Gene expression profiling of neural stem cells and their neuronal progeny reveals IGF2 as a regulator of adult hippocampal neurogenesis.

Neural stem cells (NSCs) generate neurons throughout life in the hippocampal dentate gyrus (DG). How gene expression signatures differ among NSCs and immature neurons remains largely unknown. We isolated NSCs and their progeny in the adult DG using transgenic mice expressing a GFP reporter under the control of the Sox2 promoter (labeling NSCs) and transgenic mice expressing a DsRed reporter under the control of the doublecortin (DCX) promoter (labeling immature neurons). Transcriptome analyses revealed distinct gene expression profiles between NSCs and immature neurons. Among the genes that were expressed at significantly higher levels in DG NSCs than in immature neurons was the growth factor insulin-like growth factor 2 (IGF2). We show that IGF2 selectively controls proliferation of DG NSCs in vitro and in vivo through AKT-dependent signaling. Thus, by gene expression profiling of NSCs and their progeny, we have identified IGF2 as a novel regulator of adult neurogenesis.

Directed differentiation of hippocampal stem/progenitor cells in the adult brain.

Adult neurogenesis is a lifelong feature of brain plasticity; however, the potency of adult neural stem/progenitor cells in vivo remains unclear. We found that retrovirus-mediated overexpression of a single gene, the bHLH transcription factor Ascl1, redirected the fate of the proliferating adult hippocampal stem/progenitor (AHP) progeny and lead to the exclusive generation of cells of the oligodendrocytic lineage at the expense of newborn neurons, demonstrating that AHPs in the adult mouse brain are not irrevocably specified in vivo. These data indicate that AHPs have substantial plasticity, which might have important implications for the potential use of endogenous AHPs in neurological disease.

In vivo fate analysis reveals the multipotent and self-renewal capacities of Sox2+ neural stem cells in the adult hippocampus.

To characterize the properties of adult neural stem cells (NSCs), we generated and analyzed Sox2-GFP transgenic mice. Sox2-GFP cells in the subgranular zone (SGZ) express markers specific for progenitors, but they represent two morphologically distinct populations that differ in proliferation levels. Lentivirus- and retrovirus-mediated fate-tracing studies showed that Sox2+ cells in the SGZ have potential to give rise to neurons and astrocytes, revealing their multipotency at the population as well as at a single-cell level. A subpopulation of Sox2+ cells gives rise to cells that retain Sox2, highlighting Sox2+ cells as a primary source for adult NSCs. In response to mitotic signals, increased proliferation of Sox2+ cells is coupled with the generation of Sox2+ NSCs as well as neuronal precursors. An asymmetric contribution of Sox2+ NSCs may play an important role in maintaining the constant size of the NSC pool and producing newly born neurons during adult neurogenesis.

Functional and morphological effects of NG2 proteoglycan deletion on hippocampal neurogenesis.

NG2-expressing cells are the largest proliferating cell population in the adult central nervous system. The function of NG2 proteoglycan or NG2-expressing cells in the adult brain, however, is unknown. So far, NG2-positive cells are thought to be mainly oligodendrocyte precursor cells. This view was recently challenged when NG2+/CNP-EGFP-positive cells were identified as multipotent progenitor cells in the postnatal and adult CNS (e.g., [Belachew, S., Chittajallu, R., Aguirre, A.A., Yuan, X., Kirby, M., Anderson, S., Gallo, V., 2003. Postnatal NG2 proteoglycan-expressing progenitor cells are intrinsically multipotent and generate functional neurons. J. Cell Biol. 161, 169-186]). In addition, purified NG2-expressing progenitor cells, were shown to differentiate into neurons and astrocytes in vitro [Sellers, D.L., Horner, P.J., 2005. Instructive niches: environmental instructions that confound NG2 proteoglycan expression and the fate-restriction of CNS progenitors J. Anat. 207, 727-734]. In this study, we focus on the influence of NG2 ablation on neurogenesis in the hippocampus, where putative multipotent NG2-positive cells reside, and on hippocampus-dependent behavior using NG2 knockout mice. Using the thymidine analogue bromodeoxyuridine (BrdU) to label dividing cells in vivo we show that the number of BrdU-positive cells was unchanged in the hippocampus of NG2 knockout mice 1 day after a series of BrdU injections. This finding suggests that the proliferation rate of hippocampal progenitor cells is not influenced by NG2. A few BrdU-positive cells were found in deeper layers of the granule zone 1 day after a series of BrdU injections, which is different from the wild type. The presence and the phenotype of newborn hippocampal cells were studied 4 weeks after a series of BrdU injections. The survival and differentiation of BrdU-positive cells in NG2 knockout hippocampus did not significantly differ from wild-type mice. Concurrently, the water maze task did not reveal obvious differences compared to wild-type animals. These results suggest that the null mutation for NG2 does not influence adult hippocampal neurogenesis or hippocampal-dependent behavioral tasks.

