammatory processes and neurotoxicity. Although the overall neurotoxic effect of THP-1 microglia-like cells was not affected by proBDNF, my results do show that 500 ng/ml proBDNF decreases the production of ROS by lipopolysaccharide-primed, fMLP-stimulated HL-60 microglia-like cells. The effect of proBDNF on ROS production was previously unstudied, and this result provides valuable insight into the function of proBDNF and p75NTR signaling pathways in microglia. This supports my LM11A-31 experimental data showing an effect on the production of ROS and further bolsters the conclusion that p75NTR signaling affects ROS production in HL-60 microglia-like cells. However, further research is needed to confirm that p75NTR mediates these effects of LM11A-31 and proBDNF in HL-60 microglia-like cells as well as primary human cells. Lastly, I show that proBDNF increases the phagocytic activity of lipopolysaccharide-stimulated BV-2 murine microglia. Since phagocytic activity is generally considered an anti-inflammatory process which occurs in the classically defined “M2” microglia, I show that the usual consensus of proBDNF being a pro-inflammatory molecule may not be fully supported. Previously, it was reported that treating murine microglia with 20 ng/ml proBDNF for 12 h did not affect the phagocytosis of beads nor the ramification and migration of microglia (Mizoguchi et al., 2021). These results support my observation that proBDNF did not affect the phagocytic activity of unstimulated BV-2 microglia. However, I show that proBDNF does increase the phagocytic activity of activated microglia, and therefore, this protein may be important in inflammatory processes. 55 Neuroinflammatory processes including the overall neurotoxicity, MCP-1 secretion, ROS production, RNS release, and phagocytic activity of unstimulated and stimulated microglia and microglia-like cells was assessed following treatment with TrkA/B ligands. I examined whether GK-2 affects the neurotoxic effect and secretion of MCP-1 by unstimulated and immune-activated THP-1 human monocytic cells. Cultured THP-1 cells were exposed to lipopolysaccharide and interferon-γ, or their vehicle solution, in the absence or presence of GK-2 (0.1-100 μM). Supernatant from THP-1 monocytic cells was then transferred to SH-SY5Y human neuronal cells. When measured at the time of transfer, GK-2 (0.1-100 μm) had no effect on the viability of THP-1 cells (Fig.14A, B). Figures 14C, D show that supernatant from lipopolysaccharide plus interferon-γ-stimulated THP-1 cells caused an approximately 50% decrease in SH-SY5Y viability compared to SH-SY5Y cells that received unstimulated THP-1 supernatant. Importantly, GK-2 had no effect on THP-1 cell cytotoxicity towards SH-SY5Y cells (Fig.14C, D). THP-1 cells increased the secretion of MCP-1 following stimulation with lipopolysaccharide and interferon-γ compared to unstimulated cells (Fig.14E, F). There were differences in the concentration of this chemokine in the supernatant of stimulated THP-1 cells treated with GK-2 at various concentrations, but no clear effect of GK-2 was observed (Fig.14F). Chapter 5: GK-2 and GSB-106 5.1 GK-2 and GSB-106 hypothesis 5.1.1 Hypothesis #3: TrkA/B ligands, GK-2 and GSB-106, regulate select neuroinflammatory processes of microglia and microglia-like cells. 5.2 GK-2 results 5.2.1 Cytotoxicity and chemokine secretion of human THP-1 microglia-like cells 56 Vehicle100nM1uM10uM100uM050100150% THP-1 ViabilityLPS (200 ng/ml) - - - - - IFN-γ (150 U/ml) - - - - -GK-2 (µM) 0 0.1 1 10 100N=4F=2.2P=0.1AVehicle100nM1uM10uM100uM050100150% THP-1 ViabilityLPS (200 ng/ml) + + + + + IFN-γ (150 U/ml) + + + + + GK-2 (µM) 0 0.1 1 10 100N=4F=2.4P=0.1BVehicle100nM1uM10uM100uM050100150% SH-SY5Y ViabilityN=4F=1.4P=0.3CLPS (200 ng/ml) - - - - - IFN-γ (150 U/ml) - - - - -GK-2 (µM) 0 0.1 1 10 100 Vehicle100nM1uM10uM100uM050100150% SH-SY5Y ViabilityN=4F=2.0P=0.2DLPS (200 ng/ml) + + + + + IFN-γ (150 U/ml) + + + + + GK-2 (µM) 0 0.1 1 10 100 57 Figure 14. Effect of GK-2 on secretion of MCP-1 and cytotoxicity of stimulated human THP-1 microglia-like cells towards human SH-SY5Y neuronal cells. THP-1 cell supernatant was collected from cultures that were incubated for 24 h with GK-2 (0.1-100 ng/ml) or its vehicle solution prior to being stimulated for 48 h with lipopolysaccharide (200 ng/ml) plus interferon-γ (150 U/ml). The effect of GK-2 on the viability of THP-1 cells was measured by the MTT (A, B) assay and the secretion of MCP-1 was measured by an ELISA (E, F) at the time of transfer of the supernatant to SH-SY5Y cells. Viability of human SH-SY5Y cells was measured by the MTT (C, D) assay after 72 h incubation with supernatants from THP-1 cells. Data (means ± SEM) are presented from 4 different experiments performed on different days. The dotted lines represent the LOD of the ELISAs. P and F values obtained from one-way randomized blocks ANOVA are displayed. LPS: lipopolysaccharide. IFN: interferon. Using BV-2 murine microglia, I assessed whether GK-2 affects the secretion of NO. GK-2 (0.1-100 μM) or its vehicle solution was added to BV-2 cells prior to treatment with lipopolysaccharide, for stimulated cultures, or its vehicle solution, for unstimulated cultures. Nitrite levels were below the detection limit in supernatants from unstimulated BV-2 cells but were detectable in the supernatant of BV-2 cells stimulated with lipopolysaccharide (Fig.15A, B). However, GK-2 (0.1-100 μm) had no effect on the production of nitric oxide by stimulated BV-2 murine microglia (Fig.15B). Lipopolysaccharide-stimulation caused a slight decrease in Vehicle100nM1uM10uM100uM05001000MCP-1 (pg/ml)N=4F=1.5P=0.3ELPS (200 ng/ml) - - - - - IFN-γ (150 U/ml) - - - - -GK-2 (µM) 0 0.1 1 10 100Vehicle100nM1uM10uM100uM05001000MCP-1 (pg/ml)N=4F=3.4P=0.044FLPS (200 ng/ml) + + + + + IFN-γ (150 U/ml) + + + + + GK-2 (µM) 0 0.1 1 10 1005.2.2 BV-2 Griess assay 58 BV-2 cell viability (Fig.15C, D). BV-2 cell viability was increased following treatment with 0.1 μM GK-2 but decreased following treatment with 10 and 100 μM GK-2 (Fig.15D). Figure 15. Effect of GK-2 on nitric oxide secretion by BV-2 murine microglia. Increasing concentrations of GK-2 (0.1-100 μM) or its vehicle solution were added to BV-2 murine microglia for 