Kinetic analysis reveals that independent nucleation events determine the progression of polyglutamine aggregation in C. elegans

Protein aggregation is associated with a wide range of degenerative human diseases with devastating consequences, as exemplified by Alzheimer’s, Parkinson’s, and Huntington’s diseases. In vitro kinetic studies have provided a mechanistic understanding of the aggregation process at the molecular level. However, it has so far remained largely unclear to what extent the biophysical principles of amyloid formation learned in vitro translate to the complex environment of living organisms. Here, we take advantage of the unique properties of a Caenorhabditis elegans model expressing a fluorescently tagged polyglutamine (polyQ) protein, which aggregates into discrete micrometer-sized inclusions that can be directly visualized in real time. We provide a quantitative analysis of protein aggregation in this system and show that the data are described by a molecular model where stochastic nucleation occurs independently in each cell, followed by rapid aggregate growth. Global fitting of the image-based aggregation kinetics reveals a nucleation rate corresponding to 0.01 h⁻¹ per cell at 1 mM intracellular protein concentration, and shows that the intrinsic molecular stochasticity of nucleation accounts for a significant fraction of the observed animal-to-animal variation. Our results highlight how independent, stochastic nucleation events in individual cells control the overall progression of polyQ aggregation in a living animal. The key finding that the biophysical principles associated with protein aggregation in small volumes remain the governing factors, even in the complex environment of a living organism, will be critical for the interpretation of in vivo data from a wide range of protein aggregation diseases.

Protein aggregation is a pathological hallmark of a wide range of neurodegenerative and systemic diseases (1, 2). A mechanistic understanding of the pathways of amyloid formation has been obtained in vitro by approaches of chemical kinetics, providing fundamental insights into the microscopic aggregation steps of disease-associated proteins and peptides, most notably the amyloid-β peptide associated with Alzheimer’s disease (AD) (3). These studies have been invaluable to the design of small molecules that reduce the generation of potentially toxic oligomeric species (4, 5), to understand the action of molecular chaperones in inhibiting protein aggregation (6, 7), and to rationalize the efficacy of antibodies in clinical trials for AD (8). This framework has been extended to include stochastic and spatial effects that control kinetics in small volumes (femtoliter-picoliter range) (9–11), with the premise of being applicable to protein aggregation in living cells.However, cells and organisms have evolved intricate protein homeostasis pathways to ensure correct protein folding and to suppress misfolding and aggregation (12, 13), and it has not yet been established whether a chemical kinetics approach is sufficient to describe protein aggregation in vivo. The fundamental question is whether the complex nature of the cellular and organismal environment induces a major remodeling of the aggregation network as studied in vitro, or whether the same biophysical principles remain dominant.The nematode Caenorhabditis elegans provides the high level of control and the tools necessary to perform a quantitative kinetic analysis and determine the mechanisms governing protein aggregation in a living animal. C. elegans has a well-defined anatomy, and the animals within a population are genetically identical. Perhaps the most beneficial feature of this animal model system is its optical transparency, allowing the aggregation of a fluorescently labeled protein to be directly visualized. Specifically, we take advantage of the C. elegans muscle tissue that corresponds to 95 physiologically identical cells, and propose that these postmitotic cells can be quantitatively modeled as individual “test tubes” in which the deposition of expanded polyQ takes place by a mechanism of nucleated aggregation.