A critical period in cortical interneuron neurogenesis in down syndrome revealed by human neural progenitor cells

Abstract

Down syndrome (DS) is a developmental disorder whose mental impairment is due to defective cortical development. Human neural progenitor cells (hNPCs) derived from fetal DS cortex initially produce normal numbers of neurons, but generate fewer neurons with time in culture, similar to the pattern of neurogenesis that occurs in DS in vivo. Microarray analysis of DS hNPCs at this critical time reveals gene changes indicative of defects in interneuron progenitor development. In addition, dysregulated expression of many genes involved in neural progenitor cell biology points to changes in the progenitor population and subsequent reduction in interneuron neurogenesis. Delineation of a critical period in interneuron development in DS provides a foundation for investigation of the basis of reduced neurogenesis in DS and defines a time when these progenitor cells may be amenable to therapeutic treatment.

Fig. 1.

Developmental time course of neuron production by hNPCs. Euploid and trisomy 21 cortical hNPCs expanded as neurospheres for less than 6 weeks or for more than 10 weeks were plated onto coverslips in media without mitogens for 1 week. The cells were fixed and immunostained for β-III-tubulin to label young neurons. a β-III-Tubulin-positive neurons were quantified in each culture. The graph shows mean ± SD of the percentage of neurons in 10–15 neurospheres in 3–4 separate neurosphere lines. ∗p = 0.02. b, c Immunostaining for β-tubulin 3 to label young neurons in euploid (b) and trisomy 21 (c) cultures.

Fig. 2.

Expanded hNPCs generate GABAergic neurons. Euploid hNPCs were propagated for more than 10 weeks in culture plated onto coverslips in media that promoted differentiation. After 10 weeks, the cells were fixed and immunostained for β-III-tubulin (green; color only in online version) and either vGAT (red) to identify GABAergic neurons or vGlut (red) to identify glutamatergic neurons. Arrows indicate GABAergic neurons.

Fig. 3.

Developmental time course of hNPC growth. Control (euploid) and trisomy 21 cortical hNPCs expanded as neurospheres for less than 6 weeks or for more than 10 weeks. The diameter of individual neurospheres in single wells of a 96-well plate was measured over a period of 2 weeks. The diameter measurements were converted to volumes to allow comparison between the trisomy 21 and euploid neurospheres. Points on graphs represent mean ± SEM of the percent increase in volume of 10–15 neurospheres in 2–5 separate neurosphere lines . Pictures show representative images of euploid (c) and trisomy 21 (d) neurospheres expanded for >10 weeks at endpoint. Bar = 100 μm.

Fig. 4.

qPCR confirmation of selected genes involved in interneuron progenitor cell development. a Fold change in expression of three chromosome 21 genes whose expression is upregulated >1.5-fold. b Fold change in expression of three DLX genes involved in interneuron development. c Fold change in expression of transcription factor genes involved in neural progenitor development. d Fold change in expression of three GABA receptor subunit genes. Bars are mean fold change ± SEM of 2–4 separate PCR runs on distinct cDNAs. Numbers in parentheses refer to fold changes as assessed by microarray analysis.