24 h prior to the addition of lipopolysaccharide (200 ng/ml) or its vehicle solution for 24 h. Supernatant from BV-2 microglia was removed and nitrite concentration measured using the Griess assay (A, B). Cell viability was assessed using the MTT (C, D) assay. Data (means ± SEM) are presented from 3 independent experiments performed on different days. P Vehicle100nM1uM10uM100uM01020304050Nitrite (µM)LPS (200 ng/ml) - - - - - GK-2 (µM) 0 0.1 1 10 100N=3F=3.3P=0.07AVehicle100nM1uM10uM100uM01020304050Nitrite (µM)LPS (200 ng/ml) + + + + + GK-2 (µM) 0 0.1 1 10 100N=3F=0.5P=0.7BVehicle100nM1uM10uM100uM050100150% BV-2 ViabilityN=3F=4.9P=0.027CLPS (200 ng/ml) - - - - - GK-2 (µM) 0 0.1 1 10 100 Vehicle100nM1uM10uM100uM050100150% BV-2 ViabilityN=3F=27.4P<0.001****DLPS (200 ng/ml) + + + + + GK-2 (µM) 0 0.1 1 10 100 59 and F values were calculated by one-way randomized blocks ANOVA. The dotted line represents the LOD of the Griess assay. * P<0.05, ** P<0.01 according to Dunnett’s post-hoc test. LPS: lipopolysaccharide. Here I evaluated if the highest concentration of GK-2 tested in other assays affects the phagoyctic activity of BV-2 murine microglia. GK-2 (100 μM) was added to cell cultures 15 minutes prior to 24 h stimulation with lipopolysaccharide. Figure 16A illustrates that stimulation with lipopolysaccharide caused an increase in the phagocytosis of fluorescently-labeled latex beads by BV-2 murine microglia. This same figure shows that GK-2 (100 μΜ) caused a suppression in lipopolysaccharide-induced phagocytic activity and had no effect on unstimulated cells. No significant changes in cell viability were observed as a result of lipopolysaccharide stimulation or GK-2 treatment (Fig.16B). Figure 16. Effect of GK-2 on the phagocytosis of latex beads by unstimulated and lipopolysaccharide-stimulated BV-2 murine microglia. 100 μM GK-2 or its vehicle solution was added to BV-2 cells for 15 minutes prior to the addition of lipopolysaccharide (400 ng/ml) or its vehicle solution . 24 h later cells were incubated for one h with fluorescently-labeled latex beads. Non-internalized beads were quenched with 0.05% trypan blue and the cells were imaged with widefield microscope. Fluorescence intensity of internalized beads was measured using ImageJ software (A). Cell viability was assessed using the MTT assay (B). Data (means ± SEM) are presented from 5 independent phagocytosis assays performed on different days in which 199 020000400006000080000FluorescenceLPS (400 ng/ml) - - + + GK-2 (µM) 0 100 0 100 N=199 (5)F=33.3P<0.001*******A0 µM100 µM0 µM100 µM050100% Cell ViabilityN=5F=2.0P=0.2BLPS (400 ng/ml) - - + + GK-2 (µM) 0 100 0 100 5.2.3 BV-2 phagocytic activity 60 cells total for each of the four experimental conditions were imaged and analyzed. P and F values were calculated by one-way ANOVA.* P<0.05, *** P<0.001 according to Tukey’s post-hoc test. LPS: lipopolysaccharide. I determined if GK-2 affects the production of ROS by HL-60 microglia-like cells. Differentiated HL-60 cells were treated with GK-2 (1-100 μM) or its vehicle solution prior to the addition of lipopolysaccharide to prime the cells. fMLP stimulation of unprimed (Fig.17A) and lipopolysaccharide-primed (Fig.17B) HL-60 cells resulted in a large respiratory burst in the latter case, and led to increased ROS release (Fig.17A, B). Increasing concentrations of GK-2 appear to cause a decreased release of ROS by unprimed, fMLP-stimulated HL-60 cells; however, these effects are not significant compared to the untreated control (Fig.17A). The addition of GK-2 (100 μM) 15 minutes before priming with lipopolysaccharide caused a significant decrease in ROS-induced chemiluminescence after fMLP stimulation compared to cells that were treated with its vehicle solution (Fig.17B). GK-2 also had no effect on the viability of unprimed or lipopolysaccharide-primed HL-60 microglia-like cells according to the MTT assay (Fig.17C, D). 5.2.4 HL-60 Respiratory Burst 61 Figure 17. Effect of GK-2 on the respiratory burst of differentiated human HL-60 microglia-like cells. HL-60 cells differentiated for six days with 1.3% DMSO were primed for 24 h with lipopolysaccharide (500 ng/ml) (B), or its vehicle solution in unprimed cultures (A), 15 minutes after treatment with GK-2 (1-100 μM) or its vehicle solution. A respiratory burst was stimulated with fMLP and the chemiluminescence of ROS reacting with luminol was measured (A, B). The viability of HL-60 cells was also measured in parallel wells by the MTT (C, D) assay. Data (means ± SEM) are presented from 9 and 4 independent experiments performed on 0uM 1uM 10uM100uM050000100000150000ChemiluminescenceLPS - - - -GK-2 (µM) 0 1 10 100 LPS (500 ng/ml) - - - -GK-2 (µM) 0 1 10 100 N=9F=2.1P=0.1A0uM 1uM 10uM100uM050000100000150000ChemiluminescenceLPS (500 ng/ml) + + + +GK-2 (µM) 0 1 10 100 N=9F=4.4P=0.013*B0uM1uM10uM100uM050100150% HL-60 ViabilityN=4F=2.3P=0.2CLPS (500 ng/ml) - - - -GK-2 (µM) 0 1 10 100 0uM 1uM10uM100uM050100150% HL-60 ViabilityN=4F=0.6P=0.6DLPS (500 ng/ml) + + + +GK-2 (µM) 0 1 10 100 62 different days. P and F values were calculated by one-way randomized blocks ANOVA. * P<0.05 according to Dunnett’s post hoc test. LPS: lipopolysaccharide. I examined whether GSB-106 influences the neurotoxic effect and secretion of MCP-1 by unstimulated and immune-activated THP-1 monocytes. Cultured THP-1 cells were stimulated with lipopolysaccharide and interferon-γ in the presence or absence of GSB-106 (0.1-100 μM). Supernatant from stimulated or unstimulated cells was transferred to SH-SY5Y human neuronal cells. When measured at the time of transfer, lipopolysaccharide and interferon-γ stimulation decreased THP-1 viability and GSB-106 (0.1 μM) increased the viability of unstimulated and stimulated cells compared to vehicle-treated cells (Fig.18A, B). The viability of SH-SY5Y cells was decreased approximately 50% following a 72 h incubation with stimulated THP-1 supernatant compared to unstimulated THP-1 supernatant (Fig.18C, D). GSB-106 (0.1-100 μM) affected the viability of SH-SY5Y cells that received unstimulated THP-1 supernatant, however, a consistent effect of GSB-106 was not observed. In addition, I also assessed if GSB-106 could affect the secretion of MCP-1 by THP-1 monocytic cells. The concentration of MCP-1 in THP-1 cell supernatant was increased following stimulation with lipopolysaccharide and interferon-γ and GSB-106 (0.1 μM) decreased the secretion of this chemokine (Fig.18E, F). 