Stress induced TDP-43 mobility loss independent of stress granules

Abstract

TAR DNA binding protein 43 (TDP-43) is closely related to the pathogenesis of amyotrophic lateral sclerosis (ALS) and translocates to stress granules (SGs). The role of SGs as aggregation-promoting “crucibles” for TDP-43, however, is still under debate. We analyzed TDP-43 mobility and localization under different stress and recovery conditions using live cell single-molecule tracking and super-resolution microscopy. Besides reduced mobility within SGs, a stress induced decrease of TDP-43 mobility in the cytoplasm and the nucleus was observed. Stress removal led to a recovery of TDP-43 mobility, which strongly depended on the stress duration. ‘Stimulated-emission depletion microscopy’ (STED) and ‘tracking and localization microscopy’ (TALM) revealed not only TDP-43 substructures within stress granules but also numerous patches of slow TDP-43 species throughout the cytoplasm. This work provides insights into the aggregation of TDP-43 in living cells and provide evidence suggesting that TDP-43 oligomerization and aggregation takes place in the cytoplasm separate from SGs.

Targeting the NEDP1 enzyme to ameliorate ALS phenotypes through stress granule disassembly

“…A biophysical property that defines the dynamicity and is directly related to the potential pathogenicity of SGs is the mobility of SG proteins within the condensates. Prolonged stress conditions reduce protein mobility within SGs, leading to their transition from a liquid-liquid into a liquid-solid phase that compromises SG elimination during the recovery period ( 16 , 22 ). By fluorescence recovery after photobleaching (FRAP), we found that prolonged exposure of parental cells to arsenite reduced the mobility of GFP-G3BP1 protein with concomitant increase in the half-life of recovery (Fig.…”

“…The generation and persistence of aberrant inclusions due to mutations in several genes encoding for SG proteins is regarded as hallmark in neurodegenerative diseases, such as ALS/FTD ( 14 , 15 , 17 ). However, how alterations in SG dynamics contribute to the formation of the toxic protein inclusions and the impact of aberrant SGs in the onset/progression of ALS/FTD is still unclear ( 22 , 23 ). The definition of pathways and development of approaches that promote the elimination of aberrant SGs would allow to assess their role in pathology.…”

“…As a result, SGs fail to disassemble upon stress relief, leading to protein synthesis deficiency and contributing to neuronal cell death (17). However, the direct link between aberrant SG formation and ALS pathology is still unclear, as exemplified by the study of ALS-derived mutants of TDP-43 protein that can induce cytoplasmic aggregates and ALS-related pathology, independently of SGs (21)(22)(23). Nevertheless, it is evident that, independently on whether they mainly derive from either direct deposition or maturation of SGs into an aggregated-like state, proteinaceous inclusions enriched for mutant TDP-43 or FUS are associated with cell toxicity (17,(22)(23)(24).…”

“…However, the direct link between aberrant SG formation and ALS pathology is still unclear, as exemplified by the study of ALS-derived mutants of TDP-43 protein that can induce cytoplasmic aggregates and ALS-related pathology, independently of SGs (21)(22)(23). Nevertheless, it is evident that, independently on whether they mainly derive from either direct deposition or maturation of SGs into an aggregated-like state, proteinaceous inclusions enriched for mutant TDP-43 or FUS are associated with cell toxicity (17,(22)(23)(24). Thus, acceleration of the elimination of toxic protein inclusions is regarded as an attractive approach to potentially reestablish normal neuronal function.…”

A New Phase of Networking: The Molecular Composition and Regulatory Dynamics of Mammalian Stress Granules

“…With that said, the exact relationship between SGs and ALS-FTD disease pathogenesis continues to be hotly debated. Other reports show that the RNAs enriched in SGs prevent aggregation of TDP-43 and that TDP-43 aggregation occurs outside of SGs, − disrupting the idea that SGs seed TDP-43 aggregation. Furthermore, in contrast to previous reports that observed transiently expressed TDP-43 ALS variants localizing to SGs, TDP-43 M337V is not found in SGs following heat stress that robustly induces SGs in the mouse central nervous system .…”