A019 Comparative semi-quantitative analysis of mutant huntingtin aggregation

<h3>Background</h3> Huntington’s disease (HD) is a neurodegenerative disorder characterized by a polyglutamine expansion in the huntingtin (HTT) protein. Mutant HTT is prone to aggregation, resulting in the formation of intracellular inclusions. This histological hallmark of HD coincides with advancing neuronal degeneration and compromises the protein’s physiological functions. Consequently, studies often examine HTT aggregation to assess disease progression and therapeutic efficacy. Microscopy-based analyses of HTT inclusions provide information on inclusion structure, size, number and subcellular localization. However, these methods are limited when analysing sub-microscopic HTT aggregate species or larger sample sizes. <h3>Aims</h3> We sought to reassess the applicability and reproducibility of immunodetection-based semi-quantitative methods for exploiting aggregated forms of mutant HTT as a readout parameter of disease progression and treatment efficacy. <h3>Methods</h3> Using optimized filter retardation (FT) assays and denaturing detergent agarose gel electrophoresis (DD-AGE), we compared both semi-quantitative methods to microscopy-based analysis. Through a broad selection of specific antibodies, we assessed HTT aggregation in HD cell and animal models expressing mutant full-length and exon-1 HTT. <h3>Results</h3> While microscopic analysis was insufficient for reliably detecting and quantifying mutant HTT inclusions across analysed HD models, FT assay and DD-AGE proved more sensitive and robust for semi-quantitative assessment of HTT aggregation. Moreover, through methodical establishment and selection of appropriate HTT-specific antibodies, both assays delivered reliable readouts when pharmacologically modulating HTT aggregation <h3>Conclusions</h3> Our study underscores the importance of thorough assay establishment for robust and reliable semi-quantitative and comparative readouts. It may serve as a practical guideline for future studies exploiting HTT aggregation as a quantifiable readout parameter.