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Analysis by a highly sensitive split luciferase assay of the regions involved in APP dimerization and its impact on processing.

Decock M, El Haylani L, Stanga S, Dewachter I, Octave JN, Smith SO, Constantinescu SN, Kienlen-Campard P - FEBS Open Bio (2015)

Bottom Line: Two types of lesions are found in AD brains: neurofibrillary tangles and senile plaques.We show that both non-familial and familial AD mutations in the TM GXXXG motifs strongly modulate Aβ production, but do not consistently change dimerization of the C-terminal fragments.Finally, we found for the first time that removal of intracellular domain strongly increases APP dimerization.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neuroscience, Université catholique de Louvain, Brussels 1200, Belgium.

ABSTRACT
Alzheimer's disease (AD) is a neurodegenerative disease that causes progressive loss of cognitive functions, leading to dementia. Two types of lesions are found in AD brains: neurofibrillary tangles and senile plaques. The latter are composed mainly of the β-amyloid peptide (Aβ) generated by amyloidogenic processing of the amyloid precursor protein (APP). Several studies have suggested that dimerization of APP is closely linked to Aβ production. Nevertheless, the mechanisms controlling APP dimerization and their role in APP function are not known. Here we used a new luciferase complementation assay to analyze APP dimerization and unravel the involvement of its three major domains: the ectodomain, the transmembrane domain and the intracellular domain. Our results indicate that within cells full-length APP dimerizes more than its α and β C-terminal fragments, confirming the pivotal role of the ectodomain in this process. Dimerization of the APP transmembrane (TM) domain has been reported to regulate processing at the γ-cleavage site. We show that both non-familial and familial AD mutations in the TM GXXXG motifs strongly modulate Aβ production, but do not consistently change dimerization of the C-terminal fragments. Finally, we found for the first time that removal of intracellular domain strongly increases APP dimerization. Increased APP dimerization is linked to increased non-amyloidogenic processing.

No MeSH data available.


Related in: MedlinePlus

Involvement of GXXXG motifs in CTF dimerization and Aβ production. CHO cells were transfected with C99-hGLuc1 and 2 or C83-hGLuc1 and 2 and their GXXXG Flemish (Fle) and mutant 5 (mut5) corresponding mutants. (A) Cells transfected with the control empty vector (mock), the C99-hGLuc1 and 2, C99Fle-hGLuc1 and 2 or C99mut5-hGLuc1 and 2 proteins. Protein expression was monitored in cell lysates by Western blotting with the W0-2 or hGluc antibodies (top panels). Luciferase activity was measured and expressed as RLU normalized to non-mutated C99 (bottom). Values (means ± SEM) are representative of 5 independent experiments (n = 4 in each experiment). *p < 0.05, **p < 0.01 and ***p < 0.001, as compared to C99-hGLuc1 and 2. (B) Cells transfected with the control empty vector (mock), the C83-hGLuc1 and 2, C83Fle-hGLuc1 and 2 or C83mut5-hGLuc1 and 2 proteins. Protein expression was monitored in cell lysates by Western blotting with the Cter or hGLuc antibodies (top panels). Luciferase activity was measured and expressed as RLU normalized to non-mutated C83 (bottom). Values (means ± SEM) are representative of 3 independent experiments (n = 4 in each experiment). *p < 0.05, **p < 0.01 and n.s. (non significant), as compared to C83-hGLuc1 and 2. (C) Aβ 38, 40 and 42 production for C99-hGLuc1 and 2, C99Fle-hGLuc1 and 2 or C99mut5-hGLuc1 and 2 was measured by ECLIA in the culture media and given in pg/ml. Values (means ± SEM) are representative of 3 independent experiment (n = 4 in each experiment). ***p < 0.001, as compared to non-mutated C99.
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f0030: Involvement of GXXXG motifs in CTF dimerization and Aβ production. CHO cells were transfected with C99-hGLuc1 and 2 or C83-hGLuc1 and 2 and their GXXXG Flemish (Fle) and mutant 5 (mut5) corresponding mutants. (A) Cells transfected with the control empty vector (mock), the C99-hGLuc1 and 2, C99Fle-hGLuc1 and 2 or C99mut5-hGLuc1 and 2 proteins. Protein expression was monitored in cell lysates by Western blotting with the W0-2 or hGluc antibodies (top panels). Luciferase activity was measured and expressed as RLU normalized to non-mutated C99 (bottom). Values (means ± SEM) are representative of 5 independent experiments (n = 4 in each experiment). *p < 0.05, **p < 0.01 and ***p < 0.001, as compared to C99-hGLuc1 and 2. (B) Cells transfected with the control empty vector (mock), the C83-hGLuc1 and 2, C83Fle-hGLuc1 and 2 or C83mut5-hGLuc1 and 2 proteins. Protein expression was monitored in cell lysates by Western blotting with the Cter or hGLuc antibodies (top panels). Luciferase activity was measured and expressed as RLU normalized to non-mutated C83 (bottom). Values (means ± SEM) are representative of 3 independent experiments (n = 4 in each experiment). *p < 0.05, **p < 0.01 and n.s. (non significant), as compared to C83-hGLuc1 and 2. (C) Aβ 38, 40 and 42 production for C99-hGLuc1 and 2, C99Fle-hGLuc1 and 2 or C99mut5-hGLuc1 and 2 was measured by ECLIA in the culture media and given in pg/ml. Values (means ± SEM) are representative of 3 independent experiment (n = 4 in each experiment). ***p < 0.001, as compared to non-mutated C99.

