Chronic optogenetic induction of stress granules is cytotoxic and reveals the evolution of ALS-FTD pathology

Abstract

Stress granules (SGs) are non-membrane-bound RNA-protein granules that assemble through phase separation in response to cellular stress. Disturbances in SG dynamics have been implicated as a primary driver of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), suggesting the hypothesis that these diseases reflect an underlying disturbance in the dynamics and material properties of SGs. However, this concept has remained largely untestable in available models of SG assembly, which require the confounding variable of exogenous stressors. Here we introduce a light-inducible SG system, termed OptoGranules, based on optogenetic multimerization of G3BP1, which is an essential scaffold protein for SG assembly. In this system, which permits experimental control of SGs in living cells in the absence of exogenous stressors, we demonstrate that persistent or repetitive assembly of SGs is cytotoxic and is accompanied by the evolution of SGs to cytoplasmic inclusions that recapitulate the pathology of ALS-FTD.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

Composition dependent phase separation underlies directional flux through the nucleolus

“…G3BP1/2 Knockout Cells 15 expressing, via lentiviral transfection and integration, protein constructs (either G3BP1 or optoG3BP1) were treated to a total concentration of 400uM sodium arsenite for 1 hour prior to imaging and maintained at standard conditions before and throughout imaging. For optoG3BP1 experiments, we cloned Cry2 N-terminal of G3BP1ΔNTF2 similarly as done in 25 .…”

Biomolecular condensates in neurodegeneration and cancer

“…SG hardening is expected to contribute to neurodegeneration in multiple ways (Figure C). The formation of cytotoxic aggregates is likely an extreme consequence of SG hardening that compromises quality control processes and membrane‐bound organelle function, as observed with other protein aggregates . In addition to aggregation, SG hardening is likely to prolong inhibition of global translation by delaying SG disassembly and alter global nucleocytoplasmic transport and gene expression by trapping constituent proteins involved in RNA processing and nuclear transport .…”

“…Interestingly, chaperone inhibition also delays SG disassembly post‐stress, suggesting that SG persistence and aggregation may be linked. Supporting this, constitutively inducing SG protein phase separation in cells (using optogenetic oligomerization tags) causes their aggregation . Other SG disassembly mechanisms include autophagy and microtubule dynamics, although their role in SG aggregation is not yet clear.…”

Enhanced stress tolerance through reduction of G3BP and suppression of stress granules

“…Recent efforts have focused largely on deciphering the principles that drive SG assembly as well as identifying the protein and RNA constituents of SG Khong et al, 2017;Markmiller et al, 2018;Namkoong et al, 2018;Souquere et al, 2009;Wheeler et al, 2016). G3BP1 and its paralog, G3BP2, (collectively referred to as G3BP) are the best characterized SG nucleating proteins and have been shown to be critical for SG assembly, where overexpression induces SG formation in the absence of stress and deletion ablates SG in response to arsenite (Kedersha et al, 2016;Reineke et al, 2012;Zhang et al, 2019). Although the formation of biomolecular condensates is thought to be highly dependent on factors that drive LLPS, such as protein concentrations of key nucleators, whether G3BP protein levels dictate the set point for SG assembly and the cellular stress threshold have not been determined.…”