Longitudinal Biochemical Assay Analysis of Mutant Huntingtin Exon 1 Protein in R6/2 Mice

Abstract

Background: Biochemical analysis of mutant huntingtin (mHTT) aggregation species in HD mice is a common measure to track disease. A longitudinal and systematic study of how tissue processing affects detection of conformers has not yet been reported. Understanding the homeostatic flux of mHTT over time and under different processing conditions would aid in interpretation of pre-clinical assessments of disease interventions. Objective: Provide a systematic evaluation of tissue lysis methods and molecular and biochemical assays in parallel with behavioral readouts in R6/2 mice to establish a baseline for HTT exon1 protein accumulation. Methods: Established biochemical methods were used to process tissue from R6/2 mice of specific ages following behavior tasks. Aggregation states and accumulation of mHTT exon 1 protein were evaluated using multiple break and assay methods to determine potential conformational flux assay specificity in detection of mHTT species, and tissue specificity of conformers. Results: Detection of mHTT exon 1 protein species varied based on biochemical processing and analysis providing a baseline for subsequent studies in R6/2 mice. Insoluble, high molecular weight species of mHTT exon 1 protein increased and tracked with onset of behavioral impairments in R6/2 mice using multiple assay methods. Conclusions: Conformational flux from soluble monomer to high molecular weight, insoluble species of mHTT exon 1 protein was generally consistent for multiple assay methods throughout R6/2 disease progression; however, the results support the use of multiple biochemical techniques to detect mHTT exon 1 protein species for preclinical assessments in HD mouse models expressing mHTT exon 1 protein.

Pharmacological characterization of mutant huntingtin aggregate-directed PET imaging tracer candidates

“…These results are consistent with the hypothesis that [ 3 H]CHDI-180 specifically bound mHTT, consistent with aggregates (see next section) expressed in STR, CTX and HPC from HD mice brain sections that accumulate (i.e., increase in quantity and size) in an age- and disease-progressive fashion 47 , 49 , 50 .…”

“…S5 B), which correlates well with the increase observed by IHC (mEM48 immunoreactivity) as well as ARG and RAFTA binding signals with [ 3 H]CHDI-180 (Fig. 5 ); in contrast, the soluble expanded HTT levels actually decreased over this same age series, consistent with the hypothesis that the dynamics between the soluble and aggregated expanded HTT pools are likely shifting toward the aggregation of expanded HTT as the animal ages and the disease progresses 47 – 50 . Thus, our results are consistent with CHDI-180 recognizing and binding to the higher-order aggregated forms of expanded mHTT but not to the less fibrillar, more soluble expanded HTT states.…”

“…As previously demonstrated 47 – 50 for HD mouse models and expanded upon in this report, we observed an age- (or disease-) dependent increase in brain (hippocampus, cortex and striatum) aggregate load (density and size) in progressively increasing ages of HOM zQ175 and R6/2 mice using immunochemistry with mEM48 antibody (Fig. 5 B, C ); no significant immunoreactive signal was detected at any age from age-matched wild-type controls, as these mice do not express mHTT nor HTT aggregates (Supplementary Fig.…”

“…Aggregates of mHTT accumulate in HD mouse models such as R6/2 and zQ175 (i.e. increase in quantity and size) in an age-, disease-, and region- progressive fashion 47 – 50 and hence the specific binding signal of a small molecule targeting mHTT aggregates is expected to increase with age in brains of these animals. Therefore, homogenates or slices from brains of 3-, 6-, 9- and 12-month-old HOM zQ175 and age-matched WT mice were prepared and subjected to RAFTA or ARG analysis using [ 3 H]CHDI-180; HOM zQ175 mice were used due to increased sensitivity and dynamic range compared to HET, likely reflecting the greater target load of the former versus the latter model.…”

IKKβ slows Huntington’s disease progression in R6/1 mice

“…All behavioral assessments were performed as previously described (54, 60). Specifically, body weights were measured weekly at 8 to 15 wk of age.…”