Carlos Vicario-Abejón

Targeted Genomic Disruption of H-ras and N-ras, Individually or in Combination, Reveals the Dispensability of Both Loci for Mouse Growth and Development

ABSTRACT Mammalian cells harbor three highly homologous and widely expressed members of the ras family (H-ras, N-ras, and K-ras), but it remains unclear whether they play specific or overlapping cellular roles. To gain insight into such functional roles, here we generated and analyzed H-ras null mutant mice, which were then also bred with N-ras knockout animals to ascertain the viability and properties of potential double null mutations in both loci. Mating among heterozygous H-ras+/− mice produced H-ras −/− offspring with a normal Mendelian pattern of inheritance, indicating that the loss of H-rasdid not interfere with embryonic and fetal viability in the uterus. Homozygous mutant H-ras −/− mice reached sexual maturity at the same age as their littermates, and both males and females were fertile. Characterization of lymphocyte subsets in the spleen and thymus showed no significant differences between wild-type and H-ras −/− mice. Analysis of neuronal markers in the brains of knockout and wild-type H-ras mice showed that disruption of this locus did not impair or alter neuronal development. Breeding between our H-ras mutant animals and previously available N-ras null mutants gave rise to viable double knockout (H-ras −/−/N-ras −/−) offspring expressing only K-ras genes which grew normally, were fertile, and did not show any obvious phenotype. Interestingly, however, lower-than-expected numbers of adult, double knockout animals were consistently obtained in Mendelian crosses between heterozygous N-ras/H-ras mice. Our results indicate that, as for N-ras, H-ras gene function is dispensable for normal mouse development, growth, fertility, and neuronal development. Additionally, of the three ras genes, K-ras appears to be not only essential but also sufficient for normal mouse development.

Role of neurotrophins in central synapse formation and stabilization

The neurotrophins are best known for their ability to support neuronal survival and differentiation, but a role in synapse formation and plasticity has recently emerged. For central neurons, brain-derived neurotrophic factor can increase the number of excitatory and inhibitory synapses by regulating axonal morphology or by directly promoting synapse formation. In addition, neurotrophins promote the maturation and stabilization of the cellular and molecular components that are responsible for neurotransmitter release, and this ultimately leads to an increase in the number of functional synapses. These long-term structural and molecular changes are likely to be crucial not only during development, but also during synaptic plasticity in the adult.

Locally born olfactory bulb stem cells proliferate in response to insulin-related factors and require endogenous insulin-like growth factor-I for differentiation into neurons and glia

After late embryogenesis, new neurons are continuously added to the olfactory bulb (OB) from stem cells located in the forebrain subventricular zone. Nonetheless, stem cells have not been described within the embryonic olfactory bulb. Here we report the isolation of local olfactory bulb stem cells from the embryonic day 12.5–14.5 mouse embryo. These cells were 99.2% nestin positive and proliferated extensively in culture to at least 150 cell doublings. Clonal analysis demonstrated that neurons (TuJ1+), astrocytes (GFAP+), and oligodendrocytes (O4+) could be generated from single-plated cells, indicating that they are multipotent. At least 90% of proliferating cells expressed insulin-like growth factor-I (IGF-I), (pro)insulin, and their cognate receptors; these growth factors collaborated with fibroblast growth factor-2 plus epidermal growth factor (EGF) to promote stem cell proliferation and sphere formation. Cells fromIgf-I−/−mice, however, proliferated as extensively as didIgf-I+/+ cells. Differentiation and survival of stem cell-generated neurons and glia showed strong dependence on exogenous IGF-I, but oligodendrocyte differentiation also required insulin at low concentration. Furthermore, the percentages of stem cell-generated neurons, astrocytes, and oligodendrocytes were markedly lower in the cultures prepared from theIgf-I−/−mice compared with those ofIgf-I+/+. Concordantly, lack of IGF-I resulted in abnormal formation of the olfactory bulb mitral cell layer and altered radial glia morphology. These results support the presence within the embryonic mouse olfactory bulb of stem cells with specific requirements for insulin-related growth factors for proliferation or differentiation. They demonstrate that IGF-I is an endogenous factor regulating the differentiation of stem and other precursor cells within the olfactory bulb.

