bioRxiv2026
Huntingtons disease (HD) is a repeat-associated neurodegenerative disorder traditionally characterized by toxic protein pathology resulting from expanded CAG repeats in the huntingtin (HTT) gene. In recent years, however, studies have identified repeat expansion-driven RNA pathology as an additional and potentially independent contributor to disease. In particular, mutant HTT transcripts containing expanded CAG repeats accumulate in the nucleus and form discrete RNA clusters, a feature shared with several other repeat-associated disorders. While protein aggregation and its downstream consequences have been extensively studied, our current understanding of the composition, organization, and dynamics of these nuclear mRNA clusters remains limited. Progress in this area has been constrained in part by the lack of robust methods to detect and quantify expanded HTT transcripts at single-molecule resolution within intact tissue. As a result, the contribution of RNA clustering to disease mechanisms, its relationship to repeat length, and its interaction with other pathological features of HD remain poorly defined. Here we present a high-throughput RNAscope pipeline that combines automated confocal imaging with rigorous microscope characterization to quantify both single mRNA molecules and multi-transcript clusters in fixed mouse brain tissue. Using 3D Gaussian point-spread function (PSF) fitting calibrated on 200 nm fluorescent beads and pointilistic image features from tissue data, we establish per-slide intensity thresholds from negative controls and normalize experimental signals to single-molecule reference intensities. The critical validation of our approach operates at two scales: for single molecules, the linear relationship between spot size and intensity (r2 > 0.90) reflects variable probe binding along transcripts; for clusters, the linear scaling between cluster volume and mRNA content (R2 > 0.98) confirms uniform probe accessibility and enables quantitative conversion of fluorescence intensity to absolute mRNA counts. Applied to HttQ111+/- knock-in mice across multiple ages, we analyzed thousands of fields of view (FOVs), detecting >900,000 single mRNA molecules and segmenting >1.9 million mRNA clusters using two probes targeting mouse huntingtin (Htt): one detecting the spliced transcript that uses early cryptic polyadenylation sites in intron 1 (HTT1a), and one detecting full-length Htt (fl-HTT). Our analysis reveals considerable heterogeneity in mRNA accumulation: 16-63% of Q111 FOVs are classified as "extreme" (exceeding the 95th percentile of wildtype clustered mRNA levels), with striatum showing higher prevalence than cortex for both probes (HTT1a: 63% striatum, 31% cortex; fl-HTT: 44% striatum, 16%cortex). Extreme FOVs are characterized by elevated cluster numbers (2-6x more clusters per nucleus) and higher cluster density (1.3-1.7x more mRNA per {micro}m3). Cluster localization shows nuclear bias ([~]68%) in normal FOVs, but extreme FOVs exhibit a shift toward cytoplasmic localization, particularly for fl-HTT (48% nuclear vs 68% in normal FOVs), though the interpretation of this shift requires further investigation. Despite the large dataset at the cellular level, our study included only 11 mice (9 Q111, 2 wildtype), and this limited sample size precluded robust statistical inference at the animal level. Nevertheless, these quantitative metrics provide a framework for investigating disease mechanisms and evaluating therapeutic interventions using RNAscope in future studies with larger cohorts.