Inferring Aggregation Mechanisms Of Molecules Involved In Neurodegeneration Through Quantitative Studies Of Phase Behavior
Date of Award
Doctor of Philosophy (PhD)
Chair and Committee
Polyglutamine is involved in at least nine known neurodegenerative diseases, the most prominent of which is Huntington's Disease. It is thought that polyglutamine aggregation leads to disease. The biophysical mechanism of polyglutamine aggregation remains controversial as highlighted by conflicting proposals that have been put forth in the literature ranging from homogeneous nucleation to a more complex assembly mechanism that involves heterogeneous distributions of oligomers. Converging upon an accurate framework for describing polyglutamine aggregation in vitro is an essential first step for understanding how interactions in cis i.e., flanking sequences and trans i.e., heterotypic interactions in the cellular milieu shape self-assembly and the formation of inclusions. In this work, we leverage concepts from polymer physics, to understand solution phase behavior of polyglutamine. Specifically, we first characterize water as poor solvent for polyglutamine. This classification suggests that polyglutamine forms collapsed structures in aqueous solution. At low concentrations, this will lead to homogeneously dispersed solutions of compact globules. At higher concentrations, the globules will coalesce leading to phase separation. Next, we characterize the phase behavior of polyglutamine solutions and develop a reference phase diagram for polyglutamine peptides that provides thermodynamic constraints for aggregation mechanisms. Specifically, we measure temperature-dependent saturation concentrations of aqueous polyglutamine solutions containing 30 and 40 glutamine residues and either 2 or 4 flanking lysines. We used classical Flory-Huggins theory to construct the phase diagram for partitioning between soluble and insoluble phases from the measured saturation concentrations. The low-concentration arm of the phase diagram provides a thermodynamic basis for assessing aggregation propensity. For a given chain length, aggregation propensity increases as the number of lysine residues decrease highlighting the contributions from intermolecular electrostatic repulsions. For a fixed number of lysine residues, the aggregation propensity increases with increasing chain length, highlighting the intrinsic contributions of polyglutamine length to the driving forces for aggregation. The inferred phase diagrams provide thermodynamic constraints on the kinetic mechanisms for aggregation. In addition, at physiological temperatures, the gap between the saturation curve and the instability boundary spans roughly two orders of magnitude. This suggests that the formation of metastable, higher-order clusters and conformational conversions within these clusters are likely precursors for polyglutamine aggregation thereby rationalizing a role for oligomers that have been observed in recent studies based on AFM and light scattering. Finally, we apply our knowledge of the phase behavior of polyglutamine to understand mechanisms by which amyloid beta aggregation might be modulated by cellular activities. In particular, our experiments suggest that amyloid beta is taken up from the extracellular space by neurons, trafficked into acidic vesicles, and concentrated to levels known to support aggregation based on the phase diagram.
Crick, Scott, "Inferring Aggregation Mechanisms Of Molecules Involved In Neurodegeneration Through Quantitative Studies Of Phase Behavior" (2011). All Theses and Dissertations (ETDs). 76.
Permanent URL: http://dx.doi.org/10.7936/K74Q7RZ1