A new monoclonal antibody, 4F2, specific for the oligodendroglial cell lineage, recognizes ATP-dependent RNA helicase Ddx54: possible association with myelin basic protein.

Recent research in neural development has highlighted the importance of markers to discriminate phenotypic alterations of neural cells at various developmental stages. We isolated a new monoclonal antibody, 4F2, which was shown to be specific for an oligodendrocyte lineage. In primary cultures of oligodendroglial and mixed neural cells, the 4F2 antibody labeled a large proportion of Sox2(+) , Sox10(+) , A2B5(+) , NG2(+) , Olig2(+) , O4(+) , and myelin basic protein (MBP)(+) cells but did not label any GFAP(+) or NeuN(+) cells. In immunohistochemisty of rat embryos, the 4F2 antibody labeled a portion of neuroepithelial cells of the neural tube at embryonic day 9. The 4F2-positive cells were located initially in the ventricular zone as Musashi1(+) Tuj1(-) populations and distributed throughout the striatum; thereafter, they populated the whole brain and spinal cord. These cells showed ramified processes during embryonal development. The 4F2 antigen was associated with all four isoforms of MBP in coimmunoprecipitation experiments using brain homogenates or cell lysates of cultured oligodendrocytes. Immunoscreening of a brain cDNA library identified the antigen as DEAD (Asp-Glu-Ala-Asp) box polypeptide 54 (Ddx54), a member of the DEAD box family of RNA helicases involved in RNA metabolism, transcription, and translation. Cotransfection of the Ddx54 gene with MBP isoform genes increased the nuclear localization of the 21.5-kDa MBP isoform, which has been reported to function as a nuclear signal transduction molecule. These data indicate that Ddx54 might be not only a useful marker for investigating the ontogeny of oligodendrocytes but also an important factor in oligodendrocyte differentiation and myelination.

Essential role of the Hedgehog signaling pathway in human glioma-initiating cells.

Recent findings have demonstrated that malignant tumors, including glioblastoma multiforme, contain cancer-initiating cells (also known as cancer stem cells), which self-renew and are malignant, with features of tissue-specific stem cells. As these cells are resistant to irradiation and anti-cancer drugs, it is important to characterize them and find targeting therapies. In this study, we established two primary human glioma cell lines from anaplastic oligodendroglioma and glioblastoma multiforme. These lines were enriched in glioma-initiating cells, as just 10 cells formed malignant glioma when injected into mouse brain. We used these cell lines to examine the roles of the Notch, Hedgehog and Wnt signaling pathways, which are involved in stem-cell maintenance and tumorigenesis, to determine which of these pathways are crucial to glioma-initiating cells and their regulation. Here we show that the Hedgehog pathway is indispensable for glioma-initiating cell proliferation and tumorigenesis; the Hedgehog signaling inhibitors prevented glioma-initiating cell proliferation, while signaling inhibitors for Notch or Wnt did not. Overexpression of Gli2ΔC, a C-terminal-truncated form of Gli2 that antagonizes Gli transcription factor functions, blocked glioma-initiating cell proliferation in culture and tumorigenesis in vivo. Knockdown of the Gli downstream factor Cdc2 also prevented glioma-initiating cell proliferation. Taken together, these results show that the Hedgehog→ Gli→Cdc2 signaling cascade plays a role in the proliferation and malignancy of glioma-initiating cells.

Gli2 is a novel regulator of sox2 expression in telencephalic neuroepithelial cells.

