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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

Localization of split-luciferase constructs in CHO cells. Cells were co-transfected with the two split-luciferase constructs expressing either APP C99 or C83. 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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f0015: Localization of split-luciferase constructs in CHO cells. Cells were co-transfected with the two split-luciferase constructs expressing either APP C99 or C83. 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 next compared dimerization of the APP, C99 and C83 constructs (Fig. 2B–D). Immunoblots performed with the W0-2 antibody, the hGLuc antibody or the APP C-terminal antibody (Cter) showed that all constructs were expressed at readily detectable levels. Immunolabelling of the double transfected cells indicated a vesicular localization of the APP fusion proteins (Fig. 3), particularly in the perinuclear region. APP hGLuc fusion proteins had a subcellular distribution consistent with a post-ER/cis-Golgi localization previously reported for endogenous APP and APP fragments, but also for fusion constructs used in split protein assays [27,31,40]. Signals of antibodies directed against human APP epitopes (W0-2) or hGLuc epitopes completely co-localized, excluding the hypothesis that spurious cleavage or degradation products could be responsible for bioluminescent signals.


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)

Localization of split-luciferase constructs in CHO cells. Cells were co-transfected with the two split-luciferase constructs expressing either APP C99 or C83. 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

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

f0015: Localization of split-luciferase constructs in CHO cells. Cells were co-transfected with the two split-luciferase constructs expressing either APP C99 or C83. 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 next compared dimerization of the APP, C99 and C83 constructs (Fig. 2B–D). Immunoblots performed with the W0-2 antibody, the hGLuc antibody or the APP C-terminal antibody (Cter) showed that all constructs were expressed at readily detectable levels. Immunolabelling of the double transfected cells indicated a vesicular localization of the APP fusion proteins (Fig. 3), particularly in the perinuclear region. APP hGLuc fusion proteins had a subcellular distribution consistent with a post-ER/cis-Golgi localization previously reported for endogenous APP and APP fragments, but also for fusion constructs used in split protein assays [27,31,40]. Signals of antibodies directed against human APP epitopes (W0-2) or hGLuc epitopes completely co-localized, excluding the hypothesis that spurious cleavage or degradation products could be responsible for bioluminescent signals.

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