Maribel Murillo-Carretero

Nitric Oxide Is a Physiological Inhibitor of Neurogenesis in the Adult Mouse Subventricular Zone and Olfactory Bulb

The subventricular zone of the rodent brain retains the capacity of generating new neurons in adulthood. The newly formed neuroblasts migrate rostrally toward the olfactory bulb, where they differentiate as granular and periglomerular interneurons. The reported presence of differentiated neurons expressing the neuronal isoform of nitric oxide synthase (NOS) in the periphery of the neurogenic region and the organization of their varicose axons as a network in which the precursors are immersed raised the hypothesis that endogenous nitric oxide (NO) may participate in the control of neurogenesis in the subventricular zone. Systemic administration of the NOS inhibitors Nω-nitro-l-arginine methyl ester or 7-nitroindazole to adult mice produced a dose- and time-dependent increase in the number of mitotic cells in the subventricular zone, rostral migratory stream, and olfactory bulb, but not in the dentate gyrus of the hippocampus, without affecting apoptosis. In the subventricular zone, this effect was exerted selectively on a precursor subpopulation expressing nestin but not neuronal or glial cell-specific proteins. In addition, in the olfactory bulb, analysis of maturation markers in the newly generated neurons indicated that chronic NOS inhibition caused a delay in neuronal differentiation. Postmitotic cell survival and migration were not affected when NO production was impaired. Our results suggest that NO, produced by nitrergic neurons in the adult mouse subventricular zone and olfactory bulb, exerts a negative control on the size of the undifferentiated precursor pool and promotes neuronal differentiation.

Nitric Oxide Decreases Subventricular Zone Stem Cell Proliferation by Inhibition of Epidermal Growth Factor Receptor and Phosphoinositide‐3‐Kinase/Akt Pathway

Nitric oxide (NO) inhibits proliferation of subventricular zone (SVZ) neural precursor cells in adult mice in vivo under physiological conditions. The mechanisms underlying this NO effect have now been investigated using SVZ‐derived neural stem cells, which generate neurospheres in vitro when stimulated by epidermal growth factor (EGF). In these cultures, NO donors decreased the number of newly formed neurospheres as well as their size, which indicates that NO was acting on the neurosphere‐forming neural stem cells and the daughter neural progenitors. The effect of NO was cytostatic, not proapoptotic, and did not involve cGMP synthesis. Neurosphere cells expressed the neuronal and endothelial isoforms of NO synthase (NOS) and produced NO in culture. Inhibition of NOS activity by Nω‐nitro‐l‐arginine methylester (l‐NAME) promoted neurosphere formation and growth, thus revealing an autocrine/paracrine action of NO on the neural precursor cells. Both exogenous and endogenous NO impaired the EGF‐induced activation of the EGF receptor (EGFR) tyrosine kinase and prevented the EGF‐induced Akt phosphorylation in neurosphere cells. Inhibition of the phosphoinositide‐3‐kinase (PI3‐K)/Akt pathway by LY294002 significantly reduced the number of newly formed neurospheres, which indicates that this is an essential pathway for neural stem cell self‐renewal. Chronic administration of l‐NAME to adult mice enhanced phospho‐Akt staining in the SVZ and reduced nuclear p27Kip1 in the SVZ and olfactory bulb. The inhibition of EGFR and PI3‐K pathway by NO explains, at least in part, its antimitotic effect on neurosphere cells and may be a mechanism involved in the physiological role of NO as a negative regulator of SVZ neurogenesis in adult mice.

