Biochemistry and Pharmacology - Theses
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ItemFunctional roles of serum amyloid P component in amyloid diseases(University of Melbourne, 2006)
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ItemFunctional roles of serum amyloid P component in amyloid diseases(University of Melbourne, 2006)
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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.
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ItemThe intracellular trafficking pathways of β-secretase and the amyloid precursor protein in Alzheimer’s disease( 2015)Alzheimer’s disease (AD) is characterized by the accumulation of amyloid plaques in the brain consisting of an aggregated form of β-amyloid peptide (Aβ) derived from sequential amyloidogenic processing of the Amyloid Precursor Protein (APP) by membrane-bound proteases BACE1 and γ-secretase. The trafficking of these components in the amyloidogenic pathway is thought to influence the levels of Aβ production. In fact, GWAS (Genome wide sequencing studies) has identified several AD susceptibility genes that are associated with the regulation of membrane trafficking (Harold et al., 2009, Lambert et al., 2009, Lee et al., 2011), suggesting that AD could be a result of defective membrane trafficking. The main aims of my thesis were to 1) define the intracellular trafficking itineraries of BACE1 and APP, 2) identify the sorting signals of BACE1 and the molecular machinery responsible for BACE1 endosomal transport, 3) identify the anterograde transport pathways of BACE1 and APP and lastly, 4) investigate the intracellular localization of endogenous BACE1 and APP in mouse primary cortical neurons and the influence of neuronal stimulation on their localization. Knowledge of the intracellular trafficking pathways of BACE1 and APP remain only poorly defined. Previous studies have suggested that BACE1 recycles via the trans-Golgi network (TGN) while APP is trafficked to either the TGN or the late endosome. In Chapter 3, I showed BACE1 is rapidly internalized from the plasma membrane and subsequently traffics to the early endosomes and Rab11-positive, juxtanuclear recycling endosomes in a number of cell lines, including neuronal cells whereas very little internalised BACE1 is transported to the TGN. In contrast, the majority of the internalized APP traffics to the late endosomes/ lysosomes. Events that may alter the kinetics of endosomal sorting of BACE1 or APP would alter the residency time of either protein in endosomes which may impact on Aβ production. In Chapter 4, I assessed the mechanisms that regulate endosomal sorting of BACE1. A phosphorylated DISLL motif has been identified in the cytoplasmic tail of BACE1 in vivo which may be involved in endosomal sorting (Walter et al., 2001). To explore a role for this DISLL motif in early endosome-to-recycling endosome trafficking, the impact of Ser498 phosphorylation on this trafficking step was examined. Using antibody internalization assays, the phosphomimetic S498D mutant of BACE1 was found to traffic to the recycling endosomes at a faster rate compared to wild-type BACE1 while the non-phosphorylatable S498A mutant traffics to the recycling endosomes at a slower rate. Increased residency time of BACE1 S498A in the early endosome resulted increased Aβ production while expression of BACE1 S498D reduced Aβ production. These results suggest that phosphorylation of the BACE1 sorting motif can alter residency time of BACE1 in the early endosomes which in turns affects Aβ production. Expression of BACE1 phospho-mutants in mouse primary cortical neurons showed that BACE1 S498A localized predominantly to early endosomes while BACE1 S498D localized predominantly to the recycling endosomes, a result consistent with the results obtained in HeLa cells. In Chapter 4, I have also identified the molecular machinery, Rab11 and SNX4 which are essential for the regulation of BACE1 endosomal sorting. Depletion of Rab11 resulted in a block in BACE1 transport from the recycling endosomes to the cell surface. Live cell imaging showed an increased number and half-life of dynamic tubules containing BACE1 emanating from intracellular structures towards the cell surface as compared to Rab11 positive cells. It is likely that the absent of Rab11 results in reduced fusion of the transport carriers to the plasma membrane. Depletion of SNX4, a machinery component that mediates biogenesis of transport carriers from early endosomes to recycling endosomes resulted in both BACE1 and transferrin receptor redirected to the lysosomes. Depletion of Rab11 or SNX4 resulted in decreased or increased Aβ production respectively, suggesting that functional trafficking machinery is important in preventing excessive Aβ production. Aβ can also be generated along the anterograde trafficking pathway from the ER to the cell surface; however the anterograde transport itineraries of BACE1 and APP have yet to be defined. Arl5b, a member of the ARL family of small G proteins, has recently been discovered by my laboratory to localize to the trans-Golgi network (TGN) in a GTP-dependent fashion and regulates Golgi-endosome trafficking (Houghton et al., 2012). In Chapter 5, I show that APP is transported from the TGN to the early endosome after exiting the TGN. In addition, I show that Arl5b recruits and interacts with an adaptor protein complex-4 (AP4) to regulate APP trafficking. Depletion of Arl5b or AP4 accumulates APP in the Golgi and results in increased Aβ production. Arl5b depletion did not affect BACE1 distribution, indicating that the action of Arl5b is specific for APP. It is likely that APP and BACE1 utilize distinct sets of machinery for post-Golgi transport along distinct pathways. There is increasing evidence suggesting that Aβ production is linked to the levels of neuronal activity in the brain (Kamenetz et al., 2003, Cirrito et al., 2005, Das et al., 2013). Increased neuronal activity is associated with Aβ production (Kamenetz et al., 2003). In Chapter 6, I raised rabbit polyclonal antibodies to a BACE1 peptide which is capable of recognizing endogenous BACE1 in mouse primary cortical neurons and human neuroblastoma cell line, SH-SY5Y. In resting primary neurons, I show that endogenous BACE1 and APP resided in distinct compartments with only modest co-localization. Endogenous BACE1 resided predominantly in Rab11 recycling endosomes while endogenous APP resided predominantly in Rab7 late endosome. NMDA receptor activation-induced neuronal stimulation of primary neurons increased endogenous BACE1 and APP co-localization and also increased APP co-localization with Rab11. These results demonstrate that changes in neuronal activity altered the intracellular distribution of APP to increase co-localization with BACE1. Hence it is possible that signalling may be an important factor in regulating the trafficking of BACE1 and APP and may influence the convergence of both components and total Aβ production.