Modulation of the PI 3-kinase–Akt signalling pathway by IGF-I and PTEN regulates the differentiation of neural stem/precursor cells

Neural stem cells depend on insulin-like growth factor I (IGF-I) for differentiation. We analysed how activation and inhibition of the PI 3-kinase–Akt signalling affects the number and differentiation of mouse olfactory bulb stem cells (OBSCs). Stimulation of the pathway with insulin and/or IGF-I, led to an increase in Akt phosphorylated on residues Ser473 and Thr308 (P-AktSer473 and P-AktThr308, respectively) in proliferating OBSCs, and in differentiating cells. Conversely, P-AktSer473 levels decreased by 50% in the OB of embryonic day 16.5-18.5 IGF-I knockout mouse embryos. Overexpression of PTEN, a negative regulator of the PI 3-kinase pathway, caused a reduction in the basal levels of P-AktSer473 and P-AktThr308 and a minor reduction in IGF-I-stimulated P-AktSer473. Although PTEN overexpression decreased the proportion of neurons and astrocytes in the absence of insulin/IGF-I, it did not alter the proliferation or survival of OBSCs. Accordingly, overexpression of a catalytically inactive PTEN mutant promoted OBSCs differentiation. Inhibition of PI 3-kinase by LY294002 produced strong and moderate reductions in IGF-I-stimulated P-AktSer473 and P-AktThr308, respectively. Consequently, LY294002 reduced the proliferation of OBSCs and the number of neurons and astrocytes, and also augmented cell death. These findings indicate that OBSC differentiation is more sensitive to lower basal levels of P-Akt than proliferation or death. By regulating P-Akt levels in opposite ways, IGF-I and PTEN contribute to the fine control of neurogenesis in the olfactory bulb.

IGF‐I promotes neuronal migration and positioning in the olfactory bulb and the exit of neuroblasts from the subventricular zone

While insulin‐like growth factor‐I (IGF‐I) supports neuronal and glial differentiation in the CNS, it is largely unknown whether IGF‐I also influences neuronal migration and positioning. We show here that the pattern of olfactory bulb (OB) layering is altered in Igf‐I −/− mice. In these animals, Tbr1+‐glutamatergic neurons are misplaced in the mitral cell layer (ML) and the external plexiform layer (EPL). In addition, there are fewer interneurons in the glomerular layer and the EPL of the Igf‐I −/− mice, and fewer newborn neurons are incorporated into the OB from the forebrain subventricular zone (SVZ). Indeed, neuroblasts accumulate in the postnatal/adult SVZ of Igf‐I −/− mice. Significantly, the positioning of Tbr1+‐cells in a primitive ML is stimulated by IGF‐I in cultured embryonic OB slices, an effect that is partially repressed by the phosphoinositide 3‐kinase (PI3K) inhibitor. In OB cell cultures, IGF‐I increases the phosphorylation of disabled1 (P‐Dab1), an adaptor protein that is a target of Src family kinases (SFK) in the reelin signalling pathway, whereas reduced P‐Dab1 levels were found in Igf‐I −/− mice. Neuroblast migration from the rostral migratory stream (RMS) explants of postnatal Igf‐I −/− was similar to that from Igf‐I +/+ explants. However, cell migration was significantly enhanced by IGF‐I added to the explants, an effect that was repressed by PI3K and SFK inhibitors. These findings suggest that IGF‐I promotes neuronal positioning in the OB and support a role for IGF‐I in stimulating neuroblast exit from the SVZ into the RMS, thereby promoting the incorporation of newly formed neurons into the OB.

