Dominant-Negative Effects of Adult-Onset Huntingtin Mutations Alter the Division of Human Embryonic Stem Cells-Derived Neural Cells.

Carla Lopes,Sophie Aubert,Fany Bourgois-Rocha,Nicole Déglon,Sandrine Humbert,Anselme L Perrier,Monia Barnat,Ana Cristina Rego,David Blum

doi:10.1371/journal.pone.0148680

Abstract

Mutations of the huntingtin protein (HTT) gene underlie both adult-onset and juvenile forms of Huntington’s disease (HD). HTT modulates mitotic spindle orientation and cell fate in mouse cortical progenitors from the ventricular zone. Using human embryonic stem cells (hESC) characterized as carrying mutations associated with adult-onset disease during pre-implantation genetic diagnosis, we investigated the influence of human HTT and of an adult-onset HD mutation on mitotic spindle orientation in human neural stem cells (NSCs) derived from hESCs. The RNAi-mediated silencing of both HTT alleles in neural stem cells derived from hESCs disrupted spindle orientation and led to the mislocalization of dynein, the p150Glued subunit of dynactin and the large nuclear mitotic apparatus (NuMA) protein. We also investigated the effect of the adult-onset HD mutation on the role of HTT during spindle orientation in NSCs derived from HD-hESCs. By combining SNP-targeting allele-specific silencing and gain-of-function approaches, we showed that a 46-glutamine expansion in human HTT was sufficient for a dominant-negative effect on spindle orientation and changes in the distribution within the spindle pole and the cell cortex of dynein, p150Glued and NuMA in neural cells. Thus, neural derivatives of disease-specific human pluripotent stem cells constitute a relevant biological resource for exploring the impact of adult-onset HD mutations of the HTT gene on the division of neural progenitors, with potential applications in HD drug discovery targeting HTT-dynein-p150Glued complex interactions.

Highlights

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by abnormal extension of a tract of CAG repeats in the first exon of the HTT gene [1]
Human HTT regulates spindle orientation in dividing neural stem cells derived from human embryonic stem cells
We used neural stem cells (NSCs) differentiated from control human embryonic stem cells and plasmids encoding a short hairpin RNA directed against HTT or a control shRNA to study the effect of HTT knockdown (Fig 1A) [19]

Summary

Introduction

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by abnormal extension of a tract of CAG repeats in the first exon of the HTT gene [1]. Most forms of HD patients have an onset during adulthood For such forms, the longest CAG expansion of the two HTT alleles contains 41 to 48 repeats, with a mean of 44 CAG repeats [3], [4], [5]. Onset of the symptoms, high severity and rapid disease progression are associated with the presence of larger numbers of CAG repeats [6]. Fewer than 10% of patients develop symptoms before the age of 20 years; this juvenile form of the disease is characterized by a more widespread and rapidly progressing pattern of brain degeneration associated with a larger number (> 60) of CAG repeats than for adult-onset HD [7]

Methods

Results

Conclusion