Saudi Cultural Missions Theses & Dissertations
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Item Restricted Targeting Microglia as a Therapeutic Approach to Treat Neuroinflammatory Disorders(University of Nottingham, 2025-05) Almalki, Masi; Alexander, Stephen; Bennett, AndrewNeuroinflammation, a complex immune response within the central nervous system (CNS), contributes to neurodegenerative diseases. It is mediated predominantly by microglia, the brain's resident immune cells. Existing in vitro models, however, often fail to capture the human-specific characteristics of microglial behaviour, limiting the understanding of neuroinflammatory mechanisms and therapeutic intervention points. This thesis addresses these limitations by developing a human microglia model derived from peripheral blood mononuclear cells (PBMCs), offering a physiologically and human-relevant system for exploring microglial activation and signalling pathways central to neuroinflammation. The primary objective was to create and validate a reproducible differentiation protocol for microglia-like cells from PBMCs, confirmed through microglial markers and functional assays that mimic primary microglial responses. This model was then applied to investigate how damage-associated molecular patterns (DAMPs), such as adenosine triphosphate (ATP), influence microglial activation via P2 receptors (P2X4 and P2Y12), showing distinct inflammatory roles across acute and sustained responses. Furthermore, Pattern Recognition Receptors (PRRs), including Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4), were examined for their role in responses to pathological aggregates like α-synuclein fibrils, common in neurodegenerative conditions. This study elucidates the receptor-specific mechanisms underlying microglial activation and cytokine production by simulating neuroinflammatory conditions, advancing our understanding of the molecular pathways central to disease progression. To achieve these, PBMCs were differentiated into microglia-like cells through a standardised protocol, optimised to yield cells expressing validated microglial markers, such as ionised calcium-binding adapter molecule 1 (Iba1), purinergic receptor P2Y12 (P2RY12), and transmembrane protein 119 (TMEM119). Functional assays, including cytokine profiling and phagocytosis, confirmed the cells’ responsiveness to neuroinflammatory stimuli, closely replicating primary microglial behaviours. This model enabled an in-depth investigation into receptor-specific inflammatory pathways by using real-time quantitative PCR, cytokine assays, and transcriptomic analyses, revealing interactions with pathological aggregates like α-synuclein fibrils. A major contribution of this research lies in evaluating transient receptor potential (TRP) channels, specifically transient receptor potential vanilloid 4 (TRPV4), as modulators of inflammation in microglia. Pharmacological activation of TRPV4 with GSK1016790A (GSK101) significantly reduced pro-inflammatory cytokine production (e.g., interleukin-1 beta [IL-1β], tumour necrosis factor-alpha [TNF-α]) in ATP-stimulated microglia while concurrently upregulating nuclear receptor subfamily 4 group A member 2 (Nurr1), an orphan nuclear receptor associated with neuroprotective and anti-inflammatory effects. This dual effect demonstrates that TRPV4 activation mitigates inflammatory responses and promotes a neuroprotective microglial phenotype, suggesting a promising therapeutic target for neuroinflammation-driven diseases. The TRPV4-Nurr1 axis modulation established here paves the way for future investigations into its role as a therapeutic approach, with broad implications for neuroinflammatory diseases such as Parkinson’s and Alzheimer’s. In conclusion, this thesis introduces a robust and scalable human-derived microglia model that captures human-specific inflammatory responses and receptor interactions observed in neurodegenerative diseases. This work elucidates the roles of DAMPs, P2 receptors, and TRPV4 in microglial activation, providing a foundation for developing therapeutic strategies targeting neuroinflammation. The findings suggest the potential of the TRPV4-Nurr1 pathway for managing neuroinflammation in neurodegenerative diseases.17 0Item Restricted AMIGO3 gene expression and function during oligodendrocyte differentiation in the central nervous system(University of Birmingham, 2024-04-21) Almutairi, Majed; Fulton, Daniel; Ahmed, ZubairDemyelination causes disruption to neuronal signalling and occurs in many neurodegenerative diseases or in response to brain injury. There are no cures available for