Generation of GABAergic and dopaminergic interneurons from endogenous embryonic olfactory bulb precursor cells

During the embryonic period, many olfactory bulb (OB) interneurons arise in the lateral ganglionic eminence (LGE) from precursor cells expressing Dlx2, Gsh2 and Er81 transcription factors. Whether GABAergic and dopaminergic interneurons are also generated within the embryonic OB has not been studied thoroughly. In contrast to abundant Dlx2 and Gsh2 expression in ganglionic eminences (GE), Dlx2 and Gsh2 proteins are not expressed in the E12.5-13.5 mouse OB, whereas the telencephalic pallial domain marker Pax6 is abundant. We found GABAergic and dopaminergic neurons originating from dividing precursor cells in E13.5 OB and in short-term dissociated cultures prepared from the rostral half of E13.5 OB. In OB cultures, 22% of neurons were GAD+, of which 53% were Dlx2+, whereas none expressed Gsh2. By contrast, 70% of GAD+ cells in GE cultures were Dlx2+ and 16% expressed Gsh2. In E13.5 OB slices transplanted with EGFP-labeled E13.5 OB precursor cells, 31.7% of EGFP+ cells differentiated to GABAergic neurons. OB and LGE precursors transplanted into early postnatal OB migrated and differentiated in distinct patterns. Transplanted OB precursors gave rise to interneurons with dendritic spines in close proximity to synaptophysin-positive boutons. Interneurons were also abundant in differentiating OB neural stem cell cultures; the neurons responded to the neurotrophin Bdnf and expressed presynaptic proteins. In vivo, the Bdnf receptor TrkB colocalized with synaptic proteins at the glomeruli. These findings suggest that, in addition to receiving interneurons from the LGE, the embryonic OB contains molecularly distinct local precursor cells that generate mature GABAergic and dopaminergic neurons.

IGF-I: A Key Growth Factor that Regulates Neurogenesis and Synaptogenesis from Embryonic to Adult Stages of the Brain.

The generation of neurons in the adult mammalian brain requires the activation of quiescent neural stem cells (NSCs). This activation and the sequential steps of neuron formation from NSCs are regulated by a number of stimuli, which include growth factors. Insulin-like growth factor-I (IGF-I) exert pleiotropic effects, regulating multiple cellular processes depending on their concentration, cell type, and the developmental stage of the animal. Although IGF-I expression is relatively high in the embryonic brain its levels drop sharply in the adult brain except in neurogenic regions, i.e., the hippocampus (HP) and the subventricular zone-olfactory bulb (SVZ-OB). By contrast, the expression of IGF-IR remains relatively high in the brain irrespective of the age of the animal. Evidence indicates that IGF-I influences NSC proliferation and differentiation into neurons and glia as well as neuronal maturation including synapse formation. Furthermore, recent studies have shown that IGF-I not only promote adult neurogenesis by regulating NSC number and differentiation but also by influencing neuronal positioning and migration as described during SVZ-OB neurogenesis. In this article we will revise and discuss the actions reported for IGF-I signaling in a variety of in vitro and in vivo models, focusing on the maintenance and proliferation of NSCs/progenitors, neurogenesis, and neuron integration in synaptic circuits.

Maintenance of Undifferentiated State and Self-Renewal of Embryonic Neural Stem Cells by Polycomb Protein Ring1B

Cell lineages generated during development and tissue maintenance are derived from self‐renewing stem cells by differentiation of their committed progeny. Recent studies suggest that epigenetic mechanisms, and in particular the Polycomb group (PcG) of genes, play important roles in controlling stem cell self‐renewal. Here, we address PcG regulation of stem cell self‐renewal and differentiation through inactivation of Ring1B, a histone H2A E3 monoubiquitin ligase, in embryonic neural stem cells (NSCs) from the olfactory bulb of a conditional mouse mutant line. We show that neural stem/progenitor cell proliferation in vivo and in neurosphere assays is impaired, lacking Ring1B, and their self‐renewal and multipotential abilities, assessed as sphere formation and differentiation from single cells, are severely affected. We also observed unscheduled neuronal, but not glial, differentiation of mutant stem/progenitor cells under proliferating conditions, an alteration enhanced in cells also lacking Ring1A, the Ring1B paralog, some of which turned into morphologically identifiable neurons. mRNA analysis of mutant cells showed upregulation of some neuronal differentiation–related transcription factors and the cell proliferation inhibitor Cdkn1a/p21, as well as downregulation of effectors of the Notch signaling pathway, a known inhibitor of neuronal differentiation of stem/progenitor cells. In addition, differentiation studies of Ring1B‐deficient progenitors showed decreased oligodendrocyte formation in vitro and enhanced neurogenesis and reduced gliogenesis in vivo. These data suggest a role for Ring1B in maintenance of the undifferentiated state of embryonic neural stem/progenitor cells. They also suggest that Ring1B may modulate the differentiation potential of NSCs to neurons and glia. STEM CELLS 2009;27:1559–1570

