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

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.


Western blot analysis of HTT proteins
3 µg each of MBP-tagged or thrombin digested HTT(1-97)-Q16 and Q46 were mixed with 4x Laemmli loading buffer and incubated for 5 min at 99°C. Electrophoresis was run for 24 min at 100 mA in pre-cast NuPAGE Novex 4-12% bis-tris protein gels (ThermoFisher Scientific) with MOPS running buffer (50 mM MOPS, 50 mM Tris, 0.1% SDS, 1 mM EDTA, pH 7.7). Subsequently, proteins were transferred to a PVDF membrane for 10 min at 20 V using the iBlot 2 Dry Blotting System (ThermoFisher Scientific). Membranes were probed with antibodies using the iBind system (ThermoFisher Scientific). As primary antibody EM48 (1:200) was used. The mouse monoclonal antibody EM48 (MAB5374), raised against a GST fusion protein of the first 256 amino acids from human HTT with an in-frame deletion of the poly-glutamine and poly-proline stretches [1], was obtained from Millipore. The secondary antibody was an alkaline phosphatase (AP)-conjugated antimouse IgG antibody (1:1,500; Promega). For visualization of protein bands, the membrane was treated with NBT/BCIP solution (Sigma).

Immunoprecipitation of HTT proteins
The MW8 antibody was diluted in PBS-T to obtain 2 µg antibody in 200 µL final volume.
30 µL of Dynabeads per sample were transferred to a 1.5 mL Eppendorf tube, placed on a magnet and the supernatant was removed. The beads were then washed 3x with 500 containing 5% nonfat dried milk for 1 h at room temperature and was subsequently incubated for 2 h with primary anti-HTT antibody MW8 (Developmental Studies Hybridoma Bank) diluted 1:1,000 in TBS-T containing 5% nonfat dried milk. The membrane was incubated with a second HRP-conjugated antibody for 1 h, processed for visualization using an enhanced chemiluminescence system (GE Healthcare), and exposed to medical X-ray film (Hyperfilm ECL, GE Healthcare) to obtain fluorographic images. Quantification was performed by densitometry with ImageJ.

ZFP expression in HD mouse models Animal studies
Live zQ175 C57B/L6J knock-in mice and wildtype littermates were obtained from the Jackson Laboratory (Bar Harbor, USA). Animals were housed in Eurostandard Type II long cages and given access to food and water ad libitum. Environmental conditions were maintained at a temperature of 21 ± 1°C, humidity of 55 ± 1 0% and a 12:12 h light:dark cycle, with lights on at 7 am and off at 7 pm. Animals were checked daily for health status during housing and study duration. All animal work was carried out in accordance with the regulations of the German animal welfare act and the EU legislation (EU directive 2010/63/EU). The study protocol was approved by the local Ethics committee of the Authority for Health and Consumer Protection of the city and state of Hamburg ("Behörde 6 für Gesundheit und Verbraucherschutz" BGV, Hamburg) under the file number #V11307/591 00.33.

AAV vector construction and production
For expression of ZFPs, plasmids were modified from the AAV vector pAAV-6P-SWB [3].
ZFP30640 (FLAG-tagged) was cloned after the human synapsin 1 promoter (phSyn1) to generate pAAV-6P-SWB-ZFP-30640. In addition, an inactive ZFP control construct was generated by deleting the ZFP DNA binding domain from ZFP33074 (pAAV-6P-SWB-ZFP-ΔDBD). Pseudotyped recombinant (r)AAV2/1+2 particles were produced and purified as previously described [4,5]. In brief, HEK293 cells were co-transfected with AAV vector carrying the transcription units of interest and plasmids containing rep and cap genes (pDP1rs and pDP2rs, Plasmid Factory) in equimolar ratios by polyethylenimine-mediated plasmid transfection. Cells were lysed 48 hours after transfection by three freeze-thaw cycles, and cellular debris were removed by centrifugation. The supernatant containing the viral particles was treated with benzonase, and subjected to iodixanol density centrifugation (S6, S7) at 60,000 rpm. Iodixanol was removed and viral particles were concentrated in PBS 300 MK (300 mM NaCl, 1 mM MgCl2, 2.5 mM KCl) by filter centrifugation. The remaining rAAV solution was filtered through a Millex GV 0.22 µm pore size. Sterile rAAV particles were stored at 4°C and diluted 1:1 with sterile PB buffer to obtain PBS MK (150 mM NaCl, 0.5 mM MgCl2, 1.25 mM KCl) prior to in vivo application.
AAV titers were determined using qPCR.

Histology and immunohistochemistry
Mice were euthanized 4 months post-injection at 6 months of age. For immunohistochemistry, mice were sacrificed by transcardial perfusion of anesthetized animals with 30 mL of ice-cold PBS and 50 mL of 4% paraformaldehyde using a peristaltic pump. Brain samples were removed from the skull and processed for immunohistochemistry as previously described [5].

Image acquisition and automated image analysis
Image acquisition and analysis were performed as previously described [5]. In brief, automated image acquisition was conducted using the Opera High Content Screening system and Opera software 2.0.1 (PerkinElmer Inc.) with a 40x water immersion objective (Olympus, NA 1.15, pixel size: 0.32 µm). Image analysis scripts for characterization and quantification of mHTT inclusions were developed using Acapella Studio 3.1 (PerkinElmer Inc.) and the integrated Acapella batch analysis as part of the Columbus system.