Anterograde sorting and trafficking of the β-amyloid precursor protein and β-Secretase in Alzheimer’s disease
AuthorTan, Jing Zhi Anson
AffiliationBiochemistry and Molecular Biology
Bio21 Molecular Science and Biotechnology Institute
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2020-09-14.
© 2018 Dr. Jing Zhi Anson Tan
Alzheimer’s disease (AD) is characterized by the extracellular deposition of amyloid plaques in the brain. Amyloid plaques are derived from the aggregation of pathogenic β-amyloid (Aβ) peptide, which is generated from the sequential amyloidogenic processing of the β-amyloid precursor protein (APP) by membrane-bound proteases, β-secretase (BACE1) and γ-secretase. Several AD susceptibility alleles have been associated with membrane trafficking of APP and BACE1, and the regulation of Aβ production. Although the endocytic pathway has received considerable attention, early studies indicated that the secretory/anterograde pathway can also generate Aβ. However, the anterograde transport of APP and BACE1, and particularly the mechanism of sorting of these proteins in the Golgi, has not been well-defined yet is highly relevant for understanding the pathogenesis of AD. My thesis aimed to define (1) the trafficking machineries that are required for regulating the Golgi export of APP and BACE1; (2) the post-Golgi trafficking itineraries of newly synthesized APP and BACE1; (3) the relevance of my findings for the trafficking of APP and BACE1 in primary neurons; and (4) the importance of trafficking machineries in regulating the intracellular processing of APP in the Golgi. As newly synthesized APP and BACE1 are sorted at the trans-Golgi network (TGN), dysregulation in the sorting of APP and mature BACE1 is likely to enhance convergence of the two proteins to promote amyloidogenic processing in this compartment. I investigated the sorting and trafficking of newly synthesized APP and BACE1 from the Golgi using a range of approaches including RNA interference, immunofluorescence microscopy, flow cytometry, and the retention using selective hooks (RUSH) system. In Chapter 3, I have shown that Arl5b is required for the efficient recruitment of AP4 to the TGN which regulates the export of APP, but not BACE1, in HeLa cells. From the Golgi, newly synthesized APP traffics directly to the early endosomes then the late endosomes/lysosomes. In contrast, I have demonstrated in Chapter 4 that newly synthesized BACE1 is transported directly to the PM from the TGN in HeLa cells. Moreover, the TGN export of BACE1 is regulated by AP1/Arf1/Arf4 transport machineries, which are distinct from the transport machinery required for the post-Golgi transport of APP. Depletion of either AP4/Arl5b or AP1/Arf1/Arf4 export machineries in the presence of a γ-secretase inhibitor (DAPT) increased the level of β- CTF/C99, the product of APP cleavage by BACE1, confirming that the direct cleavage of APP by endogenous BACE1 was enhanced when the Golgi export of either APP or BACE1 was impaired. In the absence of DAPT, only low levels of β-CTF/C99 were detected following depletion of the Golgi export machineries for either APP or BACE1. These findings indicate that β-CTF/C99 is rapidly processed by γ-secretase to liberate Aβ and that the biogenesis of Aβ probably occurs in the TGN. Therefore, the accumulation of APP or BACE1 in the TGN after the depletion of AP4/Arl5b or AP1/Arf1/Arf4 post- Golgi export machineries, respectively, resulted in enhanced amyloidogenic processing of APP. To determine the significance of the post-Golgi trafficking studies of APP and BACE1 conducted in HeLa cells, I generated shRNA lentivirus and successfully depleted AP4 and AP1 in primary neurons and analyzed the impact on endogenous APP trafficking. I have shown that AP4, but not AP1, is required for the trafficking of endogenous APP from the TGN in neurons. Moreover, the depletion of AP4 in primary neurons resulted in enhanced processing of APP by endogenous BACE1 in the TGN. The intracellular sites of APP cleavage by 𝛼-secretase along the protective nonamyloidogenic pathway have not been well-defined. In Chapter 5 and 6, I have demonstrated that APP can be processed by endogenous 𝛼-secretase in the TGN of both HeLa and neuronal cells. These findings are significant as the TGN had not been previously recognized as a site for 𝛼-secretase processing. Overall, the findings presented in this thesis have shown that efficient post-Golgi trafficking of newly synthesized APP and BACE1, which are regulated by distinct transport mechanisms, plays a critical role in the segregation of the two proteins. Dysregulation in the Golgi exit of either APP or BACE1 increases the residency time of these membrane cargoes in the TGN resulting in enhanced pathogenic processing of APP, most likely as a consequence of the loss of segregation and increased colocalization.
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