5.3 GSB-106 results 5.3.1 Cytotoxicity and chemokine secretion of human THP-1 microglia-like cells 63 0 µM0.1 µM 1 µΜ10 µΜ100 µΜ050100% THP-1 ViabilityLPS (200 ng/ml) - - - - -IFN-γ (150 U/ml) - - - - - GSB-106 (µM) 0 0.1 1 10 100N=4F=17.5P<0.001***A0 µM0.1 µM 1 µΜ10 µΜ100 µΜ050100% THP-1 ViabilityLPS (200 ng/ml) + + + + +IFN-γ (150 U/ml) + + + + + GSB-106 (µM) 0 0.1 1 10 100N=4F=13.2P<0.001**B0 µM0.1 µM 1 µΜ10 µΜ100 µΜ050100% SH-SY5Y ViabilityN=5F=3.0P=0.049CLPS (200 ng/ml) - - - - -IFN-γ (150 U/ml) - - - - - GSB-106 (µM) 0 0.1 1 10 1000 µM0.1 µM 1 µΜ10 µΜ100 µΜ050100% SH-SY5Y ViabilityN=5F=1.4P=0.3DLPS (200 ng/ml) + + + + +IFN-γ (150 U/ml) + + + + + GSB-106 (µM) 0 0.1 1 10 100 64 Figure 18. Effect of GSB-106 on secretion of MCP-1 and cytotoxicity of stimulated human THP-1 microglia-like cells towards human SH-SY5Y neuronal cells. THP-1 supernatant was collected from cultures that were incubated for 24 h with GSB-106 (0.1-100 ng/ml) or its vehicle solution prior to being stimulated for 48 h with lipopolysaccharide (200 ng/ml) plus interferon-γ (150 U/ml). The effect of GSB-106 on the viability of THP-1 monocytic cells and the concentration of secreted MCP-1 were assessed using the MTT (A, B) assay and an ELISA (E, F), respectively, at the time of transfer of the supernatant to SH-SY5Y cells. SH-SY5Y cell viability was assessed by the MTT (C, D) assay following a 72 h incubation with THP-1 supernatant. Data (means ± SEM) are presented from 4 or 5 different experiments performed on different days. The dotted lines represent the LOD of the ELISAs. P and F values obtained from one-way randomized blocks ANOVA are displayed. * P<0.05, ** P<0.01, *** P<0.001 according to Dunnet’s post-hoc test. LPS: lipopolysaccharide. IFN: interferon. I assessed whether GSB-106 has an effect on the secretion of nitric oxide using BV-2 murine microglia. GSB-106 (0.1-100 μM) was added prior to the addition of lipopolysaccharide or its vehicle solution to BV-2 cells. Nitrite levels were below the detection limit in unstimulated BV-2 supernatant (Fig.19A) but a large increase in nitrite concentration was detected in lipopolysaccharide-stimulated cultures (Fig.19B). GSB-106 had no clear effect on the secretion of nitric oxide by lipopolysaccharide-stimulated BV-2 cells although there may be a trend 0 µM0.1 µM 1 µΜ10 µΜ100 µΜ05001000MCP-1 (pg/ml)N=4F=0.6P=0.7ELPS (200 ng/ml) - - - - -IFN-γ (150 U/ml) - - - - - GSB-106 (µM) 0 0.1 1 10 1000 µM0.1 µM 1 µΜ10 µΜ100 µΜ05001000MCP-1 (pg/ml)N=4F=4.1P=0.025*FLPS (200 ng/ml) + + + + +IFN-γ (150 U/ml) + + + + + GSB-106 (µM) 0 0.1 1 10 1005.3.2 BV-2 Griess assay 65 towards a decrease (Fig.19B). The viability of BV-2 cells was also assessed at the time of supernatant collection and both unstimulated and lipopolysaccharide-stimulated cells showed a trend towards decreased cell viability with increasing GSB-106 concentrations. Although there are overall significant differences between the treatment groups, no single effective concentration compared to the untreated control can be identified (Fig.19C, D). Figure 19. Effect of GSB-106 on nitric oxide secretion by BV-2 murine microglia. Increasing concentrations of GSB-106 (0.1-100 μM) or its vehicle solution was added to BV-2 murine microglia prior to 24 h incubation with lipopolysaccharide (200 ng/ml) or its vehicle solution. 0 µM0.1 µM 1 µΜ10 µΜ100 µΜ01020304050Nitrite (µM)N=3F=5.8P=0.017LPS (200 ng/ml) - - - - -GSB-106 (µM) 0 0.1 1 10 100A0 µM0.1 µM 1 µΜ10 µΜ100 µΜ01020304050Nitrite (µM)N=3F=1.8P=0.2LPS (200 ng/ml) + + + + +GSB-106 (µM) 0 0.1 1 10 100B0 µM0.1 µM 1 µΜ10 µΜ100 µΜ050100% BV-2 ViabilityN=3F=4.4P=0.035CLPS (200 ng/ml) - - - - -GSB-106 (µM) 0 0.1 1 10 100 0 µM0.1 µM 1 µΜ10 µΜ100 µΜ050100% BV-2 ViabilityN=3F=4.8P=0.029DLPS (200 ng/ml) + + + + +GSB-106 (µM) 0 0.1 1 10 100 66 Supernatant from BV-2 cells was removed and measured for nitrite concentration using the Griess assay (A, B), and cell viability was assessed using the MTT (C, D) assay at the time of supernatant collection. Data (means ± SEM) are presented from 3 independent experiments performed on different days. The dotted lines represent the LOD of the Griess assay. P and F values were calculated by one-way randomized blocks ANOVA. LPS: lipopolysaccharide. Here I evaluated if GSB-106, at the highest concentration used in other assays, has an effect on the phagocytic activity of BV-2 murine microglia. Cells were treated with GSB-106 (100 μM) prior to stimulation with lipopolysaccharide. Figure 20A illustrates that stimulation with lipopolysaccharide caused a significant increase in the phagocytosis of fluorescently-labeled latex beads as shown by an increase in the internal fluorescence of BV-2 cells. This same figure shows that GSB-106 had no effect on the phagocytic activity of unstimulated or stimulated BV-2 cells. In addition, the viability of unstimulated BV-2 cells was decreased in GSB-106-treated cells compared to untreated cells (Fig.20B). Figure 20. Effect of GSB-106 on the phagocytosis of latex beads by unstimulated and stimulated BV-2 murine microglia. BV-2 cells were treated with GSB-106 (100 μM) or its vehicle solution 15 minutes prior to the addition of lipopolysaccharide (400 ng/ml) or its vehicle solution for 24 h. Cells were incubated with fluorescently-labeled latex beads for one h. Non-internalized beads were quenched with 0.05% trypan blue and the cells were imaged with a widefield microscope. Emitted light from viable cells was measured using imageJ software (A). 0 uM100 uM0 uM100 uM01000020000300004000050000FluorescenceLPS (400 ng/ml) - - + + GSB-106 (µM) 0 100 0 100 N=239 (6)F=31.3P<0.001***A***0 µM100 µM0 µM100 µM050100% Cell Viability *N=4F=9.0P=0.004BLPS (400 ng/ml) - - + + GSB-106 (µM) 0 100 0 100 5.3.3 BV-2 Phagocytic activity 67 Cell viability was also analyzed by the MTT (B) assay in parallel wells. Data (means ± SEM) are presented from 6 independent phagocytosis assays performed on different days in which 239 cells total for each of the four experimental conditions were imaged and analyzed. P and F values were calculated by one-way ANOVA.