L-DOPA-induced increase in TH-immunoreactive striatal neurons in parkinsonian mice: Insights into regulation and function

Tyrosine hydroxylase (TH)-immunoreactive (ir) neurons have been found in the striatum after dopamine depletion; however, little is known about the mechanism underlying their appearance or their functional significance. We previously showed an increase in striatal TH-ir neurons after L-DOPA treatment in mice with unilateral 6-OHDA lesions in the striatum. In the present study, we further examined the time-course and persistence of the effects of chronic L-DOPA treatment on the appearance and regulation of TH-ir neurons as well as their possible function. We found that the L-DOPA-induced increase in striatal TH-ir neurons is dose-dependent and persists for days after L-DOPA withdrawal, decreasing significantly 10 days after L-DOPA treatment ends. Using hemiparkinsonian D1 receptor knock-out (D1R-/-) and D2 receptor knock-out (D2R-/-) mice, we found that the D1R, but not the D2R, is required for the L-DOPA-induced appearance of TH-ir neurons in the dopamine-depleted striatum. Interestingly, our experiments in aphakia mice, which lack Pitx3 expression in the brain, indicate that the L-DOPA-dependent increase in the number of TH-ir neurons is independent of Pitx3, a transcription factor necessary for the development of mesencephalic dopaminergic neurons. To explore the possible function of L-DOPA-induced TH-ir neurons in the striatum, we examined dopamine overflow and forelimb use in L-DOPA-treated parkinsonian mice. These studies revealed a tight spatio-temporal correlation between the presence of striatal TH-ir neurons, the recovery of electrically stimulated dopamine overflow in the lesioned striatum, and the recovery of contralateral forelimb use with chronic L-DOPA treatment. Our results suggest that the presence of TH-ir neurons in the striatum may underlie the long-duration response to L-DOPA following withdrawal. Promotion of these neurons in the early stages of Parkinsons disease, when dopamine denervation is incomplete, may be beneficial for maintaining motor function.

Fibroblast growth factor-2 increases the expression of neurogenic genes and promotes the migration and differentiation of neurons derived from transplanted neural stem/progenitor cells

The capacity of neural stem cells (NSC) to generate different types of neurons and glia depends on the action of intrinsic determinants and extracellular signals. Here, we isolated adult olfactory bulb stem cells (aOBSC) that express nestin, RC2 and Sox2, and that have the capacity to generate neurons possessing mature features in culture and in vivo. The differentiation of aOBSC into neurons and glia, as well as their genetic profile, was compared to that of embryonic OBSC (eOBSC) and ganglionic eminence stem cells (GESC). While these eOBSC express neurogenin (Ngn) 1 and 2, two telencephalic dorsal markers, GESC only express Ngn2. Adult OBSC express either little or no detectable Ngn1 and 2, and they produced significantly fewer neurons in culture than eOBSC. By contrast, Dlx2 transcripts (a telencephalic ventral marker) were only clearly detected in GESC. When transplanted into the early postnatal P5-P7 OB, each of the three populations gave rise to cells with a distinct pattern of neuronal migration and/or dendritic arborization. Overall, these findings suggest that cultured NSC partially maintain their regional and temporal specification. Notably, significant neuronal migration and differentiation were only observed in vivo when the NSC were briefly exposed to fibroblast growth factor-2 (FGF-2) before grafting, a treatment that enhanced the neurogenin expression. Hence, the migration and maturation of neurons derived from transplanted NSC can be promoted by upregulating neurogenic gene expression with FGF-2.