No abstract

Morphologically distinct TDP‐43 aggregates occur in clinically different FTLD‐TDP subtypes, yet the mechanism of their emergence and contribution to clinical heterogeneity are poorly understood. Several lines of evidence suggest that pathological TDP‐43 follows a prion‐like cascade, but the molecular determinants of this process remain unknown. We use advanced microscopy techniques to compare the seeding properties of pathological FTLD‐TDP‐A and FTLD‐TDP‐C aggregates. Upon inoculation of patient‐derived aggregates in cells, FTLD‐TDP‐A seeds amplify in a template‐dependent fashion, triggering neoaggregation more efficiently than those extracted from FTLD‐TDP‐C patients, correlating with the respective disease progression rates. Neoaggregates are sequentially phosphorylated with N‐to‐C directionality and with subtype‐specific timelines. The resulting FTLD‐TDP‐A neoaggregates are large and contain densely packed fibrils, reminiscent of the pure compacted fibrils present within cytoplasmic inclusions in postmortem brains. In contrast, FTLD‐TDP‐C dystrophic neurites show less dense fibrils mixed with cellular components, and their respective neoaggregates are small, amorphous protein accumulations. These cellular seeding models replicate aspects of the patient pathological diversity and will be a useful tool in the quest for subtype‐specific therapeutics.

Aggregation of the RNA-binding protein TAR DNA-binding protein 43 (TDP-43) is the key neuropathological feature of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). In physiological conditions, TDP-43 is predominantly nuclear, forms oligomers, and is contained in biomolecular condensates assembled by liquid-liquid phase separation (LLPS). In disease, TDP-43 forms cytoplasmic or intranuclear inclusions. How TDP-43 transitions from physiological to pathological states remains poorly understood. Using a variety of cellular systems to express structure-based TDP-43 variants, including human neurons and cell lines with near-physiological expression levels, we show that oligomerization and RNA binding govern TDP-43 stability, splicing functionality, LLPS, and subcellular localization. Importantly, our data reveal that TDP-43 oligomerization is modulated by RNA binding. By mimicking the impaired proteasomal activity observed in ALS/FTLD patients, we found that monomeric TDP-43 forms inclusions in the cytoplasm, whereas its RNA binding-deficient counterpart aggregated in the nucleus. These differentially localized aggregates emerged via distinct pathways: LLPS-driven aggregation in the nucleus and aggresomedependent inclusion formation in the cytoplasm. Therefore, our work unravels the origins of heterogeneous pathological species reminiscent of those occurring in TDP-43 proteinopathy patients.

Aggregation of the RNA-binding protein TDP-43 is the main common neuropathological feature of TDP-43 proteinopathies. In physiological conditions, TDP-43 is predominantly nuclear and contained in biomolecular condensates formed via liquid-liquid phase separation (LLPS). However, in disease, TDP-43 is depleted from these compartments and forms cytoplasmic or, sometimes, intranuclear inclusions. How TDP-43 transitions from physiological to pathological states remains poorly understood. Here, we show that self-oligomerization and RNA binding cooperatively govern TDP-43 stability, functionality, LLPS and cellular localization. Importantly, our data reveal that TDP-43 oligomerization is connected to, and conformationally modulated by, RNA binding. Mimicking the impaired proteasomal activity observed in patients, we found that TDP-43 forms nuclear aggregates via LLPS and cytoplasmic aggregates via aggresome formation. The favored aggregation pathway depended on the TDP-43 state –monomeric/oligomeric, RNA-bound/-unbound– and the subcellular environment –nucleus/cytoplasm. Our work unravels the origins of heterogeneous pathological species occurring in TDP-43 proteinopathies.