Aggregates of the RNA-binding protein TDP-43 (TAR DNAbinding protein) are a hallmark of the overlapping neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The process of TDP-43 aggregation remains poorly understood, and whether it includes formation of intermediate complexes is unknown. Here, we analyzed aggregates derived from purified TDP-43 under semidenaturing conditions, identifying distinct oligomeric complexes at the initial time points before the formation of large aggregates. We found that this early oligomerization stage is primarily driven by TDP-43's RNA-binding region. Specific binding to GU-rich RNA strongly inhibited both TDP-43 oligomerization and aggregation, suggesting that RNA interactions are critical for maintaining TDP-43 solubility. Moreover, we analyzed TDP-43 liquid-liquid phase separation and detected similar detergentresistant oligomers upon maturation of liquid droplets into solid-like fibrils. These results strongly suggest that the oligomers form during the early steps of TDP-43 misfolding. Importantly, the ALS-linked TDP-43 mutations A315T and M337V significantly accelerate aggregation, rapidly decreasing the monomeric population and shortening the oligomeric phase. We also show that aggregates generated from purified TDP-43 seed intracellular aggregation detected by established TDP-43 pathology markers. Remarkably, cytoplasmic aggregate seeding was detected earlier for the A315T and M337V variants and was 50% more widespread than for WT TDP-43 aggregates. We provide evidence for an initial step of TDP-43 self-assembly into intermediate oligomeric complexes, whereby these complexes may provide a scaffold for aggregation. This process is altered by ALS-linked mutations, underscoring the role of perturbations in TDP-43 homeostasis in protein aggregation and ALS-FTD pathogenesis.

KEYWORDS: amyotrophic lateral sclerosis (ALS) (Lou Gehrig disease), TAR DNA-binding protein 43 (TDP-43) (TARDBP), RNA binding protein, protein aggregation, liquid droplet, frontotemporal dementia (FTD) (FTLD), aggregate propagation, ALS mutations, liquid-liquid phase separation. ABSTRACTAggregates of the RNA binding protein TDP-43 are a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which are neurodegenerative disorders with overlapping clinical, genetic and pathological features. Mutations in the TDP-43 gene are causative of ALS, supporting its central role in pathogenesis. The process of TDP-43 aggregation remains poorly understood and whether this includes formation of intermediate complexes is unknown. We characterized aggregates derived from purified TDP-43 as a function of time and analyzed them under semi-denaturing conditions. Our assays identified oligomeric complexes at the initial time points prior to the formation of large aggregates, suggesting that ordered oligomerization is an intermediate step of TDP-43 aggregation. In addition, we analyzed liquid-liquid phase separation of TDP-43 and detected similar oligomeric assembly upon the maturation of liquid droplets into solid-like fibrils. These results strongly suggest that the oligomers form during the early steps of TDP-43 misfolding. Importantly, ALSlinked mutations A315T and M337V significantly accelerate aggregation, rapidly decreasing the monomeric population and shortening the oligomeric phase. We also show that the aggregates generated from purified protein seed intracellular aggregation, which is detected by established markers of TDP-43 pathology. Remarkably, cytoplasmic aggregate propagation is detected earlier with A315T and M337V and is 50% more widespread than with wild-type aggregates. Our findings provide evidence for a controlled process of TDP-43 self-assembly into intermediate structures that provide a scaffold for aggregation. This process is altered by ALSlinked mutations, underscoring the role of perturbations in TDP-43 homeostasis in protein aggregation and ALS-FTD pathogenesis.