Mentions: Our recent work showed that mutation of the GXXXG motifs modifies TM interactions of the APP CTFs [19]. To determine whether GXXXG motifs are involved in CTFs homo-dimerization by a quantitative approach, we compared dimerization of C99 (corresponding to the βCTF of APP), C99 with the Flemish mutation or C99 mut5 (Fig. 1A). Both C99 mutants exhibited the same dimerization profile as wild-type C99 (Fig. 6A). Similar experiments were carried out with C83 constructs, corresponding to the APP αCTF. C99 and C83 constructs had similar expression profiles (Fig. 6B). Both the Flemish mutant and mut5 showed a slight but significant decrease in dimerization of C99. For C83, dimerization of the Flemish mutant was slightly but significantly increased, whereas dimerization of mut5 was not affected. Mutation of the GXXXG motifs was reported to dramatically impact Aβ production (Fig. 6C). We previously showed [19,29] that the Flemish FAD mutation increased Aβ production whereas mutant 5 strongly decreased it. Our data (Fig. 6) indicate that mutations of GXXXG motifs strongly affect APP γ-cleavage without a corresponding change in dimerization of the APP CTFs.


Analysis by a highly sensitive split luciferase assay of the regions involved in APP dimerization and its impact on processing.

Decock M, El Haylani L, Stanga S, Dewachter I, Octave JN, Smith SO, Constantinescu SN, Kienlen-Campard P - FEBS Open Bio (2015)

Involvement of GXXXG motifs in CTF dimerization and Aβ production. CHO cells were transfected with C99-hGLuc1 and 2 or C83-hGLuc1 and 2 and their GXXXG Flemish (Fle) and mutant 5 (mut5) corresponding mutants. (A) Cells transfected with the control empty vector (mock), the C99-hGLuc1 and 2, C99Fle-hGLuc1 and 2 or C99mut5-hGLuc1 and 2 proteins. Protein expression was monitored in cell lysates by Western blotting with the W0-2 or hGluc antibodies (top panels). Luciferase activity was measured and expressed as RLU normalized to non-mutated C99 (bottom). Values (means ± SEM) are representative of 5 independent experiments (n = 4 in each experiment). *p < 0.05, **p < 0.01 and ***p < 0.001, as compared to C99-hGLuc1 and 2. (B) Cells transfected with the control empty vector (mock), the C83-hGLuc1 and 2, C83Fle-hGLuc1 and 2 or C83mut5-hGLuc1 and 2 proteins. Protein expression was monitored in cell lysates by Western blotting with the Cter or hGLuc antibodies (top panels). Luciferase activity was measured and expressed as RLU normalized to non-mutated C83 (bottom). Values (means ± SEM) are representative of 3 independent experiments (n = 4 in each experiment). *p < 0.05, **p < 0.01 and n.s. (non significant), as compared to C83-hGLuc1 and 2. (C) Aβ 38, 40 and 42 production for C99-hGLuc1 and 2, C99Fle-hGLuc1 and 2 or C99mut5-hGLuc1 and 2 was measured by ECLIA in the culture media and given in pg/ml. Values (means ± SEM) are representative of 3 independent experiment (n = 4 in each experiment). ***p < 0.001, as compared to non-mutated C99.
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Related In: Results  -  Collection

