CB2 cannabinoid receptors promote neural progenitor cell proliferation via mTORC1 signaling

The endocannabinoid system is known to regulate neural progenitor (NP) cell proliferation and neurogenesis. In particular, CB(2) cannabinoid receptors have been shown to promote NP proliferation. As CB(2) receptors are not expressed in differentiated neurons, CB(2)-selective agonists are promising candidates to manipulate NP proliferation and indirectly neurogenesis by overcoming the undesired psychoactive effects of neuronal CB(1) cannabinoid receptor activation. Here, by using NP cells, brain organotypic cultures, and in vivo animal models, we investigated the signal transduction mechanism involved in CB(2) receptor-induced NP cell proliferation and neurogenesis. Exposure of hippocampal HiB5 NP cells to the CB(2) receptor-selective agonist HU-308 led to the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway, which, by inhibiting its downstream target p27Kip1, induced NP proliferation. Experiments conducted with the CB(2) receptor-selective antagonist SR144528, inhibitors of the PI3K/Akt/mTORC1 axis, and CB(2) receptor transient-transfection vector further supported that CB(2) receptors control NP cell proliferation via activation of mTORC1 signaling. Likewise, CB(2) receptor engagement induced cell proliferation in an mTORC1-dependent manner both in embryonic cortical slices and in adult hippocampal NPs. Thus, HU-308 increased ribosomal protein S6 phosphorylation and 5-bromo-2'-deoxyuridine incorporation in wild-type but not CB(2) receptor-deficient NPs of the mouse subgranular zone. Moreover, adult hippocampal NP proliferation induced by HU-308 and excitotoxicity was blocked by the mTORC1 inhibitor rapamycin. Altogether, these findings provide a mechanism of action and a rationale for the use of nonpsychotomimetic CB(2) receptor-selective ligands as a novel strategy for the control of NP cell proliferation and neurogenesis.     

Taurine induces proliferation of neural stem cells and synapse development in the developing mouse brain

Taurine is a sulfur-containing amino acid present in high concentrations in mammalian tissues. It has been implicated in several processes involving brain development and neurotransmission. However, the role of taurine in hippocampal neurogenesis during brain development is still unknown. Here we show that taurine regulates neural progenitor cell (NPC) proliferation in the dentate gyrus of the developing brain as well as in cultured early postnatal (P5) hippocampal progenitor cells and hippocampal slices derived from P5 mice brains. Taurine increased cell proliferation without having a significant effect on neural differentiation both in cultured P5 NPCs as well as cultured hippocampal slices and in vivo. Expression level analysis of synaptic proteins revealed that taurine increases the expression of Synapsin 1 and PSD 95. We also found that taurine stimulates the phosphorylation of ERK1/2 indicating a possible role of the ERK pathway in mediating the changes that we observed, especially in proliferation. Taken together, our results demonstrate a role for taurine in neural stem/progenitor cell proliferation in developing brain and suggest the involvement of the ERK1/2 pathways in mediating these actions. Our study also shows that taurine influences the levels of proteins associated with synapse development. This is the first evidence showing the effect of taurine on early postnatal neuronal development using a combination of in vitro, ex-vivo and in vivo systems.     

Fibroblast growth factor signaling is required for the proliferation and patterning of progenitor cells in the developing anterior pituitary

The proliferation and patterning of progenitor cells in the anterior pituitary require signals derived from the neuroepithelium of the juxtaposed infundibulum. The infundibulum expresses Fibroblast growth factor (Fgf) 8 and Fgf 18, and FGFs can mimic some of the activities of the infundibulum. The requirement for FGF signaling during growth and patterning of the anterior pituitary has not, however, been established. By blocking FGF receptor signaling in explants of the anterior pituitary cultured in vitro we provide evidence that FGF signaling derived from the infundibulum is required for the proliferation and patterning of progenitor cells in the anterior pituitary.     

Akhirin regulates the proliferation and differentiation of neural stem cells/progenitor cells at neurogenic niches in mouse brain

Specialized microenvironment, or neurogenic niche, in embryonic and postnatal mouse brain plays critical roles during neurogenesis throughout adulthood. The subventricular zone (SVZ) and the dentate gyrus (DG) of hippocampus in the mouse brain are two major neurogenic niches where neurogenesis is directed by numerous regulatory factors. Now, we report Akhirin (AKH), a stem cell maintenance factor in mouse spinal cord, plays a pivotal regulatory role in the SVZ and in the DG. AKH showed specific distribution during development in embryonic and postnatal neurogenic niches. Loss of AKH led to abnormal development of the ventricular zone and the DG along with reduction of cellular proliferation in both regions. In AKH knockout mice (AKH^−/−), quiescent neural stem cells (NSCs) increased, while proliferative NSCs or neural progenitor cells decreased at both neurogenic niches. In vitro NSC culture assay showed increased number of neurospheres and reduced neurogenesis in AKH^−/−. These results indicate that AKH, at the neurogenic niche, exerts dynamic regulatory role on NSC self-renewal, proliferation and differentiation during SVZ and hippocampal neurogenesis.     

