Expression of the human PAC1 receptor leads to dose-dependent hydrocephalus-related abnormalities in mice.
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Hydrocephalus is a common and potentially devastating birth defect affecting the CNS, and its relationship with G protein-coupled receptors (GPCRs) is unknown. We have expressed 2, 4, or 6 copies of a GPCR--the human PAC1 receptor with a 130-kb transgene in the mouse nervous system in a pattern closely resembling that of the endogenous gene. Consistent with PAC1 actions, PKA and PKC activity were elevated in the brains of Tg mice. Remarkably, Tg mice developed dose-dependent hydrocephalus-like characteristics, including enlarged third and lateral ventricles and reduced cerebral cortex, corpus callosum, and subcommissural organ (SCO). Neuronal proliferation and apoptosis were implicated in hydrocephalus, and we observed significantly reduced neuronal proliferation and massively increased neuronal apoptosis in the developing cortex and SCO of Tg embryos, while neurite outgrowth and neuronal migration in vitro remain uncompromised. Ventricular ependymal cilia are crucial for directing cerebrospinal fluid flow, and ependyma of Tg mice exhibited disrupted cilia with increased phospho-CREB immunoreactivity. These data demonstrate that altered neuronal proliferation/apoptosis and disrupted ependymal cilia are the main factors contributing to hydrocephalus in PAC1-overexpressing mice. This is the first report to our knowledge demonstrating that misregulation of GPCRs can be involved in hydrocephalus-related neurodevelopmental disorders. (+info)
Rat bone marrow progenitor cells transduced in situ by rSV40 vectors differentiate into multiple central nervous system cell lineages.
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Using bone marrow-directed gene transfer, we tested whether bone marrow-derived cells may function as progenitors of central nervous system (CNS) cells in adult animals. SV40-derived gene delivery vectors were injected directly into femoral bone marrow, and we examined transgene expression in blood and brain for 0-16 months thereafter by immunostaining for FLAG epitope marker. An average of 5% of peripheral blood cells and 25% of femoral marrow cells were FLAG(+) throughout the study. CNS FLAG-expressing cells were mainly detected in the dentate gyrus (DG) and periventricular subependymal zone (PSZ). Although absent before 1 month and rare at 4 months, DG and PSZ FLAG(+) cells were abundant 16 months after bone marrow injection. Approximately 5% of DG cells expressed FLAG, including neurons (48.6%) and microglia (49.7%), and occasional astrocytes (1.6%), as determined by double immunostaining for FLAG and lineage markers. These data suggest that one or more populations of cells resident within adult bone marrow can migrate to the brain and differentiate into CNS-specific cells. (+info)
High levels of Cre expression in neuronal progenitors cause defects in brain development leading to microencephaly and hydrocephaly.
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Hydrocephalus is a common and variegated pathology often emerging in newborn children after genotoxic insults during pregnancy (Hicks and D'Amato, 1980). Cre recombinase is known to have possible toxic effects that can compromise normal cell cycle and survival. Here we show, by using three independent nestin Cre transgenic lines, that high levels of Cre recombinase expression into the nucleus of neuronal progenitors can compromise normal brain development. The transgenics analyzed are the nestin Cre Balancer (Bal1) line, expressing the Cre recombinase with a nuclear localization signal, and two nestin CreER(T2) (Cre recombinase fused with a truncated estrogen receptor) mice lines with different levels of expression of a hybrid CreER(T2) recombinase that translocates into the nucleus after tamoxifen treatment. All homozygous Bal1 nestin Cre embryos displayed reduced neuronal proliferation, increased aneuploidy and cell death, as well as defects in ependymal lining and lamination of the cortex, leading to microencephaly and to a form of communicating hydrocephalus. An essentially overlapping phenotype was observed in the two nestin CreER(T2) transgenic lines after tamoxifen mediated-CreER(T2) translocation into the nucleus. Neither tamoxifen-treated wild-type nor nestin CreER(T2) oil-treated control mice displayed these defects. These results indicate that some forms of hydrocephalus may derive from a defect in neuronal precursors proliferation. Furthermore, they underscore the potential risks for developmental studies of high levels of nuclear Cre in neurogenic cells. (+info)
Subependymal giant cell astrocytoma associated with tuberous sclerosis: with special reference to cell kinetic studies--case report.
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The authors report a case of subependymal giant cell astrocytoma associated with tuberous sclerosis in a 15-year-old boy. Computed tomographic scans showed a large intraventricular mass with peritumoral calcification and a cyst in the left lateral ventricle. Left dominant unilateral hydrocephalus was also revealed. Magnetic resonance images clearly demonstrated the lesion. The tumor was subtotally removed and a ventriculoperitoneal shunt was performed because of the hydrocephalus. The proliferation potential was assessed by measuring the bromodeoxyuridine (BUdR) labeling index employing the in vitro labeling method, and determining the deoxyribonucleic acid (DNA) content by flowcytometry. BUdR-positive cells were found to be rare, and the DNA histogram demonstrated no evidence of high proliferative activity or aneuploidy. (+info)
Foetal mouse neural stem cells give rise to ependymal cells in vitro.
