Cerium oxide nanoparticles inhibit differentiation of neural stem cells.

Cerium oxide nanoparticles (nanoceria) display antioxidant properties and have shown cytoprotective effects both in vitro and in vivo. Here, we explored the effects of nanoceria on neural progenitor cells using the C17.2 murine cell line as a model. First, we assessed the effects of nanoceria versus samarium (Sm) doped nanoceria on cell viability in the presence of the prooxidant, DMNQ. Both particles were taken up by cells and nanoceria, but not Sm-doped nanoceria, elicited a temporary cytoprotective effect upon exposure to DMNQ. Next, we employed RNA sequencing to explore the transcriptional responses induced by nanoceria or Sm-doped nanoceria during neuronal differentiation. Detailed computational analyses showed that nanoceria altered pathways and networks relevant for neuronal development, leading us to hypothesize that nanoceria inhibits neuronal differentiation, and that nanoceria and Sm-doped nanoceria both interfere with cytoskeletal organization. We confirmed that nanoceria reduced neuron specific ß3-tubulin expression, a marker of neuronal differentiation, and GFAP, a neuroglial marker. Furthermore, using super-resolution microscopy approaches, we could show that both particles interfered with cytoskeletal organization and altered the structure of neural growth cones. Taken together, these results reveal that nanoceria may impact on neuronal differentiation, suggesting that nanoceria could pose a developmental neurotoxicity hazard.

Gestational Age and Sex Influence the Susceptibility of Human Neural Progenitor Cells to Low Levels of MeHg.

The developing nervous system is highly susceptible to methylmercury (MeHg), a widespread environmental neurotoxic contaminant. A wide range of morphological and functional outcomes have been described; however, there are still open questions regarding the mechanisms behind the developmental neurotoxic effects induced by low-level exposure. In the present study, we have examined the effects of nanomolar concentrations of MeHg on primary fetal human progenitor cells (hNPCs) with special focus on the role played by developmental stage and sex on the neurotoxic outcome. We found that neurospheres derived from earlier gestational time points exhibit higher susceptibility to MeHg, as they undergo apoptosis at a much lower dose (25 nM) as compared to neurospheres established from older fetuses (100 nM). At subapoptotic concentrations (10 nM), MeHg inhibited neuronal differentiation and maturation of hNPCs, as shown by a reduced number of Tuj1-positive cells and a visible reduction in neurite extension and cell migration, associated with a misregulation of Notch1 and BDNF signaling pathways. Interestingly, cells derived from male fetuses showed more severe alterations of neuronal morphology as compared to cells from females, indicating that the MeHg-induced impairment of neurite extension and cell migration is sex-dependent. Accordingly, the expression of the CDKL5 gene, a major factor regulating neurite outgrowth, was significantly more downregulated in male-derived cells. Altogether, gestational age and sex appear to be critical factors influencing in vitro hNPC sensitivity to low levels of MeHg.

Glucocorticoids alter neuronal differentiation of human neuroepithelial-like cells by inducing long-lasting changes in the reactive oxygen species balance.

Prenatal exposure to excess glucocorticoid has been shown to have adverse effects on the developing nervous system that may lead to alterations of fetal and adult neurogenesis, resulting in behavioral changes. In addition, an imbalance of the redox state, with an increased susceptibility to oxidative stress, has been observed in rodent neural stem cells exposed to the synthetic glucocorticoid analog dexamethasone (Dex). In the present study, we used the induced pluripotent stem cells (IPSC)-derived lt-NES AF22 cell line, representative of the neuroepithelial stage in central nervous system development, to investigate the heritable effects of Dex on reactive oxygen species (ROS) balance and its impact on neuronal differentiation. By analysing gene expression in daughter cells that were never directly exposed to Dex, we could observe a downregulation of four key antioxidant enzymes, namely Catalase, superoxide dismutase 1, superoxide dismutase 2 and glutathione peroxidase7, along with an increased intracellular ROS concentration. The imbalance in the intracellular REDOX state was associated to a significant downregulation of major neuronal markers and a concomitant increase of glial cells. Interestingly, upon treatment with the antioxidant N-acetyl-cysteine (NAC), the misexpression of both neuronal and glial markers analyzed was recovered. These novel findings point to the increased ROS concentration playing a direct role in the heritable alterations of the differentiation potential induced by Dex exposure. Moreover, the data support the hypothesis that early insults may have detrimental long-lasting consequences on neurogenesis. Based on the positive effects exerted by NAC, it is conceivable that therapeutic strategies including antioxidants may be effective in the treatment of neuropsychiatric disorders that have been associated to increased ROS and impaired neurogenesis.

