Increased Foxo3a nuclear translocation and activity is an early neuronal response to βγ-secretase mediated processing of the Amyloid-β-Protein Precursor Protein: Utility of an AβPP-GAL4 reporter assay

Bernard M. Law, Amy L. Guest, Matthew W.J. Pullen, Michael S. Perkinton, Robert J. Williams

Research output: Contribution to journalArticle

2 Citations (Scopus)
40 Downloads (Pure)

Abstract

Sequential cleavage of the amyloid-β protein precursor (AβPP) by BACE1 (β-secretase) followed by the γ-secretase complex, is strongly implicated in Alzheimer’s Disease (AD) but the initial cellular responses to these cleavage events are not fully defined. β-secretase-mediated AβPP processing yields an extracellular domain (sAβPPβ) and a C-terminal fragment of APP of 99 amino acids (C99). Subsequent cleavage by γ-secretase produces amyloid-β (Aβ) and an APP intracellular domain (AICD). A cellular screen based on the generation of AICD from an AβPP-Gal4 fusion protein was adapted by introducing familial AD (FAD) mutations into the AβPP sequence and linking the assay to Gal4-UAS driven luciferase and GFP expression, to identify responses immediately downstream of AβPP processing in neurons with a focus on the transcription factor Foxo3a which has been implicated in neurodegeneration. The K670N/M671L, E682K, E693G, V717I FAD mutations and the A673T protective mutation, were introduced into the AβPP sequence by site directed mutagenesis. When expressed in mouse cortical neurons AβPP-Gal4-UAS driven luciferase and GFP expression was substantially reduced by γ-secretase inhibitors, lowered by β-secretase inhibitors and enhanced by α-secretase inhibitors suggesting that AICD is a product of the β-secretase pathway. AβPP-Gal4-UAS driven GFP expression was exploited to identify individual neurons undergoing amyloidogenic AβPP processing, revealing increased nuclear localization of Foxo3a and enhanced Foxo3a-mediated transcription downstream of AICD production. Foxo3a translocation was not driven by AICD directly but correlated with reduced Akt phosphorylation. Collectively this suggests that βγ-secretase-mediated AβPP processing couples to Foxo3a which could be an early neuronal signaling response in AD.
Original languageEnglish
Pages (from-to)673-688
JournalJournal of Alzheimer's Disease
Volume61
Issue number2
DOIs
Publication statusPublished - 19 Dec 2017

Fingerprint

Amyloid Precursor Protein Secretases
Amyloid beta-Protein Precursor
Proteins
Alzheimer Disease
Luciferases
Neurons
Mutation
Amyloidogenic Proteins
Site-Directed Mutagenesis
Amyloid
Transcription Factors
Phosphorylation
Amino Acids

Cite this

Increased Foxo3a nuclear translocation and activity is an early neuronal response to βγ-secretase mediated processing of the Amyloid-β-Protein Precursor Protein: Utility of an AβPP-GAL4 reporter assay. / Law, Bernard M.; Guest, Amy L.; Pullen, Matthew W.J.; Perkinton, Michael S.; Williams, Robert J.

In: Journal of Alzheimer's Disease, Vol. 61, No. 2, 19.12.2017, p. 673-688.

Research output: Contribution to journalArticle

@article{ad70e5a930fe45c0ba589d343cdbdb8c,
title = "Increased Foxo3a nuclear translocation and activity is an early neuronal response to βγ-secretase mediated processing of the Amyloid-β-Protein Precursor Protein: Utility of an AβPP-GAL4 reporter assay",
abstract = "Sequential cleavage of the amyloid-β protein precursor (AβPP) by BACE1 (β-secretase) followed by the γ-secretase complex, is strongly implicated in Alzheimer’s Disease (AD) but the initial cellular responses to these cleavage events are not fully defined. β-secretase-mediated AβPP processing yields an extracellular domain (sAβPPβ) and a C-terminal fragment of APP of 99 amino acids (C99). Subsequent cleavage by γ-secretase produces amyloid-β (Aβ) and an APP intracellular domain (AICD). A cellular screen based on the generation of AICD from an AβPP-Gal4 fusion protein was adapted by introducing familial AD (FAD) mutations into the AβPP sequence and linking the assay to Gal4-UAS driven luciferase and GFP expression, to identify responses immediately downstream of AβPP processing in neurons with a focus on the transcription factor Foxo3a which has been implicated in neurodegeneration. The K670N/M671L, E682K, E693G, V717I FAD mutations and the A673T protective mutation, were introduced into the AβPP sequence by site directed mutagenesis. When expressed in mouse cortical neurons AβPP-Gal4-UAS driven luciferase and GFP expression was substantially reduced by γ-secretase inhibitors, lowered by β-secretase inhibitors and enhanced by α-secretase inhibitors suggesting that AICD is a product of the β-secretase pathway. AβPP-Gal4-UAS driven GFP expression was exploited to identify individual neurons undergoing amyloidogenic AβPP processing, revealing increased nuclear localization of Foxo3a and enhanced Foxo3a-mediated transcription downstream of AICD production. Foxo3a translocation was not driven by AICD directly but correlated with reduced Akt phosphorylation. Collectively this suggests that βγ-secretase-mediated AβPP processing couples to Foxo3a which could be an early neuronal signaling response in AD.",
author = "Law, {Bernard M.} and Guest, {Amy L.} and Pullen, {Matthew W.J.} and Perkinton, {Michael S.} and Williams, {Robert J.}",
year = "2017",
month = "12",
day = "19",
doi = "10.3233/JAD-170393",
language = "English",
volume = "61",
pages = "673--688",
journal = "Journal of Alzheimer's Disease",
issn = "1387-2877",
publisher = "IOS Press",
number = "2",

