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.