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. 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 Ca 2+ 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 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 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.

Labile assembly of a tardigrade protein induces biostasis

“…To ensure that the increase in survival seen in osmotically shocked cells expressing CAHS D and its variants is not due a cellular response from an induced ER Stress or unfolded protein response (UPR), we measured the level of ER stress/UPR on the cell lines using a ratiometric stress sensor (Harlen et al, 2019 ). Expression of mVenus:CAHS D or any of its variants does not induce significant levels of ER stress/UPR compared to naive HEK cells or HEK cells expressing mVenus alone (Figure S6F ).…”

“…To assess ER stress/UPR, cells were assayed using a Green/Red Ratiometric Cell Stress Assay Kit (Catalog U0901G, Montana Molecular) (Harlen et al, 2019 ).…”

Aberrant activation of hippocampal astrocytes causes neuroinflammation and cognitive decline in mice

“…The Ca 2+ levels in the primary hippocampal neurons and astrocytes were monitored by a Lionheart automated image analyzer (BioTek) using Fluo-4 AM (5 μm in 0.01% of pluronic, 30-min incubation at 25°C) as previously described [ 163 ]. The Ca 2+ levels were recorded from the hippocampal neurons or astrocyte cell body and processes, and the variations in Ca 2+ were estimated as changes in the fluorescence signal over the baseline value ( Δ F/F 0 ).…”

Mechanisms of cell death induced by hexabromocyclododecane (HBCD) involves apoptosis, autophagy, and ER stress

“…The biosensor is based on splicing of the XBP1 RNA, mediated by the ER protein IRE1 and this splicing removes an intron which results in the transcription and translation of the mNeonGreen fluorescent protein biosensor. [ 16 ]…”