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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

Effects of gamma-secretase inhibition on APP C99 and C83 dimerization. CHO cells transfected with the empty vector (mock) or the luciferase constructs expressing either APP, C99 and C83 or APP-hGLuc1 and 2, C99-hGLuc1 and 2 and C83-hGLuc1 and 2. Cells were treated with DAPT 1 μM for 18 h. (A) Expression of the non-tagged proteins and effect of the treatment on metabolism were checked in cell lysates by western blotting with APP specific antibodies (W0-2 and Cter). (B) Expression of the hGLuc fusion proteins was checked in cell lysates by Western blotting with the hGLuc antibody and APP specific antibodies (W0-2 and Cter). (C) Luciferase activity measured was expressed as RLU normalized to APP-hGLuc1 and 2 (set to 100%). Values (means ± SEM) are representative of 3 independent experiments (n = 4 in each experiment). *p < 0.05, ***p < 0.001, n.s. (non-significant), as compared to APP-hGLuc1 and 2. (D) Gamma-secretase inhibition was confirmed by monitoring Aβ38, Aβ40 and Aβ42 production by ECLIA in the culture medium of cells expressing APP-hGLuc1 and 2 or C99-hGLuc1 and 2. Results are given as Aβ levels in pg/ml. Values (means ± SEM) are representative of 3 independent experiments (n = 4 in each experiment). *p < 0.05 and ***p < 0.001, as compared to non-treated cells.
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f0025: Effects of gamma-secretase inhibition on APP C99 and C83 dimerization. CHO cells transfected with the empty vector (mock) or the luciferase constructs expressing either APP, C99 and C83 or APP-hGLuc1 and 2, C99-hGLuc1 and 2 and C83-hGLuc1 and 2. Cells were treated with DAPT 1 μM for 18 h. (A) Expression of the non-tagged proteins and effect of the treatment on metabolism were checked in cell lysates by western blotting with APP specific antibodies (W0-2 and Cter). (B) Expression of the hGLuc fusion proteins was checked in cell lysates by Western blotting with the hGLuc antibody and APP specific antibodies (W0-2 and Cter). (C) Luciferase activity measured was expressed as RLU normalized to APP-hGLuc1 and 2 (set to 100%). Values (means ± SEM) are representative of 3 independent experiments (n = 4 in each experiment). *p < 0.05, ***p < 0.001, n.s. (non-significant), as compared to APP-hGLuc1 and 2. (D) Gamma-secretase inhibition was confirmed by monitoring Aβ38, Aβ40 and Aβ42 production by ECLIA in the culture medium of cells expressing APP-hGLuc1 and 2 or C99-hGLuc1 and 2. Results are given as Aβ levels in pg/ml. Values (means ± SEM) are representative of 3 independent experiments (n = 4 in each experiment). *p < 0.05 and ***p < 0.001, as compared to non-treated cells.

Mentions: After normalization to expression levels of the fusion proteins (Fig. 4A), our results indicated that APP dimerizes significantly more (∼5 times) than its α and β C-terminal fragments (Fig. 4B). Similar results were obtained with all the antibodies used for cell lysate quantifications (W0-2, Cter or hGLuc). These observations suggest that C-terminal fragments of APP (β or αCTFs) form dimers to a smaller extent than full-length APP. We further investigated this hypothesis by treating transfected cells with DAPT, a γ-secretase allosteric inhibitor, inducing the accumulation of γ-secretase substrates in cells. DAPT effects were consistent with those observed on non-tagged human APP in the same cells (Fig. 5A) [19]. DAPT treatment resulted mainly in the accumulation of αCTFs (Fig. 5A and B). Bioluminescence measured after DAPT treatment indicated a small -although significant- decrease in APP dimerization, but importantly no increase in αCTF or βCTF dimerization was measured under the same conditions (Fig. 5C). Similar results were observed in PSdKO mouse embryonic fibroblast (MEFs) that are devoid of γ-secretase activity (data not shown). Together, these data indicate that APP CTFs dimerize much less than full-length APP. Their accumulation by a γ-secretase inhibitor did not increase dimerization, suggesting that only a pool of CTFs are forming dimers, which are not accessible to cleavage by γ-secretase. DAPT treatment efficiently inhibited the γ-cleavage of APP split luciferase constructs, as evidenced by the strong inhibition of Aβ release (Fig. 5D).


