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α-Secretase processing of the Alzheimer amyloid-β precursor protein and its homolog APLP2
Stockholm University, Faculty of Science, Department of Neurochemistry. (Kerstin Iverfeldt)
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The amyloid-β precursor protein (APP) has been widely studied due to its role in Alzheimer´s disease (AD). When APP is sequentially cleaved by β- and γ-secretase, amyloid-β (Aβ) is formed. Aβ is prone to aggregate and is toxic to neurons. However, the main processing pathway for APP involves initial cleavage at the α-site, within the Aβ region, instead generating a neuroprotective soluble fragment, sAPPα. APP is a member of a protein family, also including the proteins APLP1 and APLP2, which are processed in a similar way as APP. In addition, K/O studies in mice have shown that the three proteins have overlapping functions where APLP2 play a key physiological role. The aim of this thesis was to study mechanisms underlying the α-secretase processing of APP and APLP2. We have used the human neuroblastoma cell-line SH-SY5Y as a model system and stimulated α-secretase processing with insulin-like growth factor-1 (IGF-1) or retinoic acid (RA). Our results show that the stimulated α-site cleavage of APP and APLP2 is regulated by different signaling pathways and that the cleavage is mediated by different enzymes. APP was shown to be cleaved by ADAM10 in a PI3K-dependent manner, whereas APLP2 was cleaved by TACE in a PKC-dependent manner. We further show that protein levels and maturation of ADAM10 and TACE is increased in response to RA, mediated by a PI3K- or PKC-dependent signaling pathway, respectively. Another focus of our research has been O-GlcNAcylation, a dynamic post-translational modification regulated by the enzymes O-GlcNAc transferase and O-GlcNAcase (OGA). We show that decreased OGA activity stimulates sAPPα secretion, without affecting APLP2 processing. We further show that ADAM10 is O-GlcNAcylated. Lastly, we show that APP can be manipulated to be cleaved in a similar way as APLP2 during IGF-1 stimulation by substituting the E1 domain in APP with the E1 domain in APLP2. Together our results show distinct α-site processing mechanisms of APP and APLP2.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2013. , 57 p.
Keyword [en]
APP, APLP2, ADAM10, TACE, Alzheimer's Disease
National Category
Neurosciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-95114ISBN: 978-91-7447-732-0 (print)OAI: oai:DiVA.org:su-95114DiVA: diva2:658332
Public defence
2013-12-06, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrheniusväg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper 4: Manuscript; Paper 5: Manuscript.

Available from: 2013-11-14 Created: 2013-10-21 Last updated: 2015-03-09Bibliographically approved
List of papers
1. Insulin-like growth factor-1 (IGF-1)-induced processing of amyloid-β precursor protein (APP) and APP-like protein 2 is mediated by different metalloproteinases
Open this publication in new window or tab >>Insulin-like growth factor-1 (IGF-1)-induced processing of amyloid-β precursor protein (APP) and APP-like protein 2 is mediated by different metalloproteinases
2010 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 14, 10223-10231 p.Article in journal (Refereed) Published
Abstract [en]

α-Secretase cleavage of the amyloid precursor protein (APP) is of great interest since it prevents the formation of the Alzheimer-linked amyloid-β peptide. APP belongs to a conserved gene family including the two paralogues APP-like protein (APLP) 1 and 2. Insulin-like growth factor-1 (IGF-1) stimulates the shedding of all three proteins. IGF-1-induced shedding of both APP and APLP1 is dependent on phosphatidylinositol 3-kinase (PI3-K), whereas sAPLP2 secretion is independent of this signaling pathway. Here, we used human neuroblastoma SH-SY5Y cells to investigate the involvement of protein kinase C (PKC) in the proteolytic processing of endogenously expressed members of the APP family. Processing was induced by IGF-1 or retinoic acid, another known stimulator of APP a-secretase shedding. Our results show that stimulation of APP and APLP1 processing involves multiple signaling pathways, whereas APLP2 processing is mainly dependent on PKC. Next, we wanted to investigate if the difference in the regulation of APLP2 shedding compared to APP shedding could be due to involvement of different processing enzymes. We focused on the two major a-secretase candidates ADAM10 and TACE, which both are members of the ADAM (a disintegrin and metalloprotease) family. Shedding was analyzed in the presence of the ADAM10 inhibitor GI254023X, or after transfection with siRNA targeted against TACE. The results clearly demonstrate that different α-secretases are involved in IGF-1-induced processing. APP is mainly cleaved by ADAM10, whereas APLP2 processing is mediated by TACE. Finally, we also show that IGF-1 induces PKC-dependent phosphorylation of TACE.

Place, publisher, year, edition, pages
Bethesda: ASBMB, 2010
Keyword
Alzheimer disease, Amyloid, IGF-1, ADAM10, TACE
National Category
Physiology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-38487 (URN)10.1074/jbc.M109.038224 (DOI)000276264600006 ()
Available from: 2010-04-14 Created: 2010-04-14 Last updated: 2017-12-12Bibliographically approved
2. PI3-K- and PKC-dependent up-regulation of APP processing enzymes by retinoic acid
Open this publication in new window or tab >>PI3-K- and PKC-dependent up-regulation of APP processing enzymes by retinoic acid
Show others...
2008 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 365, no 2, 298-303 p.Article in journal (Refereed) Published
Abstract [en]

Retinoic acid stimulates α-secretase processing of amyloid precursor protein (APP) and decreases β-secretase cleavage that leads to amyloid-β formation. Here, we investigated the effect of retinoic acid on the two putative α-secretases, the disintegrin metalloproteinases ADAM10 and TACE, and the β-site cleaving enzyme BACE1, in human neuroblastoma SH-SY5Y cells. Western blot analysis showed that exposure to retinoic acid resulted in significantly increased levels of ADAM10 and TACE, suggesting that regulation of α-secretases causes the effects on APP processing. The presence of the phosphatidylinositol 3-kinase inhibitor LY 294002 selectively reduced the effect on ADAM10 protein levels but not on ADAM10 mRNA levels as determined by RT-PCR. On the other hand, the effect on TACE was shown to be dependent on protein kinase C, since it was completely blocked in the presence of the inhibitor bisindolylmaleimide XI. Our data indicate that different signalling pathways are involved in retinoic acid-induced up-regulation of the secretases.

