A007 Human huntingtin transcripts accumulate in the nucleus in the form of RNA clusters

<h3>Background</h3> YAC128 mice are transgenic for a yeast artificial chromosome carrying human HTT with an expanded CAG repeat. Our previous results indicated that human HTT undergoes alternative processing in the brains of YAC128 mice to produce HTT1a and that both full-length (FL)-HTT and HTT1a are retained in cell nuclei in the form of RNA clusters. <h3>Aim</h3> To understand the molecular characteristics of these clusters and assess whether they can be observed in human induced pluripotent stem cells (iPSC) and medium spiny neurons (MSNs) differentiated from these iPSCs as well as in human brain samples. <h3>Methods</h3> We used RNAscope combined with immunofluorescent detection of proteins of interest to interrogate mouse and human cell- and tissue-based samples. <h3>Results</h3> Microscopic analysis revealed that, similar to YAC128 animals, BAC-CAG transgenic mice, show accumulation of FL-HTT and HTT1a mRNAs in cell nuclei. In contrast, RNA clusters were not observed in zQ175 animals, where instead FL-Htt and Htt1a mRNAs were identified as single, cytoplasmic molecules. Interestingly, these clusters were enriched in neurons and formed as early as embryonic stage 14.5. RNAscope experiments using iPSCs and MSNs detected HTT1a in both cell types but the location of FL-HTT and HTT1a was strikingly different; preliminary data on post-mortem primary motor cortex from an HD patient with 63 CAGs revealed the presence of HTT1a in this cortical region. <h3>Conclusions</h3> Our data identify huntingtin RNA nuclear clusters as a novel molecular signature of Huntington’s disease. Simultaneously, we establish that HTT1a is generated in human cells and brain samples.