Nitric oxide synthesis inhibition increases proliferation of neural precursors isolated from the postnatal mouse subventricular zone

The subventricular zone (SVZ) of rodents retains the capacity to generate new neurons throughout the entire life of the animal. Neural progenitors of the SVZ survive and proliferate in vitro in the presence of epidermal growth factor (EGF). Nitric oxide (NO) has been shown to participate in neural tissue formation during development and to have antiproliferative actions, mediated in part by inhibition of the EGF receptor. Based on these findings, we have investigated the possible effects of endogenously produced and exogenously added NO on SVZ cell proliferation and differentiation. Explants were obtained from postnatal mouse SVZ and cultured in the presence of EGF. Cells migrated out of the explants and proliferated in culture, as assessed by bromodeoxyuridine (BrdU) incorporation. After 72 h in vitro, the colonies formed around the explants were constituted by cells of neuronal or glial lineages, as well as undifferentiated progenitors. Immunoreactivity for the neuronal isoform of NO synthase was observed in neuronal cells with long varicose processes. Cultures treated with the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) showed an increase in the percentage of BrdU-immunoreactive cells, whereas treatment with the NO donor diethylenetriamine-nitric oxide adduct (DETA-NO) led to a decrease in cell proliferation, without affecting apoptosis. The differentiation pattern was also altered by L-NAME treatment resulting in an enlargement of the neuronal population. The results suggest that endogenous NO may contribute to postnatal neurogenesis by modulating the proliferation and fate of SVZ progenitor cells.

Nitric Oxide and Adult Neurogenesis in Health and Disease

Adult neurogenesis may be functionally important as a mechanism of brain plasticity in physiological conditions and brain repair after injury. Nitric oxide (NO), a diffusible intracellular and intercellular messenger in the mammalian nervous system, has been shown to affect adult neurogenesis in different ways. In the normal brain, NO, synthesized by the neuronal isoform of NO synthase in nitrergic neurons, is a negative regulator of precursor cell proliferation. However, after brain damage, NO overproduction in different neural and nonneural cell types promotes neurogenesis. Recently reported results on the effects of NO on new neuron generation in the adult brain are reviewed, with special attention to the proposed mechanisms of action and functional consequences in health and disease.

Antiproliferative effect of nitric oxide on epidermal growth factor‐responsive human neuroblastoma cells

Addition of nitric oxide (NO) donors to NB69 neuroblastoma cells produced a cGMP‐independent decrease in cell proliferation, without affecting cell viability or apoptosis. The potency of short half‐life NO donors was higher when cell proliferation was stimulated by epidermal growth factor (EGF), as compared with cultures exposed to fetal calf serum (FCS). Immunoprecipitation and western blot analysis of the EGF receptor (EGFR) revealed a significant reduction of its EGF‐induced tyrosine phosphorylation in cells treated with the NO donor 2‐(N,N‐diethylamino)‐diazenolate‐2‐oxide (DEA‐NO). When total cell lysates were subjected to western blotting, we observed that DEA‐NO also reduced tyrosine phosphorylation in EGF‐activated phosphoproteins, but not in those proteins whose tyrosine phosphorylation was evident in the absence of EGF. The effect of NO on EGFR transphosphorylation was concentration‐dependent and transient, with a total recovery observed between 1.5 and 3 h after addition of DEA‐NO to the cells. When cells were incubated for 15 min with DEA‐NO and then washed, the EGFR transphosphorylation returned to control levels immediately, indicating that the interaction of NO with the receptor molecule was fully reversible. NB69 cells expressed both the neuronal and the inducible isoforms of NO synthase (NOS) when cultured in the presence of FCS; under this condition, the NOS inhibitor, Nω‐nitro‐l‐arginine methyl ester, produced a small but significant increase in cell proliferation. The results suggest that NO is an endogenous antimitotic agent and that its interaction with EGFR contributes to cytostasis in NB69 cells.

S-Nitrosylation of the epidermal growth factor receptor: A regulatory mechanism of receptor tyrosine kinase activity

