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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

Influence of the intracellular in APP dimerization. (A) Protein expression was monitored in cell lysates by Western blotting with the W0-2 or hGluc antibodies (left panel). Luciferase activity was measured and expressed as RLU normalized to APP (set to 100%, right panel). Values (means ± SEM) are representative of 2 independent experiment (n = 4 in each experiment). ***p < 0.001, as compared to APP-hGLuc1 and 2. (B) sAPPα and β production of APP-hGLuc1 and 2 and APPΔC-hGLuc1 and 2 constructs were monitored by ECLIA in the culture media of cells and are given in ng/ml. *p < 0.05 and ***p < 0.001, as compared to non-transfected cells or as indicated. Ratio of sAPPα on sAPPβ produced was calculated in the same experiments. **p < 0.01, as compared to APP-hGLuc1 and 2 (C) Immunostaining of cells co-expressing APP or APPΔC constructs of either. Nuclei were stained with DAPI, and APP fusion constructs were stained by the W0-2 and/or hGLuc antibodies. Scale bar: 5 μm.
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f0035: Influence of the intracellular in APP dimerization. (A) Protein expression was monitored in cell lysates by Western blotting with the W0-2 or hGluc antibodies (left panel). Luciferase activity was measured and expressed as RLU normalized to APP (set to 100%, right panel). Values (means ± SEM) are representative of 2 independent experiment (n = 4 in each experiment). ***p < 0.001, as compared to APP-hGLuc1 and 2. (B) sAPPα and β production of APP-hGLuc1 and 2 and APPΔC-hGLuc1 and 2 constructs were monitored by ECLIA in the culture media of cells and are given in ng/ml. *p < 0.05 and ***p < 0.001, as compared to non-transfected cells or as indicated. Ratio of sAPPα on sAPPβ produced was calculated in the same experiments. **p < 0.01, as compared to APP-hGLuc1 and 2 (C) Immunostaining of cells co-expressing APP or APPΔC constructs of either. Nuclei were stained with DAPI, and APP fusion constructs were stained by the W0-2 and/or hGLuc antibodies. Scale bar: 5 μm.

Mentions: We finally examined the role of the APP intracellular C-terminal domain in dimerization. We generated APPΔC split-luciferase constructs, corresponding to APP deleted from its intracellular region (Fig. 1). Indeed, much attention has been given so far to the contribution of the extracellular and TM domains to APP dimerization, but nothing is known about the role of its intracellular region. Both APP and APPΔC were recognized by the W0-2 and hGLuc antibodies, and both constructs were expressed at similar levels (Fig. 7A). Immunostaining (Fig. 7C) indicated that APP and APPΔC displayed similar subcellular distribution profiles. Results in Fig. 7A clearly showed that APPΔC formed more dimers than APP in cells. Expression of C99 constructs identically lacking their intracellular domains (C55) led to the same increased dimerization (data not shown). To know whether this drastic change in dimerization is related to APP metabolism, we measured sAPPα and sAPPβ levels produced by those cells. Soluble APPα and sAPPβ production are indicators of the ectodomain shedding occurring as a first step of the non-amyloidogenic and amyloidogenic processing, respectively. Cells expressing APPΔC-hGLuc showed increased sAPPα levels and reduced sAPPβ production. The ratio between sAPPα and sAPPβ significantly increased, indicating that APPΔC metabolism is shifted towards non-amyloidogenic processing.


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)

Influence of the intracellular in APP dimerization. (A) Protein expression was monitored in cell lysates by Western blotting with the W0-2 or hGluc antibodies (left panel). Luciferase activity was measured and expressed as RLU normalized to APP (set to 100%, right panel). Values (means ± SEM) are representative of 2 independent experiment (n = 4 in each experiment). ***p < 0.001, as compared to APP-hGLuc1 and 2. (B) sAPPα and β production of APP-hGLuc1 and 2 and APPΔC-hGLuc1 and 2 constructs were monitored by ECLIA in the culture media of cells and are given in ng/ml. *p < 0.05 and ***p < 0.001, as compared to non-transfected cells or as indicated. Ratio of sAPPα on sAPPβ produced was calculated in the same experiments. **p < 0.01, as compared to APP-hGLuc1 and 2 (C) Immunostaining of cells co-expressing APP or APPΔC constructs of either. Nuclei were stained with DAPI, and APP fusion constructs were stained by the W0-2 and/or hGLuc antibodies. Scale bar: 5 μm.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0035: Influence of the intracellular in APP dimerization. (A) Protein expression was monitored in cell lysates by Western blotting with the W0-2 or hGluc antibodies (left panel). Luciferase activity was measured and expressed as RLU normalized to APP (set to 100%, right panel). Values (means ± SEM) are representative of 2 independent experiment (n = 4 in each experiment). ***p < 0.001, as compared to APP-hGLuc1 and 2. (B) sAPPα and β production of APP-hGLuc1 and 2 and APPΔC-hGLuc1 and 2 constructs were monitored by ECLIA in the culture media of cells and are given in ng/ml. *p < 0.05 and ***p < 0.001, as compared to non-transfected cells or as indicated. Ratio of sAPPα on sAPPβ produced was calculated in the same experiments. **p < 0.01, as compared to APP-hGLuc1 and 2 (C) Immunostaining of cells co-expressing APP or APPΔC constructs of either. Nuclei were stained with DAPI, and APP fusion constructs were stained by the W0-2 and/or hGLuc antibodies. Scale bar: 5 μm.
Mentions: We finally examined the role of the APP intracellular C-terminal domain in dimerization. We generated APPΔC split-luciferase constructs, corresponding to APP deleted from its intracellular region (Fig. 1). Indeed, much attention has been given so far to the contribution of the extracellular and TM domains to APP dimerization, but nothing is known about the role of its intracellular region. Both APP and APPΔC were recognized by the W0-2 and hGLuc antibodies, and both constructs were expressed at similar levels (Fig. 7A). Immunostaining (Fig. 7C) indicated that APP and APPΔC displayed similar subcellular distribution profiles. Results in Fig. 7A clearly showed that APPΔC formed more dimers than APP in cells. Expression of C99 constructs identically lacking their intracellular domains (C55) led to the same increased dimerization (data not shown). To know whether this drastic change in dimerization is related to APP metabolism, we measured sAPPα and sAPPβ levels produced by those cells. Soluble APPα and sAPPβ production are indicators of the ectodomain shedding occurring as a first step of the non-amyloidogenic and amyloidogenic processing, respectively. Cells expressing APPΔC-hGLuc showed increased sAPPα levels and reduced sAPPβ production. The ratio between sAPPα and sAPPβ significantly increased, indicating that APPΔC metabolism is shifted towards non-amyloidogenic processing.

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