TY - JOUR
T1 - Meso scale discovery-based assays for the detection of aggregated huntingtin
AU - Reindl, Wolfgang
AU - Baldo, Barbara
AU - Schulz, Jana
AU - Janack, Isabell
AU - Lindner, Ilka
AU - Kleinschmidt, Markus
AU - Sedaghat, Yalda
AU - Thiede, Christina
AU - Tillack, Karsten
AU - Schmidt, Christina
AU - Cardaun, Isabell
AU - Schwagarus, Tom
AU - Herrmann, Frank
AU - Hotze, Madlen
AU - Osborne, Georgina F.
AU - Herrmann, Simone
AU - Weiss, Andreas
AU - Zerbinatti, Celina
AU - Bates, Gillian P.
AU - Bard, Jonathan
AU - Munoz-Sanjuan, Ignacio
AU - Macdonald, Douglas
N1 - Funding Information:
CHDI Foundation is a privately-funded not-for-profit biomedical research organization exclusively dedicated to discovering and developing therapeutics that slow the progression of Huntington’s Disease. CHDI Foundation conducts research in a number of different ways; for the purposes of this manuscript, research was conducted at the contract research organization Evotec AG under fee-for-service agreements. The listed authors all contributed to the conception, planning, and direction of the research, including generation, analysis, and interpretation of the data. The authors would like to acknowledge the late Paul Patterson (California Institute of Technology) for access to the MW1 and MW8 antibodies. We are also grateful to Sabine Schaertl, Manuela Heßmann, and Koen Temmerman (Evotec AG) for their input into the project. We thank Chantal Bazenet (Evotec AG) for critically reviewing the manuscript.
Publisher Copyright:
© 2019 Reindl et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2019/3
Y1 - 2019/3
N2 - 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.
AB - 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.
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U2 - 10.1371/journal.pone.0213521
DO - 10.1371/journal.pone.0213521
M3 - Article
C2 - 30913220
AN - SCOPUS:85063483968
SN - 1932-6203
VL - 14
JO - PLoS One
JF - PLoS One
IS - 3
M1 - e0213521
ER -