Coupling of cell migration with neurogenesis by proneural bHLH factors.

in Proceedings of the National Academy of Sciences of the United States of America (2006), 103(5), 1319-24

After cell birth, almost all neurons in the mammalian central nervous system migrate. It is unclear whether and how cell migration is coupled with neurogenesis. Here we report that proneural basic helix ... [more ▼]

After cell birth, almost all neurons in the mammalian central nervous system migrate. It is unclear whether and how cell migration is coupled with neurogenesis. Here we report that proneural basic helix-loop-helix (bHLH) transcription factors not only initiate neuronal differentiation but also potentiate cell migration. Mechanistically, proneural bHLH factors regulate the expression of genes critically involved in migration, including down-regulation of RhoA small GTPase and up-regulation of doublecortin and p35, which, in turn, modulate the actin and microtubule cytoskeleton assembly and enable newly generated neurons to migrate. In addition, we report that several DNA-binding-deficient proneural genes that fail to initiate neuronal differentiation still activate migration, whereas a different mutation of a proneural gene that causes a failure in initiating cell migration still leads to robust neuronal differentiation. Collectively, these data suggest that transcription programs for neurogenesis and migration are regulated by bHLH factors through partially distinct mechanisms. [less ▲]

p27kip1 independently promotes neuronal differentiation and migration in the cerebral cortex.

in Genes & Development (2006), 20(11), 1511-24

The generation of neurons by progenitor cells involves the tight coordination of multiple cellular activities, including cell cycle exit, initiation of neuronal differentiation, and cell migration. The ... [more ▼]

The generation of neurons by progenitor cells involves the tight coordination of multiple cellular activities, including cell cycle exit, initiation of neuronal differentiation, and cell migration. The mechanisms that integrate these different events into a coherent developmental program are not well understood. Here we show that the cyclin-dependent kinase inhibitor p27(Kip1) plays an important role in neurogenesis in the mouse cerebral cortex by promoting the differentiation and radial migration of cortical projection neurons. Importantly, these two functions of p27(Kip1) involve distinct activities, which are independent of its role in cell cycle regulation. p27(Kip1) promotes neuronal differentiation by stabilizing Neurogenin2 protein, an activity carried by the N-terminal half of the protein. p27(Kip1) promotes neuronal migration by blocking RhoA signaling, an activity that resides in its C-terminal half. Thus, p27(Kip1) plays a key role in cortical development, acting as a modular protein that independently regulates and couples multiple cellular pathways contributing to neurogenesis. [less ▲]

Proneural bHLH and Brn proteins coregulate a neurogenic program through cooperative binding to a conserved DNA motif.

in Developmental Cell (2006), 11(6), 831-44

Proneural proteins play a central role in vertebrate neurogenesis, but little is known of the genes that they regulate and of the factors that interact with proneural proteins to activate a neurogenic ... [more ▼]

Proneural proteins play a central role in vertebrate neurogenesis, but little is known of the genes that they regulate and of the factors that interact with proneural proteins to activate a neurogenic program. Here, we demonstrate that the proneural protein Mash1 and the POU proteins Brn1 and Brn2 interact on the promoter of the Notch ligand Delta1 and synergistically activate Delta1 transcription, a key step in neurogenesis. Overexpression experiments in vivo indicate that Brn2, like Mash1, regulates additional aspects of neurogenesis, including the division of progenitors and the differentiation and migration of neurons. We identify by in silico screening a number of additional candidate target genes, which are recognized by Mash1 and Brn proteins through a DNA-binding motif similar to that found in the Delta1 gene and present a broad range of activities. We thus propose that Mash1 synergizes with Brn factors to regulate multiple steps of neurogenesis. [less ▲]

Developmental regulation of beta-carboline-induced inhibition of glycine-evoked responses depends on glycine receptor beta subunit expression

in Molecular Pharmacology (2005), 67(5), 1783-1796

In this work, we show that beta-carbolines, which are known negative allosteric modulators of GABA A receptors, inhibit glycine-induced currents of embryonic mouse spinal cord and hippocampal neurons. In ... [more ▼]

