A020 Characterization of protein aggregates in cell models of polyglutamine diseases

<h3>Background</h3> Huntington’s disease (HD), spinocerebellar ataxia type 3 (SCA3) and 17 (SCA17) are devastating neurodegenerative disorders, resulting from abnormally expanded polyglutamine (polyQ) tracts within their respective disease proteins Huntingtin (HTT), ataxin-3 (ATXN3), and TATA-box-binding protein (TBP). The formation of histopathological protein aggregates has long been recognized as a common hallmark of polyQ diseases. However, decades of research have not provided a conclusive linkage between aggregate assembly and neurodegeneration. The prevailing scientific consensus suggests that toxic protein fragments act as primary drivers of aggregation. <h3>Aims</h3> Comparative investigation of aggregates formed by fragment and full-length polyQ-expanded proteins to decipher aggregation processes and the contribution of intracellular interactions. <h3>Methods</h3> Cell lines overexpressing a broad selection of proteolysis- and missplicing-derived fragment or full-length constructs of polyQ-expanded HTT, ATXN3, and TBP were utilized. Protein aggregation was assessed via filter retardation assays (FRA) and detergent-denaturing agarose gel electrophoresis (DDAGE). Aggregate structure and composition were determined by epi- and confocal fluorescence microscopy as well as transmission-electron microscopy. <h3>Results</h3> FRA, DDAGE-, and microscopy-based analysis proved that full-length HTT and TBP assemble into aggregates. We demonstrate that HTT, ATXN3 and TBP inclusions exhibit shared structural features, including a fibrillar core/shell architecture. Interestingly, we describe a novel double-shell/core inclusion organization of HTT and TBP aggregates. Additionally, mitochondria were found sequestered into inclusion bodies. <h3>Conclusions</h3> Full-length HTT and TBP assemble into intracellular aggregates sharing prominent structural similarities with polyQ fragment inclusions. The observed double-shell/core inclusion organization hints at a complex compositional and structural uniqueness of the outer aggregate periphery, inner inclusion shell, and compact aggregate core.