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)

Effects of gamma-secretase inhibition on APP C99 and C83 dimerization. CHO cells transfected with the empty vector (mock) or the luciferase constructs expressing either APP, C99 and C83 or APP-hGLuc1 and 2, C99-hGLuc1 and 2 and C83-hGLuc1 and 2. Cells were treated with DAPT 1 μM for 18 h. (A) Expression of the non-tagged proteins and effect of the treatment on metabolism were checked in cell lysates by western blotting with APP specific antibodies (W0-2 and Cter). (B) Expression of the hGLuc fusion proteins was checked in cell lysates by Western blotting with the hGLuc antibody and APP specific antibodies (W0-2 and Cter). (C) Luciferase activity measured was expressed as RLU normalized to APP-hGLuc1 and 2 (set to 100%). Values (means ± SEM) are representative of 3 independent experiments (n = 4 in each experiment). *p < 0.05, ***p < 0.001, n.s. (non-significant), as compared to APP-hGLuc1 and 2. (D) Gamma-secretase inhibition was confirmed by monitoring Aβ38, Aβ40 and Aβ42 production by ECLIA in the culture medium of cells expressing APP-hGLuc1 and 2 or C99-hGLuc1 and 2. Results are given as Aβ levels in pg/ml. Values (means ± SEM) are representative of 3 independent experiments (n = 4 in each experiment). *p < 0.05 and ***p < 0.001, as compared to non-treated cells.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588712&req=5

f0025: Effects of gamma-secretase inhibition on APP C99 and C83 dimerization. CHO cells transfected with the empty vector (mock) or the luciferase constructs expressing either APP, C99 and C83 or APP-hGLuc1 and 2, C99-hGLuc1 and 2 and C83-hGLuc1 and 2. Cells were treated with DAPT 1 μM for 18 h. (A) Expression of the non-tagged proteins and effect of the treatment on metabolism were checked in cell lysates by western blotting with APP specific antibodies (W0-2 and Cter). (B) Expression of the hGLuc fusion proteins was checked in cell lysates by Western blotting with the hGLuc antibody and APP specific antibodies (W0-2 and Cter). (C) Luciferase activity measured was expressed as RLU normalized to APP-hGLuc1 and 2 (set to 100%). Values (means ± SEM) are representative of 3 independent experiments (n = 4 in each experiment). *p < 0.05, ***p < 0.001, n.s. (non-significant), as compared to APP-hGLuc1 and 2. (D) Gamma-secretase inhibition was confirmed by monitoring Aβ38, Aβ40 and Aβ42 production by ECLIA in the culture medium of cells expressing APP-hGLuc1 and 2 or C99-hGLuc1 and 2. Results are given as Aβ levels in pg/ml. Values (means ± SEM) are representative of 3 independent experiments (n = 4 in each experiment). *p < 0.05 and ***p < 0.001, as compared to non-treated cells.
Mentions: After normalization to expression levels of the fusion proteins (Fig. 4A), our results indicated that APP dimerizes significantly more (∼5 times) than its α and β C-terminal fragments (Fig. 4B). Similar results were obtained with all the antibodies used for cell lysate quantifications (W0-2, Cter or hGLuc). These observations suggest that C-terminal fragments of APP (β or αCTFs) form dimers to a smaller extent than full-length APP. We further investigated this hypothesis by treating transfected cells with DAPT, a γ-secretase allosteric inhibitor, inducing the accumulation of γ-secretase substrates in cells. DAPT effects were consistent with those observed on non-tagged human APP in the same cells (Fig. 5A) [19]. DAPT treatment resulted mainly in the accumulation of αCTFs (Fig. 5A and B). Bioluminescence measured after DAPT treatment indicated a small -although significant- decrease in APP dimerization, but importantly no increase in αCTF or βCTF dimerization was measured under the same conditions (Fig. 5C). Similar results were observed in PSdKO mouse embryonic fibroblast (MEFs) that are devoid of γ-secretase activity (data not shown). Together, these data indicate that APP CTFs dimerize much less than full-length APP. Their accumulation by a γ-secretase inhibitor did not increase dimerization, suggesting that only a pool of CTFs are forming dimers, which are not accessible to cleavage by γ-secretase. DAPT treatment efficiently inhibited the γ-cleavage of APP split luciferase constructs, as evidenced by the strong inhibition of Aβ release (Fig. 5D).

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