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Nuclear localization of amyloid-beta precursor protein-binding protein Fe65 is dependent on regulated intramembrane proteolysis
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0002-8268-3006
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-6461-451X
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0002-1007-747X
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Number of Authors: 62017 (English)In: PLOS ONE, 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.

Place, publisher, year, edition, pages
2017. Vol. 12, no 3, article id e0173888
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-143465DOI: 10.1371/journal.pone.0173888ISI: 000399089000040PubMedID: 28323844OAI: oai:DiVA.org:su-143465DiVA, id: diva2:1104984
Available from: 2017-06-02 Created: 2017-06-02 Last updated: 2022-03-23Bibliographically approved
In thesis
1. The amyloid-β precursor protein (APP)-binding protein Fe65 and APP processing
Open this publication in new window or tab >>The amyloid-β precursor protein (APP)-binding protein Fe65 and APP processing
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
Keywords
APP, Fe65, ADAM10, Alzheimer's disease
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-149906 (URN)978-91-7797-112-2 (ISBN)978-91-7797-113-9 (ISBN)
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: 2022-02-28Bibliographically approved
2. Phosphorylation regulates APP and Fe65, two key players in Alzheimer’s disease
Open this publication in new window or tab >>Phosphorylation regulates APP and Fe65, two key players in Alzheimer’s disease
2018 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Alzheimer’s disease (AD) is a slow progressive neurodegenerative disease characterized by the accumulation of toxic amyloid beta (Aβ) peptide within the brain. APP plays an important role in AD, as the Aβ is formed when APP is sequentially cleaved by β- and γ-secretase. This is known as amyloidogenic processing of APP. However, non-amyloidogenic processing, in which APP is cleaved by α-secretases in the middle of the Aβ sequence, giving rise to the neuroprotective fragment sAPPα is also possible. In addition to amyloidogenic and non-amyloidogenic processing, APP can be processed along non-canonical pathways by δ, η , capase or Meprin β, resulting in numerous fragments which may have different functional properties. In paper 1, we for the first time show that phosphorylation of APP at Ser675 alters APP processing resulting in a significant decrease in the release of total sAPP and sAPPα, without affecting the plasma membrane level of APP. We further show an increased level of a slower migrating APP-CTF, similar to the expected size of β-secretase generated C99-CTF. However, no expression of the major β-secretase BACE1 was found in the model used and in the presence of metalloprotease inhibitors, generation of the slower migrating CTF was blocked. Taken together these findings suggest that Ser675 phosphorylation might promote APP processing by the metalloprotease Meprin β, an alternative β-secretase localized at the plasma membrane. How Ser675 phosphorylation could promote Meprin β cleavage is unclear, but an altered APP protein interaction could be involved as we found that mutation of Ser675 increased APP interaction with the adaptor protein Fe65. In paper 2, we wanted to elucidate more about how Fe65 is regulated and found that phosphorylated forms of Fe65 preferentially localize to the cytoplasm. Furthermore we showed that the Fe65 PTB2 domain, rather than the WW domain, plays an important role in localizing Fe65 to the nucleus. Together our results show that phosphorylation regulates both APP and Fe65, two important proteins linked to AD.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2018
Keywords
Alzheimer's disease, APP, Fe65, phosphorylation, RIP
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-160321 (URN)
Presentation
2018-10-19, C458, Heilbronnsalen, Svante Arrhenius väg 16, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2019-01-17 Created: 2018-09-18 Last updated: 2022-02-26Bibliographically approved
3. The amyloid-β precursor protein (APP) and its adaptor protein Fe65: Two key players in Alzheimer’s disease
Open this publication in new window or tab >>The amyloid-β precursor protein (APP) and its adaptor protein Fe65: Two key players in Alzheimer’s disease
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
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:nbn:se:su:diva-181020 (URN)978-91-7911-172-4 (ISBN)978-91-7911-173-1 (ISBN)
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: 2022-02-26Bibliographically approved
4. Links between plasma apoE and glucose metabolism, brain insulin signaling, and synaptic integrity: Relevance to Alzheimer’s disease pathophysiology
Open this publication in new window or tab >>Links between plasma apoE and glucose metabolism, brain insulin signaling, and synaptic integrity: Relevance to Alzheimer’s disease pathophysiology
2021 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Human apolipoprotein E (apoE) exists as three main isoforms called apoE2, apoE3, and apoE4, of which the E4 isoform is associated with increased Alzheimer’s disease (AD) risk. Brain glucose hypometabolism, linked to synaptic dysfunction, occurs years before symptom onset in AD, especially in APOEε4-carriers. An association between a higher ratio of plasma apoE4 to apoE3 levels and cerebral glucose hypometabolism was recently discovered in cognitively healthy APOEε3/ε4 subjects. A lower plasma apoE level, regardless of isoform, is linked to increased AD risk. How the plasma apoE level affects neurodegenerative processes in the brain is poorly understood, given that apoE doesn’t cross the blood-brain barrier (BBB). The main aim of this thesis was therefore to investigate a relationship between plasma apoE and features of AD pathophysiology. We explored plasma apoE levels and dimer/monomer formation in APOEε3 and APOEε4 homozygous controls, in patients with mild cognitive impairment (MCI) and AD. In APOEε4-carriers versus non-carriers, plasma apoE levels were lower and significantly correlated with AD biomarkers. ApoE3 homodimers were less in AD patients than in controls. We next examined potential links between plasma apoE, glucose, and insulin levels in the previously examined cognitively healthy APOEε3/ε4 subjects. Lower plasma apoE3 was associated with higher glucose levels in males and subjects with body max index above 25. Plasma glucose levels were negatively correlated with the cerebral metabolic rate of glucose and neuropsychological test scores. To explore the potential effects of a hepatic APOEε4 phenotype on the brain, we compared liver humanized mice with an APOEε4/ε4 versus an APOEε2/ε3 genotype. Mice with an APOEε4/ε4 liver exhibited reduced endogenous mouse apoE in the brain, accompanied by changes in markers of synaptic integrity and insulin signaling. Plasma apoE4 levels were negatively associated with some of the assessed markers. We further explored the effects of a high-fat diet (HFD) in mice with livers humanized with the AD risk-neutral APOEε3/ε3 genotype. Endogenous mouse apoE was increased in the hippocampus following an HFD, with concomitant effects on levels of synaptic markers. In the cortex, we found altered levels of insulin signaling and synaptic markers. Together, our findings indicate that alterations in apoE levels or distribution, hepatic APOEε4 phenotype, and HFD contribute to AD-related pathological processes.

