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The amyloid-β precursor protein (APP)-binding protein Fe65 and APP processing
Stockholm University, Faculty of Science, Department of Neurochemistry. (Anna-Lena Ström)ORCID iD: 0000-0002-8268-3006
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by abnormal deposition of neurotoxic amyloid-β (Aβ) peptide. Aβ is generated by sequential cleavage of the amyloid-β precursor protein (APP) by β- and then γ-secretase. However, APP can also be processed by α- and γ-secretase, instead resulting in generation of neuroprotective sAPPα. Increased APP phosphorylation and altered expression levels of the brain enriched Fe65 protein have been observed in the brains of AD patients. Fe65 can not only interact with membrane tethered APP, but can also localized into the nucleus and act as a transcriptional regulator together with the APP intracellular domain (AICD), generated after γ-secretase processing. How APP processing, APP/Fe65 interaction, and the nuclear AICD/Fe65 complex is regulated has not yet been fully understood. The aim of this thesis was therefore to further elucidate how Fe65 is regulated and how APP Ser675 phosphorylation affects APP processing.

We could identify several factors regulating Fe65. First, we identified that neuronal differentiation induces Fe65 phosphorylation (paper I), and that phosphorylated forms of Fe65 were preferentially localized outside the nucleus (paper II). Second, we found that the APP binding PTB2 domain of Fe65, rather than the previously proposed N-terminal WW domain, is important for the nuclear localization of Fe65 (paper II). In addition, we surprisingly found that mutation of S228 in the Fe65 N-terminus could increase the APP/Fe65 interaction (paper III). Third, both α- and γ-secretase inhibitors decreased Fe65 nuclear localization similarly, indicating an important role of α-secretase in regulating Fe65 nuclear localization (papers II and III). Lastly, we could in paper IV for the first time show that phosphorylation of APP at Ser675 regulates APP processing at the plasma membrane, resulting in reduced levels of sAPPα. These results, together with the observation that APP Ser675 phosphorylation occur in AD brains, suggest that Ser675 phosphorylation could contribute to AD pathology by decreasing α-secretase processing and instead increasing the levels of Aβ.

In summary these studies have contributed to understanding of APP processing and the interplay between Fe65 and APP, two suggested key players in AD. 

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2018. , p. 89
Keyword [en]
APP, Fe65, ADAM10, Alzheimer's disease
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-149906ISBN: 978-91-7797-112-2 (print)ISBN: 978-91-7797-113-9 (electronic)OAI: oai:DiVA.org:su-149906DiVA, id: diva2:1164644
Public defence
2018-02-02, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2018-01-10 Created: 2017-12-11 Last updated: 2018-01-11Bibliographically approved
List of papers
1. Phosphorylation of Fe65 amyloid precursor protein-binding protein in response to neuronal differentiation
Open this publication in new window or tab >>Phosphorylation of Fe65 amyloid precursor protein-binding protein in response to neuronal differentiation
2016 (English)In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 613, p. 54-59Article in journal (Refereed) Published
Abstract [en]

Fe65 is a brain enriched multi domain adaptor protein involved in diverse cellular functions. One of its binding partners is the amyloid-beta (A beta) precursor protein (APP), which after sequential proteolytic processing by secretases gives rise to the Alzheimer's A beta peptide. Fe65 binds to the APP intracellular domain (AICD). Several studies have indicated that Fe65 binding promotes the amyloidogenic processing of APP. It has previously been shown that expression of APP increases concomitantly with a shift of its processing to the non-amyloidogenic pathway during neuronal differentiation. In this study we wanted to investigate the effects of neuronal differentiation on Fe65 expression. We observed that differentiation of SH-SY5Y human neuroblastoma cells induced by retinoic acid (RA), the phorbol ester PMA, or the gamma-secretase inhibitor DAPT resulted in an electrophoretic mobility shift of Fe65. Similar effects were observed in rat PC6.3 cells treated with nerve growth factor. The electrophoretic mobility shift was shown to be due to phosphorylation. Previous studies have shown that Fe65 phosphorylation can prevent the APP-Fe65 interaction. We propose that phosphorylation is a way to modify the functions of Fe65 and to promote the non-amyloidogenic processing of APP during neuronal differentiation.

