Naslov
Protein Proximity of APP and BACE1 and Membrane Stiffness Is Altered in Different Membranes in Niemann-Pick Type C Disease Cells

Autori
Von Arnim, Christine ; von Einem, Bjorn ; Weber, Petra ; Wagner, Michael ; Malnar, Martina ; Košiček, Marko ; Hećimović, Silva ; Schneckenburger, Herbert

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Skup
AD/PD 2013 The 11th International Conference On Alzheimer's & Parkinson's Disease

Mjesto i datum
Firenca, Italija, 06.03.2013. - 10.03.2013

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
FRET; BACE1; APP; Niemann-Pick Type C Disease

Sažetak
Objectives: Förster resonance energy transfer (FRET) between beta-secretase-GFP (BACE1-GFP) and amyloid precursor protein-mRFP (APP-mRFP) has recently been proven in U373 glioblastoma cells, and the possible role of APP-BACE1 proximity in Alzheimer's disease (AD) pathogenesis has been discussed. FRET was found to depend on intracellular cholesterol levels and associated alterations in membrane stiffness. Here, NPC1 null cells (NPC1-/-), exhibiting increased cholesterol levels and disturbed cholesterol transport found in Niemann-Pick type C disease (NPC), were used to analyze the influence of altered cholesterol levels on APP-BACE1 proximity. Methods: Fluorescence lifetime measurements of whole CHO-wild type (WT) and CHO-NPC1-/- cells (epi-illumination microscopy) as well as their plasma membranes (total internal reflection fluorescence microscopy, TIRFM) were performed. Additionally, generalized polarization (GP) measurements of CHO-WT and CHO-NPC1-/- cells incubated with the fluorescence marker laurdan were performed to determine membrane stiffness of plasma- and intracellular membranes. Results: CHO-NPC1-/- cells showed higher membrane stiffness at intracellular- but not plasma membranes, equivalent to cholesterol accumulation in late endosomes/lysosomes. Along with higher membrane stiffness, the FRET efficiency between BACE1-GFP and APP-mRFP was reduced at intracellular membranes, but not within the plasma membrane of CHO-NPC1-/- . Conclusions: Our data show that FRET combined with TIRF is a powerful tool to determine protein proximity and membrane fluidity in models of neurodegenerative diseases.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

POVEZANOST RADA