Amyloidogenic processing of the amyloid precursor protein (APP) gives rise to Aβ peptides that assemble into the Aβ plaques found in AD. The binding of the adaptor protein Fe65, through its PTB2, to APP might enhance amyloidogenic APP processing. Fe65 is localized both in the cytoplasm and in the nucleus, with compartment-specific biological functions. Mechanisms affecting Fe65 subcellular localization are poorly understood. We explored the impact of the different Fe65 interaction domains WW and PTB2 and APP processing on Fe65 cellular localization. By transfecting Fe65-domain deletion constructs into neuroblastoma cell lines, we found that deleting the PTB2 domain almost abolished nuclear localization. Upon pharmacological inhibition of APP secretases, we found decreased Fe65 localization to the nucleus. To conclude, Fe65-APP interaction and APP processing may be important factors governing the Fe65 cellular localization.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2021. p. 69
Keywords
Alzheimer’s disease, apolipoprotein E, insulin, metabolism, Amyloid precursor protein (APP), Fe65
National Category
Neurosciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-191938 (URN)978-91-7911-422-0 (ISBN)978-91-7911-423-7 (ISBN)
Public defence
2021-05-24, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, and online via Zoom, public link is available at the department website, Stockholm, 14:00 (English)
Opponent
Supervisors
Available from: 2021-04-28 Created: 2021-04-06 Last updated: 2022-02-25Bibliographically approved

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Koistinen, Niina A.Edlund, Anna K.Menon, Preeti K.Ivanova, Elena V.Iverfeldt, Kerstin

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