GABAA receptor signaling induces osmotic swelling and cell cycle activation of neonatal prominin+ precursors.

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(2+)](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(+) stem cells. Finally, we show that as in in vitro also in in vivo a short administration of diazepam promotes EGFR expression in prominin(+) 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.

Expression of Tlx in both stem cells and transit amplifying progenitors regulates stem cell activation and differentiation in the neonatal lateral subependymal zone.

Niche homeostasis in the postnatal subependymal zone of the lateral ventricle (lSEZ) requires coordinated proliferation and differentiation of neural progenitor cells. The mechanisms regulating this balance are scarcely known. Recent observations indicate that the orphan nuclear receptor Tlx is an intrinsic factor essential in maintaining this balance. However, the effect of Tlx on gene expression depends on age and cell-type cues. Therefore, it is essential to establish its expression pattern at different developmental ages. Here, we show for the first time that in the neonatal lSEZ activated neural stem cells (NSCs) and especially transit-amplifying progenitors (TAPs) express Tlx and that its expression may be regulated at the posttranscriptional level. We also provide evidence that in both cell types Tlx affects gene expression in a positive and negative manner. In activated NSCs, but not in TAPs, absence of Tlx leads to overexpression of negative cell cycle regulators and impairment of proliferation. Moreover, in both cell types, the homeobox transcription factor Dlx2 is downregulated in the absence of Tlx. This is paralleled by increased expression of Olig2 in activated NSCs and glial fibrillary acidic protein in TAPs, indicating that in both populations Tlx decreases gliogenesis. Consistent with this, we found a higher proportion of cells expressing glial makers in the neonatal lSEZ of mutant mice than in the wild type counterpart. Thus, Tlx playing a dual role affects the expression of distinct genes in these two lSEZ cell types.

Analysis of stem cell lineage progression in the neonatal subventricular zone identifies EGFR+/NG2- cells as transit-amplifying precursors.

In the adult subventricular zone (SVZ), astroglial stem cells generate transit-amplifying precursors (TAPs). Both stem cells and TAPs form clones in response to epidermal growth factor (EGF). However, in vivo, in the absence of sustained EGF receptor (EGFR) activation, TAPs divide a few times before differentiating into neuroblasts. The lack of suitable markers has hampered the analysis of stem cell lineage progression and associated functional changes in the neonatal germinal epithelium. Here we purified neuroblasts and clone-forming precursors from the neonatal SVZ using expression levels of EGFR and polysialylated neural cell adhesion molecule (PSANCAM). As in the adult SVZ, most neonatal clone-forming precursors did not express the neuroglia proteoglycan 2 (NG2) but displayed characteristics of TAPs, and only a subset exhibited antigenic characteristics of astroglial stem cells. Both precursors and neuroblasts were PSANCAM(+); however, neuroblasts also expressed doublecortin and functional voltage-dependent Ca(2+) channels. Neuroblasts and precursors had distinct outwardly rectifying K(+) current densities and passive membrane properties, particularly in precursors contacting each other, because of the contribution of gap junction coupling. Confirming the hypothesis that most are TAPs, cell tracing in brain slices revealed that within 2 days the majority of EGFR(+) cells had exited the cell cycle and differentiated into a progenitor displaying intermediate antigenic and functional properties between TAPs and neuroblasts. Thus, distinct functional and antigenic properties mark stem cell lineage progression in the neonatal SVZ.

Lot1 is a key element of the pituitary adenylate cyclase-activating polypeptide (PACAP)/cyclic AMP pathway that negatively regulates neuronal precursor proliferation.

The tumor suppressor gene Lot1 is highly expressed during brain development. During cerebellar development, Lot1 is expressed by proliferating granule cells with a time course matching the expression of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor, a neuropeptide receptor that plays an important role in the regulation of granule cell proliferation/survival. Although it has become clear that Lot1 is a negative regulator of cell division in tumor cells, its role in neuronal proliferation is not understood. We previously demonstrated that in cerebellar granule cells Lot1 expression is regulated by the PACAP/cAMP system. The aim of this study was to investigate the role played by Lot1 in neuron proliferation/survival and to identify the molecular mechanisms underlying its actions. Using a Lot1-inducible expression system, we found that in PC12 cells Lot1 negatively regulates proliferation and favors differentiation by up-regulating the expression of the PACAP receptor. In cerebellar granule cells in culture, an increase in Lot1 expression was paralleled by inhibition of proliferation and up-regulation of the PACAP receptor, which in turn positively regulated Lot1 expression. Silencing of Lot1 leads to an increase in granule cell proliferation and a reduction in survival. Confirming the in vitro results, in vivo experiments showed that PACAP induced an increase in Lot1 expression that was paralleled by inhibition of cerebellar granule cell proliferation. These data show that Lot1 is a key element of the PACAP/cAMP pathway that negatively regulates neuronal precursor proliferation. The existence of a PACAP receptor/Lot1-positive feedback loop may powerfully regulate neural proliferation during critical phases of cerebellar development.

Cell cycle elongation impairs proliferation of cerebellar granule cell precursors in the Ts65Dn mouse, an animal model for Down syndrome.

