Fluc‐EGFP reporter mice reveal differential alterations of neuronal proteostasis in aging and disease

Abstract

The cellular protein quality control machinery is important for preventing protein misfolding and aggregation. Declining protein homeostasis (proteostasis) is believed to play a crucial role in agerelated neurodegenerative disorders. However, how neuronal proteostasis capacity changes in different diseases is not yet sufficiently understood, and progress in this area has been hampered by the lack of tools to monitor proteostasis in mammalian models. Here, we have developed reporter mice for in vivo analysis of neuronal proteostasis. The mice express EGFP-fused firefly luciferase (Fluc-EGFP), a conformationally unstable protein that requires chaperones for proper folding, and that reacts to proteotoxic stress by formation of intracellular Fluc-EGFP foci and by reduced luciferase activity. Using these mice, we provide evidence for proteostasis decline in the aging brain. Moreover, we find a marked reaction of the Fluc-EGFP sensor in a mouse model of tauopathy, but not in mouse models of Huntington's disease. Mechanistic investigations in primary neuronal cultures demonstrate that different types of protein aggregates have distinct effects on the cellular protein quality control. Thus, Fluc-EGFP reporter mice enable new insights into proteostasis alterations in different diseases.

Biosensors for Studying Neuronal Proteostasis

“…Cellular compartments and organelles such as the nucleus, mitochondria, and endoplasmic reticulum have evolved distinct proteostasis pathways, and recent studies have highlighted the impact of these organelle-specific mechanisms on cellular proteostasis (Walter and Ron, 2011 ; Kirstein et al, 2015 ; Miller et al, 2015 ; Frakes and Dillin, 2017 ; Shpilka and Haynes, 2018 ; Frottin et al, 2019 ; Moehle et al, 2019 ). Some of the existing proteostasis sensors have already been targeted to different cellular compartments (Winkler et al, 2010 ; Frottin et al, 2019 ; Blumenstock et al, 2021 ; Melo et al, 2021 ; Raeburn et al, 2021 ). A set of spectrally distinct, compartment-specific sensors that could be combined with each other would be a helpful resource for elucidating intra-compartmental crosstalk.…”

“…While proteostasis sensors have proven very useful in cell culture and C. elegans models, investigating neuronal proteostasis in higher organisms has for a long time remained a challenge. The generation of proteostasis reporter mice (Blumenstock et al, 2021 ) opens exciting new avenues for fundamental insights into in vivo mechanisms of protein misfolding. Thus, transgenic Fluc-EGFP mice will allow longitudinal investigations of the dynamics of proteostasis impairments during disease progression at the single-cell level with modern imaging methods such as in vivo two-photon microscopy.…”

“…The functionality of this sensor has been proven not only in cell lines and in C. elegans , but also in primary murine neurons. Moreover, a transgenic mouse with Fluc-EGFP expression in the nervous system has been generated, which gives a unique opportunity to investigate in vivo proteostasis changes in normal aging and in mouse disease models (Blumenstock et al, 2021 ). Thus, the sensor revealed proteostasis impairments in a tauopathy model at an early stage, preceding neuronal cell death and behavioral symptoms.…”

“…These findings point to proteostasis alterations as an early hallmark of neurodegeneration in vivo . Of note, while the bioluminescence readout showed very high sensitivity in cell lines, it proved less sensitive in bulk brain tissue of proteinopathy model mice (Gupta et al, 2011 ; Blumenstock et al, 2021 ), possibly due to the heterogeneity of neuronal cell types that differ in their reactions to misfolding. This underlines the importance of single-cell sensor readouts in order to capture the complexity of cell types in vivo .…”

Molecular mechanisms of amyloid formation in living systems

“…156,157 Similar discoveries were recently made in the mouse brain. 158 Widespread changes in the proteome have also been found to take place during the reproductive phase of C. elegans, followed by the aggregation of many endogenous proteins at a later stage. 142 The ability to mount robust stress responses such as the heat shock response deteriorates even earlier in the life cycle of the nematode, at the onset of the reproductive phase.…”