Nitric oxide (NO) donors inhibit the epidermal growth factor (EGF)-dependent auto(trans)phosphorylation of the EGF receptor (EGFR) in several cell types in which NO exerts antiproliferative effects. We demonstrate in this report that NO inhibits, whereas NO synthase inhibition potentiates, the EGFR tyrosine kinase activity in NO-producing cells, indicating that physiological concentrations of NO were able to regulate the receptor activity. Depletion of intracellular glutathione enhanced the inhibitory effect of the NO donor 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO) on EGFR tyrosine kinase activity, supporting the notion that such inhibition was a consequence of an S-nitrosylation reaction. Addition of DEA/NO to cell lysates resulted in the S-nitrosylation of a large number of proteins including the EGFR, as confirmed by the chemical detection of nitrosothiol groups in the immunoprecipitated receptor. We prepared a set of seven EGFR(C --> S) substitution mutants and demonstrated in transfected cells that the tyrosine kinase activity of the EGFR(C166S) mutant was completely resistant to NO, whereas the EGFR(C305S) mutant was partially resistant. In the presence of EGF, DEA/NO significantly inhibited Akt phosphorylation in cells transfected with wild-type EGFR, but not in those transfected with C166S or C305S mutants. We conclude that the EGFR can be posttranslationally regulated by reversible S-nitrosylation of C166 and C305 in living cells.

Homocysteine inhibits proliferation of neuronal precursors in the mouse adult brain by impairing the basic fibroblast growth factor signaling cascade and reducing extracellular regulated kinase 1/2-dependent cyclin E expression

Hyperhomocysteinemia (HHcy)—abnormally elevated plasma levels of homocysteine (Hcy)—has been associated with the development of neurodegenerative dementia and mild cognitive impairment. This association suggests that HHcy might facilitate memory loss in the elderly. As memory loss can occur through a deteriorated neurogenic capacity, we have studied the effects of Hcy on neural progenitor cells (NPCs) both in vitro and in vivo. We show that Hcy exerts an antiproliferative effect on basic fibroblast growth factor (bFGF) ‐stimulated NPCs isolated from the postnatal subventricular zone (SVZ), accompanied by inactivation of the extracellular signal‐regulated kinase (Erk1/2) and inhibition of Erk1/ 2‐dependent expression of cyclin E. Using a mice model we show that, under normal folate conditions, HHcy exerts an inhibitory effect on adult brain neurogenesis. This inhibition occurs in the caudal areas of the dentate gyrus (DG) of the hippocampus, a neurogenic area mainly involved in learning and memory performance, and in the SVZ, recently implicated in olfactory learning performance. In both areas reduced number of proliferative neuroblasts were found. Since neuroblasts are primarily bFGF‐responsive progenitors already committed to a neuronal phenotype, our results strongly suggest that excess Hcyinhibits neurogenesis in the DG and SVZ byinhibiting the bFGF‐dependent activation of Erk1/2 in these cells.— Rabaneda, L. G., Carrasco, M., Lopez‐Toledano, M. A., Murillo‐Carretero, M., Ruiz, F. A., Estrada, C., Castro, C. Homocysteine inhibits proliferation of neuronal precursors in the mouse adult brain by impairing the basic fibroblast growth factor signaling cascade and reducing extracellular regulated kinase 1/2‐dependent cyclin E expression. FASEB J. 22, 3823–3835 (2008)

Glycine N-methyltransferase expression in the hippocampus and its role in neurogenesis and cognitive performance

The hippocampus is a brain area characterized by its high plasticity, observed at all levels of organization: molecular, synaptic, and cellular, the latter referring to the capacity of neural precursors within the hippocampus to give rise to new neurons throughout life. Recent findings suggest that promoter methylation is a plastic process subjected to regulation, and this plasticity seems to be particularly important for hippocampal neurogenesis. We have detected the enzyme GNMT (a liver metabolic enzyme) in the hippocampus. GNMT regulates intracellular levels of SAMe, which is a universal methyl donor implied in almost all methylation reactions and, thus, of prime importance for DNA methylation. In addition, we show that deficiency of this enzyme in mice (Gnmt−/−) results in high SAMe levels within the hippocampus, reduced neurogenic capacity, and spatial learning and memory impairment. In vitro, SAMe inhibited neural precursor cell division in a concentration‐dependent manner, but only when proliferation signals were triggered by bFGF. Indeed, SAMe inhibited the bFGF‐stimulated MAP kinase signaling cascade, resulting in decreased cyclin E expression. These results suggest that alterations in the concentration of SAMe impair neurogenesis and contribute to cognitive decline.

