The CB1 cannabinoid receptor mediates excitotoxicity-induced neural progenitor proliferation and neurogenesis

Endocannabinoids are lipid signaling mediators that exert an important neuromodulatory role and confer neuroprotection in several types of brain injury. Excitotoxicity and stroke can induce neural progenitor (NP) proliferation and differentiation as an attempt of neuroregeneration after damage. Here we investigated the mechanism of hippocampal progenitor cell engagement upon excitotoxicity induced by kainic acid administration and the putative involvement of the CB1 cannabinoid receptor in this process. Adult NPs express kainate receptors that mediate proliferation and neurosphere generation in vitro via CB1 cannabinoid receptors. Similarly, in vivo studies showed that excitotoxicity-induced hippocampal NPs proliferation and neurogenesis are abrogated in CB1-deficient mice and in wild-type mice administered with the selective CB1 antagonist rimonabant (N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazolecarboxamide; SR141716). Kainate stimulation increased basic fibroblast growth factor (bFGF) expression in cultured NPs in a CB1-dependent manner as this response was prevented by rimonabant and mimicked by endocannabinoids. Likewise, in vivo analyses showed that increased hippocampal expression of bFGF, as well as of brain-derived neurotrophic factor and epidermal growth factor, occurs upon excitotoxicity and that CB1 receptor ablation prevents this induction. Moreover, excitotoxicity increased the number of CB1+ bFGF+ cells, and this up-regulation preceded NP proliferation. In summary, our results show the involvement of the CB1 cannabinoid receptor in NP proliferation and neurogenesis induced by excitotoxic injury and support a role for bFGF signaling in this process.     

Curcumin stimulates proliferation of embryonic neural progenitor cells and neurogenesis in the adult hippocampus

Curcumin is a natural phenolic component of yellow curry spice, which is used in some cultures for the treatment of diseases associated with oxidative stress and inflammation. Curcumin has been reported to be capable of preventing the death of neurons in animal models of neurodegenerative disorders, but its possible effects on developmental and adult neuroplasticity are unknown. In the present study, we investigated the effects of curcumin on mouse multi-potent neural progenitor cells (NPC) and adult hippocampal neurogenesis. Curcumin exerted biphasic effects on cultured NPC; low concentrations stimulated cell proliferation, whereas high concentrations were cytotoxic. Curcumin activated extracellular signal-regulated kinases (ERKs) and p38 kinases, cellular signal transduction pathways known to be involved in the regulation of neuronal plasticity and stress responses. Inhibitors of ERKs and p38 kinases effectively blocked the mitogenic effect of curcumin in NPC. Administration of curcumin to adult mice resulted in a significant increase in the number of newly generated cells in the dentate gyrus of hippocampus, indicating that curcumin enhances adult hippocampal neurogenesis. Our findings suggest that curcumin can stimulate developmental and adult hippocampal neurogenesis, and a biological activity that may enhance neural plasticity and repair.     

Taurine stimulates proliferation and promotes neurogenesis of mouse adult cultured neural stem/progenitor cells

This study reports an effect of taurine (1-10 mM) increasing markedly (120%) the number of neural precursor cells (NPCs) from adult mouse subventricular zone, cultured as neurospheres. This effect is one of the highest reported for adult neural precursor cells. Taurine-containing cultures showed 73-120% more cells than controls, after 24 and 96 h in culture, respectively. Taurine effect is due to enhanced proliferation as assessed by BrdU incorporation assays. In taurine cultures BrdU incorporation was markedly higher than controls from 1.5 to 48 h, with the maximal difference found at 1.5 h. This effect of taurine reproduced at every passage with the same window time. Taurine effects are not mimicked by glycine, alanine or GABA. Clonal efficiency values of 3.6% for taurine cultures and 1.3% for control cultures suggest a taurine influence on both, progenitor and stem cells. Upon differentiation, the proportion of neurons in control and taurine cultures was 3.1% (±0.5) and 10.2% (±0.8), respectively. These results are relevant for taurine implication in brain development as well as in adult neurogenesis. Possible mechanisms underlying taurine effects on cell proliferation are discussed.     

Chondroitin sulfate glycosaminoglycans control proliferation, radial glia cell differentiation and neurogenesis in neural stem/progenitor cells

Although the local environment is known to regulate neural stem cell (NSC) maintenance in the central nervous system, little is known about the molecular identity of the signals involved. Chondroitin sulfate proteoglycans (CSPGs) are enriched in the growth environment of NSCs both during development and in the adult NSC niche. In order to gather insight into potential biological roles of CSPGs for NSCs, the enzyme chondroitinase ABC (ChABC) was used to selectively degrade the CSPG glycosaminoglycans. When NSCs from mouse E13 telencephalon were cultivated as neurospheres, treatment with ChABC resulted in diminished cell proliferation and impaired neuronal differentiation, with a converse increase in astrocytes. The intrauterine injection of ChABC into the telencephalic ventricle at midneurogenesis caused a reduction in cell proliferation in the ventricular zone and a diminution of self-renewing radial glia, as revealed by the neurosphere-formation assay, and a reduction in neurogenesis. These observations suggest that CSPGs regulate neural stem/progenitor cell proliferation and intervene in fate decisions between the neuronal and glial lineage.     

