Francesca Ciccolini

Activity requires soluble amyloid precursor protein α to promote neurite outgrowth in neural stem cell‐derived neurons via activation of the MAPK pathway

It is known that activity modulates neuronal differentiation in the adult brain but the signalling mechanisms underlying this process remain to be identified. We show here that activity requires soluble amyloid precursor protein (sAPP) to enhance neurite outgrowth of young neurons differentiating from neural stem cells. Inhibition of sAPP secretion and anti‐APP antibodies both abolished the effect of depolarization on neurite outgrowth, whereas exogenous sAPPα, similar to depolarization, induced neurite elongation. Depolarization and sAPPα both required active N‐methyl‐d‐aspartic acid receptor (NMDAR) and mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK) recruitment to induce neurite outgrowth. However, depolarization and sAPPα played different roles in modulating this signalling cascade. Depolarization induced ERK phosphorylation with fast kinetics via activation of NMDAR. By contrast, acute application of sAPPα did not lead to ERK activation. However, continuous generation of sAPPα was necessary for depolarization‐induced ERK phosphorylation, indicating that sAPPα promotes MAPK/ERK recruitment by an indirect mechanism. In addition, we found that blockade of NMDAR down‐regulated APP expression, whereas depolarization increased sAPPα, suggesting that activity may also act upstream of sAPP signalling by regulating the amount of cellular APP and extracellular sAPPα. Finally, we show that soluble amyloid precursor‐like protein 2 (sAPLP2), but not sAPLP1, is functionally redundant to sAPP in promoting neurite outgrowth and that soluble members of the APP family require membrane‐bound APP to enhance neurite outgrowth. In summary, these experiments indicate a novel role of APP family members in activity‐dependent neuronal differentiation.

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 Ca2+ 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. STEM CELLS 2009;27:1443–1454

Interaction between DLX2 and EGFR regulates proliferation and neurogenesis of SVZ precursors.

In the postnatal subventricular zone (SVZ) neural stem cells (NSCs) give rise to transit-amplifying precursors (TAPs) expressing high levels of epidermal growth factor receptor (EGFR) that in turn generate neuroblasts. Both TAPs and neuroblasts express distal-less (DLX)2 homeobox transcription factor but the latter proliferate less. Modulation of its expression in vivo has revealed that DLX2 affects both neurogenesis and proliferation in the postnatal SVZ. However, the mechanisms underlying these effects are not clear. To investigate this issue we have here forced the expression of DLX2 in SVZ isolated NSCs growing in defined in vitro conditions. This analysis revealed that DLX2 affects the proliferation of SVZ precursors by regulating two distinct steps of neural lineage progression. Firstly, it promotes the lineage transition from NSCs to TAPs. Secondly it enhances the proliferative response of neuronal progenitors to EGF. Thus DLX2 and EGFR signalling interact at multiple levels to coordinate proliferation in the postnatal SVZ.

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 (GABAARs) 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 GABAARs. However, unlike in neuroblasts, where it induced depolarization‐mediated [Ca2+]i increase, GABAARs activation in precursors caused hyperpolarization. This resulted in osmotic swelling and increased surface expression of epidermal growth factor receptors (EGFRs). Furthermore, activation of GABAARs 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 GABAARs modulators during neurodevelopment. STEM CELLS 2011;29:307–319

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. STEM CELLS 2011; 29:1415–1426

Growth/differentiation factor 15 promotes EGFR signalling, and regulates proliferation and migration in the hippocampus of neonatal and young adult mice

The activation of epidermal growth factor receptor (EGFR) affects multiple aspects of neural precursor behaviour, including proliferation and migration. Telencephalic precursors acquire EGF responsiveness and upregulate EGFR expression at late stages of development. The events regulating this process and its significance are still unclear. We here show that in the developing and postnatal hippocampus (HP), growth/differentiation factor (GDF) 15 and EGFR are co-expressed in primitive precursors as well as in more differentiated cells. We also provide evidence that GDF15 promotes responsiveness to EGF and EGFR expression in hippocampal precursors through a mechanism that requires active CXC chemokine receptor (CXCR) 4. Besides EGFR expression, GDF15 ablation also leads to decreased proliferation and migration. In particular, lack of GDF15 impairs both processes in the cornu ammonis (CA) 1 and only proliferation in the dentate gyrus (DG). Importantly, migration and proliferation in the mutant HP were altered only perinatally, when EGFR expression was also affected. These data suggest that GDF15 regulates migration and proliferation by promoting EGFR signalling in the perinatal HP and represent a first description of a functional role for GDF15 in the developing telencephalon.