* P<0.05, ** P<0.01 *** P<0.001 according to Tukey’s post-hoc test. LPS: lipopolysaccharide. I determined if GSB-106 has an effect on the production of ROS by HL-60 microglia-like cells. Differentiated HL-60 cells were treated with GSB-106 (1-100 μM) or its vehicle solution prior to the addition of lipopolysaccharide to prime the cells. fMLP stimulation of unprimed (Fig.21A) and lipopolysaccharide-primed (Fig.21B) HL-60 cells resulted in a large respiratory burst response in the latter case, and led to increased ROS release (Fig.21A, B). The addition of GSB-106 (10-100 μM) to unprimed cells caused a significant decrease in ROS release following fMLP stimulation as observed by decreased chemiluminescence (Fig.21A). Also, in lipopolysaccharide-primed cells, GSB-106 (100 μM) caused a significant reduction in ROS release following fMLP stimulation (Fig.21B). GSB-106 had no clear impact on the viability of unprimed or lipopolysaccharide-primed HL-60 cells according to the MTT assay (Fig.21C, D). 5.3.4 HL-60 Respiratory Burst 68 Figure 21. Effect of GSB-106 on the respiratory burst of differentiated human HL-60 microglia-like cells. Following a six day differentiation period with DMSO, HL-60 microglia-like cells were cultured in the presence or absence of GSB-106 (1-100 μM) for 15 minutes prior to being primed with lipopolysaccharide (500 ng/ml) (B) or kept unprimed by incubating with the vehicle solution (A) for 24 h. The respiratory burst was stimulated with fMLP and the chemiluminescence of the luminol-ROS reaction was measured on a microplate reader (A, B). Cell viability was quantified by the MTT (C, D) assay in parallel wells. Data (means ± SEM) are presented from 8 and 4 independent experiments performed on different days. P and F values 0 µM1 µΜ10 µΜ100 µΜ050000100000150000200000ChemiluminescenceLPS (500 ng/ml) - - - -GSB-106 (µM) 0 1 10 100N=8F=4.7P=0.011**A0 µM1 µΜ10 µΜ100 µΜ050000100000150000200000ChemiluminescenceLPS (500 ng/ml) + + + +GSB-106 (µM) 0 1 10 100N=8F=6.8P=0.002**B0 µM1 µΜ10 µΜ100 µΜ050100150% Cell ViabilityLPS (500 ng/ml) - - - -GSB-106 (µM) 0 1 10 100N=4F=1.7P=0.2C0 µM1 µΜ10 µΜ100 µΜ050100150% Cell ViabilityN=4F=5.5P=0.020DLPS (500 ng/ml) + + + +GSB-106 (µM) 0 1 10 100 69 were calculated by one-way randomized blocks ANOVA. * P<0.05 **P<0.01 according to Dunnett’s post hoc test. LPS: lipopolysaccharide. GK-2 is effective at protecting and supporting the survival of human and murine neurons following insult with many toxic or damaging molecules (Antipova et al., 2011; Genrikhs et al., 2018; T. A. Gudasheva et al., 2014; Seredenin & Gudasheva, 2015; Stelmashook et al., 2015, 2020). Furthermore, GK-2 reduces neurodegeneration and neurological deficit in several disease models involving neuroinflammation including AD, PD, middle cerebral artery occlusion, cardiac arrest and haemorrhagic stroke (Kraineva et al., 2013; Povarnina et al., 2013, 2017; Seredenin & Gudasheva, 2015; Zarzhetskii et al., 2015). In addition to promoting neurogenesis and synaptogenesis, GK-2 may also effect glial cells and increase gliogenesis (T. A. Gudasheva et al., 2019). GK-2 behaves similar to NGF and binds to TrkA; however, it is shown to only activate the PI3K/AKT pathway, and not the MAPK/ERK signaling pathway in neurons. As a result, GK-2 causes similar neuroprotective effects to those observed in NGF signaling but not the analgesic effects of NGF (T. A. Gudasheva et al., 2014, 2018, 2019). I studied if GK-2 alters the function and decreases the neurotoxicity of microglia. GK-2 had no effect on the neurotoxicity of immune-stimulated THP-1 microglia-like cells. Likewise, GK-2 had no effect on the production of MCP-1 by lipopolysaccharide plus interferon-γ- stimulated THP-1 microglia-like cells. In murine BV-2 microglia, GK-2 affected the viability but not the production of nitric oxide following 200 ng/ml lipopolysaccharide stimulation. However, GK-2 decreases the phagocytic activity of BV-2 microglia stimulated with higher concentrations (400 ng/ml) of lipopolysaccharide. The effect of GK-2 on phagocytic activity has not previously been studied for any cell type to the best of my knowledge. Previous research suggests that NGF increases the macropinocytosis of soluble amyloid-β in primary mouse microglia but does not increase microglial phagocytosis of latex beads or fibrillar Αβ (Rizzi et al., 2018). However, this group does not study whether NGF affects the phagocytosis of latex beads in BV-2 microglia or in lipopolysaccharide-stimulated microglia. My results indicate that GK-2 may decrease the activation of lipopolysaccharide-stimulated BV-2 microglia leading to a decrease in phagocytic activity. GK-2 also decreases the production of ROS by lipopolysaccharide-primed, fMLP-stimulated HL-60 microglia-like cells. This effect is not observed in cells not primed with 5.4 GK-2 discussion 70 lipopolysaccharide. This further supports my hypothesis that GK-2 decreases microglial activation and affects the immune function of microglia. It is well known that TrkA binding by NGF results in increased PI3K/AKT activity, a pathway that GK-2 also activates. AKT prevents NF-κB nuclear translocation and activation in monocytes which is a fundamental process in pro-inflammatory signaling caused by lipopolysaccharide binding to TLR4 (Minnone et al., 2017). NF-κB activation is also important for the production of ROS in microglia [reviewed by (Akhmetzyanova et al., 2019)]; therefore, AKT activation by GK-2 likely reduces ROS production by inhibiting NF-κB in lipopolysaccharide-primed HL-60 microglia-like cells. Activation of MAPK/ERK signaling in microglia enhances phagocytosis [reviewed by (Akhmetzyanova et al., 2019)]. GK-2 does not activate this pathway, which may explain why my results show that GK-2 did not cause a significant increase in the phagocytic activity of BV-2 microglia. There is contrasting information regarding PI3K/AKT pathway signaling and its effect on the inflammatory activation state of microglia [reviewed by (Akhmetzyanova et al., 2019)]. My data suggest that GK-2 signals through PI3K/AKT and inhibits NF-κB activation, causing a reduction in “M1-like” microglia activation induced by the pro-inflammatory molecule lipopolysaccharide (Lang et al., 2021; D. Yin et al., 2018; L. Zhang et al., 2018). The effect of GK-2 may also be due to inhibition of macrophage antigen complex-1 (MAC1) signaling in microglia and microglia-like cells. MAC1 is an important TLR-independent receptor for lipopolysaccharide (Pei et al., 2007). MAC1 signaling pathways increase the production of ROS and microglial phagocytic activity (Akhmetzyanova et al., 2019; D. Zhang et al., 2011). My results show that GK-2 decreases ROS production and phagocytosis by lipopolysaccharide-stimulated microglia and microglia-like cells, suggesting a possible inhibition of MAC1 signaling pathways by GK-2 activated signaling intermediates. Activation of PI3K is an important step in MAC1 signaling and the production of ROS by NADPH oxidase. An interaction between GK-2 and MAC1 signaling pathways may occur at PI3K but further research is required to determine this. In my experiments, GK-2 was used at a slightly higher concentration (100 μΜ) than was the typical effective dosage in studies on immortalized HT-22 neuronal cells (10 μM). However, higher concentrations of GK-2 were not tested in these previous experiments as this research aimed to observe the effects of low concentrations of this compound in vitro (Antipova et al., 2011). Also, GK-2 has already been used at a wide range of concentrations from 10(-13) M to 10(- 71 5) M, and the effective concentration may be cell type dependent. Lastly, GK-2 may also increase the production of BDNF in human neurons similarly to mouse neurons, which may further enhance the neuroprotective effects of GK-2 (Zarzhetskii et al., 2015). Further research is needed to confirm the mechanism of action of GK-2 and whether microglial production of BDNF may be responsible for the effects observed in this study. GSB-106 protects and increases the survival of neuronal cells exposed to the damaging cytotoxins hydrogen peroxide or glutamate (Gudasheva et al., 2013; Logvinov et al., 2013). GSB-106 has many BDNF-like properties such as increasing neuronal survival and neurogenesis and anti-depressant effects in murine models of CNS pathologies including ischemic stroke (T. A. Gudasheva et al., 2017, 2018). However, to my knowledge, no research on the effect of GSB-106 on microglia has been completed. Considering the anti-inflammatory effect of BDNF on microglia and the evidence that GSB-106 may participate in gliogenesis, I studied the effect of GSB-106 on microglia by using in vitro neuroinflammation models. In my experiments, GSB-106 did not influence the cytotoxicity of lipopolysaccharide plus interferon-γ-stimulated THP-1 microglia-like supernatant following a transfer to SH-SY5Y neuronal cells. Although the overall neurotoxicity of stimulated THP-1 cells was not affected, I explored whether GSB-106 had an effect on the production of certain cytotoxic molecules by microglia and microglia-like cells. In lipopolysaccharide-stimulated BV-2 microglia cultures, the production of nitric oxide was unchanged following GSB-106 treatment. I observed a decrease in MCP-1 concentration in the supernatant of lipopolysaccharide plus interferon-γ-stimulated THP-1 cells following treatment with GSB-106 (0.1 μM). Considering 0.1 μM GSB-106 also increases the viability of THP-1 microglial-like cells, it is worth noting that this effect may be even more significant if the data was corrected for the concentration of cells in the culture wells. These results warrant further research on the effect of GSB-106 on the expression of other pro-inflammatory cytokines in THP-1 microglial-like cells as well as other microglial models. Also, in fMLP-stimulated HL-60 microglia-like cells, a decrease in ROS production is observed in unprimed cultures treated with GSB-106 (10-100 μM) and lipopolysaccharide-primed cultures treated with GSB-106 (100 μM). These results show that GSB-106 may affect the inflammatory activation and response of microglia, which should be analyzed in future experiments. The effect 5.5 GSB-106 discussion 72 of GSB-106 on microglia, to my knowledge, has not been studied previously. Here I show that GSB-106 may mimic certain neuroprotective effects of BDNF in microglia as seen by a decrease in the production of the pro-inflammatory chemokine MCP-1 and a decrease in ROS production by microglia-like cells. GSB-106, similar to GK-2, activates TrkB and the PI3K/AKT signaling pathway which may be responsible for the decrease in ROS production through the inhibition of NF-κB activation by AKT (Gudasheva et al., 2016). Further research to determine which receptors and signaling pathways are activated in microglia by GSB-106 and if this compound affects MAC1 signaling pathways is recommended, as was already suggested for GK-2. I observed no changes in the phagocytic activity of BV-2 murine microglia treated with GSB-106. However, there was a possible trend towards an increase in the phagocytic activity of unstimulated BV-2 microglia. GSB-106 activates the MAPK/ERK pathway in many cell types. Activation of this pathway is associated with increased phagocytic activity in microglia; therefore, lack of significant change in the phagocytosis of latex beads was not expected. Perhaps this insignificant result was due to a decrease of viability in unstimulated BV-2 microglia following treatment with GSB-106. It is worth mentioning that further research should also explore the effects of GSB-106 on cell viability since my experiments produced inconclusive data showing GSB-106 causing an increase in unstimulated THP-1 microglia-like cell viability and a decrease in unstimulated BV-2 murine microglia viability. I show that GSB-106 may decrease the pro-inflammatory effects of microglia-like cells. Considering GSB-106 also promotes the growth and survival of neurons in neuroinflammatory pathology or insult models, it must be analyzed whether these effects are a result of direct interactions between GSB-106 and neurons or if other cell types such as microglia are involved. I show that GSB-106 decreases the pro-inflammatory activation of microglia-like cells. Therefore, it is likely that in neuroinflammatory pathologies and conditions, such as middle cerebral artery occlusion, the survival and growth promoting effects of GSB-106 may be due to decreased neurotoxicity caused by microglia. 