A member of a novel family of yeast 'zn-finger' proteins mediates the transition from stationary phase to cell proliferation.

The cloning and molecular characterization of the GCS1 gene from the budding yeast Saccharomyces cerevisiae show that stationary phase is in fact a unique developmental state, with requirements to resume cell proliferation that can be distinct from those for maintenance of proliferation. Deletion of the GCS1 gene products a novel phenotype: stationary-phase mutant cells do not resume proliferation at a restrictive temperature of 15 degrees C, but mutant cells lacking Gcs1p that are proliferating at the permissive temperature of 29 degrees C continue to proliferate after transfer to 15 degrees C as long as nutrients are available. The GCS1 gene sequence predicts a 39 kDa polypeptide with a novel 'Zn-finger' motif. A point mutation within the finger motif produces a phenotype that mimics that of deletion of the GCS1 gene, showing that the finger motif is essential for full Gcs1p activity. Gcs1p and the products of two newly identified genes, SPS18 and GLO3, constitute a family of novel Zn-finger proteins.     

Keeping neural progenitor cells on a short leash during Drosophila neurogenesis

The developmental potential of stem cells and progenitor cells must be functionally distinguished to ensure the generation of diverse cell types while maintaining the stem cell pool throughout the lifetime of an organism. In contrast to stem cells, progenitor cells possess restricted developmental potential, allowing them to give rise to only a limited number of post-mitotic progeny. Failure to establish or maintain restricted progenitor cell potential can perturb tissue development and homeostasis, and probably contributes to tumor initiation. Recent studies using the developing fruit fly Drosophila larval brain have provided molecular insight into how the developmental potential is restricted in neural progenitor cells.     

Borna disease virus infects human neural progenitor cells and impairs neurogenesis

Understanding the complex mechanisms by which infectious agents can disrupt behavior represents a major challenge. The Borna disease virus (BDV), a potential human pathogen, provides a unique model to study such mechanisms. Because BDV induces neurodegeneration in brain areas that are still undergoing maturation at the time of infection, we tested the hypothesis that BDV interferes with neurogenesis. We showed that human neural stem/progenitor cells are highly permissive to BDV, although infection does not alter their survival or undifferentiated phenotype. In contrast, upon the induction of differentiation, BDV is capable of severely impairing neurogenesis by interfering with the survival of newly generated neurons. Such impairment was specific to neurogenesis, since astrogliogenesis was unaltered. In conclusion, we demonstrate a new mechanism by which BDV might impair neural function and brain plasticity in infected individuals. These results may contribute to a better understanding of behavioral disorders associated with BDV infection.     

Involvement of gangliosides in proliferation of immortalized neural progenitor cells

The CNS consists of neuronal and glial cells generated from common neural progenitor cells during development. Cellular events for neural progenitor cells, such as proliferation and differentiation, are regulated by multiple intrinsic and extrinsic cell signals. Although much is known on the importance of the proteinous factors in regulating the fate of neural progenitor cells, the involvement of other molecules such as gangliosides, sialic acid-containing glycosphingolipids, remains to be clarified. To elucidate the biological functions of gangliosides in neural progenitor cells, we transfected an immortalized neural progenitor cell line, C17.2, which does not express GD3 ganglioside, with a fusion protein of GD3-synthase (ST-II) and enhanced green fluorescent protein (ST-II-EGFP). Analysis of the ST-II transfectants revealed the ectopic expression of b- and c-series gangliosides. In the ST-II transfectants, proliferation induced by epidermal growth factor (EGF) was severely retarded. EGF-induced proliferation of C17.2 cells was dependent on the Ras-mitogen-activated protein kinase (Ras-MAPK) pathway, and the EGF-induced activation of this pathway was significantly repressed in the transfectants. Thus, ST-II overexpression retarded proliferation of C17.2 cells via repression of the Ras-MAPK pathway. The result supports the concept that gangliosides may play an important role in regulating the proliferation of neural progenitor cells.     

