Abstract
Many neurodegenerative diseases induce high levels of sustained cellular stress and alter a number of cellular processes. To examine how different mutations associated with neurodegenerative disease affect cell stress and signaling, we created live-cell assays for endoplasmic reticulum (ER)-mediated cell stress and second messenger signaling. We first examined neurodegenerative mutations associated with direct ER stress by exploring the effect of rhodopsin mutations on ER stress and Ca2+ signaling. The rhodopsin P23H mutation, the most common mutation in autosomal dominant Retinitis Pigmentosa (RP), produced increased ER stress levels compared to wild type (WT) rhodopsin. Moreover, this increase in cell stress correlated with blunted Ca2+ signaling in a stress-dependent manner. Analysis of single-cell Ca2+ signaling profiles revealed unique Ca2+ signaling responses exist in cells expressing WT or P23H rhodopsin, consistent with the idea that second messenger signaling is affected by cell stress. To explore the use of the ER-stress biosensor in neurodegenerative diseases that may not have a direct effect on ER-mediated cell stress, we examined how various mutants of α-synuclein and TDP-43 affected ER stress. Mutants of both α-synuclein and TDP-43 associated with Parkinson’s disease (PD) and Amyotrophic lateral sclerosis (ALS) demonstrated increased ER stress compared to WT proteins. To examine the effect of α-synuclein and TDP-43 mutants on cellular signaling, we created a second live-cell assay to monitor changes in cAMP signaling during expression of various forms of α-synuclein and TDP-43. The increased cell stress caused by expression of the mutant proteins 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 for cellular stress and signaling can provide nuanced, new views of neurodegenerative disease processes.
Highlights
Genetically-encoded fluorescent biosensors are powerful tools that have provided new views of how circuits in the brain operate, and how cells and networks of cells process and respond to stimuli (Chen et al, 2017)
Ɑ-synuclein and TAR DNA binding protein (TDP-43) we show that Ca2+ and cyclic AMP (cAMP) signaling is altered under cell stress
Upon detection of misfolded proteins within the lumen of the endoplasmic reticulum (ER), IRE1ɑ is activated and carries out an unconventional cytoplasmic splicing of the XBP1 transcript. This splicing leads to a frame shift in the XBP1 open reading frame, creating a functional transcription factor which in turn activates a host of stress response genes (Grootjans et al, 2016)
Summary
Genetically-encoded fluorescent biosensors are powerful tools that have provided new views of how circuits in the brain operate, and how cells and networks of cells process and respond to stimuli (Chen et al, 2017) Many of these biosensors detect small molecule analytes, like Ca2+, cyclic AMP (cAMP) and diacylglycerol (Broussard et al, 2014; Tewson et al, 2012, 2016; Zhao et al, 2011) that change in concentration during a cell signaling events. Biosensors for apoptosis (Xu et al, 1998), cell cycle state (Sakaue-Sawano et al, 2008), autophagy (Katayama et al, 2011), and cell stress (Iwawaki et al, 2004; Roy et al, 2017) have been developed to detect broad changes to cellular states. In RP, rod photoreceptors slowly degrade over time, eventually degrading the cone photoreceptors as well, leading to blindness (Ferrari et al, 2011; Hartong et al, 2006)
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