ADAM‐17/Tumor Necrosis Factor‐α‐Converting Enzyme Inhibits Neurogenesis and Promotes Gliogenesis from Neural Stem Cells

Neural precursor cells (NPCs) are activated in central nervous system injury. However, despite being multipotential, their progeny differentiates into astrocytes rather than neurons in situ. We have investigated the role of epidermal growth factor receptor (EGFR) in the generation of non‐neurogenic conditions. Cultured mouse subventricular zone NPCs exposed to differentiating conditions for 4 days generated approximately 50% astrocytes and 30% neuroblasts. Inhibition of EGFR with 4‐(3‐chloroanilino)‐6,7‐dimethoxyquinazoline significantly increased the number of neuroblasts and decreased that of astrocytes. The same effects were observed upon treatment with the metalloprotease inhibitor galardin, N‐[(2R)‐2‐(hydroxamidocarbonylmethyl)‐4‐methylpentanoyl]‐L‐tryptophan methylamide (GM 6001), which prevented endogenous transforming growth factor‐α (TGF‐α) release. These results suggested that metalloprotease‐dependent EGFR‐ligand shedding maintained EGFR activation and favored gliogenesis over neurogenesis. Using a disintegrin and metalloprotease 17 (ADAM‐17) small interference RNAs transfection of NPCs, ADAM‐17 was identified as the metalloprotease involved in cell differentiation in these cultures. In vivo experiments revealed a significant upregulation of ADAM‐17 mRNA and de novo expression of ADAM‐17 protein in areas of cortical injury in adult mice. Local NPCs, identified by nestin staining, expressed high levels of ADAM‐17, as well as TGF‐α and EGFR, the three molecules necessary to prevent neurogenesis and promote glial differentiation in vitro. Chronic local infusions of GM6001 resulted in a notable increase in the number of neuroblasts around the lesion. These results indicate that, in vivo, the activation of a metalloprotease, most probably ADAM‐17, initiates EGFR‐ligand shedding and EGFR activation in an autocrine manner, preventing the generation of new neurons from NPCs. Inhibition of ADAM‐17, the limiting step in this sequence, may contribute to the generation of neurogenic niches in areas of brain damage. STEM CELLS 2011;29:1628–1639

12-Deoxyphorbols Promote Adult Neurogenesis by Inducing Neural Progenitor Cell Proliferation via PKC Activation.

Background: Neuropsychiatric and neurological disorders frequently occur after brain insults associated with neuronal loss. Strategies aimed to facilitate neuronal renewal by promoting neurogenesis constitute a promising therapeutic option to treat neuronal death-associated disorders. In the adult brain, generation of new neurons occurs physiologically throughout the entire life controlled by extracellular molecules coupled to intracellular signaling cascades. Proteins participating in these cascades within neurogenic regions constitute potential pharmacological targets to promote neuronal regeneration of injured areas of the central nervous system. Methodology: We have performed in vitro and in vivo approaches to determine neural progenitor cell proliferation to understand whether activation of kinases of the protein kinase C family facilitates neurogenesis in the adult brain. Results: We have demonstrated that protein kinase C activation by phorbol-12-myristate-13-acetate induces neural progenitor cell proliferation in vitro. We also show that the nontumorogenic protein kinase C activator prostratin exerts a proliferative effect on neural progenitor cells in vitro. This effect can be reverted by addition of the protein kinase C inhibitor G06850, demonstrating that the effect of prostratin is mediated by protein kinase C activation. Additionally, we show that prostratin treatment in vivo induces proliferation of neural progenitor cells within the dentate gyrus of the hippocampus and the subventricular zone. Finally, we describe a library of diterpenes with a 12-deoxyphorbol structure similar to that of prostratin that induces a stronger effect than prostratin on neural progenitor cell proliferation both in vitro and in vivo. Conclusions: This work suggests that protein kinase C activation is a promising strategy to expand the endogenous neural progenitor cell population to promote neurogenesis and highlights the potential of 12-deoxyphorbols as pharmaceutical agents to facilitate neuronal renewal.