Detection of Mutant Huntingtin Aggregation Conformers and Modulation of SDS-Soluble Fibrillar Oligomers by Small Molecules

Abstract

The Huntington’s disease (HD) mutation leads to a complex process of Huntingtin (Htt) aggregation into multimeric species that eventually form visible inclusions in cytoplasm, nuclei and neuronal processes. One hypothesis is that smaller, soluble forms of amyloid proteins confer toxic effects and contribute to early cell dysfunction. However, analysis of mutant Htt aggregation intermediates to identify conformers that may represent toxic forms of the protein and represent potential drug targets remains difficult. We performed a detailed analysis of aggregation conformers in multiple in vitro, cell and ex vivo models of HD. Conformation-specific antibodies were used to identify and characterize aggregation species, allowing assessment of multiple conformers present during the aggregation process. Using a series of assays together with these antibodies, several forms could be identified. Fibrillar oligomers, defined as having a β-sheet rich conformation, are observed in vitro using recombinant protein and in protein extracts from cells in culture or mouse brain and shown to be globular, soluble and non-sedimentable structures. Compounds previously described to modulate visible inclusion body formation and reduce toxicity in HD models were also tested and consistently found to alter the formation of fibrillar oligomers. Interestingly, these compounds did not alter the rate of visible inclusion formation, indicating that fibrillar oligomers are not necessarily the rate limiting step of inclusion body formation. Taken together, we provide insights into the structure and formation of mutant Htt fibrillar oligomers that are modulated by small molecules with protective potential in HD models.

Mutant Huntingtin Disrupts the Nuclear Pore Complex

“…We previously showed the presence of an insoluble HMW mHtt species along with other modified proteins in the detergentinsoluble fraction from R6/2 striatum (Ochaba et al, 2016), therefore detergent-soluble and detergent-insoluble proteins were evaluated. The detergent-soluble fraction contains mainly cytoplasmic proteins, monomeric forms of Htt (including the R6/2 mHTT fragment encoding human transgene), endogenous mouse full-length Htt, and soluble oligomeric species of Htt, which do not fully resolve on standard PAGE gels (O'Rourke et al, 2013; Sontag et al, 2012). In contrast, the detergent-insoluble fraction contains primarily nuclear proteins such as HMW mHtt species (likely multimers or potentially insoluble oligomers and fibrils), and insoluble accumulated forms of SUMO-and ubiquitin-modified proteins.…”

“…Membrane was then washed 3× with 0.1% SDS and then blocked in 5% milk and subject to western blot analysis (previously described by (Sontag et al, 2012; Wanker et al, 1999). …”

Prefibrillar huntingtin oligomers isolated from HD brain potently seed amyloid formation

“…In contrast to prefibrillar HTT oligomers, fibrillar HTT oligomers appear to be relatively unstable and SDS-soluble [39]. Compounds which block formation of fibrillar HTT oligomers do not halt inclusion body formation, suggesting fibrillar HTT oligomers are off the amyloid formation pathway [39]. The resilience of prefibrillar oligomers to strong denaturing conditions suggests possible structural stabilization by covalent bonds, as hypothesized to be present in HTT amyloids [27], implying that such covalent bonds are an early step in fibril formation.…”

“…Fully formed HTT amyloids are notoriously stable, remaining insoluble in boiling SDS [45]; the observation that amyloidogenic oligomeric precursors share this biochemical feature suggests a fundamental structural commonality. In contrast to prefibrillar HTT oligomers, fibrillar HTT oligomers appear to be relatively unstable and SDS-soluble [39]. Compounds which block formation of fibrillar HTT oligomers do not halt inclusion body formation, suggesting fibrillar HTT oligomers are off the amyloid formation pathway [39].…”

Self-assembly of mutant huntingtin exon-1 fragments into large complex fibrillar structures involves nucleated branching

“…Evidence has been provided that N-terminal huntingtin exon-1 (HTTex1) fragments with pathogenic polyglutamine (polyQ) tracts spontaneously self-assemble into insoluble aggregates with fibrillar morphology [24][25][26][27][28][29][30]. This indicates that formation of amyloid-like HTTex1 aggregates is mechanistically similar to -synuclein or amyloid- aggregation observed in various model systems [26].…”