73 Table 1 summarizes the effects of IL-34, LM11A-31, proBDNF, GK-2, and GSB-106 on microglia and microglia-like cells. Viability data is not included in this table, but only summaries of specific effects on the immune functions of microglia and microglia-like cells revealed by assays analyzing their cytotoxicity, MCP-1 production, nitrite secretion, phagocytic activity, and ROS release. The immune activation states of the cells in each assay are denoted including unstimulated (U), stimulated (S), unprimed (UP), and primed (P). Arrows (↑ and ↓) indicate a significant increase or decrease in microglial function in the presence of IL-34, LM11A-31, proBDNF, GK-2, or GSB-106 for each assay, whereas “X” indicates no effect was observed. Colours (green and red) indicate if the effects are anti-inflammatory or pro-inflammatory, respectively. Possible effects or trends that are not significant are not included in the table. Chapter 6: Conclusion 6.1 Summary table 74 Table 1. The significant effects of IL-34, LM11A-31, proBDNF, GK-2 and GSB-106 on immune functions of microglia and microglia-like cells. IL-34 LM11A-31 proBDNF GK-2 GSB-106 Cytotoxicity of THP-1 microglia-like cells toward SH-SY5Y neuronal cells U: X U: X U: ↓ U: X U: X S: X S: X S: ↑ S: X S: X MCP-1 production by THP-1 microglia-like cells U: X U: X U: X U: X U: X S: ↓ S: X S: X S: X S: ↓ Nitrite secretion by BV-2 murine microglia U: ↑ U: X U: X U: X U: X S: X S: X S: X S: X S: X Phagocytic activity of BV-2 murine microglia U: ↑ U: X U: X U: X U: X S: ↑ S: X S: ↑ S: ↓ S: X ROS release by HL-60 microglia-like cells UP: X UP: X UP: X UP: X UP: ↓ P: ↓ P: ↓ P: ↓ P: ↓ P: ↓ 6.2 Hypotheses addressed This study contributes to the understanding of the effects that cytokine IL-34 and NTR ligands GK-2, GSB-106, LM11A-31 and proBDNF have on the inflammatory response of microglia. In literature, there are conflicting reports surrounding the effect of IL-34 on microglia. Previous evidence shows that in different types of microglial cells IL-34 increases pro-inflammatory gene expression (Walker et al., 2017), but, in other studies, also reduces pro-inflammatory cytokine secretion and increases the expression of anti-inflammatory and amyloid clearance enzymes (P. W. Lee et al., 2020; Ma et al., 2012; Mizuno et al., 2011). My study was completed to understand the effect of IL-34 on neurotoxicity, phagocytosis, and the production or release of ROS, RNS, and MCP-1 in human and murine in vitro microglial models. I 75 determined that IL-34 increases the phagocytic activity of unstimulated and stimulated murine microglia, decreases the release of ROS by primed microglia-like cells, decreases the production of MCP-1 by stimulated microglia-like cells, and increases the production of RNS by unstimulated murine microglia. IL-34 did not have an effect on the overall neurotoxic effect of microglia-like cells. As a result, it can be concluded that IL-34 has a pro-inflammatory effect in unstimulated microglial cells and an anti-inflammatory effect in stimulated microglial cells. Although significant effects of IL-34 were not observed in all experimental assays, these results support my hypothesis #1 because IL-34 regulates select neuroinflammatory processes of microglia and microglia-like cells. The p75NTR is implicated in the regulation of microglial immune responses. p75NTR ligand LM11A-31 reduces microglial recruitment in feline immunodeficiency virus infected animals (Meeker et al., 2012) and lowers the levels of microglial activation and release of pro-inflammatory cytokines IL-6, TNF, CXCL10, IL-2, IL-1β, and IL-5 in R6/2 and BACHD mouse models of Huntington’s disease (Simmons et al., 2018, 2021). However, human peripheral monocytes treated with the p75NTR ligand, proBDNF, have upregulated expression of pro-inflammatory cytokines, IL-1β, IL-6 and TNF (W. Shen et al., 2020). Mouse microglia react to proBDNF by increasing intracellular calcium levels and p75NTR expression but no changes in the expression of pro-inflammatory markers TNF, IL-1β, IL-6 and CD-45 or anti-inflammatory markers IL-10, TGF-β, and arginase are observed. In addition, p75NTR KO mice have increased expression of major histocompatibility complex II in microglia but no changes in their phagocytic activity or expression of iNOS are observed (Düsedau et al., 2019). Therefore, I sought to investigate if p75NTR ligands modulate the neurotoxicity of microglia and microglia-like cells in vitro as well as their phagocytic activity and production of inflammatory molecules such as ROS, RNS and MCP-1. LM11A-31 and proBDNF decreased the release of ROS by primed microglia-like cells, and proBDNF also increased the phagocytic activity of stimulated murine microglia. No changes to the overall neurotoxicity of stimulated microglia-like cells or the production of RNS or MCP-1 was observed following treatment with LM11A-31 and proBDNF. As a result, it can be concluded that the p75NTR ligands LM11A-31 and proBDNF modulate the immune response of microglia under certain conditions, but further research is needed to analyze these effects in human neuroinflammation and disease. Although significant effects of LM11A-31 and proBDNF were not observed in all experimental assays, these results 76 show that my hypothesis #2 was supported because p75NTR ligands, LM11A-31 and proBDNF, regulate select neuroinflammatory processes of microglia and microglia-like cells. NTR ligands, GK-2 and GSB-106, which activate TrkA and TrkB receptors, respectively, have shown neuroprotection in animal models of CNS neurotoxicity and pathology (Antipova et al., 2011; Genrikhs et al., 2018; T. A. Gudasheva et al., 2013; Logvinov et al., 2013; Seredenin & Gudasheva, 2015; Stelmashook et al., 2015, 2020; Zainullina et al., 2021). However, it is unclear to what extent microglia may have a role in mediating these neuroprotective or anti-inflammatory effects of the NTR ligands. I observed that the release of ROS by primed microglia-like cells was decreased following treatment with GK-2 and GSB-106. The production of ROS by unprimed microglia-like cells was also decreased following GSB-106 treatment. GK-2 decreased the phagocytic activity of stimulated