Biochemistry and Pharmacology - Theses
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ItemTrafficking and Processing of Amyloid Precursor Protein in Alzheimer’s Disease and the Secretory Network in Human Neurons( 2023-02)Alzheimer’s disease, a prevalent neurodegenerative disorder, is the leading cause of dementia. The production of amyloid-beta (A-beta) describes the most commonly accepted disease progression mechanism of Alzheimer’s disease, which involves initial cleavage of the amyloid precursor protein (APP) by the beta-secretase BACE1. APP and BACE1 are both transmembrane proteins; cleavage events only occur when BACE1 co-localises with APP in the same membrane compartment. Hence, intracellular trafficking of APP is critical in regulating APP processing and A-beta production. However, the intracellular sites of APP cleavage are often inferred from detection of APP proteolytic products under steady-state conditions, and have not been previously described in real-time. In this thesis, using an advanced method to synchronise protein transport, the “RUSH” system, I have established a profile of APP transport and cleavage along its trafficking pathway using stable cell lines expressing fluorescently tagged RUSH-APP constructs. The intracellular location of APP and the timing of APP cleavage have been linked directly to define the spatio-temporal regulation of APP processing. Previous work in the laboratory has shown that under physiological conditions, APP and BACE1 are segregated in the Golgi apparatus and sorted into different pathways from the trans-Golgi network. However, it is not known if APP trafficking is altered under disease conditions which may impact on the level of APP processing. In chapter 4, I compared the trafficking and processing kinetics between wild type APP (APPwt) and APP bearing disease-related familial mutations using the RUSH system. Familial disease-related APP mutants displayed distinct Golgi trafficking kinetics and beta-secretase processing. APP with the pathogenic Swedish mutation (APPswe) is transported less efficiently through the Golgi and is associated with enhanced beta-secretase processing and A-beta secretion. In contrast, APP with the protective Icelandic mutation (APPice) is transported more rapidly through the Golgi and is associated with low levels of beta-secretase processing. beta-secretase processing of APP was demonstrated to occur in the secretory pathway before APP is delivered from the TGN, and likely to play an important role in A-beta production associated with disease pathogenesis. In contrast with non-polarised cells, neurons are highly specialised with multiple branched dendrites and a long axon. The large area of the neuronal cell surface poses great challenges in protein trafficking: newly synthesised axonal and dendritic proteins from the soma need to be delivered across long distances to their functional destinations to maintain synaptic activities. A solution to the quandary is “local” protein synthesis and trafficking in dendrites away from the central cell body, which was proposed after the identification of endoplasmic reticulum (ER) and Golgi outpost structures in the dendrites of rodent and Drosophila neurons. However, little is known about the dynamics of the local secretory system and whether the organelles are transient or stable structures. Moreover, it is not known whether the secretory system of human neurons is similar or different to rodents and/or whether there are unique adaptations of the secretory system in human neurons. In chapter 5, I have generated human neurons from induced pluripotent stem cells (iPSC) and mapped for the first time the local secretory network in the dendrites of human iPSC-derived neurons. In early neuronal development, the entire Golgi apparatus transiently translocates from the soma into the neurites. In mature neurons, dynamic Golgi elements, containing cis- and trans-cisternae, are transported from the soma along the dendrites in an actin-dependent process. Dendritic Golgi outposts in human neurons are dynamic and display bidirectional movement, which is different from rodent or Drosophila models. Using the RUSH system, Golgi resident proteins were shown to be transported efficiently into Golgi outposts from the endoplasmic reticulum. Findings in chapter 5 have revealed unique features of the secretory system in human neurons and identified a spatial map for investigating dendrite trafficking in human neurons. Overall, findings in this thesis have illustrated that alternations in intracellular trafficking of APP are associated with dysregulated APP beta-secretase cleavage, and traffic of APP through the Golgi plays an important role in its beta-secretase processing. In human neurons, specialised trafficking organelles, in particular the dendritic Golgi structures, are likely to enable additional local protein trafficking pathways in dendrites away from the central soma machinery. The work in this thesis contributed to better understanding of molecular pathogenesis of Alzheimer’s disease and laid foundations for investigating membrane trafficking in human neurons in healthy and disease conditions.
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ItemMicroglia and type-I interferons: emerging actors in Alzheimer’s disease( 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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ItemNo Preview AvailableUnderstanding the Role of the Innate Immune System in Alzheimer’s and Parkinson’s Disease Pathogenesis( 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.
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ItemInvestigating the functional roles of microRNA-29b and microRNA-146a in prion diseases( 2018)Neurodegenerative diseases such as Alzheimer’s disease and prion disease are closely related with specific gene and protein dysfunction. Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are characterized by the structural transformation of the cellular prion protein (PrPC) to the disease associated isoform (PrPSc). One hallmark of Alzheimer’s disease is the accumulation of beta amyloid (Aβ) plaques in the brain, resulting from the pathological cleavage of the amyloid precursor protein (APP) by β-secretase (BACE1) and the γ-secretase complex. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene or protein expression by targeting mRNAs and triggering either translational repression or mRNA degradation. Distinct expression levels of miRNAs, including miR-29b and miR-146a, have been detected in various biological fluids and tissues from prion disease and Alzheimer’s disease patients, as well as in cell and animal models. These miRNAs could be potential diagnostic biomarkers of these diseases, suggesting that investigating the miRNA functional roles and miRNA-target regulation pathways will improve our understanding of the disease regulation networks. The first aspect of the thesis utilized CRISPR/Cas9 gene editing to knockdown miR-29b and miR-146a respectively in a number of cell lines, and cell clones with stable miRNA knockdown were generated. Off-target analysis of the cell clones revealed the high specificity of CRISPR/Cas9 editing of miRNAs. Common and distinct pathways and novel targets of miR-29b were also identified in two cell lines using transcriptome profiling, and potential miR-29b targets in Alzheimer’s and prion diseases were revealed. In the second aspect of the thesis, miR-29b was shown to positively regulate prion protein levels in both miR-29b stable knockdown cell clones and miR-29b overexpressed cells. This regulation is not mediated through miR-29b target SP1 or potential target PPP2CA, which can interact with prion protein or was implicated in prion pathogenesis. miR-29b could further affect PrPSc generation through regulating prion protein levels and potentially affect prion progression. miR-29b was also revealed to regulate APP and BACE1, the two key proteins in Alzheimer’s disease, in in vitro models. Lastly, the dual roles of miR-146a in regulating prion protein and inflammatory pathways were revealed in prion disease. miR-146a can upregulate prion protein levels in both overexpressed and stably downregulated cell models, as well as in miR-146a transgenic mice generated using CRISPR/Cas9 gene editing. miR-146a overexpression also resulted in the decreased formation of PrPSc in prion cell models. The miR-29b/miR-146a-PrP-PrPSc pathways possibly share a similar mechanism involving the interaction of prion protein with Argonaute protein – the key component of miRNA induced silencing complex (miRISC). Prion protein was demonstrated to be a direct target of miR-146a. miR-146a can also target inflammatory regulator TRAF6 in both prion infected cell models and in miR-146a transgenic mice. The findings from this thesis have important implications for the comprehensive understanding of prion disease pathogenesis. The miR-29b/miR-146a-PrP-PrPSc pathways and miR-146a mediated inflammatory pathway are added to the regulation network of prion disease. miRNAs represent novel regulators in prion diseases and other neurodegenerative disorders and hold promise to be future therapeutics to cure prion disease.