Dataset Information

Development of novel bioassays to detect soluble and aggregated Huntingtin proteins on three technology platforms.

ABSTRACT: Huntington's disease is caused by a CAG / polyglutamine repeat expansion. Mutated CAG repeats undergo somatic instability, resulting in tracts of several hundred CAGs in the brain; and genetic modifiers of Huntington's disease have indicated that somatic instability is a major driver of age of onset and disease progression. As the CAG repeat expands, the likelihood that exon 1 does not splice to exon 2 increases, resulting in two transcripts that encode full-length huntingtin protein, as well as the highly pathogenic and aggregation-prone exon 1 huntingtin protein. Strategies that target the huntingtin gene or transcripts are a major focus of therapeutic development. It is essential that the levels of all isoforms of huntingtin protein can be tracked, to better understand the molecular pathogenesis, and to assess the impact of huntingtin protein-lowering approaches in preclinical studies and clinical trials. Huntingtin protein bioassays for soluble and aggregated forms of huntingtin protein are in widespread use on the homogeneous time-resolved fluorescence and Meso Scale Discovery platforms, but these do not distinguish between exon 1 huntingtin protein and full-length huntingtin protein. In addition, they are frequently used to quantify huntingtin protein levels in the context of highly expanded polyglutamine tracts, for which appropriate protein standards do not currently exist. Here, we set out to develop novel huntingtin protein bioassays to ensure that all soluble huntingtin protein isoforms could be distinguished. We utilized the zQ175 Huntington's disease mouse model that has ?190 CAGs, a CAG repeat size for which protein standards are not available. Initially, 30 combinations of six antibodies were tested on three technology platforms: homogeneous time-resolved fluorescence, amplified luminescent proximity homogeneous assay and Meso Scale Discovery, and a triage strategy was employed to select the best assays. We found that, without a polyglutamine-length-matched standard, the vast majority of soluble mutant huntingtin protein assays cannot be used for quantitative purposes, as the highly expanded polyglutamine tract decreased assay performance. The combination of our novel assays, with those already in existence, provides a tool-kit to track: total soluble mutant huntingtin protein, soluble exon 1 huntingtin protein, soluble mutant huntingtin protein (excluding the exon 1 huntingtin protein) and total soluble full-length huntingtin protein (mutant and wild type). Several novel aggregation assays were also developed that track with disease progression. These selected assays can be used to compare the levels of huntingtin protein isoforms in a wide variety of mouse models of Huntington's disease and to determine how these change in response to genetic or therapeutic manipulations.

SUBMITTER: Landles C

PROVIDER: S-EPMC7878250 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Development of novel bioassays to detect soluble and aggregated Huntingtin proteins on three technology platforms.

Landles Christian C Milton Rebecca E RE Jean Alexandre A McLarnon Stuart S McAteer Sean J SJ Taxy Bridget A BA Osborne Georgina F GF Zhang Chuangchuang C Duan Wenzhen W Howland David D Bates Gillian P GP

Brain communications 20210105 1

Huntington's disease is caused by a CAG / polyglutamine repeat expansion. Mutated CAG repeats undergo somatic instability, resulting in tracts of several hundred CAGs in the brain; and genetic modifiers of Huntington's disease have indicated that somatic instability is a major driver of age of onset and disease progression. As the CAG repeat expands, the likelihood that exon 1 does not splice to exon 2 increases, resulting in two transcripts that encode full-length huntingtin protein, as well as ...[more]

PMID: 33604571

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Project description:Huntington's disease (HD) is a monogenic neurodegenerative disorder caused by an expansion of the CAG trinucleotide repeat domain in the huntingtin (HTT) gene, leading to an expanded poly-glutamine (polyQ) stretch in the HTT protein. This mutant HTT (mHTT) protein is highly prone to intracellular aggregation, causing significant damage and cellular loss in the striatal, cortical, and other regions of the brain. Therefore, modulation of mHTT levels in these brain regions in order to reduce intracellular mHTT and aggregate levels represents a direct approach in the development of HD therapeutics. To this end, assays that can be used to detect changes in HTT levels in biological samples are invaluable tools to assess target engagement and guide dose selection in clinical trials. The Meso Scale Discovery (MSD) ELISA-based assay platform is a robust and sensitive method previously employed for the quantification of HTT. However, the currently available MSD assays for HTT are primarily detecting the monomeric soluble form of the protein, but not aggregated species. In this study, we describe the development of novel MSD assays preferentially detecting mHTT in an aggregated form. Recombinant monomeric HTT(1-97)-Q46, which forms aggregates in a time-dependent manner, was used to characterize the ability of each established assay to distinguish between HTT monomers and HTT in a higher assembly state. Further validation of these assays was performed using brain lysates from R6/2, zQ175 knock-in, and BACHD mouse models, to replicate a previously well-characterized age-dependent increase in brain aggregate signals, as well as a significant reduction of aggregate levels in the striatum following mHTT knockdown with a CAG-directed allele-specific zinc-finger repressor protein (ZFP). Lastly, size exclusion chromatography was used to separate and characterize HTT species from brain tissue lysates to demonstrate specificity of the assays for the fractions containing aggregated HTT. In summary, we demonstrate that the newly developed assays preferentially detect aggregated HTT with improved performance in comparison to previous assay technologies. These assays complement the existing MSD platform assays specific for soluble HTT monomers, allowing for a more comprehensive analysis of disease-relevant HTT species in preclinical models of HD.

Project description:BACKGROUND:Research on the development and functioning of technology platforms specifically for health applications in sub-Saharan Africa (SSA), is limited. The healthcare sector has also been resistant to platform adoption due to characteristics such as sensitive data and high cost of failure. A framework for the design, development and implementation of technology platforms in the South African health context could therefore contribute to the gap in research as well as provide a practical tool that platform owners could use to potentially increase the adoption of platforms in this context. METHODS:The research design for this study was based on the Grounded Theory Conceptual Framework Analysis process. The process focused on mapping and investigating data sources, categorising and integrating concepts, synthesising these concepts into a framework and iteratively evaluating the framework. The first stage of the evaluation process was a preliminary evaluation exploring an existing Health platform in South Africa (MomConnect). The second evaluation stage included local and international interviews with nine experts to identify any missing concepts in the framework. Stage three included a case study and case study interviews which led to the formulation of the final framework and management tool. RESULTS:The developed and evaluated framework comprised three components, namely the pre-use component, which includes considerations the platform owner should be aware of prior to using the framework. The framework comprises of two dimensions, 1) an ecosystem dimension to guide the platform owner to consider different ecosystem actors before embarking on designing a platform 2) a platform development dimension that include typical platform development components and presents an interpretation of the viewpoints included in the ecosystem levels. CONCLUSIONS:The final framework can be used by platform owners as a management tool. A unique contribution of this study is that the framework draws from two platform perspectives, namely the engineering and the economic perspectives to provide a holistic understanding of platforms. Finally, a contribution of this article is the tailoring of the framework for the South African health context.

Dataset Information

Development of novel bioassays to detect soluble and aggregated Huntingtin proteins on three technology platforms.

Publications

Development of novel bioassays to detect soluble and aggregated Huntingtin proteins on three technology platforms.

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