PI3K‐p110‐alpha‐subtype signalling mediates survival,proliferation and neurogenesis of cortical progenitor cells via activation of mTORC2

Development of the cerebral cortex is controlled by growth factors among which transforming growth factor beta (TGFβ) and insulin‐like growth factor 1 (IGF1) have a central role. The TGFβ‐ and IGF1‐pathways cross‐talk and share signalling molecules, but in the central nervous system putative points of intersection remain unknown. We studied the biological effects and down‐stream molecules of TGFβ and IGF1 in cells derived from the mouse cerebral cortex at two developmental time points, E13.5 and E16.5. IGF1 induces PI3K, AKT and the mammalian target of rapamycin complexes (mTORC1/mTORC2) primarily in E13.5‐derived cells, resulting in proliferation, survival and neuronal differentiation, but has small impact on E16.5‐derived cells. TGFβ has little effect at E13.5. It does not activate the PI3K‐ and mTOR‐signalling network directly, but requires its activity to mediate neuronal differentiation specifically at E16.5. Our data indicate a central role of mTORC2 in survival, proliferation as well as neuronal differentiation of E16.5‐derived cortical cells. mTORC2 promotes these cellular processes and is under control of PI3K‐p110‐alpha signalling. PI3K‐p110‐beta signalling activates mTORC2 in E16.5‐derived cells but it does not influence cell survival, proliferation and differentiation. This finding indicates that different mTORC2 subtypes may be implicated in cortical development and that these subtypes are under control of different PI3K isoforms.

     

Intercellular signalling in the transition from stem cells to organogenesis in meristems

Meristems continuously produce new cells to sustain plant growth. Stem cells are maintained in the centre of the meristem and provide the precursor cells for the initiation of new organs and tissues in the periphery. The structure of the meristem is maintained while cells are constantly displaced by new divisions. Recent advances have been made in understanding the intercellular signals that maintain meristem structure by adjusting gene expression according to cell position. In addition to refinements in our understanding of how the position and size of the stem-cell population is regulated, there have been advances in understanding how the location of new organ primordia is controlled and how the meristem influences organ polarity.     

A key role for Pak4 in proliferation and differentiation of neural progenitor cells

The Pak4 serine/threonine kinase regulates cytoskeletal organization, and controls cell growth, proliferation, and survival. Deletion of Pak4 in mice results in embryonic lethality prior to embryonic day 11.5. Pak4 knockout embryos exhibit abnormalities in the nervous system, the heart, and other tissues. In this study a conditional deletion of Pak4 was generated in order to study the function of Pak4 in the development of the brain. Nervous system-specific conditional deletion of Pak4 was accomplished by crossing mice with a floxed allele of Pak4 with transgenic mice expressing Cre recombinase under the control of the nestin promoter. The conditional Pak4 knockout mice were born normally, but displayed growth retardation and died prematurely. The brains showed a dramatic decrease in proliferation of cortical and striatal neuronal progenitor cells. In vitro analyses revealed a reduced proliferation and self-renewing capacity of neural progenitor cells isolated from Pak4 knockout brains. The mice also exhibited cortical thinning, impaired neurogenesis and loss of neuroepithelial adherens junctions. By the time the mice died, by 4 weeks after birth, severe hydrocephalus could also be seen. These results suggest that Pak4 plays a critical role in the regulation of neural progenitor cell proliferation and in establishing the foundation for development of the adult brain.     

p38 MAP kinase mediates nitric oxide-induced apoptosis of neural progenitor cells

Neural progenitor cells (NPC) can proliferate, differentiate into neurons or glial cells, or undergo a form of programmed cell death called apoptosis. Although death of NPC occurs during development of the nervous system and in the adult, the underlying mechanisms are unknown. Here we show that nitric oxide (NO) can induce death of C17.2 NPC by a mechanism requiring activation of p38 MAP kinase, poly(ADP-ribose) polymerase, and caspase-3. Nitric oxide causes release of cytochrome c from mitochondria, and Bcl-2 protects the neural progenitor cells against nitric oxide-induced death, consistent with a pivotal role for mitochondrial changes in controlling the cell death process. Inhibition of p38 MAP kinase by SB203580 abolished NO-induced cell death, cytochrome c release, and activation of caspase-3, indicating that p38 activation serves as an upstream mediator in the cell death process. The anti-apoptotic protein Bcl-2 protected NPC against nitric oxide-induced apoptosis and suppressed activation of p38 MAP kinase. The ability of nitric oxide to trigger death of NPC by a mechanism involving p38 MAP kinase suggests that this diffusible gas may regulate NPC fate in physiological and pathological settings in which NO is produced.     

