Examining the role of small heat shock proteins in regulating the phase separation and aggregation of TDP-43

TAR DNA-binding protein 43 (TDP-43) is a nuclear RNA-binding protein that undergoes liquid-liquid phase separation (LLPS) to form dynamic biomolecular condensates that are essential for its native function. Aberrant phase transitions of TDP-43 can occur which eventuate in pathological aggregation, a process that is implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTD). Molecular chaperones, a class of proteins that maintain protein folding and prevent aggregation, perform an essential role in modulating phase separation of TDP-43 and other proteins linked to neurodegenerative disease. Indeed, recent studies have revealed that chaperones from the Hsp40, Hsp70, Hsp90 and small Hsp (sHsp) classes can prevent TDP-43 aggregation by maintaining it in a soluble state or by inhibiting the formation of aberrant condensates. However, molecular chaperones are highly dynamic and heterogeneous, as are their substrate proteins. As such, different chaperones, even those within the same class, may interact differently with the same substrate protein. The work in this thesis aimed to evaluate interactions between TDP-43 and the two human sHsps HspB1 and HspB5. Studies were performed using purified proteins and in human osteosarcoma U2OS cells. Further, this work sought to establish a system for optogenetic induction of stress granule (SG) assembly with greater light sensitivity than previously published systems.This work first sought to evaluate the phase separation and aggregation of the TDP-43 low complexity domain (TDP-43LCD) in the presence of HspB1 and HspB5. Using confocal microscopy, spectrophotometry, biochemical techniques, and advanced microscopy, divergent impacts were observed for each chaperone upon the phase separation and aggregation of TDP-43LCD. HspB1 was found to promote TDP-43LCD phase separation through interactions involving its disordered N- and C-terminal extensions and specifically partitioned with TDP-43LCD in condensates, where it enhanced the dynamic exchange of protein with the surrounding medium. Though HspB5 did not promote TDP-43LCD phase separation, it localised within condensates and inhibited the reduction in dynamic exchange associated with condensate ‘ageing’. Further, HspB5 was found to strongly inhibit the kinetics of TDP-43LCD aggregation in a concentration-dependent manner, delaying fibril formation, slowing the rate of fibril elongation, and reducing the relative amount of fibril present. HspB1 was less effective at preventing TDP-43LCD aggregation. Notably, the effects of both chaperone upon TDP-43LCD aggregation involve direct interaction with TDP-43LCD oligomers through multiple functional domains of HspB1 and HspB5. These findings provide insight into the mechanisms by which different sHsps bind to the same client protein to regulate its phase separation and aggregation.Similar divergent effects were observed for HspB1 and HspB5 when overexpressed in cells expressing different TDP-43 variants. High-throughput image analysis and fluorescence recovery after photobleaching (FRAP) revealed that overexpression of a phosphomimetic mutant form of HspB1 (S15D, S78D, S82D, herein referred to as HspB13D) alters the abundance of cytoplasmic TDP-43 aggregates, and modifies their morphology, composition, and dynamic exchange properties. Similar analysis for phosphomimetic mutant HspB5 (S19D, S45D, S59D, herein referred to as HspB53D) revealed that its overexpression broadly reduces the accumulation of TDP-43 into condensates and aggregates and alters their dynamic exchange properties. Notably, this effect of HspB53D was most apparent for TDP-43 isoforms that are unable to bind RNA regardless of localisation to the nucleus or cytoplasm, suggesting that HspB5 binding to TDP-43 is modulated by RNA. Furthermore, it was found that overexpression of either chaperone had different effects against different C-terminal fragments of TDP-43; in general the sHsps reduced TDP-35 phase separation but enhanced TDP-20 aggregation. Thus, the differences observed here can inform on the mechanisms by which aggregated TDP-43 escapes the proteostasis machinery in ALS and FTD.Finally, this thesis presents an evaluation of a modified version of the OptoG3BP1 system for optogenetic control of SG assembly, in which the cryptochrome 2 photolyase homology region (CRY2PHR) is replaced with an isoform termed CRY2clust, which offers stronger clustering upon blue light exposure. Here, cells stably expressing CRY2clust-mCherry-G3BP1 assemble light-induced optogranules that colocalise with canonical SG markers PABP1 and TDP-43, confirming these as SGs. Further, these optogranules are assembled more rapidly in cells stably expressing CRY2clust-mCherry-G3BP1 compared to cells stably expressing CRY2PHR-mCherry-G3BP1. These findings indicate that CRY2clust-mCherry-G3BP1 is a suitable system for investigating pathological phase transitions in SGs and provides a platform for evaluating modifiers of SG dynamics in cells.Together, the results presented in this thesis demonstrate that the sHsp molecular chaperones HspB1 and HspB5 differentially modify TDP-43 phase separation and aggregation, thereby highlighting mechanisms by which cells may regulate TDP-43 dynamics. Determining how these chaperones integrate within the proteostasis network will contribute to understanding the fundamental mechanisms underlying ALS and FTD and the potential development of targeted therapeutics to protect against protein aggregation.