Research Article: Meso scale discovery-based assays for the detection of aggregated huntingtin

Date Published: March 26, 2019

Publisher: Public Library of Science

Author(s): Wolfgang Reindl, Barbara Baldo, Jana Schulz, Isabell Janack, Ilka Lindner, Markus Kleinschmidt, Yalda Sedaghat, Christina Thiede, Karsten Tillack, Christina Schmidt, Isabell Cardaun, Tom Schwagarus, Frank Herrmann, Madlen Hotze, Georgina F. Osborne, Simone Herrmann, Andreas Weiss, Celina Zerbinatti, Gillian P. Bates, Jonathan Bard, Ignacio Munoz-Sanjuan, Douglas Macdonald, Xiao-Jiang Li.

http://doi.org/10.1371/journal.pone.0213521

Abstract

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.

Partial Text

Huntington’s disease (HD) is a neurodegenerative genetic disorder that leads to motor dysfunction and cognitive decline [1]. The neuropathology is most prominently characterized by neuronal loss and atrophy of the caudate nucleus and putamen regions of the striatum [2, 3]. HD is caused by the abnormal expansion of CAG trinucleotide repeats within exon 1 of the huntingtin (HTT) gene, which leads to the expansion of the poly-glutamine (polyQ) stretch located in the N-terminus of the HTT protein [4].

The evaluation of current HTT-lowering approaches for treatment of HD requires the availability of assays that are capable of measuring HTT levels in relevant HD model or patient samples. Multiple assay technologies are available to measure different monomeric HTT species [16–18, 20]. For example, MSD assays have been described for detection of mouse HTT, human HTT or mutant HTT [19]. Besides the presence of the pathogenic monomeric mHTT protein, aggregation of HTT protein is another hallmark of HD. However, only very few assays have been described that specifically target HTT aggregates [22, 23]. To complement the available set of MSD assays for detection of different HTT species, we developed two assays that preferentially detect aggregated HTT. The described MW8 / 4C9-ST and MW8 / MW8-ST MSD assays were evaluated using both recombinant HTT proteins and different tissue samples from HD mouse models. Each of the performed tests showed a distinct detection pattern in comparison to MSD monomer assays and confirmed that the two novel assays are preferentially detecting HTT in an aggregated form. Both assays show similar overall performance, with the MW8 / 4C9-ST assay being slightly more sensitive. When set against existing HTT aggregate assays, our assays showed comparable results with equal or improved assay performance. The novel MSD assays are clearly more sensitive in the detection of HTT aggregates in comparison to existing TR-FRET assays with more than 1000-fold lower LLODs. The Seprion ELISA is carried out in 96-well format. The described MSD assays are run in 384-well format and therefore allow for a higher throughput. Seprion ligands have been described to also detect non-HTT aggregates like prions and amyloid-β [42, 43]. Our data suggest that the two novel MSD assays described here do not detect other protein oligomeric or aggregated species, such as Aβ fibrils (S5 Fig) for which no signals were detected. Lastly, for the analysis of musculus tibialis anterior samples the MSD assays showed improved sensitivities (Fig 8C).

 

Source:

http://doi.org/10.1371/journal.pone.0213521

 

Leave a Reply

Your email address will not be published.