Keyword
Fe65, Neuroblastoma cells, Phorbol ester, Phosphorylation, Retinoic acid, gamma-secretase
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-127350 (URN)10.1016/j.neulet.2015.12.050 (DOI)000369463200010 ()26742640 (PubMedID)
Available from: 2016-03-11 Created: 2016-03-02 Last updated: 2017-12-14Bibliographically approved
2. Nuclear localization of amyloid-beta precursor protein-binding protein Fe65 is dependent on regulated intramembrane proteolysis
Open this publication in new window or tab >>Nuclear localization of amyloid-beta precursor protein-binding protein Fe65 is dependent on regulated intramembrane proteolysis
Show others...
2017 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 3, article id e0173888Article in journal (Refereed) Published
Abstract [en]

Fe65 is an adaptor protein involved in both processing and signaling of the Alzheimer-associated amyloid-beta precursor protein, APP. Here, the subcellular localization was further investigated using TAP-tagged Fe65 constructs expressed in human neuroblastoma cells. Our results indicate that PTB2 rather than theWWdomain is important for the nuclear localization of Fe65. Electrophoretic mobility shift of Fe65 caused by phosphorylation was not detected in the nuclear fraction, suggesting that phosphorylation could restrict nuclear localization of Fe65. Furthermore, both ADAM10 and gamma-secretase inhibitors decreased nuclear Fe65 in a similar way indicating an important role also of alpha-secretase in regulating nuclear translocation.

National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-143465 (URN)10.1371/journal.pone.0173888 (DOI)000399089000040 ()28323844 (PubMedID)
Available from: 2017-06-02 Created: 2017-06-02 Last updated: 2017-12-14Bibliographically approved
3. ADAM10 dependent nuclear localization of the amyloid-β precursor protein-binding protein Fe65 is attenuated in neuronally differentiated SH-SY5Y cells
Open this publication in new window or tab >>ADAM10 dependent nuclear localization of the amyloid-β precursor protein-binding protein Fe65 is attenuated in neuronally differentiated SH-SY5Y cells
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Fe65 is a brain enriched adaptor protein involved in various cellular processes. These processes may include regulated intramembrane proteolysis (RIP) of the amyloid-β precursor protein (APP) and transcriptional activation. However, much still needs to be learned regarding the regulation of Fe65 functions throughout the cell. In this study we therefore investigated the role of Fe65 Ser228 phosphorylation and α-secretase processing of proteins like APP undergoing RIP, in the regulation of Fe65 nuclear localization. We found that although Ser228 phosphorylation is not a major regulator of Fe65 nuclear localization, mutation of Ser228 results in an increased interaction with APP, suggesting that the N-terminal domain of Fe65 may have a more prominent role in mediating the Fe65-APP interaction than previously thought.  Moreover, we found that α-secretase processing play a key role in promoting Fe65 nuclear localization, but while ADAM10 play a considerable role in undifferentiated cells, other α-secretases take a more prominent part in releasing Fe65 from the plasma membrane in differentiated cells.      

Keyword
ADAM10, Fe65, APP
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-149905 (URN)
Available from: 2017-12-11 Created: 2017-12-11 Last updated: 2017-12-14Bibliographically approved
4. APP Ser675 phosphorylation affects α-secretase processing resulting in decreased secretion of the neuroprotective ectodomain sAPPα
Open this publication in new window or tab >>APP Ser675 phosphorylation affects α-secretase processing resulting in decreased secretion of the neuroprotective ectodomain sAPPα
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by abnormal deposition of the amyloid-β (Aβ) peptide. Aβ is produced after amyloidogenic (β-secretase) processing of the transmembrane amyloid precursor protein (APP). However, APP can also be processed by α-secretases, instead resulting in release of neuroprotective sAPPα.  Growing evidence indicate that aberrant post-translational modifications of APP may play a pivotal role in AD pathogenesis by dysregulating APP processing. APP Ser675 phosphorylation occurs in AD brains and here we for the first time show that this phosphorylation decreases the release of sAPPα, while the level of an alternative APP-C83-CTF fragment is increased. Moreover, we found that while APP Ser675 phosphorylation increased the APP-Fe65 interaction, the level of APP at the plasma membrane were unaltered. Taken together these results suggest that APP Ser675 phosphorylation alters the α-secretase processing of APP at the plasma membrane. As α-secretase processing of APP is an essential step in decreasing the generation of Aβ these results suggest that Ser675 phosphorylation could contribute to AD pathology.

Keyword
Alzheimer's disease, Amyloid precursor protein
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-149903 (URN)
Available from: 2017-12-11 Created: 2017-12-11 Last updated: 2017-12-14Bibliographically approved

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