Insoluble protein aggregates are the hallmarks of many neurodegenerative diseases. For example, aggregates of TDP-43 occur in nearly all cases of amyotrophic lateral sclerosis (ALS). However, whether aggregates cause cellular toxicity is still not clear, even in simpler cellular systems. We reasoned that deep mutagenesis might be a powerful approach to disentangle the relationship between aggregation and toxicity. We generated >50,000 mutations in the prion-like domain (PRD) of TDP-43 and quantified their toxicity in yeast cells. Surprisingly, mutations that increase hydrophobicity and aggregation strongly decrease toxicity. In contrast, toxic variants promote the formation of dynamic liquid-like condensates. Mutations have their strongest effects in a hotspot that genetic interactions reveal to be structured in vivo, illustrating how mutagenesis can probe the in vivo structures of unstructured proteins. Our results show that aggregation of TDP-43 is not harmful but protects cells, most likely by titrating the protein away from a toxic liquid-like phase.

Plaques of the amyloid beta (Aβ) peptide are a pathological hallmark of Alzheimer's Disease (AD), the most common form of dementia. Mutations in Aβ also cause familial forms of AD (fAD). Here we use deep mutational scanning to quantify the effects of >14,000 mutations on the aggregation of Aβ. The resulting genetic landscape reveals mechanistic insights into fibril nucleation, including the importance of charge and gatekeeper residues in the disordered region outside of the amyloid core in preventing nucleation. Strikingly, unlike computational predictors and previous measurements, the empirical nucleation scores accurately identify all known dominant fAD mutations in AB42, genetically validating that the mechanism of nucleation in a cell-based assay is likely to be very similar to the mechanism that causes the human disease. These results provide the first comprehensive atlas of how mutations alter the formation of any amyloid fibril and a resource for the interpretation of genetic variation in Aβ.

Amyloid fibrils are associated with many human diseases but how mutations alter the propensity of proteins to form fibrils has not been comprehensively investigated and is not well understood. Alzheimer's Disease (AD) is the most common form of dementia with amyloid plaques of the amyloid beta (Aβ) peptide a pathological hallmark of the disease. Mutations in Aβ also cause familial forms of AD (fAD). Here we use deep mutational scanning to quantify the effects of >14,000 mutations on the aggregation of Aβ. The resulting genetic landscape reveals fundamental mechanistic insights into fibril nucleation, including the importance of charge and gatekeeper residues in the disordered region outside of the amyloid core in preventing nucleation. Strikingly, unlike computational predictors and previous measurements, the in vivo nucleation scores accurately identify all known dominant fAD mutations, validating this simple cell-based assay as highly relevant to the human genetic disease and suggesting accelerated fibril nucleation is the ultimate cause of fAD. Our results provide the first comprehensive map of how mutations alter the formation of any amyloid fibril and a validated resource for the interpretation of genetic variation in Aβ.

Specific insoluble protein aggregates are the hallmarks of many neurodegenerative diseases 1-5 . For example, cytoplasmic aggregates of the RNA-binding protein are observed in 97% of cases of Amyotrophic Lateral Sclerosis (ALS) 6,7 . However, it is still unclear for ALS and other diseases whether it is the insoluble aggregates or other forms of the mutated proteins that cause these diseases that are actually toxic to cells [8][9][10][11][12][13] . Here we address this question for TDP-43 by systematically mutating 14 the protein and quantifying the effects on cellular toxicity. We generated >50,000 mutations in the intrinsically disordered prion-like domain (PRD) and observed that changes in hydrophobicity and aggregation potential are highly predictive of changes in toxicity. Surprisingly, however, increased hydrophobicity and cytoplasmic aggregation actually reduce cellular toxicity. Mutations have their strongest effects in a central region of the PRD, with variants that increase toxicity promoting the formation of more dynamic liquidlike condensates. The genetic interactions in double mutants reveal that specific structures exist in this 'unstructured' region in vivo. Our results demonstrate that deep mutagenesis is a powerful approach for probing the sequence-function relationships of intrinsically disordered proteins as well as their in vivo structural conformations.Moreover, we show that aggregation of TDP-43 is not harmful but actually protects cells, most likely by titrating the protein away from a toxic liquid-like phase.3 Main textThe conversion of specific proteins into insoluble aggregates is a hallmark of many neurodegenerative disorders, including Alzheimer's, Parkinson's, Huntington's, and Amyotrophic Lateral Sclerosis (ALS) with dominantly inherited mutations in aggregateforming proteins causing rare familial forms of these diseases 3,6,15 . However, both in humans and in animal models, there is often only a weak association between the presence of aggregates and disease progression 16,17 . Indeed, many therapeutic approaches that reduce the formation of aggregates have failed at different stages of development 12,18,19 . On the other hand, there is increasing evidence that alternative protein assemblies generated during or in parallel to the aggregation process, may be toxic [8][9][10][11]20 . Despite evidence that cellular damage may be induced either before, after or independent of the formation of insoluble aggregates, the latter are still widely assumed to be pathogenic in many neurodegenerative diseases 21,22 .For many proteins, aggregation depends critically on intrinsically disordered regions with a low sequence complexity resembling that of infectious yeast prions. These prion-like domains (PRDs) are also enriched in proteins that can form liquid-like cellular condensates 23-25 with the PRDs necessary and sufficient for liquid-demixing 26,27 . At least in vitro, insoluble aggregates can nucleate from more liquid phases 27-29 , leading to the suggestion that liquid de-mixed states can ma...