Molecular hydrogen reduces LPS-induced neuroinflammation and promotes recovery from sickness behaviour in mice.

Molecular hydrogen has been shown to have neuroprotective effects in mouse models of acute neurodegeneration. The effect was suggested to be mediated by its free-radical scavenger properties. However, it has been shown recently that molecular hydrogen alters gene expression and protein phosphorylation. The aim of this study was to test whether chronic ad libitum consumption of molecular hydrogen-enriched electrochemically reduced water (H-ERW) improves the outcome of lipopolysaccharide (LPS)-induced neuroinflammation. Seven days after the initiation of H-ERW treatment, C57Bl/6 mice received a single injection of LPS (0.33 mg/kg i.p.) or an equivalent volume of vehicle. The LPS-induced sickness behaviour was assessed 2 h after the injection, and recovery was assessed by monitoring the spontaneous locomotor activity in the homecage for 72 h after the administration of LPS. The mice were killed in the acute or recovery phase, and the expression of pro- and antiinflammatory cytokines in the hippocampus was assessed by real-time PCR. We found that molecular hydrogen reduces the LPS-induced sickness behaviour and promotes recovery. These effects are associated with a shift towards anti-inflammatory gene expression profile at baseline (downregulation of TNF- α and upregulation of IL-10). In addition, molecular hydrogen increases the amplitude, but shortens the duration and promotes the extinction of neuroinflammation. Consistently, molecular hydrogen modulates the activation and gene expression in a similar fashion in immortalized murine microglia (BV-2 cell line), suggesting that the effects observed in vivo may involve the modulation of microglial activation. Taken together, our data point to the regulation of cytokine expression being an additional critical mechanism underlying the beneficial effects of molecular hydrogen.

Inherited effects of low-dose exposure to methylmercury in neural stem cells.

Methylmercury (MeHg) is an environmental contaminant with recognized neurotoxic effects, particularly to the developing nervous system. In the present study, we show that nanomolar concentrations of MeHg can induce long-lasting effects in neural stem cells (NSCs). We investigated short-term direct and long-term inherited effects of exposure to MeHg (2.5 or 5.0 nM) using primary cultures of rat embryonic cortical NSCs. We found that MeHg had no adverse effect on cell viability but reduced NSC proliferation and altered the expression of cell cycle regulators (p16 and p21) and senescence-associated markers. In addition, we demonstrated a decrease in global DNA methylation in the exposed cells, indicating that epigenetic changes may be involved in the mechanisms underlying the MeHg-induced effects. These changes were observed in cells directly exposed to MeHg (parent cells) and in their daughter cells cultured under MeHg-free conditions. In agreement with our in vitro data, a trend was found for decreased cell proliferation in the subgranular zone in the hippocampi of adult mice exposed to low doses of MeHg during the perinatal period. Interestingly, this impaired proliferation had a measurable impact on the total number of neurons in the hippocampal dentate gyrus. Importantly, this effect could be reversed by chronic antidepressant treatment. Our study provides novel evidence for programming effects induced by MeHg in NSCs and supports the idea that developmental exposure to low levels of MeHg may result in long-term consequences predisposing to neurodevelopmental disorders and/or neurodegeneration.

Dickkopf 1 mediates glucocorticoid-induced changes in human neural progenitor cell proliferation and differentiation.

Glucocorticoids (GC) are critical for normal development of the fetal brain, and alterations in their levels can induce neurotoxicity with detrimental consequences. Still, there is little information available on the effects of GC on human neural stem/progenitor cells (hNPC). In the present study, we have investigated the effects of the synthetic GC dexamethasone (Dex) on hNPC grown as neurospheres, with special focus on their proliferation and differentiation capacity and the underlying molecular mechanisms. Immunocytochemical stainings showed that Dex markedly decreases proliferation and neuronal differentiation while promoting glia cell formation. Analysis of pathway-specific genes revealed that Dex induces an upregulation of the Wnt-signaling antagonist DKK1. Moreover, Dex- or DKK1-treated hNPCs showed reduced transcriptional levels of the two canonical Wnt target genes cyclin D1 and inhibitor of DNA binding 2 (ID2). Chromatin immunoprecipitation showed that Dex, via the glucocorticoid receptor, interacts with the DKK1 promotor. Treatment of hNPC with recombinant DKK1 or neutralizing antibodies indicated that DKK1 has a critical role in the Dex-induced inhibition of proliferation and neuronal differentiation with a concomitant increase in glial cells. Taken together, our findings show that GC reduce proliferation and interfere with differentiation of hNPCs via the canonical Wnt-signaling pathway.

