Live-cell assays for cell stress responses reveal new patterns of cell signaling caused by mutations in rhodopsin, α-synuclein and TDP-43

Abstract

Many neurodegenerative diseases induce high levels of sustained cellular stress and alter a number of cellular processes. Genetically-encoded fluorescent biosensors are effective tools to examine neuronal activity and signaling in living cells. To examine how different mutations associated with neurodegenerative disease affect cell stress and signaling we created live-cell assays for ER-mediated cell stress and second messenger signaling. Analysis of the rhodopsin P23H mutation, the most common mutation in autosomal dominant Retinitis Pigmentosa, revealed increased cell stress levels compared to wild type rhodopsin. Moreover, this increase in cell stress correlated with blunted Ca 2+ signaling in a stress dependent manner. Analysis of single cell Ca 2+ signaling profiles revealed unique Ca 2+ signaling responses exist in cells expressing wild type or P23H mutants, further supporting the notion that second messenger signaling is affected by cell stress. To explore the use of the ER-stress biosensor in other neurodegenerative diseases we examined how various mutants of ɑ-synuclein and TDP-43 affected ER-mediated cell stress. Mutants of both ɑ-synuclein and TDP-43 associated with Parkinson's Disease and ALS demonstrated increases in ER-mediated cell stress. This increased cell stress was accompanied by changes in phosphodiesterase activity. Both HEK293T and SH-SY5Y cells expressing these proteins displayed a shift towards increased cAMP degradation rates, likely due to increased phosphodiesterase activity. Together these data illustrate how biosensors can provide nuanced, new views of neurodegenerative disease processes.