Oxidation Promotes Distinct Huntingtin Aggregates in the Presence and Absence of Membranes.

Expansion of a polyglutamine (polyQ) domain within the first exon of the huntingtin (htt) protein is the underlying cause of Huntington's disease, a genetic neurodegenerative disorder. PolyQ expansion triggers htt aggregation into oligomers, fibrils, and inclusions. The 17 N-terminal amino acids (Nt17) of htt-exon1, which directly precede the polyQ domain enhances polyQ fibrillization and functions as a lipid-binding domain. A variety of post-translational modifications occur within Nt17, including oxidation of two methionine residues. Here, the impact of oxidation within Nt17 on htt aggregation both in the presence and absence of lipid membranes was investigated. Treatment with hydrogen peroxide (H2O2) reduced fibril formation in a dose-dependent manner, resulting in shorter fibrils and an increased oligomer population. With excessive H2O2 treatments, fibrils developed a unique morphological feature around their periphery. In the presence of total brain lipid vesicles, H2O2 impacted fibrillization in a similar manner. That is, oligomerization was promoted at the expense of fibril elongation. The interaction of unoxidized and oxidized htt with supported lipid bilayers was directly observed using in situ atomic force microscopy. Without oxidation, granular htt aggregates developed on the bilayer surface. However, in the presence of H2O2, distinct plateau-like regions initially developed on the bilayer surface that gave way to rougher patches containing granular aggregates. Collectively, these observations suggest that oxidation of methionine residues within Nt17 plays a crucial role in both the aggregation of htt and its ability to interact with lipid surfaces.

Macromolecular crowding in solution alters huntingtin interaction and aggregation at interfaces.

Huntington's disease (HD) is a fatal neurodegenerative disease caused by an extended polyglutamine (polyQ) domain within the first exon of the huntingtin protein (htt). PolyQ expansion directly invokes the formation of a heterogenous mixture of toxic htt aggregates, including fibrils and oligomers. While htt is a cytosolic protein, it also associates with numerous membranous surfaces within the cell, leading to altered organelle morphology and dysfunction. Here, the impact of macromolecular crowding on htt aggregation in bulk solution and at solid/liquid or membrane/liquid interfaces was investigated. Dextran, Ficoll, and polyethylene glycol (PEG) were used as crowding agents. In bulk solution, crowding enhanced the heterogeneity of non-fibrillar aggregate species formed in a crowder dependent manner. However, crowding agents interfered with the deposition of htt fibrils on mica, suggesting that a crowded aqueous phase influences the interaction of htt with interfaces. By use of in situ atomic force microcopy (AFM), the aggregation of htt directly at mica and bilayer interfaces was tracked. The predominate aggregates type observed to form at the mica interface was fibrillar, but oligomeric aggregates of various stabilities were also observed. Crowding in the aqueous phase suppressed deposition and formation of htt aggregates on mica. In contrast, the addition of crowders enhanced deposition of htt aggregates onto supported total brain lipid extract (TBLE) bilayers. Different crowding agents led to distinct htt aggregates on supported bilayers with unique morphological impact on bilayer integrity. Collectively, these observations point to the complexity of htt aggregation at interfaces and that crowding in the aqueous phase profoundly influences this process.