THE HUNTINGTIN CAG REPEATS AS 'FINE-TUNING KNOB' FOR PROTEIN NEURAL FUNCTION

Introduction. Huntingtin gene (HTT) encodes for a high molecular weight protein (HTT) that is essential during gastrulation and in forebrain formation. The N-terminal region of the protein has been recently associated to in vitro pro-neurulation activity. This region includes a peculiar CAG repeat stretch whose expansion results in an inherited neurodegenerative disorder named Huntington’s disease (HD). The disease itself, its penetrance and age of onset are dependent on the length of CAG stretch that encodes for a polyglutamine (polyQ) tract. Evolutionary studies showed that this polyQ tract appeared for the first time in echinoderms, then increased in length during deuterostome evolution in parallel to the emergence of progressively more complex nervous systems. Despite these indications, the functional significance of the polyQ variability in normal HTT remains to be explored. Aim. Here we investigate whether the polyQ tract in HTT is biologically active during early neurogenesis. In particular, as a tool to study in vitro neurogenesis, we adopted the rosette assay, an in vitro assay that measures the ability of ES cell-derived neural progenitors to form radial arrangements of columnar cells (named neural rosettes) that mimic neural tube formation in vivo. Additionally, we worked on designing a novel automated method for faster and unbiased quantifications of the rosette phenotype. Methods. We produced a mouse embryonic stem (mES) cell-based platform for complementation assays with HTT N-terminal portion bearing different polyQ repeats, their deletion and substitutions (0Q, 2Q, 4Q, 7Q, 15Q, 128Q, Q3Q(CAA)Q3, and Q3PQ3). The newly generated cells were exposed to a neural induction protocol to assess their rosette formation potential. Results. We report that the pro-neurulation activity, but not the antiapoptotic function, of HTT is impaired by the sole polyQ domain deletion from the N-terminal portion of HTT. Moreover, the CAG tract length affects rosette formation potential in a linear fashion. Conversely, either a pathological CAG expansion or a CAG interruption causes loss of HTT N-terminal pro-neurulation activity. Finally, to further characterize this phenotype in an unbiased and quantitative way, we developed and tested a novel automated system, based on high-content imaging approach that allowed us to pinpoint this trait with unparalleled precision and efficiency. Conclusions. Overall, these results demonstrate that HTT ability to promote rosette formation is linked to a persistent and uninterrupted CAG tract, while HTT antiapoptotic activity lays outside HTT polyQ domain.