Aloufi, Rawan
(2024)
Design and Evaluation of a Human Microglia Model: Drug Delivery and the Inhibitory Impact of PPARβ Agonist on Inflammation.
PhD thesis, University of Nottingham.
Abstract
Neuroinflammation is a hallmark of various neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and multiple sclerosis. Microglia and astrocytes, the primary immune cells of the central nervous system (CNS), play a crucial role in the initiation and progression of neuroinflammation. Peroxisome proliferator-activated receptors (PPARs) and fatty acid-binding proteins (FABPs), which are thought to deliver ligands to PPARs, have emerged as important regulators of glial cell functions and neuroinflammation, making them attractive therapeutic targets for neurodegenerative diseases. However, the specific roles of PPARs in microglia and astrocytes, as well as the impact of age and inflammatory stimuli on their expression and function, remain largely unexplored.
Treatment of neuroinflammatory conditions, such as Alzheimer's disease and Parkinson’s disease, has been hampered by a lack of translational models and effective drug targets, as well as limited access of drugs through the blood-brain barrier. This thesis aims to elucidate the expression patterns and functions of PPARs and FABPs in microglia and astrocytes and to investigate the therapeutic potential of targeting PPARs for the treatment of neuroinflammatory and neurodegenerative disorders. This research is focused particularly on the role of PPARβ, the most abundant PPAR isoform in the CNS, and its role in modulating glial cell activation and neuroinflammation.
To address these objectives, this study employed a combination of ex vivo and in vitro approaches, including primary rat microglia and astrocyte cultures and human monocyte-derived microglia (iMG) cell models. TaqMan quantitative PCR (qPCR) was employed to characterize the expression of PPARs and FABPs in microglia and astrocytes under basal conditions and in response to inflammatory stimuli, such as lipopolysaccharide (LPS) and adenosine triphosphate (ATP). The findings revealed that PPARα, PPARβ, and PPARγ were expressed in both neonatal and adult microglia in the rat brain and spinal cord. Notably, neonatal microglia expressed higher levels of FABP5, FABP7, PPARα, and PPARβ compared to adult microglia in cortical and spinal cord cultures. Additionally, age-related and region-specific differences in microglia were observed in response to LPS and ATP, highlighting the importance of considering developmental stage and CNS region when studying microglial function.
To further investigate the role of PPARβ in regulating microglial activation and neuroinflammation, human iMG cells were developed which closely resemble primary human microglia in terms of morphology, gene expression, and functional properties. This study validated the iMG model by demonstrating the expression of key microglial markers, such as TMEM119, P2RY12, and HEXB, and their responsiveness to inflammatory stimuli. Using RNA sequencing (RNA-seq) analysis, iMG cells exhibit a transcriptional profile distinct from peripheral monocytes and macrophages and more closely resembling that of primary human microglia.
Treatment with the PPARβ agonist GW0742 exerted potent anti- inflammatory effects in iMG cells, reducing the production of pro-inflammatory cytokines, such as TNF-α and IL-1β, in response to LPS and ATP stimulation. RNA-seq analysis revealed that PPARβ activation in ATP-stimulated iMG cells resulted in the upregulation of genes involved in neuroprotection, such as ATG3, RB1CC1, and BNIP3L, while downregulating inflammation-related pathways. Moreover, this study demonstrated that the anti-inflammatory effects of GW0742 were mediated through the modulation of microglia metabolism, autophagy, and the inhibition of NF-κB signalling. Additionally, the role of SUMOylation in regulating PPARβ activity was investigated, as PPARβ was found to be minimally SUMOylated compared to the Liver X receptor – a nuclear receptor which has been shown to be a target of SUMOylation, and this modification was reduced in the presence of the PPARβ agonist GW0742, suggesting that ligand-induced SUMOylation is not a major mechanism in terms of the anti-inflammatory actions of PPARβ. This study also assessed whether PPAR ligands function as substrates for the ABCG2 efflux transporter, which could hinder their passage through the blood-brain barrier, affecting their ability to reach therapeutic levels within the CNS. By employing a cell line overexpressing ABCG2, the potential of PPAR ligands to interact with this transporter and be effluxed was evaluated, with data indicating that none of the PPAR ligands tested were ligands for ABCG2.
In conclusion, this thesis provides valuable insights into the role of PPARs in regulating microglial and astrocytic functions and highlights the therapeutic potential of targeting PPARβ for the treatment of neuroinflammatory and neurodegenerative diseases. The findings, obtained through Taq-Man qPCR, RNAseq analysis, and functional assays, contribute to a better understanding of the molecular mechanisms underlying glial cell activation and neuroinflammation. The validation of the iMG model and the demonstration of the anti-inflammatory effects of PPARβ activation in these cells provide a promising platform for future drug discovery and translational research. Further studies should focus on elucidating the complex interactions between PPARs, FABPs, and other signalling pathways in the CNS, as well as translating these findings into clinical applications for the treatment of age-related neuro-degenerative disorders.
| Item Type: |
Thesis (University of Nottingham only)
(PhD)
|
| Supervisors: |
Andrew, Bennett
Stephen, Alexander |
| Keywords: |
neuroinflammation, Peroxisome proliferator-activated receptors (PPARs), fatty acid-binding proteins (FABPs), microglia, astrocytes, neuroinflammation, neurodegenerative
diseases, PPARβ, GW0742, monocyte-derived microglia, RNA sequencing, SUMOylation, NF-κB signaling, pro-inflammatory cytokines, neuroprotection |
| Subjects: |
Q Science > QP Physiology > QP351 Neurophysiology and neuropsychology
R Medicine > RC Internal medicine > RC 321 Neuroscience. Biological psychiatry. Neuropsychiatry |
| Faculties/Schools: |
UK Campuses > Faculty of Medicine and Health Sciences > School of Health Sciences
UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences |
| Item ID: |
79469 |
| Depositing User: |
Aloufi, Rawan
|
| Date Deposited: |
11 Dec 2024 04:40 |
| Last Modified: |
11 Dec 2024 04:40 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/79469 |
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