In this work, we show that beta-carbolines, which are known negative allosteric modulators of GABA A receptors, inhibit glycine-induced currents of embryonic mouse spinal cord and hippocampal neurons. In both cell types, beta-carboline-induced inhibition of glycine receptor (GlyR)-mediated responses decreases with time in culture. Single-channel recordings show that the major conductance levels of GlyR unitary currents shifts from high levels (>= 50 pS) in 2 to 3 days in vitro (DIV) neurons to low levels (< 50 pS) in 11 to 14 DIV neurons, assessing the replacement of functional homomeric GlyR by heteromeric GlyR. In cultured spinal cord neurons, the disappearance of beta-carboline inhibition of glycine responses and high conductance levels is almost complete in mature neurons, whereas a weaker decrease in beta-carboline-evoked glycine response inhibition and high conductance level proportion is observed in hippocampal neurons. To confirm the hypothesis that the decreased sensitivity of GlyR to beta-carbolines depends on beta subunit expression, Chinese hamster ovary cells were permanently transfected either with GlyR alpha 2 subunit alone or in combination with GlyR beta subunit. Single-channel recordings revealed that the major conductance levels shifted from high levels (>= 50 pS) in GlyR-alpha 2-transfected cells to low levels (< 50 pS) in GlyR-alpha 2-containing cells. Consistently, both picrotoxinand beta-carboline-induced inhibition of glycine-gated currents were significantly decreased in GlyR-alpha 2-transfected cells compared with GlyR-alpha 2-containing cells. In summary, we demonstrate that the incorporation of beta subunits in GlyRs confers resistance not only to picrotoxin but also to beta-carbolineinduced inhibition. Furthermore, we also provide evidence that hippocampal neurons undergo in vitro a partial maturation process of their GlyR-mediated responses. [less ▲]

Phosphorylation of Neurogenin2 specifies the migration properties and the dendritic morphology of pyramidal neurons in the neocortex.

in Neuron (2005), 48(1), 45-62

The molecular mechanisms specifying the dendritic morphology of different neuronal subtypes are poorly understood. Here we demonstrate that the bHLH transcription factor Neurogenin2 (Ngn2) is both ... [more ▼]

The molecular mechanisms specifying the dendritic morphology of different neuronal subtypes are poorly understood. Here we demonstrate that the bHLH transcription factor Neurogenin2 (Ngn2) is both necessary and sufficient for specifying the dendritic morphology of pyramidal neurons in vivo by specifying the polarity of its leading process during the initiation of radial migration. The ability of Ngn2 to promote a polarized leading process outgrowth requires the phosphorylation of a single tyrosine residue at position 241, an event that is neither involved in Ngn2 direct transactivation properties nor its proneural function. Interestingly, the migration defect observed in the Ngn2 knockout mouse and in progenitors expressing the Ngn2(Y241F) mutation can be rescued by inhibiting the activity of the small-GTPase RhoA in cortical progenitors. Our results demonstrate that Ngn2 coordinates the acquisition of the radial migration properties and the unipolar dendritic morphology characterizing pyramidal neurons through molecular mechanisms distinct from those mediating its proneural activity. [less ▲]

The Inhibition of Cyclin-Dependent Kinases Induces Differentiation of Supernumerary Hair Cells and Deiters' Cells in the Developing Organ of Corti

in FASEB Journal (2003), 17(14), 2136-8

In the embryonic day 19 organs of Corti, we showed that roscovitine, a chemical inhibitor of cyclin-dependent kinases (CDKs), significantly increased the number of hair cells (HCs) and corresponding ... [more ▼]

