Reduced cell size, chromosomal aberration and altered proliferation rates are characteristics and confounding factors in the STHdh cell model of Huntington disease

Abstract

Huntington disease is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the gene encoding the huntingtin protein. Expression of the mutant protein disrupts various intracellular pathways and impairs overall cell function. In particular striatal neurons seem to be most vulnerable to mutant huntingtin-related changes. A well-known and commonly used model to study molecular aspects of Huntington disease are the striatum-derived STHdh cell lines generated from wild type and huntingtin knock-in mouse embryos. However, obvious morphological differences between wild type and mutant cell lines exist, which have rarely been described and might not have always been considered when designing experiments or interpreting results. Here, we demonstrate that STHdh cell lines display differences in cell size, proliferation rate and chromosomal content. While the chromosomal divergence is considered to be a result of the cells’ tumour characteristics, differences in size and proliferation, however, were confirmed in a second non-immortalized Huntington disease cell model. Importantly, our results further suggest that the reported phenotypes can confound other study outcomes and lead to false conclusions. Thus, careful experimental design and data analysis are advised when using these cell models.

A Patient-Derived Cellular Model for Huntington’s Disease Reveals Phenotypes at Clinically Relevant CAG Lengths

“…Quantification of cell surface area shows that HD cells are smaller than control cells ( Figure 2C). Therefore, consistent with numerous independent previous reports, huntingtin may be involved in cytoskeletal regulation 15,[70][71][72] .…”

“…To confirm karyotypic stability in TruHD cells after 25+ passages, we compared the karyotypes of ST Hdh cells and TruHD cells. Large chromosomal abnormalities were detected in transformed HD mouse striatal derived cells (ST Hdh Q111/Q111 ) cells (Supplemental Figure 1A, Table 2), consistent with a recently published study 15 . No chromosomal changes were recorded for control TruHD-Q21Q18F, and minor chromosomal changes were recorded in TruHD-Q43Q17M and TruHD-Q50Q40F cell lines ( Figure 1C, Table 2).…”

“…Consistent with previous reports, we found that HD cells could be distinguished from wild type based on morphology 7,15 , size 15 , growth rate 20,41,42 , sensitivity to stress 9,[43][44][45] , aberrant ADP/ATP ratios 7,46,47 , hypophosphorylated huntingtin protein [48][49][50] and altered ROS-dependent localization to nuclear speckles 51 . We have therefore generated and characterized a panel of clinically relevant cellular models for investigating disease mechanisms in HD.…”

“…Mutant ST Hdh cells exhibit altered morphology 7,15,44 . To probe whether TruHD cells could be distinguished by their morphology, nuclei were stained with Hoechst and immunofluorescence was performed with antibodies against phosphorylated huntingtin at serines 13 and 16 (N17-phospho) and beta-tubulin ( Figure 2A Figure 2A, where beta-tubulin alone showed the greatest separation in the clusters compared to Hoechst and N17-phospho.…”

“…The functional implications of this expanded, mutant huntingtin are not fully understood. Much of the existing research on HD cell biology in relevant neuronal cell types has been limited to primary post-mitotic neurons from murine brain tissue or transformed cell lines, which have several limitations, including the use of synthetically long CAG lengths in order to mimic human disease in mice [2][3][4][5][6][7][8][9][10][11][12][13][14][15] . These alleles actually genetically model juvenile or Westphal variant HD, which are not age-onset diseases.…”