demyelinating diseases, including multiple sclerosis (MS), the most common autoimmune inflammatory disease in the central nervous system (CNS). Current approaches to treat MS focus on anti-inflammatory drugs, which prevent demyelination progression and reduce clinical relapses, but do not allow repair or replacement myelin. A natural repair process called remyelination can occur following demyelination. Remyelination can help to repair demyelinating plaques and protect axons from degeneration, but endogenous remyelination is inefficient and fails with ageing and disease progression, Improving remyelination is therefore an important for the focus for the development of MS therapies. Studies of animal models have to led to the discovery of new therapeutic targets which can promote endogenous remyelination. An example is the leucine rich repeat protein (LRRP) leucine rich repeat and immunoglobulin-like domain containing protein 1 (LINGO1). Inhibition of LINGO1 promoted remyelination in several animal models of demyelination, but clinical inhabitation of LINGO1 showed unsuccessful results in clinical trials. We considered the idea that the LINGO1 clinical trials failed due to compensation from another LRRP, amphoterin-induced gene and open reading fram-3 (AMIGO3). AMIGO3 is upregulated more rapidly than LINGO1 after spinal cord injury (SCI) in animal models, and also increases in expression rapidly after axotomy in culture of dorsal root ganglion neurons and retinal ganglion cells. Importantly, AMIGO3 expression is rapidly increased in the CNS during postnatal development when myelinating oligodendrocyte (OL) are generated, and decreases again quickly after myelination begins. AMIGO3 may therefore act a regulator of OL differentiation, and its inhibition could be an alternative therapeutic target for demyelinating diseases. Considering the above, the first aim of this project was to study the function of AMIGO3 using in vitro models of OL differentiation. For this work, a primary mixed glial culture system was developed to study OL differentiation during early stage of CNS development. Using this system we found that AMIGO3 is downregulated in mixed glial cultures during OL differentiation. We also found that downregulation of AMIGO3 expression by siRNA resulted in the upregulation of OL maturation. On the other hand, increased AMIGO3 levels produced by treatment with recombinant AMIGO3 reduced OL maturation, and activated RhoA GTP activity, a molecule involved in AMIGO3 intracellular signalling. These findings suggest that AMIGO3 downregulates OL differentiation via RhoA GTP signalling. AMIGO3 is known to engage in homotypic interactions, thus expression on different glial cells could allow cell to cell signalling. Also, AMIGO3 is expressed in cells of the OL lineage and astrocytes, thus AMIGO3 expression in non-OL glia could provide a signal to regulate OL differentiation. Considering this idea, the second aim in this thesis was to compare the expression of AMIGO3 and LINGO1 in primary cultures of astrocytes and microglia. Using Western blot and immunocytochemistry we found strong expression of AMIGO3 proteins in astrocytes, but not in microglia. Surprisingly, we also found that astrocytes express LINGO1 proteins. This work shows that astrocytes, but not microglia, are an abundant source of both AMIGO3 and LINGO1, which together could influence OL differentiation and myelin formation and repair.114 0Item Restricted ANGIOTENSIN II TYPE 2 RECEPTOR AGONIST PREVENTS THE PRO- INFLAMMATORY RESPONSE IN LPS TREATED HUMAN MACROPHAGES(ProQuest, 2020-08) Alshammari, Abdulkarim; Fagan, SusanStroke is a leading cause of long term disability and is associated with a 30% incidence of severe cognitive impairment. Sustained pro-inflammatory microglia activation contributes to, and our lab has shown that the Angiotensin II type 2 receptor (AT2R) agonist, compound 21 (C21), can prevent the development of, PSCI. We hypothesized that activation of pro-inflammatory microglia and macrophages can be prevented with C21. This was assessed using a microglial cell line (C8-B4) and THP-1 derived macrophages. The reduction in the pro- inflammatory cell markers was assessed via RT-qPCR using the following genes, IL-1b, TNFa, and NOS2. Cells were either pre-treated, prior to LPS exposure, or post-treated after LPS treatment, with C21 (100 uM). C21 effectively reduced the expression of IL-1b in a concentration-dependent manner. Both pre- and post- treatment with C21 significantly reduced the expression of pro-inflammatory markers after LPS exposure in a mouse microglial cell line and human macrophages.36 0