« Back
Analysis of the Effect of Aggregated β-Amyloid on Cellular Signaling Pathways Critical for Memory in Alzheimer’s Disease
Analysis of the Effect of Aggregated β-Amyloid on Cellular Signaling Pathways Critical for Memory in Alzheimer’s Disease
Submitted on 11 Jul 2017

Brad Larson1, Arturo Gonzalez-Moya2, Alexandra Wolff3 , Wini Luty3
1BioTek Instruments, Inc. Winooski, VT USA • 2Cisbio Bedford, MA USA • 3Enzo Life Sciences Farmingdale, NY USA
Poster Views: 470
View poster »
Poster Abstract
According to literature references, Extracellular-Signal Regulated Kinase (ERK) signaling has been linked to memory and regulated by environmental stimuli. In Alzheimer’s disease, progressive cognitive impairment is seen as a classic characteristic. These deficits are believed to be the result of progressive synaptic dysfunction initiated by aggregated β–amyloid peptide 1-42 (Aβ). Zhu et al. (2002) showed that Aβ induced disruption of kinases critical for memory. In late stages of Alzheimer’s disease, ERK activation is suppressed relative to early stages and controls (Webster et al. 2006). In vitro studies have also shown that under certain conditions Aβ or fragments inhibit ERK or the downstream cAMPresponse element-binding protein (CREB) in neuronal cell models (Daniels et al. 2001).

Here we evaluate the ability to detect changes in phosphorylation levels of ERK and CREB following treatment with Aβ using the SH-SY5Y neuroblastoma cell line. Aβ was oligomerized using a method previously described in the literature (FA et al. 2010). A two-step HTRFR assay process was incorporated such that cell plating and treatment are carried out in a 96-well clear-bottom imaging plate. Following lysis, aliquots were transferred to separate LV384-well assay plates to perform the phospho and total ERK and CREB assays. A neutralizing antibody was also tested for its capacity to counteract the inhibitory effects of Aβ. Aβ binding and antibody neutralization were detected via immunocytochemisty and microscopic analysis. All microplate reading and cellular imaging steps were performed using a novel cell imaging multi-mode reader. The combination provides an efficient, robust method for testing of new molecules to combat the degenerative effects of the disease.

1. The β-Amyloid (1-42) Peptide, Amyloid β A4 (CT, 1-42) monoclonal antibody, and Goat anti-mouse ATTO 590 conjugated IgG1 secondary antibody from Enzo Life Sciences represent a sensitive method to reproduce and monitor β–amyloid peptide binding in an in vitro setting.
2. Cisbio HTRF phospho and total protein assays allow the effect of peptide binding to be easily determined for specific targets. Multiple targets can be assessed from the same well, reducing plate to plate variability.
3. The combined filter-based microplate reading, fluorescent imaging, and image analysis capabilities of the Cytation 5 and Gen5 Data Analysis Software, allow sample processing and calculations to be performed by a single instrument
4. The combination of microplate reading and imaging based assay methodologies and instrumentation create robust in vitro methods to make phenotypic and target-based determinations that can further the understanding of Alzheimer’s diseas
Report abuse »
Ask the author a question about this poster.
Ask a Question »

Creative Commons

Related Posters

The added value of traditional bulk sample measurements in single cell RT-qPCR experiments
Lukas Valihrach1, David Dzamba2, Peter Androvic1, Miroslava Anderova2, Mikael Kubista1,3

Different tolerance of loop-mediated isothermal amplification and polymerase chain reaction to inhibitors in chicken carcass rinse and feces for detecting Campylobacter jejuni
Hongsheng Huang, Beverley Phipps-Todd, Courtney Chew Leung and Dina Elleithy

A quick potency assay for osteogenic and chondrogenic differentiation and evaluation of donor variability of adipose derived stem cells
Eleni Oberbauer1,3, Susanne Wolbank1,3, Carolin Steffenhagen1,3, Ara Hacobian1,3, Georg Feichtinger1,3, Christian Gabriel2,3, Heinz Redl1,3, Florian Hildner2,3

Highly Automated “Sample-to-Result” NGS and PCR Workflow for Molecular Diagnostics
Elian Rakhmanaliev, Tatiana Ivanova, Yin Kum Ng, Alex Yeo, Harry Suhardi, Caroline Villy, Mei Qi Yee, Kevin Tan, Amanda Fan, Andy Soh, Pramila Ariyaratne, Si Kee Tan, Jocelyn Wong, Jiang Xia, Grace Tan, Jasmin Ang, John Ong, Gerd Michel, Charlie Lee, Wen Huang

Characterizing GPCR Activation Using Automated Live Cell Imaging
Joe Clayton and Peter Banks