Multipotential neural stem cells (NSCs) in the central nervous system (CNS) proliferate indefinitely and give rise to neurons, astrocytes, and oligodendrocytes. As NSCs hold promise for CNS regeneration, it is important to understand how their proliferation and differentiation are controlled. We show here that the expression of sox2 gene, which is essential for the maintenance of NSCs, is regulated by the Gli2 transcription factor, a downstream mediator of sonic hedgehog (Shh) signaling: Gli2 binds to an enhancer that is vital for sox2 expression in telencephalic neuroepithelial (NE) cells, which consist of NSCs and neural precursor cells. Overexpression of a truncated form of Gli2 (Gli2DeltaC) or Gli2-specific short hairpin RNA (Gli2 shRNA) in NE cells in vivo and in vitro inhibits cell proliferation and the expression of Sox2 and other NSC markers, including Hes1, Hes5, Notch1, CD133, and Bmi1. It also induces premature neuronal differentiation in the developing NE cells. In addition, we show evidence that Sox2 expression decreases significantly in the developing neuroepithelium of Gli2-deficient mice. Finally, we demonstrate that coexpression of Gli2DeltaC and Sox2 can rescue the expression of Hes5 and prevent premature neuronal differentiation in NE cells but cannot rescue its proliferation. Thus these data reveal a novel transcriptional cascade, involving Gli2 --> Sox2 --> Hes5, which maintains the undifferentiated state of telencephalic NE cells.

Influence of LIF and BMP-2 on differentiation and development of glial cells in primary cultures of embryonic rat cerebral hemisphere.

Cells prepared from the cerebral hemisphere of embryonic Day 18 rats were maintained for 2 days in serum-free modified Bottenstein-Sato (mBS) medium containing thyroid hormone (TH), with or without leukemia inhibitory factor (LIF) or bone morphogenetic protein (BMP)-2, and these influences on the differentiation and development of glial cells were investigated using the cells maintained in mBS medium containing TH as controls. The levels of glial fibrillary acidic protein (GFAP) expression and the number of GFAP-positive astrocytes increased markedly with the addition of LIF or BMP-2, and were enhanced further with the addition of both LIF and BMP-2. The number of O1-positive oligodendrocytes increased with the addition of LIF, whereas it decreased with the addition of BMP-2. The number did not change with the addition of both cytokines. Using antibody against platelet-derived growth factor (PDGF), we then excluded indirect effects of these cytokines through PDGF, which would increase by accelerated astrocyte development. When PDGF was neutralized, the number of oligodendrocytes increased under all conditions examined. As a result of the neutralization, the effect of BMP-2 on oligodendrocyte differentiation was eliminated, although LIF remained effective. These results suggest that the differentiation of oligodendrocytes was delayed partially by PDGF even in control cultures. It is also suggested that LIF and BMP-2, each of which accelerates the differentiation and development of astrocytes, would seem to have different effects on oligodendrocyte differentiation, i.e., LIF would directly affect oligodendrocyte differentiation, whereas BMP-2 would affect it mainly through PDGF.

Beta-naphthoflavone disturbs astrocytic differentiation of C6 glioma cells by inhibiting autocrine interleukin-6.

Regulation of astrocyte differentiation is a key process in the development of the central nervous system (CNS), and disturbance of the differentiation can lead to brain system dysfunction. Here we show that beta-naphthoflavone (betaNF), an agonist of the aryl hydrocarbon receptor (AhR), disturbed the cAMP-induced astrocytic differentiation of C6 glioma by inhibiting autocrine interleukin-6 (IL-6). Treatment of cells with betaNF reduced the induction of an astrocyte marker glial fibrillary acidic protein (GFAP). This was caused by the inactivation of its upstream transcription factor signal transducer and activator of transcription 3 (STAT3) by betaNF. In addition, betaNF attenuated the induction of the IL-6 gene, which leads to the activation of STAT3. Most importantly, the inhibitory effect of betaNF on GFAP promoter activity was recovered by the addition of recombinant IL-6. Taken together, these results indicate that the inhibitory effect of betaNF on IL-6 induction suppresses STAT3 activation. These processes subsequently lead to the attenuation of GFAP induction.

cAMP-induced astrocytic differentiation of C6 glioma cells is mediated by autocrine interleukin-6.

