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The amyloid-β precursor protein (APP) and its adaptor protein Fe65: Two key players in Alzheimer’s disease
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0001-6461-451x
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by the abnormal accumulation and aggregation of amyloid beta (Aβ) peptides within the brain. Generation of Aβ occur when the amyloid-beta precursor protein (APP) is proteolytically processed by β- and then γ-secretase in the amyloidogenic pathway. However, if APP instead is cleaved by α- and γ-secretase in the non-amyloidogenic pathway, Aβ formation is prevented and neuroprotective sAPPα is generated. In addition to these canonical processing pathways, APP can also be cleaved along non-canonical pathways by Δ, η, caspase or Meprinβ, resulting in numerous fragments that have different functional properties. The trafficking and processing of APP is a complex process and can be regulated by the adaptor protein Fe65. Following γ-secretase mediated cleavage of APP, the intracellular domain of APP and Fe65 can together translocate into the nucleus and regulate nuclear signaling. However, the exact mechanisms of how APP processing and APP/Fe65 nuclear signaling are regulated is still unclear.  

The aim of this thesis was to study different factors that may influence the regulation of APP processing and Fe65 nuclear localization. We found that phosphorylation of APP at Ser675 alters APP processing resulting in reduced levels of sAPPα and total sAPP, without affecting the plasma membrane level of APP. We could further observe an increased level of a slower migrating C99 like CTF, which was not generated by β-secretase cleavage of APP as there was no expression of BACE1 in the cell model used. Instead, generation of this CTF was blocked upon Meprinβ siRNA knockdown. Taken together these findings suggest that APP-Ser675 phosphorylation promotes Meprinβ processing of APP. In another study, we found that mutation of Ser228 at the Fe65 N-terminal dramatically increased the interaction between Fe65 and full-length APP. Moreover, this enhanced interaction resulted in decreased levels of non-amyloidogenic processing of APP and thus neuroprotective sAPPα. This suggest that the level of Fe65-APP interaction is important in regulating APP processing. Therefore, we also wanted to elucidate more about how the adaptor protein Fe65 is regulated. We found that Fe65 is likely phosphorylated on several residues in the N-terminus and that these phosphorylated forms preferentially localized in the cytoplasm. In addition, we could show that the nuclear level and nuclear/cytoplasmic ratio of Fe65 was decreased upon mutation of Fe65-Ser228 to glutamic acid, mimicking phosphorylation. Taken together this suggest that phosphorylation of Ser228 together with other residues in the N-terminus of Fe65 negatively regulate the Fe65 nuclear localization. In a third study, we could also show that the Fe65 PTB2 domain, rather than the WW domain, plays an important role in localizing Fe65 to the nucleus. Lastly, using different inhibitors, we found that blocking α-secretase processing decrease the Fe65 nuclear localization to the same extent as γ-secretase inhibition in both undifferentiated and RA or PMA differentiated cells. This suggest that α-secretase processing of APP or other Fe65 interacting transmembrane proteins play a more important role in regulation of Fe65 nuclear localization than previously thought. Interestingly, while ADAM10 was the most important α-secretase mediating this effect in undifferentiated cells, other α-secretases, likely ADAM17, played a more important role in RA or PMA differentiated neuroblastoma cells.

In summary, the results obtained in this thesis have increased the understanding of APP processing and how the adaptor protein Fe65 may act as a molecular switch altering APP cleavage.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2020. , p. 81
Keywords [en]
Alzheimer's disease, APP, Fe65, Meprinβ, α-secretase, APP processing
National Category
Biochemistry and Molecular Biology Neurosciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-181020ISBN: 978-91-7911-172-4 (print)ISBN: 978-91-7911-173-1 (electronic)OAI: oai:DiVA.org:su-181020DiVA, id: diva2:1426285
Public defence
2020-06-12, 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: 2020-05-20 Created: 2020-04-24 Last updated: 2020-05-25Bibliographically approved
List of papers
1. Phosphorylation of the amyloid precursor protein (APP) at Ser-675 promotes APP processing involving meprin β
Open this publication in new window or tab >>Phosphorylation of the amyloid precursor protein (APP) at Ser-675 promotes APP processing involving meprin β
2019 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 294, no 47, p. 17768-17776Article in journal (Refereed) Published
Abstract [en]

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by abnormal deposition of ß-amyloid (Aß) peptides. Aß is a cleavage product of the amyloid precursor protein (APP), and aberrant posttranslational modifications of APP can alter APP processing and increase Aß generation. In the AD brain, seven different residues, including Ser-675 (APP(695) numbering) in the APP cytoplasmic domain has been found to be phosphorylated. Here, we show that expression of a phosphomimetic variant of Ser-675 in APP (APP-S675E), in human neuroblastoma SK-N-AS cells, reduces secretion of the soluble APP ectodomain (sAPPα), even though the total plasma membrane level of APP was unchanged compared with APP levels in cells expressing APPwt or APP-S675A. Moreover, the level of an alternative larger C-terminal fragment (CTF) increased in the APP-S675E cells, whereas the CTF form that was most abundant in cells expressing APPwt or APP-S675A decreased in the APP-S675E cells. Upon siRNA-mediated knockdown of the astacin metalloprotease meprin ß, the levels of the alternative CTF decreased and the CTF ratio was restored back to APPwt levels. Our findings suggest that APP Ser-675 phosphorylation alters the balance of APP processing, increasing meprin ß mediated and decreasing α-secretase mediated processing of APP at the plasma membrane. As meprin ß cleavage of APP has been shown to result in formation of highly aggregation-prone, truncated Aß40/42 peptides, enhanced APP processing by this enzyme could contribute to AD pathology. We propose that it would be of interest to clarify in future studies how APP Ser-675 phosphorylation promotes meprin ß  mediated APP cleavage.