microglia. In addition, a low concentration of GSB-106 decreased the production of MCP-1 in stimulated microglia-like cells. Although significant effects of GK-2 and GSB-106 were not observed in all experimental assays, these results support my hypothesis #3 because TrkA/B ligands, GK-2 and GSB-106, regulate select neuroinflammatory processes of microglia and microglia-like cells. My experiments show that IL-34 can have anti-inflammatory or pro-inflammatory effects in microglia, depending on their activation state. In my experimental models, stimulated microglial cells mimic microglia found in neuroinflammatory disease. I showed that the pro-inflammatory effects of stimulated microglia are reduced by IL-34. In the future, it should be studied whether IL-34 causes similar effects in other neuroinflammatory models including 2D and 3D mixed cultures or brain organoids. Finally, these studies may then be extended to animal models and human brains. If IL-34 lowers the production of ROS and MCP-1 and increases phagocytic activity of stimulated human microglia in vivo, it is likely that IL-34 has high therapeutic potential for neuroinflammatory diseases such as AD and PD. IL-34 has roles in various diseases including neurological disorders such as AD. These roles encompass restoring cytokine-induced blood-brain barrier disruption, degradation and phagocytosis of oligomeric amyloid-β, and reducing neuronal damage [reviewed by (Chhetri, 2023)].Therefore, increasing the concentration of IL-34 by exogenous treatment may be a viable therapeutic intervention. However, it must also be studied if IL-34 causes an increase in RNS production in unstimulated 6.3 Future directions 77 human microglia in vivo. If this is observed, IL-34 may only be beneficial as a therapeutic agent that is effective in stimulated microglia and a specific method of targeting stimulated microglia with IL-34 would be required. Considering that the expression of CSF-1R is upregulated in certain CNS injuries and pathologies (Murphy et al., 2000; Raivich et al., 1998), it is important to confirm that the effects of IL-34 on microglia are mediated through CSF-1R and to discover how increased expression of CSF-1R may affect IL-34 signaling. NTR ligands LM11A-31 and proBDNF bind and activate the p75NTR. Both LM11A-31 and proBDNF modulate certain inflammatory functions of primed or stimulated microglia-like cells as they both cause a decrease in the production of ROS, and proBDNF causes an increase in phagocytic activity. In the future, it would be beneficial to study whether similar effects occur in 2D or 3D mixed cultures or brain organoids, which more closely represent the extracellular environment of the human brain. The use of animal models may also further increase understanding surrounding the effects of LM11A-31 and proBDNF. My results show that the p75NTR is an important receptor in the modulation of microglial immune responses. However, to confirm this, ligand binding assays or receptor knockout studies should be completed to confirm that LM11A-31 and proBDNF bind to p75NTR. It would also be worthwhile comparing the signaling pathways that are activated by these two p75NTR ligands since slightly differing effects are observed when using proBDNF and LM11A-31. It is possible that each ligand may activate different signaling pathways such as the NF-κB, AKT, or JNK pathways or that one ligand may enhance or suppress NT-mediated Trk receptor activity by causing p75NTR to function as a Trk co-receptor (Roux & Barker, 2002). NTR ligands, GK-2 and GSB-106, modulate specific functions of microglia-like cells in different activation states. In stimulated or primed microglia-like cells, I observed that these NTR ligands reduce certain pro-inflammatory functions such as ROS and MCP-1 production. However, GK-2 also causes a decrease in the phagocytic activity of stimulated murine microglia, a function which is most commonly referred to as anti-inflammatory and beneficial. Considering these NTR ligands function to reduce only select pro- and anti-inflammatory functions of microglia-like cells, it must be studied whether similar effects are observed in 2D or 3D mixed cell cultures or brain organoids, which have extracellular environments more similar to that of human brains. In addition, animal models may be utilized to further understand the effects of GK-2 and GSB-106 in neuroinflammation. More research should also be completed to confirm 78 that the observed effects occur through the interactions of GK-2/GSB-106 with TrkA/B, such as TrkA/B knockout studies, ligand binding analysis, or receptor inhibition experiments. Lastly, to fully elucidate the effect of these NTR ligands it should be discovered whether signaling occurs through the RAS, PLC-γ1 or PI3K pathways. All my studies were performed on cancerous or immortalized cell lines that had undergone viral gene transformation. Although these cell lines serve as excellent models of microglia, they differ genotypically and phenotypically from primary microglia, which may affect their function (Horvath, Nutile-McMenemy, et al., 2008; Pan et al., 2009). I also utilized murine BV-2 microglia, which express different genes and respond to stimuli differently than do human microglia. The use of BV-2 microglia allowed the observation of nitric oxide release by microglia, which was not possible with the other cell types in this study. Nitric oxide release by BV-2 cells may be similar to the release of nitric oxide by human microglia in vivo (Horvath, Nutile-McMenemy, et al., 2008; Stansley et al., 2012). It is also worth noting that all microglia-like immune cells used in this study, except BV-2 cells, were not microglia that originate from the CNS, but were isolated from the periphery. Lastly, all cell cultures and assays utilized cells grown as a monoculture. Although monocultures allow the direct study of particular cell types, many important intercellular signaling pathways would not be present in these monocultures. Therefore, these cell lines serve as a great tool to understand the function and responses of microglia; however, all studies performed should be replicated in mixed 2D and 3D cultures or brain organoids to observe whether this effect occurs in tissue