Elevation in the level of intracellular cAMP is known to induce the astrocytic differentiation of C6 glioma cells by unknown mechanisms. In this report, we show that cAMP-induced autocrine interleukin 6 (IL-6) promoted astrocytic differentiation of C6 cells. Treatment of cells with N(6),2'-O-dibutyryl cAMP (Bt(2)AMP) and theophylline caused the delayed phosphorylation of signal transducer and activator of transcription 3 (STAT3), as well as the expression of an astrocyte marker, glial fibrillary acidic protein (GFAP). Overexpression of the dominant-negative form of STAT3 leads to the suppression of GFAP promoter activity, suggesting that STAT3 activity was essential for cAMP-induced GFAP promoter activation. On the other hand, the IL-6 gene was quickly induced by Bt(2)AMP/theophylline, and subsequent IL-6 protein secretion was stimulated. In addition, recombinant IL-6 induced GFAP expression and STAT3 phosphorylation. Most importantly, treatment with IL-6-neutralizing antibody dramatically reduced the cAMP-induced GFAP expression and STAT3 phosphorylation and reversed the cellular morphological changes that had been caused by Bt(2)AMP/theophylline. Taken together, these results indicated that Bt(2)AMP/theophylline lead to delayed STAT3 activation via autocrine IL-6. These processes subsequently led to the induction of GFAP. IL-6 secretion is thus thought to be a key event in controlling the astrocytic differentiation of C6 cells.

Selective disappearance of an axonal protein, 440-kDa ankyrinB, associated with neuronal degeneration induced by methylmercury.

The 440-kDa isoform of brain ankyrin, 440-kDa ankyrinB, is a neuron-specific protein and is confined to axons. Cerebellum is one of the areas characteristically altered by methylmercury intoxication both in the adult and during development. When rat cerebellar neurons matured for 7 days in vitro were exposed to methylmercury at 0.03 microM for 48 hr, viability of the cells was unaffected. However, the immunocytochemical staining of 440-kDa ankyrinB diminished drastically, whereas that of microtubule-associated protein-2, which is localized in dendrites and cell bodies, and of glial fibrillary acidic protein (GFAP), a marker for astroglial cells coexisting in the culture, remained unchanged. To confirm these observations, a simplified dot blot assay was established to determine 440-kDa ankyrinB and several other marker proteins in cultured cell samples. With this assay, we found that methylmercury at a submicromolar range induced a decrease of 440-kDa ankyrinB and an increase of GFAP in a dose-dependent manner in cerebellar cells in primary culture. Surprisingly, another axonal protein, tau, remained mostly in its intact molecular sizes even in the presence of 0.3-1.0 microM methylmercury, though its immunocytochemical localization was substantially altered. These results indicate that selective loss of the axonal protein 440-kDa ankyrinB is associated with the early stage of degeneration of cerebellar neurons induced by methylmercury. Therefore, 440-kDa ankyrinB should be useful as a specific and sensitive marker for the neurotoxicity of methylmercury.

Influence of cell density and thyroid hormone on glial cell development in primary cultures of embryonic rat cerebral hemisphere.

The influence of cell density and thyroid hormone (TH) on the development of astrocytes and oligodendrocytes was investigated in primary cultures prepared from rat cerebral hemisphere on embryonic day (E)18. At the beginning of the culture, most of the cells were microtubule-associated protein 2 (MAP2)-positive neurons, whereas O1-positive oligodendrocytes and glial fibrillary acidic protein (GFAP)-positive astrocytes were rarely observed. After the cells were maintained in serum-free defined medium, astrocytes developed at high cell density but rarely at a low one. When leukemia inhibitory factor (LIF) was supplemented in low-density cultures, the levels of GFAP expression markedly increased to almost the same extent as in high-density culture without TH. This suggests that, in low-density cultures, astrocyte progenitors could not differentiate because of insufficient astrocyte-inducing factors. Interestingly, the addition of TH increased GFAP expression levels only at high density. The number of oligodendrocytes increased with TH addition at both cell densities, although the effects were more remarkable at high density. These results suggest that cell density and TH are pivotal factors in the development of both astrocytes and oligodendrocytes. It is also suggested that the effects of TH on glial cell development could be accelerated via cell-cell communications.