In the embryonic day 19 organs of Corti, we showed that roscovitine, a chemical inhibitor of cyclin-dependent kinases (CDKs), significantly increased the number of hair cells (HCs) and corresponding supporting cells (SCs) by triggering differentiation of precursor cells without interacting with cell proliferation. The effect of roscovitine was mimicked by other CDK1, 2, 5, and 7 inhibitors but not by CDK4/6 and mitogen-activated protein kinase pathway antagonists. Immunohistochemical analysis indicated that roscovitine-specific intracellular targets, CDK1, 2, 5, and 7, were expressed in the organ of Corti and especially in Hensen's cells. Affinity chromatography studies showed a tight correlation between the protein levels of CDK1/2 and 5 and the rate of roscovitine-induced supernumerary cells in the organ of Corti. In addition, we demonstrated that basal CDK activity was higher and more roscovitine-sensitive at developmental stages that are selectively permissive for the emergence of supernumerary cells. These results suggest that CDKs are involved in the normal development of the organ of Corti and that, at least in E19 embryos, inhibition of CDKs is sufficient to trigger the differentiation of HCs and corresponding SCs, presumably from the Hensen's cell progenitors and/or from progenitors located in the greater epithelial ridge area. [less ▲]

Untangling the functional potential of PSA-NCAM-expressing cells in CNS development and brain repair strategies

in Current Medicinal Chemistry (2003), 10(20), 2185-2196

Central nervous system (CNS) neural stem cells (NSCs), which are mostly defined by their ability to self-renew and to generate the three main cell lineages of the CNS, were isolated from discrete regions ... [more ▼]

Central nervous system (CNS) neural stem cells (NSCs), which are mostly defined by their ability to self-renew and to generate the three main cell lineages of the CNS, were isolated from discrete regions of the adult mammalian CNS including the subventricular zone (SVZ) of the lateral ventricle and the dentate gyrus in the hippocampus. At early stages of CNS cell fate determination, NSCs give rise to progenitors that express the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). PSA-NCAM(+) cells persist in adult brain regions where neuronal plasticity and sustained formation of new neurons occur. PSA-NCAM, has been shown to be involved in the regulation of CNS myelination as well as in changes of cell morphology that are necessary for motility, axonal guidance, synapse formation, and functional plasticity in the CNS. Although being preferentially committed to a restricted either glial or neuronal fate, cultured PSA-NCAM(+) progenitors do preserve a relative degree of multipotentiality. Considering that PSA-NCAM(+) cells can be neatly used for brain repair purposes, there is much interest for studying signaling factors regulating their development. With this regard, it is noteworthy that neurotransmitters, which belong to the micro-environment of neural cells in vivo, regulate morphogenetic events preceding synaptogenesis such as cell proliferation, migration, differentiation and death. Consistently, several ionotropic but also G-protein-coupled neurotransmitter receptors were found to be expressed in CNS embryonic and postnatal progenitors. In the present review, we outlined the ins and outs of PSA-NCAM(+) cells addressing to what extent our understanding of extrinsic and in particular neurotransmitter-mediated signaling in these CNS precursor cells might represent a new leading track to develop alternative strategies to stimulate brain repair. [less ▲]

Autocrine/paracrine activation of the GABA(A) receptor inhibits the proliferation of neurogenic polysialylated neural cell adhesion molecule-positive (PSA-NCAM+) precursor cells from postnatal striatum.

in Journal of Neuroscience (2003), 23(8), 3278-94

GABA and its type A receptor (GABA(A)R) are present in the immature CNS and may function as growth-regulatory signals during the development of embryonic neural precursor cells. In the present study, on ... [more ▼]

