Structural basis of proteasome impairment by protein aggregates in ALS/FTD

Abstract text (incl. figure legends and references)

Protein aggregation is a hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, the mechanisms linking aggregation to neurotoxicity remain poorly understood, partly because only limited information is available on the native structure of protein aggregates inside cells. We address this pressing issue utilizing the latest developments in cryo-electron tomography (cryo-ET). We use cryo-focused ion beam to prepare thin lamellas of vitrified cells containing protein aggregates, and subsequently image them in three dimensions by cryo-ET. This allows us to analyse aggregate structure within pristinely preserved cellular environments and at molecular resolution.

Using this approach, we analysed neuronal poly-GA and TDP-43 aggregates. The expansion of a GGGGCC repeat in the C9orf72 gene is the most common cause of familial ALS/FTD. This mutation leads to the translation of poly-GA and other dipeptide repeat proteins, which aggregate in neurons. On the other hand, most ALS/FTD cases are sporadic and display aggregation of C-terminal fragments of TDP-43. Our in situ cryo-ET studies show that both poly-GA [ref #1; Fig.1] and TDP-43 [ref #2, Fig.2] aggregates recruit large amounts of proteasomes in neurons. Subtomogram averaging revealed that association with aggregates alters the conformational landscape of the proteasome. This leads to defects in proteasomal degradation and accumulation of proteasomal substrates in the cells. To understand these processes with mechanistic detail, we reconstituted the poly-GA/proteasome interaction in vitro [ref #3]. Combined single-particle, cryo-ET and biochemical analyses suggest a structural basis of proteasome impairment by poly-GA. Altogether, our studies highlight proteasome inhibition as an important factor of neurotoxicity in ALS/FTD, pointing to possible therapeutic avenues.

References

[1] Guo et al., Cell (2018) 172 (4), 696-705, DOI: 10.1016/j.cell.2017.12.030

[2] Riemenschneider et al., EMBO Rep (2022) 23 (6), e53890, DOI: 10.15252/embr.202153890

[3] Qu et al., manuscript in preparation

Legends

Fig 1. C9orf72 poly-GA aggregates consist of polymorphic twisted ribbons (red) that recruit high numbers of proteasomes (light blue). Other large macromolecules, such as ribosomes (gold) or TRiC/CCT (purple) are mostly excluded from the aggregate and accumulate at its periphery. Further analysis of proteasome structure by subtomogram averaging suggests that poly-GA-associated proteasomes are functionally impaired. Guo et al., Cell (2018).

Fig 2. Aggregates formed by C-terminal fragments of TDP-43 ("TDP-25") appear amorphous (red) and recruit high numbers of proteasomes (light blue). Other large macromolecules, such as ribosomes (gold) or TRiC/CCT (green) are mostly excluded from the aggregate and accumulate at its periphery. Further analysis of proteasome structure by subtomogram averaging suggests that poly-GA-associated proteasomes are functionally impaired. Riemenschneider et al., EMBO Rep (2022).