Multipotent precursors in the anterior and hippocampal subventricular zone display similar transcription factor signatures but their proliferation and maintenance are differentially regulated

Precursors within the subventricular zone (SVZ) exhibit regional variations in the expression of transcription factors important for the regulation of their proliferation and differentiation. In the anterior SVZ (aSVZ) the homeobox transcription factor distalless (Dlx)2 modulates both processes by promoting neural stem cell (NSC) activation as well as neurogenesis. Activated NSCs and transit-amplifying precursors (TAPs) in the aSVZ both express high levels of epidermal growth factor receptor (EGFR(high)) and form clones in response to exogenous EGF. EGF-responsive cells are also present in the hippocampal subependyma (hSVZ). However, it is not clear whether they represent NSCs or TAPs and whether their proliferation and differentiation are regulated as in the aSVZ. Here we have purified EGFR(high) cells from both the aSVZ and hSVZ at different ages. When isolated from perinatal tissue both populations were enriched in multipotent clonogenic precursors, which generated GABAergic neurons. Although they differed in absolute expression levels, activated NSCs and TAPs in both regions displayed similar signatures of transcription factor expression. However, activated NSCs were less frequent in the hSVZ than in the aSVZ. Furthermore, increasing age had a greater inhibitory effect on NSC proliferation in the hSVZ than in the aSVZ. This suggests that NSC activation is differentially regulated in the two regions. Consistent with this hypothesis, we found that in hippocampal precursors Dlx2 promoted neurogenesis but not NSC activation. Thus, most clonogenic EGFR(high) precursors in the hSVZ represent TAPs and NSC proliferation in the aSVZ and hSVZ is regulated by different mechanisms.

GABA Not Only a Neurotransmitter: Osmotic Regulation by GABAAR Signaling

Mature macroglia and almost all neural progenitor types express γ-aminobutyric (GABA) A receptors (GABAARs), whose activation by ambient or synaptic GABA, leads to influx or efflux of chloride (Cl−) depending on its electro-chemical gradient (ECl). Since the flux of Cl− is indissolubly associated to that of osmotically obliged water, GABAARs regulate water movements by modulating ion gradients. In addition, since water movements also occur through specialized water channels and transporters, GABAAR signaling could affect the movement of water by regulating the function of the channels and transporters involved, thereby affecting not only the direction of the water fluxes but also their dynamics. We will here review recent observations indicating that in neural cells GABAAR-mediated osmotic regulation affects the cellular volume thereby activating multiple intracellular signaling mechanisms important for cell proliferation, maturation, and survival. In addition, we will discuss evidence that the osmotic regulation exerted by GABA may contribute to brain water homeostasis in physiological and in pathological conditions causing brain edema, in which the GABAergic transmission is often altered.

Inducible gene expression in GFAP+ progenitor cells of the SGZ and the dorsal wall of the SVZ—A novel tool to manipulate and trace adult neurogenesis

In the adult mammalian brain, neurogenesis originates from astrocyte‐like stem cells. We generated a transgenic mouse line in which the tetracycline dependent transactivator (tTA) is expressed under the control of the murine GFAP promoter. In this mouse line, inducible gene expression targets virtually all GFAP‐expressing stem‐like cells in the dentate gyrus and a subset of GFAP‐expressing progenitors located primarily in the dorsal wall/dorsolateral corner of the subventricular zone. Outside the neurogenic zones, astrocytes are infrequently targeted. We introduce a panel of transgenic mice which allow both inducible expression of candidate genes under control of the murine GFAP promoter and, at the same time, lineage tracing of all cells descendant from the original GFAP‐positive cell. This new mouse line represents a versatile tool for functional analysis of neurogenesis and lineage tracing.

Depolarization promotes GAD 65-mediated GABA synthesis by a post-translational mechanism in neural stem cell-derived neurons.

Neuronal activity regulates neurogenesis and neuronal differentiation in the mammalian brain. The commencement of neurotransmitter expression establishes the neuronal phenotype and enables the formation of functional connectivity between neurons. In addition, release of neurotransmitters from differentiating neurons may modulate the behaviour of neural precursors. Here, we show that neuronal activity regulates γ‐aminobutyric acid (GABA) expression in neurons generated from stem cells of the striatum and adult subventricular zone (SVZ). Differentiating neurons display spontaneous Ca2+ events, which are voltage‐gated calcium channel (VGCC) dependent. Depolarization increases both the frequency of Ca2+ transients and the amount of Ca2+ influx in differentiating neurons. We show that depolarization‐dependent GABA expression is regulated by the amplitude and not by the frequency of Ca2+ influx. Brief activation of VGCCs leads to Ca2+ influx that in turn promotes a rapid expression of GABA. Depolarization‐dependent GABA expression does not require changes in gene expression. Instead, it involves cAMP‐dependent protein kinase (PKA) and Ca2+ and phospholipid‐dependent protein kinase (PKC) signalling. Activity increases the number of glutamic acid decarboxylase (GAD) 65‐immunoreactive neurons in a PKA‐dependent manner, without altering the expression of GAD 65, suggesting that depolarization promotes recruitment of GAD 65 by a post‐translational mechanism. In line with this, depolarization does not permanently increase the expression of GABA in neurons derived from neural stem cells of the embryonic striatum, cortex and adult SVZ. Thus, neuronal activity does not merely accelerate neuronal differentiation but it may alter the mechanism of GABA synthesis in newly generated neurons.