GABA and its type A receptor (GABA(A)R) are present in the immature CNS and may function as growth-regulatory signals during the development of embryonic neural precursor cells. In the present study, on the basis of their isopycnic properties in a buoyant density gradient, we developed an isolation procedure that allowed us to purify proliferative neural precursor cells from early postnatal rat striatum, which expressed the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). These postnatal striatal PSA-NCAM+ cells were shown to proliferate in the presence of epidermal growth factor (EGF) and formed spheres that preferentially generated neurons in vitro. We demonstrated that PSA-NCAM+ neuronal precursors from postnatal striatum expressed GABA(A)R subunits in vitro and in situ. GABA elicited chloride currents in PSA-NCAM+ cells by activation of functional GABA(A)R that displayed a typical pharmacological profile. GABA(A)R activation in PSA-NCAM+ cells triggered a complex intracellular signaling combining a tonic inhibition of the mitogen-activated protein kinase cascade and an increase of intracellular calcium concentration by opening of voltage-gated calcium channels. We observed that the activation of GABA(A)R in PSA-NCAM+ neuronal precursors from postnatal striatum inhibited cell cycle progression both in neurospheres and in organotypic slices. Furthermore, postnatal PSA-NCAM+ striatal cells synthesized and released GABA, thus creating an autocrine/paracrine mechanism that controls their proliferation. We showed that EGF modulated this autocrine/paracrine loop by decreasing GABA production in PSA-NCAM+ cells. This demonstration of GABA synthesis and GABA(A)R function in striatal PSA-NCAM+ cells may shed new light on the understanding of key extrinsic cues that regulate the developmental potential of postnatal neuronal precursors in the CNS. [less ▲]

Identification of factors that maintain mammalian outer hair cells in adult organ of Corti explants

in Hearing Research (2002), 170(1-2), 48-58

Both outer hair cells (OHCs) and inner hair cells (IHCs) survive and mature in 3 days old rat organ of Corti explants cultured for I month in a minimal essential medium. In contrast. under the same ... [more ▼]

Both outer hair cells (OHCs) and inner hair cells (IHCs) survive and mature in 3 days old rat organ of Corti explants cultured for I month in a minimal essential medium. In contrast. under the same culture conditions, only IHCs survive in explants from adult guinea pig organ of Corti while many of the OHCs are lost within the first 48 It. Hair cell Count,, show OHCs loss to be greater in the lower portion (i.e. middle turn) of the cochlea than Lit the apex. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) indicates that there is DNA damage in adult OHCs, within 8 h of explantation. Treatment of the adult organ of Corti explants with either actinomycin D (10(-7) M) or cycloheximide (10(-6) M) prevents most OHC losses . According to these results apoptosis may be the mechanism of OHC loss in adult organ of Corti explants, Stable membrane potentials recorded from the OHCs in both uncultured and actinomycin D-treated organ of Corti explants cultured for 72 h demonstrate the functional integrity of these hair cells. OHC losses in the adult guinea pig, organ of Corti cultures can also be prevented by treatment with several of the growth factors tested. i.e. acidic fibroblast growth factor (aFGF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), transforming growth factor-beta1 (TGF-beta1). and glial cell-derived neurotrophic factor (GDNF). The results of this study suggest that growth factor therapy may be applicable to the treatment of some hearing disorders. (C) 2002 Elsevier Science B.V. All rights reserved. [less ▲]

Functional glycine receptors are expressed by postnatal nestin-positive neural stem/progenitor cells

in European Journal of Neuroscience (2002), 15(8), 1299-1305

Multipotent neural stem and progenitor cells (NS/PCs) are well-established cell subpopulations occurring in the developing, and also in the mature mammalian nervous systems. Trophic and transcription ... [more ▼]

Multipotent neural stem and progenitor cells (NS/PCs) are well-established cell subpopulations occurring in the developing, and also in the mature mammalian nervous systems. Trophic and transcription factors are currently the main signals known to influence the development and the commitment of NS/PCs and their progeny. However, recent studies suggest that neurotransmitters could also contribute to neural development. In that respect, rodent-cultured embryonic NS/PCs have been reported to express functional neurotransmitter receptors. No similar investigation has, however, been made in postnatal and/or in adult rodent brain stem cells. In this study, using RT-PCR and immunocytochemical methods, we show that alpha(1) -, alpha(2) - and beta-subunit mRNAs and alpha-subunit proteins of the glycine ionotropic receptor are expressed by 80.5 +/- 0.9% of postnatal rat striatum-derived, nestin-positive cells within cultured neurospheres. Whole-cell patch-clamp experiments further demonstrated that glycine triggers in 33.5% of these cells currents that can be reversibly blocked by strychnine and picrotoxin. This demonstrates that NS/PCs express functional glycine receptors, the consequence(s) of their activation remaining unknown. [less ▲]