Keyword
ADAM10, APP, BACE1, BDNF, PI3-kinase, Protein kinase C, Retinoic acid, TACE
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-19985 (URN)10.1016/j.bbrc.2007.10.167 (DOI)000251494000015 ()
Available from: 2009-01-23 Created: 2009-01-23 Last updated: 2017-12-13Bibliographically approved
3. O-GlcNAcylation increases non-amyloidogenic processing of the amyloid-beta precursor protein (APP)
Open this publication in new window or tab >>O-GlcNAcylation increases non-amyloidogenic processing of the amyloid-beta precursor protein (APP)
2011 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 404, no 3, 882-886 p.Article in journal (Refereed) Published
Abstract [en]

The amyloid-beta precursor protein (APP) was shown to be O-GlcNAcylated 15 years ago, but the effect of this modification on APP processing and formation of the Alzheimer's disease associated amyloid-beta (A beta) peptide has so far not been investigated. Here, we demonstrate with pharmacological tools or siRNA that O-GlcNAcase and O-GlcNAc transferase regulate the level of O-GlcNAcylated APP. We also show that O-GlcNAcylation increases non-amyloidogenic alpha-secretase processing, resulting in increased levels of the neuroprotective sAPP alpha fragment and decreased A beta secretion. Our results implicate O-GlcNAcylation as a potential therapeutic target for Alzheimer's disease.

Keyword
Alzheimer's disease, Amyloid-beta, APP processing, O-linked glycosylation, alpha-Secretase
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-67334 (URN)10.1016/j.bbrc.2010.12.080 (DOI)000286848100023 ()
Note

authorCount :2

Available from: 2011-12-28 Created: 2011-12-28 Last updated: 2017-12-08Bibliographically approved
4. O-GlcNAcylation of the α-secretase ADAM10 selectively affects APP processing in neuron-like cells
Open this publication in new window or tab >>O-GlcNAcylation of the α-secretase ADAM10 selectively affects APP processing in neuron-like cells
(English)Manuscript (preprint) (Other academic)
Abstract [en]

α-Secretase processing of APP has recently gained more interest, highlighting its potential as a therapeutic target to prevent Alzheimer’s disease (AD). We have previously shown that O-GlcNAcylation stimulates α-secretase processing of APP, concomitantly with decreased Aβ secretion. O-GlcNAcylation has previously been linked to AD since the levels of O-GlcNAcylated proteins are decreased in AD brains. Here, we have further investigated the mechanism behind α-secretase processing of APP in response to increased O-GlcNAcylation. Our results shown that APP is not O-GlcNAcylated during the conditions used in this study. Instead, we demonstrate that the α-secretase ADAM10 is O-GlcNAcylated and that APP cell surface localization is enhanced in response to increased O-GlcNAcylation. Furthermore, the effects of O-GlcNAcylation on APP processing are cell-type specific, only affecting sAPPα secretion in neuroblastoma cell-lines.

Keyword
APP, ADAM10, O-GlcNAcylation
National Category
Biological Sciences Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-95071 (URN)
Available from: 2013-10-21 Created: 2013-10-21 Last updated: 2016-01-29Bibliographically approved
5. The E1 domain of APP and APLP2 determines α-secretase specificity
Open this publication in new window or tab >>The E1 domain of APP and APLP2 determines α-secretase specificity
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The α-secretase cleavage of the amyloid-β precursor protein (APP) precludes the formation of amyloid-β (Aβ), the main constituent of senile plaques in Alzheimer´s disease (AD). Stimulation of α-secretase processing may thereby constitute an important therapeutical strategy. APP belongs to a conserved protein family including the APP-like protein 2 (APLP2). Although the proteins are sequentially processed in a similar way, we have previously shown that insulin-like factor-1 (IGF-1) - and retinoic acid (RA)-induced α-secretase processing of APP and APLP2 is mediated by different enzymes. APP was shown to be cleaved by the α-secretase ADAM10 in a PI3K-dependent manner, whereas APLP2 was cleaved by the α-secretase TACE in a PKC-dependent manner. To better understand the mechanism underlying this difference in α-secretase processing between these two homologous proteins, we aimed to determine which part of APP that was essential for making it a better substrate for ADAM10 than for TACE during stimulated conditions. We constructed a chimeric protein, were the E1 domain of APP was substituted with the corresponding domain of APLP2. Our results demonstrate that the APP/E1/APLP2 chimer is successfully expressed and secreted into the cell medium of transiently transfected SH-SY5Y cells. Pharmacological inhibition demonstrate that the IGF-1-induced processing of the chimer is PI3K-independent but dependent on PKC, as previously shown for the APLP2 processing. Our result indicates that the E1 domain in APP determines its specificity towards ADAM10 over TACE.

Keyword
APP, APLP2, ADAM10, TACE
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-95072 (URN)
Available from: 2013-10-21 Created: 2013-10-21 Last updated: 2016-01-29Bibliographically approved

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