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(1018-D) Live Cell Assays Reveal New Patterns of Cell Signaling in Neurodegenerative Disease Mutants


Authors:

Anne Marie Quinn – CEO, Montana Molecular


Co-Authors:

Thom Hughes - Chief Scientific Officer, Montana Molecular
Anne Marie Quinn - CEO, Montana Molecular
Michael Hendrickson - Project Manager, BrainXell

Abstract:

Neurodegenerative diseases are notoriously difficult to study and treat. The neurons burdened by a mutant protein often survive for many decades before finally succumbing. These are often rare cells that die before the surrounding ones do, even though all cells express the mutant protein. Because cells destined to die are rare, and difficult to identify or purify, it is difficult to study the underlying biology of their disease state: are the changes they are going through irreversible? Can they still respond to drugs that target unique receptors and enzymes they express? 


Our goal was to develop live cell assays that can detect the stressed cells, before they are destined to die, and probe their ability to respond to receptor-driven changes in cell signaling. 

We targeted two different classes of neurodegenerative diseases, one that causes a misfolded protein in the ER - Retinitis Pigmentosa - and the other that causes misfolded, aggregated proteins in the cytoplasm- ɑ-synuclein and TDP-43. For Retinitis Pigmentosa, we created a two color fluorescent biosensor that could report both cytosolic Ca2+ signaling as well as ER stress caused by misfolded protein. Using high content imaging, we identified the cells in which the P23H mutant rhodopsin causes ER stress. This revealed that the stressed cells, unlike the surrounding ones, had elevated resting Ca2+ levels in the cytosol. Challenged with hM1 receptor activation, the changes in cytosolic Ca2+ were blunted in comparison with surrounding healthy cells. While these observations are made in cultured, transformed cells, they reproduce what is seen in animal models (Shinde et al., 2016)


Cells expressing the mutant ɑ-synuclein or TDP-43 proteins had a very different signal transduction; they responded poorly and partially to stimulation of Gs-coupled receptors and elevation of cAMP levels was modest. To allow rapid, real-time, quantitation of cAMP regulation in cells expressing mutants of ɑ-synuclein and TDP-43, we created a completely optical-based assay. By monitoring cAMP degradation in thousands of single cells we discovered that cells displayed either rapid, intermediate, or delayed cAMP degradation kinetics. Mutants in both ɑ-synuclein and TDP-43 shifted the cAMP degradation towards more rapid degradation kinetics, indicating that mutants in both proteins affect regulation of cAMP levels within the cell. 


Shinde, V., Kotla, P., Strang, C., and Gorbatyuk, M. (2016). Unfolded protein response-induced dysregulation of calcium homeostasis promotes retinal degeneration in rat models of autosomal dominant retinitis pigmentosa. Cell Death Dis. 7, e2085.

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