Isolation of a novel rat neural progenitor clone that expresses dlx family transcription factors and gives rise to functional gabaergic neurons in culture

Abstract

GABAergic interneurons are lost in conditions including epilepsy and CNS injury, but there are few culture models available to study their function. Towards the goal of obtaining renewable sources of GABAergic neurons, we used the molecular profile of a functionally-incomplete GABAergic precursor clone to screen 17 new clones isolated from GFP+ rat E14.5 cortex and ganglionic eminence (GE) that were generated by viral introduction of v-myc. The clones grow as neurospheres in medium with FGF2, and after withdrawal of FGF2 they exhibit varying patterns of differentiation. Transcriptional profiling and qPCR indicated that one clone (GE6) expresses high levels of mRNAs encoding Dlx1, 2, 5 and 6, glutamate decarboxylases, and presynaptic proteins including neuropeptide Y and somatostatin. Protein expression confirmed that GE6 is a progenitor with restricted differentiation giving rise mostly to neurons with GABAergic markers. In co-cultures with hippocampal neurons, GE6 neurons became electrically excitable and received both inhibitory and excitatory synapses. After withdrawal of FGF2 in cultures of GE6 alone, neurons matured to express βIII-tubulin, and staining for synaptophysin and vesicular GABA transporter (VGAT) were robust after 1-2 weeks of differentiation. GE6 neurons also became electrically excitable and displayed synaptic activity, but synaptic currents were carried by chloride and were blocked by bicuculline. The results suggest that the GE6 clone, which is ventrally derived from the GE, resembles GABAergic interneuron progenitors that migrate into the developing forebrain. This is the first report of a relatively stable fetal clone that can be differentiated into GABAergic interneurons with functional synapses.

Glutamate dehydrogenase 1 and SIRT4 regulate glial development

“…We cultured the three cell types, attempting to keep them as homogenous as culturing methods would permit. The CTX8 radial glial cell line, which possesses the capability to differentiate into either glia or neurons, can be kept in an “undifferentiated” radial glial fate (Li et al, 2012). Cultures of E18 cortical neurons treated with AraC represented pure neurons.…”

“…These GFP-positive cells were isolated from E14.5 cortices of rats expressing GFP under control of the β-actin promoter and were selected for their radial glial characteristics, which can be maintained in medium containing basic fibroblast growth factor (Li et al, 2012). CTX8 cells can also differentiate into both neurons and astroglia.…”

“…CTX8 cells can differentiate into both Tuj1+ and GFAP+ cells (Li et al, 2012). As such, we further characterized CTX8 cells before using them to alter GDH1 or SIRT4 levels.…”

“…Here we show that GDH1 and SIRT4 are localized to mitochondria within the brain, expressed at high levels in astrocytes in the postnatal brain and radial glia in embryonic tissues, and expression decreases during development. Using cultures of CTX8 cells, a novel radial glial cell line (Li et al, 2012), we further show that factors known to allosterically regulate GDH1 alter gliogenesis and that SIRT4 and GDH1 overexpression play antagonistic roles in regulating gliogenesis. Importantly a mutant variant of GDH1 found in HI/HA patients promotes glial development of cultured CTX8 radial glia cells.…”

An interneuron progenitor maintains neurogenic potential in vivo and differentiates into GABAergic interneurons after transplantation in the postnatal rat brain

“…Our previous report has demonstrated that GE6 cells proliferate rapidly in culture in the presence of FGF2 and differentiate into primarily neurons with little astroglia upon FGF2 withdrawal 30 . In the current study, we aim to determine if this distinct neurogenic potential of GE6 still holds after transplantation into the postnatal brain.…”