Oligomeric assemblies formed from a variety of disease-associated peptides and proteins have been strongly associated with toxicity in many neurodegenerative conditions, such as Alzheimer's disease. The precise nature of the toxic agents, however, remains still to be established. We show that prefibrillar aggregates of E22G (arctic) variant of the Abeta(1-42) peptide bind strongly to 1-anilinonaphthalene 8-sulfonate and that changes in this property correlate significantly with changes in its cytotoxicity. Moreover, we show that this phenomenon is common to other amyloid systems, such as wild-type Abeta(1-42), the I59T variant of human lysozyme and an SH3 domain. These findings are consistent with a model in which the exposure of hydrophobic surfaces as a result of the aggregation of misfolded species is a crucial and common feature of these pathogenic species.

SummaryMultiple human diseases are associated with a liquid-to-solid phase transition resulting in the formation of amyloid fibers or protein aggregates. Here, we present an alternative mechanism for cellular toxicity based on a concentration-dependent liquid-liquid demixing. Analyzing proteins that are toxic when their concentration is increased in yeast reveals that they share physicochemical properties with proteins that participate in physiological liquid-liquid demixing in the cell. Increasing the concentration of one of these proteins indeed results in the formation of cytoplasmic foci with liquid properties. Demixing occurs at the onset of toxicity and titrates proteins and mRNAs from the cytoplasm. Focus formation is reversible, and resumption of growth occurs as the foci dissolve as protein concentration falls. Preventing demixing abolishes the dosage sensitivity of the protein. We propose that triggering inappropriate liquid phase separation may be an important cause of dosage sensitivity and a determinant of human disease.

In recent genome-wide association studies, the extracellular chaperone protein, clusterin, has been identified as a novel risk factor in Alzheimer's disease (AD). We have examined the interactions between clusterin and the AD-associated amyloid-β 1-40 peptide (Aβ 1-40 ) which is prone to aggregate into an ensemble of oligomeric intermediates implicated in both the proliferation of amyloid fibrils and in neuronal toxicity. Using highly sensitive single molecule fluorescence methods, we have found that Aβ 1-40 forms a heterogeneous distribution of small oligomers (from dimers to 50mers), all of which interact with clusterin to form long-lived, stable complexes. Consequently, clusterin is able to influence strongly both the aggregation and disaggregation of Aβ 1-40 by sequestration of the Aβ oligomers. These results not only elucidate the protective role of clusterin but also provide a molecular basis for the genetic link between clusterin and AD.

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.