Aktuelle Neurologie 2007; 34 - P674
DOI: 10.1055/s-2007-987945

Assessment of the interaction between presenilin 1 (PS1) and ubiquilin 1 splice variants in intact cells

AV Thomas 1, L Herl 1, M Hiltunen 1, PB Jones 1, RE Tanzi 1, BT Hyman 1, O Berezovska 1
  • 1Köln; Charlestown, USA

Background/ojective: Ubiquilin 1 splice variants are differentially expressed in the brains of Alzheimer patients. We have previously shown that endogenous presenilin 1 (PS1) and ubiquilin 1 come into close proximity in murine primary neurons. The objective of this study was to determine whether PS1 comes into close proximity with each of the different ubiquilin 1 splice variants and whether there are differences in their interactions.

Methods: Chinese Hamster Ovary (CHO) cells were co-transfected with a wild type PS1 construct and either one of the four, v5-tagged ubiquilin 1 splice variants (TV1-V5, TV2-V5, TV3-V5, TV5-V5). Cells were double immunostained with antibodies against the V5-tag and the PS1 C-Terminus. Secondary antibodies were Alexa 430 and Cy3. Proximity between the epitopes, i.e. interaction between the proteins, was assessed using a high-throughput plate reader by measuring the lifetimes of the donor fluorophore Alexa 430. Localization of the interaction was assessed by Fluorescence Lifetime Imaging Microscopy using the same immunostaining protocol, with Alexa488 as donor fluorophore. Statistical analysis was performed using student's t-test.

Results: In the high-throughput system used in this study, it was shown that the lifetime of Alexa 430 on the C-terminus of either ubiquilin 1 splice variant was around 3800–4100 psec. Immunolabeling of both each of the ubiquilin 1 splice variants with Alexa 430 and PS1 with Cy3 resulted in the appearance of a second lifetime of around 1400 psec in the cells, indicating that the two proteins come into close proximity to each other. There were no differences in proximity between PS1 and each of the ubiquilin 1 splice variants, pointing towards the general potential of every splice variant to interact with PS1. Using Fluorescence Lifetime Imaging Microscopy, the obtained lifetimes were displayed on a pixel-by-pixel basis in a pseudocolored image. It was shown that the fastest lifetimes occur in distal subcellular compartments, indicating that ubiquilin 1 and PS1 come into closest proximity near the cell surface.

Conclusions: Using fluorescence lifetime measurements in intact cells, we have shown that PS1 comes into close proximity to four different ubiquilin 1 splice variants near the cellular surface. These data indicate that neither differences in the proximity nor localization of the interaction between PS1 and the ubiquilin 1 splice variants seem to be of relevance for Alzheimer's Disease.