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NSCs are responsible for the generation of CNS cell types derived from the neural tube. Published data resulting from experiments studying the differentiation of NSCs in vitro or in vivo have confirmed their spontaneous tripotency, i.e. their ability to generate cells of the neuronal, astroglial and oligodendroglial lineages. The relationship between NSCs generated in vitro and ependymal cells has not yet been studied. To confirm that ependymal cells can also be produced by NSCs, we utilized the neurosphere assay, which permits isolation and cultivation of NSCs. Cells from the forebrain of E14-15 Balb/c foetuses were grown in DMEM/F12-N2 medium supplemented with EGF and FGF-2 to form multicellular neurospheres. After 3 to 8 passages, neurospheres were plated on surfaces coated with poly-L-lysine, polyornithine and/or laminin in dishes containing the same medium where cytokines were replaced with serum. Under these conditions, neurosphere cells spread over the surface forming a cellular layer consisting of beta-III tubulin+ neuronal, GFAP+ astroglial and O4+ oligodendroglial cells. When these cells were cultivated for prolonged periods, they formed islands of epitheloid cells. Following 2 to 3 weeks in vitro, ependymal cells with beating cilia appeared among these cells. Ciliated ependymal cells were observed in small clusters or as single cells scattered in certain areas. Confocal microscopy confirmed the presence of alpha-tubulin-immunoreactive cilia arranged in tufts located on the apical surface of epitheloid cells. Our data indicate that ependymal cells are spontaneously derived from NSCs. (+info)
A common epitope is shared by ciliary rootlets and cell-cell adherens junctions in ciliated ependymal cells.
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Using immunoblot, light and electron immunocytochemistry, we investigated the presence and the localization of polypeptides cross-reacting with the monoclonal antibody CC.310 (mAb CC.310), which is mainly directed against a 175K (K = 10(3) Mr) ciliary rootlet protein. In hypothalamic ependymal cultures, the unique antigen recognized by mAb CC.310 was associated with the Triton X-100-insoluble fraction in these cultures and electrophoretically migrated to these cultures and electrophoretically migrated to 94K. mAb CC.310, which appears to be a very suitable marker for ciliated ependymocytes, allowed us to observe ciliogenesis during the growth of the ependymal cultures, from a single spot in each undifferentiated ependymal cell to a massive labeling in ciliated ependymal cells. In fully differentiated ciliated ependymocytes, mAb CC.310 strongly reacted with fibrous structures corresponding to ciliary rootlets, as confirmed by ultrastructural observations. In addition, a weaker immunostaining was also found along the intercellular junctions, and showed that proteins sharing a common epitope are located in ependymal ciliary rootlets and near adherens-type junctional complexes. Immunofluorescence studies confirmed the presence of positive labeling at the level of junctional complexes between cells in two epithelial lines, HeLa and PtK2, in which mAb CC.310 mainly reacted with one polypeptide of 85K. (+info)
Photoperiodic regulation of cellular retinol binding protein, CRBP1 [corrected] and nestin in tanycytes of the third ventricle ependymal layer of the Siberian hamster.
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Tanycytes in the ependymal layer of the third ventricle act both as a barrier and a communication gateway between the cerebrospinal fluid, brain and portal blood supply to the pituitary gland. However, the range, importance and mechanisms involved in the function of tanycytes remain to be explored. In this study, we have utilized a photoperiodic animal to examine the expression of three unrelated gene sequences in relation to photoperiod-induced changes in seasonal physiology and behaviour. We demonstrate that cellular retinol binding protein [corrected] (CRBP1), a retinoic acid transport protein, GPR50, an orphan G-protein-coupled receptor and nestin, an intermediate filament protein, are down-regulated in short-day photoperiods. The distribution of the three sequences is very similar, with expression located in cells with tanycyte morphology in the region of the ependymal layer where tanycytes are located. Furthermore, CRBP1 expression in the ependymal layer is shown to be independent of a circadian clock and altered testosterone levels associated with testicular regression in short photo-period. Pinealectomy of Siberian hamsters demonstrates CRBP1 expression is likely to be dependent on melatonin output from the pineal gland. This provides evidence that tanycytes are seasonally responsive cells and are likely to be an important part of the mechanism to facilitate seasonal physiology and behaviour in the Siberian hamster. (+info)
Postnatal deletion of Numb/Numblike reveals repair and remodeling capacity in the subventricular neurogenic niche.
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Neural stem cells are retained in the postnatal subventricular zone (SVZ), a specialized neurogenic niche with unique cytoarchitecture and cell-cell contacts. Although the SVZ stem cells continuously regenerate, how they and the niche respond to local changes is unclear. Here we generated nestin-creER(tm) transgenic mice with inducible Cre recombinase in the SVZ and removed Numb/Numblike, key regulators of embryonic neurogenesis from postnatal SVZ progenitors and ependymal cells. This resulted in severe damage to brain lateral ventricle integrity and identified roles for Numb/Numblike in regulating ependymal wall integrity and SVZ neuroblast survival. Surprisingly, the ventricular damage was eventually repaired: SVZ reconstitution and ventricular wall remodeling were mediated by progenitors that escaped Numb deletion. Our results show a self-repair mechanism in the mammalian brain and may have implications for both niche plasticity in other areas of stem cell biology and the therapeutic use of neural stem cells in neurodegenerative diseases. (+info)