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.     

Endogenous erythropoietin signaling is required for normal neural progenitor cell proliferation

Erythropoietin (Epo) and its receptor (EpoR), critical for erythropoiesis, are expressed in the nervous system. Prior to death in utero because of severe anemia EpoR-null mice have fewer neural progenitor cells, and differentiated neurons are markedly sensitive to hypoxia, suggesting that during development Epo stimulates neural cell proliferation and prevents neuron apoptosis by promoting oxygen delivery to brain or by direct interaction with neural cells. Here we present evidence that neural progenitor cells express EpoR at higher levels compared with mature neurons; that Epo stimulates proliferation of embryonic neural progenitor cells; and that endogenous Epo contributes to neural progenitor cell proliferation and maintenance. EpoR-null mice were rescued with selective EpoR expression driven by the endogenous EpoR promoter in hematopoietic tissue but not in brain. Although these mice exhibited normal hematopoiesis and erythrocyte production and survived to adulthood, neural cell proliferation and viability were affected. Embryonic brain exhibited increased neural cell apoptosis, and neural cell proliferation was reduced in the adult hippocampus and subventricular zone. Neural cells from these animals were more sensitive to hypoxia/glutamate neurotoxicity than normal neurons in culture and in vivo. These observations demonstrate that endogenous Epo/EpoR signaling promotes cell survival in embryonic brain and contributes to neural cell proliferation in adult brain in regions associated with neurogenesis. Therefore, Epo exerts extra-hematopoietic function and contributes directly to brain development, maintenance, and repair by promoting cell survival and proliferation independent of insult, injury, or ischemia.     

Non-canonical Wnt signaling enhances differentiation of human circulating progenitor cells to cardiomyogenic cells

Human endothelial circulating progenitor cells (CPCs) can differentiate to cardiomyogenic cells during co-culture with neonatal rat cardiomyocytes. Wnt proteins induce myogenic specification and cardiac myogenesis. Here, we elucidated the effect of Wnts on differentiation of CPCs to cardiomyogenic cells. CPCs from peripheral blood mononuclear cells were isolated from healthy volunteers and co-cultured with neonatal rat cardiomyocytes. 6-10 days after co-culture, cardiac differentiation was determined by alpha-sarcomeric actinin staining of human lymphocyte antigen-positive cells (fluorescence-activated cell-sorting analysis) and mRNA expression of human myosin heavy chain and atrial natriuretic peptide. Supplementation of co-cultures with Wnt11-conditioned medium significantly enhanced the differentiation of CPCs to cardiomyocytes (1.7+/-0.3-fold), whereas Wnt3A-conditioned medium showed no effect. Cell fusion was not affected by Wnt11-conditioned medium. Because Wnts inhibit glycogen synthase kinase-3beta, we further determined whether the glycogen synthase kinase-3beta inhibitor LiCl also enhanced cardiac differentiation of CPCs. However, LiCl (10 mM) did not affect CPC differentiation. In contrast, Wnt11-conditioned medium time-dependently activated protein kinase C (PKC). Moreover, the PKC inhibitors bisindolylmaleimide I and III significantly blocked differentiation of CPCs to cardiomyocytes. PKC activation by phorbol 12-myristate 13-acetate significantly increased CPC differentiation to a similar extent as compared with Wnt11-conditioned medium. Our data demonstrate that Wnt11, but not Wnt3A, augments cardiomyogenic differentiation of human CPCs. Wnt11 promotes cardiac differentiation via the non-canonical PKC-dependent signaling pathway.     

Aberrant Otx2 expression enhances migration and induces ectopic proliferation of hindbrain neuronal progenitor cells

Dysregulation of Otx2 is a hallmark of the pediatric brain tumor medulloblastoma, yet its functional significance in the establishment of these tumors is unknown. Here we have sought to determine the functional consequences of Otx2 overexpression in the mouse hindbrain to characterize its potential role in medulloblastoma tumorigenesis and identify the cell types responsive to this lineage-specific oncogene. Expression of Otx2 broadly in the mouse hindbrain resulted in the accumulation of proliferative clusters of cells in the cerebellar white matter and dorsal brainstem of postnatal mice. We found that brainstem ectopia were derived from neuronal progenitors of the rhombic lip and that cerebellar ectopia were derived from granule neuron precursors (GNPs) that had migrated inwards from the external granule layer (EGL). These hyperplasias exhibited various characteristics of medulloblastoma precursor cells identified in animal models of Shh or Wnt group tumors, including aberrant localization and altered spatiotemporal control of proliferation. However, ectopia induced by Otx2 differentiated and dispersed as the animals reached adulthood, indicating that factors restricting proliferative lifespan were a limiting factor to full transformation of these cells. These studies implicate a role for Otx2 in altering the dynamics of neuronal progenitor cell proliferation.     

