GABAA Receptor Signaling Induces Osmotic Swelling and Cell Cycle Activation of Neonatal Prominin+ Precursors

Abstract

Signal-regulated changes in cell size affect cell division and survival and therefore are central to tissue morphogenesis and homeostasis. In this respect, GABA receptors (GABA A Rs) are of particular interest because allowing anions flow across the cell membrane modulates the osmolyte flux and the cell volume. Therefore, we have here investigated the hypothesis that GABA may regulate neural stem cell proliferation by inducing cell size changes. We found that, besides neuroblasts, also neural precursors in the neonatal murine subependymal zone sense GABA via GABA A Rs. However, unlike in neuroblasts, where it induced depolarization-mediated [Ca 21 ] i increase, GABA A Rs activation in precursors caused hyperpolarization. This resulted in osmotic swelling and increased surface expression of epidermal growth factor receptors (EGFRs). Furthermore, activation of GABA A Rs signaling in vitro in the presence of EGF modified the expression of the cell cycle regulators, phosphatase and tensin homolog and cyclin D1, increasing the pool of cycling precursors without modifying cell cycle length. A similar effect was observed on treatment with diazepam. We also demonstrate that GABA and diazepam responsive precursors represent prominin 1 stem cells. Finally, we show that as in in vitro also in in vivo a short administration of diazepam promotes EGFR expression in prominin 1 stem cells causing activation and cell cycle entry. Thus, our data indicate that endogenous GABA is a part of a regulatory mechanism of size and cell cycle entry of neonatal stem cells. Our results also have potential implications for the therapeutic practices that involve exposure to GABA A Rs modulators during neurodevelopment. STEM CELLS 2011;29:307-319 Disclosure of potential conflicts of interest is found at the end of this article.

γ-Aminobutyric A Receptor (GABAAR) Regulates Aquaporin 4 Expression in the Subependymal Zone

“…The AQP4 knock-out (KO) mice were generated as described previously (24). The whole SEZ of the lateral ventricle was dissected and dissociated in sucrose solution (150 mM sucrose, 125 mM NaCl, 3.5 mM KCl, 1.2 mM NaH 2 PO 4 , 2.4 mM CaCl 2 , 1.3 mM MgCl 2 , 6.65 mM glucose, and 2 mM HEPES, pH 6.9, osmolarity 326 mM, all from Sigma), and cells were processed for sorting as already described (17). Briefly, after enzymatic tissue dissociation, cells were incubated with prominin-1-APC antibodies (Miltenyi Biotec) and then stained with Alexa488-tagged EGF (EGF-A; 20 ng/ml) (Molecular Probes) and 1 g/ml propidium iodide, 0.001% DNase (all from Sigma).…”

“…After embedding in 4% low melting agarose, 30-m thick coronal vibratome sections were cut and processed for immunohistochemistry. Image acquisition and analysis were done as described previously (17).…”

“…Neurosphere cultures were established from the dissociated SEZ of WT and AQP4 KO neonatal mice in NBA medium supplemented with 2% B27, 10 ng/ml FGF-2, and 20 ng/ml EGF-2 (PeproTech), as described previously (17).…”

“…As we had previously found that neural precursors display benzodiazepine-sensitive GABA A Rs, we next investigated by qRT-PCR the expression of GABA A R ␣ subunits (gabra) 1, 2, 3, and 5, which confer high affinity binding of benzodiazepines, and of gabra 4 and GABA A R␦ subunits (gabrd), which are not associated with benzodiazepine sensitivity. We selected these sub-units because our previous analyses (17) had revealed that they were all expressed at very low levels in neural precursors. Indeed, transcript levels for all genes analyzed were significantly lower within the remaining populations than in purified P Ϫ /E low neuroblasts.…”

“…However, the distribution of AQP4 in the different precursor types of the SEZ is still unknown. To investigate this issue, we used flow cytometry to measure expression levels of EGFR and prominin-1 and purify the following cell populations from the SEZ of neonatal mice: P ϩ /E low cells consisting of ependymal cells and quiescent NSCs; P ϩ /E high cells being highly enriched in activated NSCs; P Ϫ /E high cells enriched in TAPs and pre-neuroblasts; and P Ϫ /E low cells representing mostly neuroblasts (7,9,17).…”