Lack of huntingtin promotes neural stem cells differentiation into glial cells while neurons expressing huntingtin with expanded polyglutamine tracts undergo cell death

Huntington's disease (HD) is a neurodegenerative disorder that affects muscle coordination and diminishes cognitive abilities. The genetic basis of the disease is an expansion of CAG repeats in the Huntingtin (Htt) gene. Here we aimed to generate a series of mouse neural stem (NS) cell lines that carried varying numbers of CAG repeats in the mouse Htt gene (Hdh CAG knock-in NS cells) or that had Hdh null alleles (Hdh knock-out NS cells). Towards this end, Hdh CAG knock-in mouse ES cell lines that carried an Htt gene with 20, 50, 111, or 140 CAG repeats or that were Htt null were neuralized and converted into self-renewing NS cells. The resulting NS cell lines were immunopositive for the neural stem cell markers NESTIN, SOX2, and BLBP and had similar proliferative rates and cell cycle distributions. After 14 days in vitro, wild-type NS cells gave rise to cultures composed of 70% MAP2+ neurons and 30% GFAP+ astrocytes. In contrast, NS cells with expanded CAG repeats underwent neuronal cell death, with only 38% ± 15% of the MAP2+ cells remaining at the end of the differentiation period. Cell death was verified by increased caspase 3/7 activity on day 14 of the neuronal differentiation protocol. Interestingly, Hdh knock-out NS cells treated using the same neuronal differentiation protocol showed a dramatic increase in the number of GFAP+ cells on day 14 (61% ± 20% versus 24% ± 10% in controls), and a massive decrease of MAP2+ neurons (30% ± 11% versus 64% ± 17% in controls). Both Hdh CAG knock-in NS cells and Hdh knock-out NS cells showed reduced levels of Bdnf mRNA during neuronal differentiation, in agreement with data obtained previously in HD mouse models and in post-mortem brain samples from HD patients. We concluded that Hdh CAG knock-in and Hdh knock-out NS cells have potential as tools for investigating the roles of normal and mutant HTT in differentiated neurons and glial cells of the brain.

► Hdh Knock-in NS cells generated from corresponding ES cells are a population of neurogenic radial glia-like stem cells.
► Mutant Hdh Knock-in NS cells exhibit cell death during neuronal differentiation.
► Absence of correlation between CAG repeat length and disease phenotypes.
► Normal Hdh directs NS cells toward neuronal fate while its absence causes glial differentiation. differentiation in vitro.