Mental retardation, the hallmark of Down syndrome (DS), has been attributed to the reduced number of neurons populating the DS brain. The Ts65Dn mouse model of DS displays several anomalies analogous to those in individuals with DS, including neurogenesis impairment. The goal of the current study was to determine whether cell cycle alterations underlie neurogenesis impairment in the cerebellum of the Ts65Dn mouse and to identify the molecular mechanisms responsible for this defect. In neonatal (2-day old) Ts65Dn mice, cerebellar granule cell precursors exhibited a reduced proliferation rate (-40%) and a notable elongation (+45%) of the cell cycle. Alteration of cell cycle rate was due to elongation of the G(2) and G(1) phases. Microarray screening of cell cycle regulatory genes showed that Ts65Dn mice had a decreased expression of Cyclin B1 and Skp2, two key regulators of G(2)/M and G(1)/S transition. Results point at cell cycle elongation as major determinant of neurogenesis reduction in the cerebellum of Ts65Dn mice and suggest that this defect is specifically linked to an altered expression of two cell-cycle regulatory genes, Cyclin B1 and Skp2. These findings may establish the basis for a therapeutic approach aimed at restoring neurogenesis in the DS brain.

Cell cycle alteration and decreased cell proliferation in the hippocampal dentate gyrus and in the neocortical germinal matrix of fetuses with Down syndrome and in Ts65Dn mice.

Down syndrome (DS), the leading genetic cause of mental retardation, is characterized by reduced number of cortical neurons and brain size. The occurrence of these defects starting from early life stages points at altered developmental neurogenesis as their major determinant. The goal of our study was to obtain comparative evidence for impaired neurogenesis in the hippocampal dentate gyrus (DG) of DS fetuses and Ts65Dn mice, an animal model for DS. Cell proliferation in human fetuses was evaluated with Ki-67 (a marker of cells in S + G(2) + M phases of cell cycle) and cyclin A (a marker of cells in S phase) immunohistochemistry. We found that in the DG of DS fetuses the number of proliferating cells was notably reduced when compared with controls. A similar reduction was observed in the germinal matrix of the lateral ventricle. In both structures, DS fetuses showed a reduced ratio between cyclin A- and Ki-67-positive cells when compared with controls, indicating that they had a reduced number of cycling cells in S phase. In the DG of P2 Ts65Dn mice cell proliferation, assessed 2 h after an injection of bromodeoxyuridine (BrdU), was notably reduced, similarly to DS fetuses. After 28 days, Ts65Dn mice had still less BrdU-positive cells than controls. Phenotypic analysis of the surviving cells showed that Ts65Dn mice had a percent number of cells with astrocytic phenotype larger than controls. Using phospho-histone H3 immunohistochemistry we found that both DS fetuses and P2 Ts65Dn mice had a higher number of proliferating cells in G(2) and a smaller number of cells in M phase of cell cycle. Results provide novel evidence for proliferation impairment in the hippocampal DG of the DS fetal brain, comparable to that of the P2 mouse model, and suggest that cell cycle alterations may be critical determinants of the reduced proliferation potency.

Choline acetyltransferase activity at different ages in brain of Ts65Dn mice, an animal model for Down's syndrome and related neurodegenerative diseases.

Ts65Dn mice, trisomic for a portion of chromosome 16 segmentally homologous to human chromosome 21, are an animal model for Down's syndrome and related neurodegenerative diseases, such as dementia of the Alzheimer type. In these mice, cognitive deficits and alterations in number of basal forebrain cholinergic neurons have been described. We have measured in Ts65Dn mice the catalytic activity of the cholinergic marker, choline acetyltransferase (ChAT), as well as the activity of the acetylcholine-degrading enzyme acetylcholinesterase (AChE), in the hippocampus and in cortical targets of basal forebrain cholinergic neurons. In mice aged 10 months, ChAT activity was significantly higher in Ts65Dn mice, compared to 2N animals, in the hippocampus, olfactory bulb, olfactory cortex, pre-frontal cortex, but not in other neocortical regions. At 19 months of age, on the other hand, no differences in ChAT activity were found. Thus, alterations of ChAT activity in these forebrain areas seem to recapitulate those recently described in patients scored as cases of mild cognitive impairment or mild Alzheimer's disease. Other neurochemical markers putatively associated with the disease progression, such as those implicating astrocytic hyperactivity and overproduction of amyloid precursor protein family, were preferentially found altered in some brain regions at the oldest age examined (19 months).

Cyclic AMP-mediated regulation of transcription factor Lot1 expression in cerebellar granule cells.

Lot1, a zinc finger transcription factor acting as a tumor suppressor gene on tumoral cells, is highly expressed during brain development. In developing rat cerebellum, Lot1 expression is high in cerebellar granule cells (CGC), a neuronal population undergoing postnatal neurogenesis. The time course of Lot1 cerebellar expression closely matches the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors coupled to adenylyl cyclase. The aim of this study was to ascertain whether Lot1 expression is regulated by cAMP-dependent pathways and to identify mechanisms of Lot1 activation in CGC cultures. Our results show that Lot1 expression in CGC is cAMP-dependent, as treatments with either forskolin or PACAP-38 induced an increase in its expression at both the mRNA and protein levels. This effect on Lot1 expression was mimicked by dibutyryl cAMP and suppressed by protein kinase A and MEK inhibitors. In parallel, we found that treatments with forskolin and PACAP-38 in precursor CGC inhibited bromodeoxyuridine incorporation by 25 and 35%, respectively, indicating a negative effect on neuronal precursor proliferation. Luciferase reporter analysis and mutagenesis of the Lot1 promoter region indicated a crucial role of the AP1-binding site (located at -268 bp) in cAMP-induced Lot1 transcription. In addition, cotransfection experiments indicated that the c-Fos/c-Jun heterodimer is responsible for cAMP-dependent Lot1 transcriptional activation. In conclusion, our data demonstrate that, in CGC, Lot1 is under the transcriptional control of cAMP through an AP1 site regulated by the c-Fos/c-Jun heterodimer and suggest that this gene may be an important element of the cAMP-mediated pathway that regulates neuronal proliferation through the protein kinase A-MEK signaling cascade.