Keywords
amyloid precursor protein (APP), amyloid-beta (Aβ), ADAM, Alzheimer's disease, neurodegeneration, α-secretase 1 (BACE1), APP-CTF, meprin Β, proteolytic processing
National Category
Biochemistry and Molecular Biology Neurosciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-178571 (URN)10.1074/jbc.RA119.008310 (DOI)000504206800006 ()31604820 (PubMedID)
Available from: 2020-02-06 Created: 2020-02-06 Last updated: 2020-05-05Bibliographically 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: 2020-04-24Bibliographically approved
3. Mutation of Fe65-Ser228 enhances the Fe65-APP interaction and decreases non-amyloidogenic processing of APP
Open this publication in new window or tab >>Mutation of Fe65-Ser228 enhances the Fe65-APP interaction and decreases non-amyloidogenic processing of APP
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Alzheimer’s disease is a neurodegenerative disease characterized by aberrant proteolysis of the transmembrane protein APP. The brain enriched adaptor protein Fe65 interacts with APP and participates together with APP and/or APP fragments in a number of cytoplasmic and nuclear functions. However, how the Fe65 subcellular localization, interaction with APP/APP fragments are regulated, as well as how Fe65 influences APP processing, is still not fully understood. In this study, we investigated the effect of Fe65 Ser-228 phosphorylation on Fe65 nuclear localization, APP interaction and APP processing. We show that although a Ser-228 phosphomimetic variant of Fe65 (Fe65-S2285E) was not excluded from the nucleus, a clear reduction of the nuclear level and the nuclear/cytoplasmic ratio of Fe65-S228E could be observed, suggesting that phosphorylation of Ser-288 could participate in regulation of the Fe65 subcellular localization. Interestingly, we found that not only Fe65-S2285E, but also mutation of Ser-228 to alanine (Fe65-S228A) resulted in a similar and dramatic increase of the Fe65 interaction with full-length APP. Moreover, we found that this increased APP interaction resulted in reduced α-secretase processing of APP and thus less generation of the neuroprotective sAPPα fragment. This suggest that the N-terminal domain of Fe65 may have a more prominent role in mediating the Fe65-APP interaction and regulating APP processing than previously thought.

Keywords
Alzheimer's disease, Fe65, amyloid precursor protein
National Category
Cell and Molecular Biology Neurosciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-181017 (URN)
Funder
Swedish Research Council, 521-2012-2367
Available from: 2020-04-24 Created: 2020-04-24 Last updated: 2020-04-24Bibliographically approved
4. Neuronal differentiation attenuates ADAM10 dependent nuclear localization of the amyloid-β precursor protein-binding protein Fe65
Open this publication in new window or tab >>Neuronal differentiation attenuates ADAM10 dependent nuclear localization of the amyloid-β precursor protein-binding protein Fe65
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Fe65 is a brain enriched adaptor protein involved in various cellular processes, including actincytoskeleton regulation, DNA repair and transcription. A well-studied interacting partner of Fe65 is the transmembrane amyloid-β precursor protein (APP), which can undergo regulatedintramembrane proteolysis (RIP). Binding of Fe65 to APP is thought to “activate” Fe65 andfollowing β- and γ-secretase mediated RIP, the released APP intracellular domain (AICD)-Fe65 complex is believed to translocate to the nucleus and regulate transcription. In this study, we investigated if and to what extent Fe65 nuclear localization can also be regulated by different α-secretases, also known to participate in RIP of APP and other transmembrane proteins. We found that in both PMA and RA differentiated neuroblastoma cells, a strong negative impact on Fe65 nuclear localization, equal to the effect observed upon γ-secretase inhibition, could be observed following inhibition of all three ADAM9, ADAM10 and ADAM17 α-secretases. Consistent with our previous study showing that α-secretase processing regulate Fe65 nuclear localization in undifferentiated cells. However, in contrast to what we found in undifferentiated cells, the major constitutive APP α-secretase ADAM10, had little or no role in differentiated neuroblastoma cells. Instead, other α-secretases, likely ADAM17, played a more important role. Taken together this suggest that α-secretase processing of APP or other Fe65 interacting transmembrane proteins play an important role in regulating Fe65 nuclear translocation and functions.

Keywords
Alzheimer's disease, alpha-secretase, ADAM, differentiation
National Category
Biochemistry and Molecular Biology Neurosciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-181016 (URN)
Funder
Swedish Research Council, 521-2012-2367
Available from: 2020-04-24 Created: 2020-04-24 Last updated: 2020-04-24Bibliographically approved

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1314151617181916 of 28
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