environments more similar to the human CNS. A technical limitation that I encountered is the inconsistency of data generated by the PI assay. As shown in Figure 5 (C-F), IL-34 has no effect on the viability of unprimed or lipopolysaccharide-primed HL-60 cells. In these results, I included data from the PI cell death assays of unprimed and lipopolysaccharide-primed HL-60 cells, in addition to the MTT assay measurements. In some instances, the results of the cell viability assays and cell death assays supported each other. For example, in some experiments where cultures underwent a 30% reduction in cell viability, a 30% increase in cell death was also observed. However, as is the case in Figure 5 (C-D), occasionally the cell death data did not support the viability data in terms 6.4 Study limitations 79 of overall values or the effect of specific treatments. Here, DMSO differentiation of HL-60 cells likely resulted in non-immortalized cultures and increased cell death, leading to increased levels of free DNA in cell cultures. PI and DNA were no longer separated by intact cell membranes and the assay yielded high fluorescence values. As a result, the percentage of cell death was skewed, and any trends observed in the cell viability assay were not noticeable in the cell death assay. Due to this lack of reliability and much variability between different days in which the assay was performed, the error values associated with the PI assay were very high. Therefore, I decided to abandon the cell death assays in subsequent experiments. Another major limitation in my research is that microglia were studied through the lens of the traditional anti-inflammatory/pro-inflammatory classification. However, recent evidence shows that human microglia exist in temporal and spatial heterogeneity. Microglial states are influenced by both extrinsic and intrinsic factors and their reaction to challenges in the brain. In human disease, reactive microglia show plasticity and acquire unique disease-related signatures, rather than the classical M1 or M2 phenotype [reviewed by (Gao et al., 2023)] Therefore, more research is needed to understand if IL-34 or NTR ligands can affect certain disease-specific microglial states, and whether they could possibly be used as a therapeutic in certain neurodegenerative diseases. To achieve this, populations of microglia must be isolated from the specific brain areas affected by neurodegenerative diseases and treated with IL-34 or NTR ligands. If these compounds can modulate the phagocytic activity and production of MCP-1, RNS and ROS by the adversely activated microglial cells, it may have major implications in neurodegenerative disease treatment. I determined that IL-34 and NTR ligands, LM11A-31, proBDNF, GK-2, and GSB-106, can be used to modulate select neuroimmune functions of microglia. These findings contribute to the expanding knowledge of microglia immune function and specific molecules that may affect their immune processes. Microglia, as the main resident immune cells in the CNS, employ and control many neuroinflammatory mechanisms which may be dysregulated in certain neurodegenerative diseases such as AD and PD. Therefore, understanding how microglial functions are modulated could lead to identification of therapeutic targets that may be beneficial in treating or curing AD or PD, for which there currently are no effective medications. 6.5 Research significance 80 By completing this study, I used murine microglia and human microglia-like cells to address several knowledge gaps surrounding the effects of these compounds, which may further extend to human microglia. I showed that IL-34 increases the phagocytic activity of BV-2 murine microglia and decreases the release of ROS by stimulated human HL-60 microglia-like cells which corroborates previous results in primary mouse microglia cultures. In addition, I showed that IL-34 reduces the secretion of the inflammatory chemokine MCP-1 by immune-activated human THP-1 microglia-like cells and increases the production of nitrite by unstimulated BV-2 murine microglia. These actions were previously unstudied, and by including immune-activated microglia and microglia-like cells, further knowledge was gained concerning the effect of IL-34 in normal physiological conditions but also in models of neuroinflammation. My results showed that IL-34 may also have similar effects on select immune functions of human microglia. LM11A-31 was previously shown to reduce microglial activation leading to neuroprotection in animal models, and my research may have narrowed the knowledge gap by showing a possible mechanism for this effect, which is decreased ROS production by microglia, as was observed in human HL-60 microglia-like cells. Previous research shows that proBDNF may cause microglia to acquire a pro-inflammatory phenotype, but also that low levels of proBDNF are observed in early AD. My research contributed to the knowledge of the effects of proBDNF in human microglia by observing that stimulated THP-1 microglia-like cells were more cytotoxic to SH-SY5Y neuronal cells following treatment with proBDNF. Contrarily, in unstimulated microglia-like cells, proBDNF treatment decreased the cytotoxicity towards these neuronal cells. proBDNF also increased the phagocytic activity of stimulated BV-2 murine microglia and decreased the release of ROS by primed HL-60 microglia-like cells. Therefore, proBDNF may be capable of steering human microglia towards a neuroprotective phenotype. Effects of GK-2 and GSB-106 were not previously studied in microglia models, therefore, my research may show a mechanism for the observed neuroprotective effects of these NTR ligands in animal models. I showed that GK-2 and GSB-106 reduced the release of ROS by HL-60 microglia-like cells and GSB-106 lowered the secretion of MCP-1 by human THP-1 microglia-like cells. 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Thesis/Dissertation
eng
Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Klegeris, Andis
Neurotrophin receptor ligands LM11A-31, pro-brain-derived neurotrophic factor, GK-2, and GSB-106, and growth factor interleukin-34 modulate select immune functions of microglia
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