The polyglutamine domain is the primary driver of seeding in huntingtin aggregation.

Huntington's Disease (HD) is a fatal, neurodegenerative disease caused by aggregation of the huntingtin protein (htt) with an expanded polyglutamine (polyQ) domain into amyloid fibrils. Htt aggregation is modified by flanking sequences surrounding the polyQ domain as well as the binding of htt to lipid membranes. Upon fibrillization, htt fibrils are able to template the aggregation of monomers into fibrils in a phenomenon known as seeding, and this process appears to play a critical role in cell-to-cell spread of HD. Here, exposure of C. elegans expressing a nonpathogenic N-terminal htt fragment (15-repeat glutamine residues) to preformed htt-exon1 fibrils induced inclusion formation and resulted in decreased viability in a dose dependent manner, demonstrating that seeding can induce toxic aggregation of nonpathogenic forms of htt. To better understand this seeding process, the impact of flanking sequences adjacent to the polyQ stretch, polyQ length, and the presence of model lipid membranes on htt seeding was investigated. Htt seeding readily occurred across polyQ lengths and was independent of flanking sequence, suggesting that the structured polyQ domain within fibrils is the key contributor to the seeding phenomenon. However, the addition of lipid vesicles modified seeding efficiency in a manner suggesting that seeding primarily occurs in bulk solution and not at the membrane interface. In addition, fibrils formed in the presence of lipid membranes displayed similar seeding efficiencies. Collectively, this suggests that the polyQ domain that forms the amyloid fibril core is the main driver of seeding in htt aggregation.

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.

Mitochondrial membranes modify mutant huntingtin aggregation.

Huntington's disease (HD) is a neurodegenerative disease caused by the expansion of a polyglutamine (polyQ) tract near the N-terminus of the huntingtin (htt) protein. Expanded polyQ tracts are prone to aggregate into oligomers and insoluble fibrils. Mutant htt (mhtt) localizes to variety of organelles, including mitochondria. Specifically, mitochondrial defects, morphological alteration, and dysfunction are observed in HD. Mitochondrial lipids, cardiolipin (CL) in particular, are essential in mitochondria function and have the potential to directly interact with htt, altering its aggregation. Here, the impact of mitochondrial membranes on htt aggregation was investigated using a combination of mitochondrial membrane mimics and tissue-derived mitochondrial-enriched fractions. The impact of exposure of outer and inner mitochondrial membrane mimics (OMM and IMM respectively) to mhtt was explored. OMM and IMM reduced mhtt fibrillization, with IMM having a larger effect. The role of CL in mhtt aggregation was investigated using a simple PC system with varying molar ratios of CL. Lower molar ratios of CL (<5%) promoted fibrillization; however, increased CL content retarded fibrillization. As revealed by in situ AFM, mhtt aggregation and associated membrane morphological changes at the surface of OMM mimics was markedly different compared to IMM mimics. While globular deposits of mhtt with few fibrillar aggregates were observed on OMM, plateau-like domains were observed on IMM. A similar impact on htt aggregation was observed with exposure to purified mitochondrial-enriched fractions. Collectively, these observations suggest mitochondrial membranes heavily influence htt aggregation with implication for HD.