Biochemistry and Pharmacology - Theses

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End date: 2022 × Start date: 2020 × Type: PhD thesis × Subject: Alzheimer's disease ×

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    Microglia and type-I interferons: emerging actors in Alzheimer’s disease
    Moore, Zachery ( 2021)
    Alzheimer’s disease (AD) remains the most common cause of dementia worldwide. Post-mortem brains of AD individuals reveal classical pathological hallmarks of amyloid plaques and hyper-phosphorylated tau throughout the regions of the cortex and hippocampus. However, there have been few successes in therapeutics targeting these pathologies to treat AD, therefore suggesting new approaches are required. Recent evidence suggests that neuroinflammation, once thought inconsequential, contributes to AD progression. AD brains display enhanced gliosis surrounding plaques and elevated levels of pro-inflammatory cytokines. The characterisation of central nervous system (CNS) cell types and specific mediators of this neuroinflammatory response is critical in the identification of much- needed therapeutic targets to limit the damage in the AD brain. Our laboratory has previously identified that the type-I interferons (IFNs) are critical mediators of neuroinflammation contributing to the cognitive decline in a murine AD model. This thesis aimed to further characterise the specific effects of the type-I IFNs on microglia, a resident CNS immune cell. This thesis posits that type-I IFN mediated neuroinflammation modulates microglial phenotype contributing to the progression of AD pathology. This thesis aimed to investigate how type-I IFNs alter microglial phenotype and function in response to AB1-42, the key component of amyloid plaques, using both in vitro and in vivo murine models. From this study it was confirmed that microglia mount both pro-inflammatory and type-I IFN responses to AB1-42, of which both are ablated in Ifnar1-/-microglia, which lack the type-I IFN receptor. This finding was then extended to investigate if this phenotypic change manifested in altered functional changes in the ability of microglia to phagocytose or take up and internalise particles. Both IFNa and IFNb decreased the ability of microglia in vitro to phagocytose both bio-particles and fluorescently tagged AB1-42. This was investigated further in vivo, in mice that had received intra-hippocampal injections of AB1-42. No changes were observed in measures of AB uptake at 1-, 2- or 4-week post injection between wildtype and Ifnar1-/- mice. Mice with reduced type-I IFN signalling did, however, display decreased expression levels of Il6 and an altered astrocytic response within the hippocampus, suggestive of a lessened inflammatory response to AB1-42. Type-I IFNs are known to be elevated in aging, the largest risk factor for AD. This thesis took an in silico approach to compare type-I IFN expression between “normal” ageing and AD to gain a greater understanding of its role in driving the pathological changes in the AD brain. Using publicly available RNASeq datasets, this confirmed significant overlaps between type-I IFN related genes upregulated throughout both ageing and AD. This was most evident when examining a dataset containing purified cell types where there is a distinct lack of differences between controls and AD individuals. One of these genes, ISG20 was validated in a post-mortem human AD cohort and found to be significantly upregulated by QPCR and western blot analyses, supporting future investigations into its role in the disease pathogenesis. This thesis has built on previous findings from both our lab and others on the diverse roles of the type-I IFNs within the CNS and in AD. Specifically, it has identified and characterised a novel role for the type-I IFNs in modulating phagocytosis, a cellular process important in not only AD but several other neurodegenerative disorders. This thesis took a bioinformatic approach to identify a novel gene involved in AD, highlighting the increasing power of these tools and data when combined with traditional wet lab approaches to identify potential new therapeutic targets. Ultimately, this thesis forms a solid foundation to build upon and aid in the development of novel and much needed therapeutic approaches to slow AD progression.
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    Understanding the Role of the Innate Immune System in Alzheimer’s and Parkinson’s Disease Pathogenesis
    Markulic, Jasmina ( 2021)
    Alzheimer’s (AD) and Parkinson’s disease (PD) are the two most common neurodegenerative diseases causing dementia. Microglia, a resident macrophage of the brain, have been implicated in disease pathogenesis, playing key roles in clearance of toxic protein aggregates and inflammation. Recently, genetic studies have identified microglial surface receptors as risk factors for AD and PD susceptibility. The focus of this thesis was to determine the structural and functional properties of two of those proteins: human cluster of differentiation 33 (hCD33) and human triggering receptor expressed on myeloid cells 2 (hTREM2). The structure of the ligand binding domain (also known as the V-set immunoglobulin (IgV) domain) of hCD33 was expressed and purified from a bacterial expression system and solved to 2.8 A resolution. Oligomerisation of hCD33 at the plasma membrane was dependent on the integrity of the ligand binding domain; mutation of a conserved arginine residue (R119A) responsible for ligand interaction compromised hCD33 oligomerisation. Activation of hCD33 signalling appeared to be dependent on a functional ligand binding domain; hCD33 wild-type (WT) expressing cells had elevated levels of SHP-2 protein, a key phosphatase recruited to activated hCD33, whereas this was lessened in hCD33 R119A and dIgV expressing cells. Activation of WT hCD33 by 6’-sialyllactose led to upregulation of SHP-1, SHP-2 and SYK expression over time; this was not observed in hCD33 R119A or dIgV expressing cells. Hence, these results support a mechanism by which hCD33 potentiates its own downstream intracellular signalling pathways, possibly resulting in amplification of hCD33 signalling. The functional activity of hCD33 may be dictated by its cellular localisation; qualitative results showed hCD33 WT and R119A presentation to the plasma membrane, while dIgV remained in intracellular compartments. These observations suggest that the IgV domain may be involved in controlling the trafficking of hCD33, and thus its ability to be available to ligand and activated. A bacterial expression system was used to purify the hTREM2 ligand binding domain, resulting in the production of natively folded, disulfide bonded protein and extensive biophysical analysis of the IgV domain. Ligand binding assays identified one novel low affinity binder of hTREM2. Disease-associated variants of hTREM2 altered its interactions with human DAP12 (hDAP12); a reduction in hDAP12 association was observed for the hTREM2 R47H mutant, a key residue involved in ligand binding, and for the K186N mutant, a critical residue responsible for association with hDAP12. The partnership with hDAP12 may also be affected by cellular localisation; qualitative results showed that while hTREM2 WT and R47H presented at the plasma membrane, K186N remained predominately intracellular. While hDAP12 appeared to co-localise at the plasma membrane with WT hTREM2, hDAP12 was predominately intracellular with both R47H and K186N co-expression. These observations suggest that hDAP12 translocation to the plasma membrane is dependent on functional native hTREM2. The cell surface presentation of hTREM2, and interaction with hDAP12, appeared to be essential for activation of hTREM2-hDAP12 signalling; activation of Akt, a downstream kinase in the hTREM2-hDAP12, was observed in both WT hTREM2 and WT hTREM2-hDAP12 expressing cells, but not for R47H or K186N expressing cells. Together, the results from these studies demonstrate that specific residues in the ligand binding domains of the microglial surface receptors, hCD33 and hTREM2, play key roles in intracellular signalling.