The brain tumor gene negatively regulates neural progenitor cell proliferation in the larval central brain of Drosophila

Brain development in Drosophila is characterized by two neurogenic periods, one during embryogenesis and a second during larval life. Although much is known about embryonic neurogenesis, little is known about the genetic control of postembryonic brain development. Here we use mosaic analysis with a repressible cell marker (MARCM) to study the role of the brain tumor (brat) gene in neural proliferation control and tumour suppression in postembryonic brain development of Drosophila. Our findings indicate that overproliferation in brat mutants is due to loss of proliferation control in the larval central brain and not in the optic lobe. Clonal analysis indicates that the brat mutation affects cell proliferation in a cell-autonomous manner and cell cycle marker expression shows that cells of brat mutant clones show uncontrolled proliferation, which persists into adulthood. Analysis of the expression of molecular markers, which characterize cell types in wild-type neural lineages, indicates that brat mutant clones comprise an excessive number of cells, which have molecular features of undifferentiated progenitor cells that lack nuclear Prospero (Pros). pros mutant clones phenocopy brat mutant clones in the larval central brain, and targeted expression of wild-type pros in brat mutant clones promotes cell cycle exit and differentiation of brat mutant cells, thereby abrogating brain tumour formation. Taken together, our results provide evidence that the tumour suppressor brat negatively regulates cell proliferation during larval central brain development of Drosophila, and suggest that Prospero acts as a key downstream effector of brat in cell fate specification and proliferation control.     

Cdk2 is critical for proliferation and self-renewal of neural progenitor cells in the adult subventricular zone

We investigated the function of cyclin-dependent kinase 2 (Cdk2) in neural progenitor cells during postnatal development. Chondroitin sulfate proteoglycan (NG2)–expressing progenitor cells of the subventricular zone (SVZ) show no significant difference in density and proliferation between Cdk2^−/− and wild-type mice at perinatal ages and are reduced only in adult Cdk2^−/− mice. Adult Cdk2^−/− SVZ cells in culture display decreased self-renewal capacity and enhanced differentiation. Compensatory mechanisms in perinatal Cdk2^−/− SVZ cells, which persist until postnatal day 15, involve increased Cdk4 expression that results in retinoblastoma protein inactivation. A subsequent decline in Cdk4 activity to wild-type levels in postnatal day 28 Cdk2^−/− cells coincides with lower NG2⁺ proliferation and self-renewal capacity similar to adult levels. Cdk4 silencing in perinatal Cdk2^−/− SVZ cells abolishes Cdk4 up-regulation and reduces cell proliferation and self- renewal to adult levels. Conversely, Cdk4 overexpression in adult SVZ cells restores proliferative capacity to wild-type levels. Thus, although Cdk2 is functionally redundant in perinatal SVZ, it is important for adult progenitor cell proliferation and self-renewal through age-dependent regulation of Cdk4.     

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.     

Gene therapy of experimental brain tumors using neural progenitor cells

Glioblastomas, the most frequent and malignant of primary brain tumors, have a very poor prognosis. Gene therapy of glioblastomas is limited by the short survival of viral vectors and by their difficulty in reaching glioblastoma cells infiltrating the brain parenchyma. Neural stem/progenitor cells can be engineered to produce therapeutic molecules and have the potential to overcome these limitations because they may travel along the white matter, like neoplastic cells, and engraft stably into the brain. Retrovirus-mediated transfer of the gene for interleukin-4 is an effective treatment for rat brain glioblastomas. Here, we transferred the gene for interleukin-4 into C57BL6J mouse primary neural progenitor cells and injected those cells into established syngeneic brain glioblastomas. This led to the survival of most tumor-bearing mice. We obtained similar results by implanting immortalized neural progenitor cells derived from Sprague-Dawley rats into C6 glioblastomas. We also documented by magnetic resonance imaging the progressive disappearance of large tumors, and detected 5-bromodeoxyuridine-labeled progenitor cells several weeks after the injection. These findings support a new approach for gene therapy of brain tumors, based on the grafting of neural stem cells producing therapeutic molecules.     

HMGB1 enhances embryonic neural stem cell proliferation by activating the MAPK signaling pathway

Neural stem cells (NSCs) are involved in neural tube formation. As the high-mobility group box 1 (HMGB1) protein is involved in neurulation and is present at elevated levels in neural tube defects (NTDs) induced by hyperthermia, we have now investigated the effects of HMGB1 on proliferation, differentiation, and MAPK signaling pathways of NSCs in vitro. We constructed a lentivirus vector with HMGB1 siRNA and used it to infect NSCs. Down-regulation of HMGB1 expression was confirmed. Proliferation of NSCs was determined by MTS and nestin/BrdU double-labeling. Differentiation of NSCs was assessed using β-tubulinIII and GFAP. Knockdown of HMGB1 significantly suppressed NSC proliferation but hardly affected differentiation, which was regulated by decreased expression of MAPK signaling pathways. Thus, HMGB1 has beneficial effects on neurulation and may serve as a new target for the prevention of NTDs.