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siRNA knock-down of Fe65 in SH-SY5Y cells decreases the levels of C-terminal fragments of APP without any effect on sAPPα secretion
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0002-8268-3006
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0002-0308-1964
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Fe65 is an adaptor protein that binds to the amyloid precursor protein (APP) within the 82YENPTY687 motif of APP which is important for amyloid β (Aβ) production. Considering that Fe65 binds to this motif, it can be hypothesized that Fe65 may influence the trafficking of APP and hence its processing by α- and/or β-secretase. Therefore in this study we wanted to determine how knock-down of Fe65 effects the processing of endogenous APP in human SH-SY5Y neuroblastoma cells. Our results showed that Fe65 knock-down did not have any effect on sAPPα secretion in SH-SY5Y cells. However, decreased levels of membrane-bound APP stubs C83 and C99 were observed, suggesting that Fe65 has a stabilizing effect on the C-terminal fragments. Furthermore, we wanted to investigate effects of retinoic acid (RA)-induced neronal differentiation on Fe65 expression. It has previously been shown that under these conditions mRNA and protein levels of APP increase concomitant with increased secretion of sAPPα, shifting the processing of APP to the more non-amyloidogenic pathway. We observed that RA-induced neuronal differentiation increases the protein levels of Fe65 in SH-SY5Y cells and gives rise to an electrophoretic mobility shift due to increased phosphorylation. The increased expression levels of Fe65 during neuronal differentiation concomitant with the increase of Fe65 phosphorylation, suggest that Fe65 and its phosphorylation may play a role during neuronal differentiation.

National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-100830OAI: oai:DiVA.org:su-100830DiVA: diva2:696966
Available from: 2014-02-17 Created: 2014-02-17 Last updated: 2016-01-29Bibliographically approved
In thesis
1. The adaptor protein Fe65 and APP processing
Open this publication in new window or tab >>The adaptor protein Fe65 and APP processing
2014 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

The amyloid precursor protein (APP) protein has been in the limelight of research on Alzheimer´s disease (AD) pathogenesis because its proteolytic processing gives rise to the neurotoxic amyloid β (Aβ) peptide, the main constituent of amyloid plaques in the brains of AD patients. APP is sequentially processed by at least three different proteases termed α-, β-, and γ-secretases. The proteolytic processing of APP can be divided into two different pathways, the non-amyloidogenic and the amyloidogenic. Whether APP is processed by the non-amyloidogenic or the amyloidogenic pathway is highly dependent on colocalization of APP with the different processing enzymes. Hence, understanding the mechanism underlying regulation of APP trafficking and its related secretases is of great importance in our understanding of AD and AD pathogenesis. The aim of this thesis was to study the processing and trafficking of APP, how it may be regulated by the interaction with the adaptor protein, Fe65, and by a novel type of posttranslational modification, O-GlcNAcylation. We have used the human neuroblastoma cell line SH-SY5Y as a modell system to investigate the effect of Fe65 knock-down on APP processing. Our results showed that Fe65 knockdown did not have any effect on sAPPα secretion. However, decreased levels of C83 and C99 were observed, suggesting that Fe65 has a stabilizing effect on the C-terminal fragments. Furthermore, we investigated the effects of RA-induced neronal differentiation on Fe65 expression. We observed increased protein levels of Fe65 and an electrophoretic mobility shift due to increased phosphorylation of Fe65. O-GlcNAcylation is a dynamic posttranslational modification regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). To investigate the effect of O-GlcNAcylation on APP trafficking and processing, SH-SY5Y cells were treated with PUGNAc, an OGA inhibitor, to increase the cellular levels of O-GlcNAc. The results revealed that cell surface localization of mature APP was significantly enhanced without any affect on the total levels of APP. We further show evidence that ADAM10 is O-GlcNAcylated and that the effect of O-GlcNAcylation on APP processing is neuron-specific.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2014. 55 p.
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-100483 (URN)978-91-7447-863-1 (ISBN)
Presentation
2014-02-25, Heillbronnsalen C 458, Department of Neurochemistry, Stockholm, 14:00 (English)
Opponent
Supervisors
Available from: 2014-02-18 Created: 2014-02-04 Last updated: 2015-03-13Bibliographically approved

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