Detection of GFP during nervous system development in Drosophila melanogaster.

Using the vital marker GFP and its spectral variants, it is possible to visualize multiple proteins in individual cells and thereby monitor embryonic development on a cellular and molecular level. In the following chapter we describe how to prepare Drosophila embryos or larvae for live imaging or immunohistochemical staining and provide some guidelines for optimal GFP detection.

Prospero acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells.

Stem cells have the remarkable ability to give rise to both self-renewing and differentiating daughter cells. Drosophila neural stem cells segregate cell-fate determinants from the self-renewing cell to the differentiating daughter at each division. Here, we show that one such determinant, the homeodomain transcription factor Prospero, regulates the choice between stem cell self-renewal and differentiation. We have identified the in vivo targets of Prospero throughout the entire genome. We show that Prospero represses genes required for self-renewal, such as stem cell fate genes and cell-cycle genes. Surprisingly, Prospero is also required to activate genes for terminal differentiation. We further show that in the absence of Prospero, differentiating daughters revert to a stem cell-like fate: they express markers of self-renewal, exhibit increased proliferation, and fail to differentiate. These results define a blueprint for the transition from stem cell self-renewal to terminal differentiation.

Ephrin-A2 reverse signaling negatively regulates neural progenitor proliferation and neurogenesis.

The number of cells in an organ is regulated by mitogens and trophic factors that impinge on intrinsic determinants of proliferation and apoptosis. We here report the identification of an additional mechanism to control cell number in the brain: EphA7 induces ephrin-A2 reverse signaling, which negatively regulates neural progenitor cell proliferation. Cells in the neural stem cell niche in the adult brain proliferate more and have a shorter cell cycle in mice lacking ephrin-A2. The increased progenitor proliferation is accompanied by a higher number of cells in the olfactory bulb. Disrupting the interaction between ephrin-A2 and EphA7 in the adult brain of wild-type mice disinhibits proliferation and results in increased neurogenesis. The identification of ephrin-A2 and EphA7 as negative regulators of progenitor cell proliferation reveals a novel mechanism to control cell numbers in the brain.

Effects of IL-1beta, IL-6 or LIF on rat sensory neurons co-cultured with fibroblast-like cells.

Inflammation may affect the local presence of sensory nerve fibers in situ and inflammatory mediators influence sensory neurons in vitro. In the present study we have investigated effects of the cytokines interleukin-1beta (IL-1beta, interleukin-6 (IL-6), and leukemia inhibitory factor (LIF) on survival of and neurite growth from neonatal rat sensory neurons co-cultured with fibroblast-like cells prepared from neonatal rat skin (sFLCs) or perichondrium (pFLCs). The results showed that both FLC types expressed receptors for all three cytokines. Five ng/ml of either cytokine, but not lower or higher concentrations, supported survival of DRG neurons co-cultured with sFLCs. Neuronal survival was also enhanced by addition of the soluble IL-6 receptor (rsIL-6R) with or without IL-6. In co-cultures with pFLCs neuronal survival was promoted by IL-6, increasing with cytokine concentration. Addition of rsIL-6R without IL-6 did also stimulate neuronal survival. The growth of neurites from DRG neurons co-cultured with sFLCs was stimulated by 0.5 ng/ml LIF, unaffected by 5 ng/ml LIF and inhibited by 50 ng/ml LIF. Considering DRG neurons co-cultured with pFLCs, 50 ng/ml of either of the three cytokines, as well as rsIL-6R conditioned medium, stimulated neurite outgrowth. Some of the cytokine effects observed were reduced by application of antibodies against nerve growth factor (NGF). We conclude that that the cytokines examined affect DRG neurons in terms of survival or neuritogenesis, that the effects are influenced by cytokine concentration and the origin of the FLCs and that some of the effects are indirect, probably being mediated by factors released from FLCs.