}

TY - JOUR

T1 - Increased Foxo3a nuclear translocation and activity is an early neuronal response to βγ-secretase mediated processing of the Amyloid-β-Protein Precursor Protein: Utility of an AβPP-GAL4 reporter assay

AU - Law, Bernard M.

AU - Guest, Amy L.

AU - Pullen, Matthew W.J.

AU - Perkinton, Michael S.

AU - Williams, Robert J.

PY - 2017/12/19

Y1 - 2017/12/19

N2 - Sequential cleavage of the amyloid-β protein precursor (AβPP) by BACE1 (β-secretase) followed by the γ-secretase complex, is strongly implicated in Alzheimer’s Disease (AD) but the initial cellular responses to these cleavage events are not fully defined. β-secretase-mediated AβPP processing yields an extracellular domain (sAβPPβ) and a C-terminal fragment of APP of 99 amino acids (C99). Subsequent cleavage by γ-secretase produces amyloid-β (Aβ) and an APP intracellular domain (AICD). A cellular screen based on the generation of AICD from an AβPP-Gal4 fusion protein was adapted by introducing familial AD (FAD) mutations into the AβPP sequence and linking the assay to Gal4-UAS driven luciferase and GFP expression, to identify responses immediately downstream of AβPP processing in neurons with a focus on the transcription factor Foxo3a which has been implicated in neurodegeneration. The K670N/M671L, E682K, E693G, V717I FAD mutations and the A673T protective mutation, were introduced into the AβPP sequence by site directed mutagenesis. When expressed in mouse cortical neurons AβPP-Gal4-UAS driven luciferase and GFP expression was substantially reduced by γ-secretase inhibitors, lowered by β-secretase inhibitors and enhanced by α-secretase inhibitors suggesting that AICD is a product of the β-secretase pathway. AβPP-Gal4-UAS driven GFP expression was exploited to identify individual neurons undergoing amyloidogenic AβPP processing, revealing increased nuclear localization of Foxo3a and enhanced Foxo3a-mediated transcription downstream of AICD production. Foxo3a translocation was not driven by AICD directly but correlated with reduced Akt phosphorylation. Collectively this suggests that βγ-secretase-mediated AβPP processing couples to Foxo3a which could be an early neuronal signaling response in AD.

AB - Sequential cleavage of the amyloid-β protein precursor (AβPP) by BACE1 (β-secretase) followed by the γ-secretase complex, is strongly implicated in Alzheimer’s Disease (AD) but the initial cellular responses to these cleavage events are not fully defined. β-secretase-mediated AβPP processing yields an extracellular domain (sAβPPβ) and a C-terminal fragment of APP of 99 amino acids (C99). Subsequent cleavage by γ-secretase produces amyloid-β (Aβ) and an APP intracellular domain (AICD). A cellular screen based on the generation of AICD from an AβPP-Gal4 fusion protein was adapted by introducing familial AD (FAD) mutations into the AβPP sequence and linking the assay to Gal4-UAS driven luciferase and GFP expression, to identify responses immediately downstream of AβPP processing in neurons with a focus on the transcription factor Foxo3a which has been implicated in neurodegeneration. The K670N/M671L, E682K, E693G, V717I FAD mutations and the A673T protective mutation, were introduced into the AβPP sequence by site directed mutagenesis. When expressed in mouse cortical neurons AβPP-Gal4-UAS driven luciferase and GFP expression was substantially reduced by γ-secretase inhibitors, lowered by β-secretase inhibitors and enhanced by α-secretase inhibitors suggesting that AICD is a product of the β-secretase pathway. AβPP-Gal4-UAS driven GFP expression was exploited to identify individual neurons undergoing amyloidogenic AβPP processing, revealing increased nuclear localization of Foxo3a and enhanced Foxo3a-mediated transcription downstream of AICD production. Foxo3a translocation was not driven by AICD directly but correlated with reduced Akt phosphorylation. Collectively this suggests that βγ-secretase-mediated AβPP processing couples to Foxo3a which could be an early neuronal signaling response in AD.

U2 - 10.3233/JAD-170393

DO - 10.3233/JAD-170393

M3 - Article

VL - 61

SP - 673

EP - 688

JO - Journal of Alzheimer's Disease

JF - Journal of Alzheimer's Disease

SN - 1387-2877

IS - 2

ER -