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Show All Figures
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f0030: Involvement of GXXXG motifs in CTF dimerization and Aβ production. CHO cells were transfected with C99-hGLuc1 and 2 or C83-hGLuc1 and 2 and their GXXXG Flemish (Fle) and mutant 5 (mut5) corresponding mutants. (A) Cells transfected with the control empty vector (mock), the C99-hGLuc1 and 2, C99Fle-hGLuc1 and 2 or C99mut5-hGLuc1 and 2 proteins. Protein expression was monitored in cell lysates by Western blotting with the W0-2 or hGluc antibodies (top panels). Luciferase activity was measured and expressed as RLU normalized to non-mutated C99 (bottom). Values (means ± SEM) are representative of 5 independent experiments (n = 4 in each experiment). *p < 0.05, **p < 0.01 and ***p < 0.001, as compared to C99-hGLuc1 and 2. (B) Cells transfected with the control empty vector (mock), the C83-hGLuc1 and 2, C83Fle-hGLuc1 and 2 or C83mut5-hGLuc1 and 2 proteins. Protein expression was monitored in cell lysates by Western blotting with the Cter or hGLuc antibodies (top panels). Luciferase activity was measured and expressed as RLU normalized to non-mutated C83 (bottom). Values (means ± SEM) are representative of 3 independent experiments (n = 4 in each experiment). *p < 0.05, **p < 0.01 and n.s. (non significant), as compared to C83-hGLuc1 and 2. (C) Aβ 38, 40 and 42 production for C99-hGLuc1 and 2, C99Fle-hGLuc1 and 2 or C99mut5-hGLuc1 and 2 was measured by ECLIA in the culture media and given in pg/ml. Values (means ± SEM) are representative of 3 independent experiment (n = 4 in each experiment). ***p < 0.001, as compared to non-mutated C99.
Mentions: Our recent work showed that mutation of the GXXXG motifs modifies TM interactions of the APP CTFs [19]. To determine whether GXXXG motifs are involved in CTFs homo-dimerization by a quantitative approach, we compared dimerization of C99 (corresponding to the βCTF of APP), C99 with the Flemish mutation or C99 mut5 (Fig. 1A). Both C99 mutants exhibited the same dimerization profile as wild-type C99 (Fig. 6A). Similar experiments were carried out with C83 constructs, corresponding to the APP αCTF. C99 and C83 constructs had similar expression profiles (Fig. 6B). Both the Flemish mutant and mut5 showed a slight but significant decrease in dimerization of C99. For C83, dimerization of the Flemish mutant was slightly but significantly increased, whereas dimerization of mut5 was not affected. Mutation of the GXXXG motifs was reported to dramatically impact Aβ production (Fig. 6C). We previously showed [19,29] that the Flemish FAD mutation increased Aβ production whereas mutant 5 strongly decreased it. Our data (Fig. 6) indicate that mutations of GXXXG motifs strongly affect APP γ-cleavage without a corresponding change in dimerization of the APP CTFs.

Bottom Line: Two types of lesions are found in AD brains: neurofibrillary tangles and senile plaques.We show that both non-familial and familial AD mutations in the TM GXXXG motifs strongly modulate Aβ production, but do not consistently change dimerization of the C-terminal fragments.Finally, we found for the first time that removal of intracellular domain strongly increases APP dimerization.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neuroscience, Université catholique de Louvain, Brussels 1200, Belgium.

ABSTRACT
Alzheimer's disease (AD) is a neurodegenerative disease that causes progressive loss of cognitive functions, leading to dementia. Two types of lesions are found in AD brains: neurofibrillary tangles and senile plaques. The latter are composed mainly of the β-amyloid peptide (Aβ) generated by amyloidogenic processing of the amyloid precursor protein (APP). Several studies have suggested that dimerization of APP is closely linked to Aβ production. Nevertheless, the mechanisms controlling APP dimerization and their role in APP function are not known. Here we used a new luciferase complementation assay to analyze APP dimerization and unravel the involvement of its three major domains: the ectodomain, the transmembrane domain and the intracellular domain. Our results indicate that within cells full-length APP dimerizes more than its α and β C-terminal fragments, confirming the pivotal role of the ectodomain in this process. Dimerization of the APP transmembrane (TM) domain has been reported to regulate processing at the γ-cleavage site. We show that both non-familial and familial AD mutations in the TM GXXXG motifs strongly modulate Aβ production, but do not consistently change dimerization of the C-terminal fragments. Finally, we found for the first time that removal of intracellular domain strongly increases APP dimerization. Increased APP dimerization is linked to increased non-amyloidogenic processing.

No MeSH data available.


Related in: MedlinePlus