Gi2 signaling enhances proliferation of neural progenitor cells in the developing brain

Our previous study showed that the pertussis toxin-sensitive G protein, Gi2, is selectively localized in the ventricular zone of embryonic brains, where the neuroepithelial cells undergo active proliferation. In order to clarify the role of Gi2 in this site, we first administered pertussis toxin by an exo-utero manipulation method into the lateral ventricle of mouse brain at embryonic day 14.5. Examination at embryonic day 18.5 revealed that pertussis toxin-injected embryos had brains with thinner cerebral cortices, made up of fewer constituent cells. Bromodeoxyuridine labeling revealed fewer numbers of bromodeoxyuridine-positive cells in the cerebral cortices of pertussis toxin-injected embryos, suggesting impaired proliferation of neuroepithelial cells. Next we cultured neural progenitor cells from rat embryonic brains and evaluated the mitogenic effects of agonists for several Gi-coupled receptors that are known to be expressed in the ventricular zone. Among agonists tested, endothelin most effectively stimulated the incorporation of [3H]thymidine in the presence of fibronectin, via the endothelin-B receptor. This was associated with phosphorylation of extracellular signal-regulated kinase, and pertussis toxin partially inhibited both endothelin-stimulated DNA synthesis and phosphorylation of extracellular signal-regulated kinase. Injection of endothelin-3 into the ventricle of embryonic brains increased numbers of bromodeoxyuridine-positive cells in the cerebral cortex, whereas injection of an endothelin-B receptor antagonist decreased them. These findings indicate that Gi2 mediates signaling from receptors such as the endothelin-B receptor to maintain mitogenic activity in the neural progenitor cells of developing brain.     

DISP3 promotes proliferation and delays differentiation of neural progenitor cells

DISP3 (PTCHD2), a sterol-sensing domain-containing protein, is highly expressed in neural tissue but its role in neural differentiation is unknown. In the present study we used a multipotent cerebellar progenitor cell line, C17.2, to investigate the impact of DISP3 on the proliferation and differentiation of neural precursors. We found that ectopically expressed DISP3 promotes cell proliferation and alters expression of genes that are involved in tumorigenesis. Finally, the differentiation profile of DISP3-expressing cells was altered, as evidenced by delayed expression of neural specific markers and a reduced capacity to undergo neural differentiation.     

Differentiation of insulin-producing cells from human neural progenitor cells

BackgroundSuccess in islet-transplantation-based therapies for type 1 diabetes, coupled with a worldwide shortage of transplant-ready islets, has motivated efforts to develop renewable sources of islet-replacement tissue. Islets and neurons share features, including common developmental programs, and in some species brain neurons are the principal source of systemic insulin.Methods and FindingsHere we show that brain-derived human neural progenitor cells, exposed to a series of signals that regulate in vivo pancreatic islet development, form clusters of glucose-responsive insulin-producing cells (IPCs). During in vitro differentiation of neural progenitor cells with this novel method, genes encoding essential known in vivo regulators of pancreatic islet development were expressed. Following transplantation into immunocompromised mice, IPCs released insulin C-peptide upon glucose challenge, remained differentiated, and did not form detectable tumors.ConclusionProduction of IPCs solely through extracellular factor modulation in the absence of genetic manipulations may promote strategies to derive transplantable islet-replacement tissues from human neural progenitor cells and other types of multipotent human stem cells.     

Glutamate receptor properties of human mesencephalic neural progenitor cells: NMDA enhances dopaminergic neurogenesis in vitro

Human midbrain‐derived neural progenitor cells (NPCs) may serve as a continuous source of dopaminergic neurons for the development of novel regenerative therapies in Parkinson’s disease. However, the molecular and functional characteristics of glutamate receptors in human NPCs are largely unknown. Here, we show that differentiated human mesencepahlic NPCs display a distinct pattern of glutamate receptors. In whole‐cell patch‐clamp recordings, l ‐glutamate and NMDA elicited currents in 93% of NPCs after 3 weeks of differentiation in vitro. The concentration‐response plots of differentiated NPCs yielded an EC₅₀ of 2.2 μM for glutamate and an EC₅₀ of 36 μM for NMDA. Glutamate‐induced currents were markedly inhibited by memantine in contrast to 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) suggesting a higher density of functional NMDA than alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)/kainate receptors. NMDA‐evoked currents and calcium signals were blocked by the NR2B‐subunit specific antagonist ifenprodil indicating functional expression of NMDA receptors containing subunits NR1 and NR2B. In calcium imaging experiments, the blockade of voltage‐gated calcium channels by verapamil abolished AMPA‐induced calcium responses but only partially reduced NMDA‐evoked transients suggesting the expression of calcium‐impermeable, GluR2‐containing AMPA receptors. Quantitative real‐time PCR showed a predominant expression of subunits NR2A and NR2B (NMDA), GluR2 (AMPA), GluR7 (kainate), and mGluR3 (metabotropic glutamate receptor). Treatment of NPCs with 100 μM NMDA in vitro during proliferation (2 weeks) and differentiation (1 week) increased the amount of tyrosine hydroxylase‐immunopositive cells significantly, which was reversed by addition of memantine. These data suggest that NMDA receptors in differentiating human mesencephalic NPCs are important regulators of dopaminergic neurogenesis in vitro.