Differential properties of adult rat and mouse brain-derived neural stem/progenitor cells.

Adult neurogenesis from neural stem/progenitor cells occurs in discrete regions of the central nervous system of all mammals, but the mechanisms regulating endogenous neurogenesis are poorly understood. Advances in understanding the neurogenesis depend on knowing their intrinsic properties and responses to environmental signals that control their behavior. Before these issues can be addressed, it is necessary to know whether there are significant species-specific differences in the properties of the stem/progenitor cells derived from CNS of two commonly studied model systems, mouse and rat. We found major differences between rat and mouse stem/progenitor cell proliferation in response to various substrates, mitogenic growth factors and heparin and to the influence of differentiation factors on generation of neurons and glia. Thus, extrapolation of cell properties from one species to another based on studies of these cells should be made with caution.

Ex vivo and in vivo gene delivery to the brain.

This unit describes methods for grafting genetically modified cells for ex vivo delivery of specific genes into the rat brain and direct delivery of transgenes to brain cells in vivo using recombinant viral vectors. These methods assess the function of a gene in the brain. The ex vivo approach of gene transfer to the nervous system depends on genetic manipulation of cells in vitro prior to grafting of the cells into the brain to enable production of a transgene at physiologically significant levels for a long period of time. This unit also includes procedures for transcardial perfusion to fix the tissue prior to analysis of expression and for sectioning of brains by use of a freezing sledge microtome. Thionine staining of tissue sections is also described.

Quantitative analysis of gene expression in living adult neural stem cells by gene trapping.

The potential of neural stem cells (NSCs) for the treatment of neurodegenerative diseases makes the identification and characterization of genes involved in neural stem cell responses therapeutically important. Although technologies exist for measuring gene expression in cells, they often provide only a representative expression profile specific to a stimulus and time. We developed a complementary technology based on a retroviral-vector gene-trap approach that uses beta-lactamase-induced disruption of fluorescence resonance energy transfer in the fluorophore CCF-2/AM. A library of 'tagged' adult rat NSCs was generated by transduction with gene-trap virus produced from a single-integrant packaging cell line that allowed us to quantitatively analyze dynamic gene expression changes in real time in living NSCs. Using this library we identified previously unknown genes regulated by oxidative stress, indomethacin and factors that induce differentiation, and show that one of the trapped genes, Sox6, is sufficient to induce astrocytic differentiation when overexpressed.

Expression and function of orphan nuclear receptor TLX in adult neural stem cells.

The finding of neurogenesis in the adult brain led to the discovery of adult neural stem cells. TLX was initially identified as an orphan nuclear receptor expressed in vertebrate forebrains and is highly expressed in the adult brain. The brains of TLX-null mice have been reported to have no obvious defects during embryogenesis; however, mature mice suffer from retinopathies, severe limbic defects, aggressiveness, reduced copulation and progressively violent behaviour. Here we show that TLX maintains adult neural stem cells in an undifferentiated, proliferative state. We show that TLX-expressing cells isolated by fluorescence-activated cell sorting (FACS) from adult brains can proliferate, self-renew and differentiate into all neural cell types in vitro. By contrast, TLX-null cells isolated from adult mutant brains fail to proliferate. Reintroducing TLX into FACS-sorted TLX-null cells rescues their ability to proliferate and to self-renew. In vivo, TLX mutant mice show a loss of cell proliferation and reduced labelling of nestin in neurogenic areas in the adult brain. TLX can silence glia-specific expression of the astrocyte marker GFAP in neural stem cells, suggesting that transcriptional repression may be crucial in maintaining the undifferentiated state of these cells.

The immunological properties of adult hippocampal progenitor cells.

Adult hippocampal progenitor cells (AHPCs) derived from mature rats were studied in mixed co-cultures and shown not to elicit a proliferative response from human peripheral blood mononuclear cells (PBMCs) or allogeneic spleen cells. FACS analysis revealed low class I and no detectable class II (Ia) MHC expression by these cells. RT-PCR showed that AHPCs express the anti-inflammatory cytokine TGF-beta1. AHPCs did not, however, significantly impede the proliferation of OKT3- or PHA-stimulated PBMCs. Taken together, these results indicate that AHPCs are non-immunogenic in vitro. This is consistent with their pattern of MHC expression and does not require an active immunosuppressive mechanism.