In Alzheimer’s disease, the beta-amyloid (Aβ) protein accumulates within neurons prior to amyloid plaque development. Recent evidence suggests that intracellular Aβ accumulation, rather than extracellular plaques, correlates best with disease progression. Aβ is generated by proteolytic processing of the amyloid precursor protein (APP) but the intracellular site of Aβ production is unclear. Aβ has been localised to multivesicular endosomes/bodies (MVBs), which have many cellular functions, including delivery of contents to the lysosome and release of intralumenal vesicles (ILVs) into the extracellular space. Several MVB and ILV populations exist that are formed by distinct mechanisms and have different fates. This study aimed to determine to which MVB population APP traffics, the role of MVBs in Aβ production and the functional consequences of Aβ production and accumulation within MVBs. Immunofluorescence and immuno-EM showed that APP trafficks to the ILVs of an EGF receptor-containing subpopulation of MVBs in a neuroglioma cell line overexpressing APP. To determine the mechanism of APP ILV targeting, we first characterized the effects of inhibition of ESCRT-dependent and ESCRT-independent mechanisms of ILV formation in HeLa cells. We found that EGF stimulation, which promotes ESCRT-dependent ILV formation, causes the generation of enlarged ILVs, whilst depletion of the ESCRT-0 component, Hrs, leads to the reduction of ILV number and size. In neuroblastoma cells overexpressing APP, APP is ubiquitinated and depletion of Hrs inhibits sorting of APP onto ILVs and Aβ secretion, implicating the ESCRT machinery in traffic of APP and Aβ generation. To analyse the effects of Aβ accumulation in the endocytic pathway, we determined the effects of exogenous Aβ endocytosed by HeLa cells and primary neurons, and demonstrated that Aβ can cause disruption of endosome-lysosome morphology and membrane impermeability. These results indicate that APP traffic to MVBs and generation of Aβ within them could contribute to early AD pathology.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:587688 |
| Date | January 2013 |
| Creators | Edgar, J. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1382533/ |
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