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.

BDE-47 and 6-OH-BDE-47 modulate calcium homeostasis in primary fetal human neural progenitor cells via ryanodine receptor-independent mechanisms.

Polybrominated diphenyl ethers (PBDEs) are bioaccumulating flame retardants found in rising concentrations in human tissue. Epidemiological and animal studies have raised concern for their potential to induce developmental neurotoxicity (DNT). Considering the essential role of calcium homeostasis in neurodevelopment, PBDE-induced disturbance of intracellular calcium concentration ([Ca(2+)]i) may underlie PBDE-induced DNT. To test this hypothesis, we investigated acute effects of BDE-47 and 6-OH-BDE-47 on [Ca(2+)]i in human neural progenitor cells (hNPCs) and unraveled involved signaling pathways. Short-time differentiated hNPCs were exposed to BDE-47, 6-OH-BDE-47, and multiple inhibitors/stimulators of presumably involved signaling pathways to determine possible effects on [Ca(2+)]i by single-cell microscopy with the fluorescent dye Fura-2. Initial characterization of calcium signaling pathways confirmed the early developmental stage of hNPCs. In these cells, BDE-47 (2 µM) and 6-OH-BDE-47 (0.2 µM) induce [Ca(2+)]i transients. This increase in [Ca(2+)]i is due to extracellular Ca(2+) influx and intracellular release of Ca(2+), mainly from the endoplasmic reticulum (ER). While extracellular Ca(2+) seems to enter the cytoplasm upon 6-OH-BDE-47 by interfering with the cell membrane and independent of Ca(2+) ion channels, ER-derived Ca(2+) is released following activation of protein lipase C and inositol 1,4,5-trisphosphate receptor, but independently of ryanodine receptors. These findings illustrate that immature developing hNPCs respond to low concentrations of 6-OH-BDE-47 by an increase in [Ca(2+)]i and provide new mechanistic explanations for such BDE-induced calcium disruption. Thus, these data support the possibility of a critical window of PBDE exposure, i.e., early human brain development, which has to be acknowledged in risk assessment.

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.

Neural stem cells for developmental neurotoxicity studies.

The developing nervous system is particularly susceptible to toxicants, and exposure during development may result in long-lasting neurological impairments. The damage can range from subtle to severe, and it may impose substantial burdens on affected individuals, their families, and society. Given the little information available on developmental neurotoxicity (DNT) and the growing number of chemicals that need to be tested, new testing strategies and approaches are necessary to identify developmental neurotoxic agents with speed, reliability, and respect for animal welfare. So far, there are no validated alternative methods for DNT testing. Recently, neural stem/progenitor cells have been proposed as relevant models for alternative DNT testing. In this chapter, we provide detailed protocols for culturing neural stem cells (NSCs), in vitro experimental models, including primary cultures of rat and human embryonic NSCs, rat and mouse adult NSCs, as well as the mouse NSC line C17.2 that we have implemented and successfully used for neurotoxicity studies.

Methylmercury-induced neurotoxicity and apoptosis.

Methylmercury is a widely distributed environmental toxicant with detrimental effects on the developing and adult nervous system. Due to its accumulation in the food chain, chronic exposure to methylmercury via consumption of fish and sea mammals is still a major concern for human health, especially developmental exposure that may lead to neurological alterations, including cognitive and motor dysfunctions. Mercury-induced neurotoxicity and the identification of the underlying mechanisms has been a main focus of research in the neurotoxicology field. Three major mechanisms have been identified as critical in methylmercury-induced cell damage including (i) disruption of calcium homeostasis, (ii) induction of oxidative stress via overproduction of reactive oxygen species or reduction of antioxidative defenses and (iii) interactions with sulfhydryl groups. In vivo and in vitro studies have provided solid evidence for the occurrence of neural cell death, as well as cytoarchitectural alterations in the nervous system after exposure to methylmercury. Signaling cascades leading to cell death induced by methylmercury involve the release of mitochondrial factors, such as cytochrome c and AIF with subsequent caspase-dependent or -independent apoptosis, respectively; induction of calcium-dependent proteases calpains; interaction with lysosomes leading to release of cathepsins. Interestingly, several pathways can be activated in parallel, depending on the cell type. In this paper, we provide an overview of recent findings on methylmercury-induced neurotoxicity and cell death pathways that have been described in neural and endocrine cell systems.

Polybrominated diphenyl ethers induce developmental neurotoxicity in a human in vitro model: evidence for endocrine disruption.

BACKGROUND: Polybrominated diphenyl ethers (PBDEs) are persistent and bioaccumulative flame retardants, which are found in rising concentrations in human tissues. They are of concern for human health because animal studies have shown that they possess the potential to be developmentally neurotoxic. OBJECTIVE: Because there is little knowledge of the effects of PBDEs on human brain cells, we investigated their toxic potential for human neural development in vitro. Moreover, we studied the involvement of thyroid hormone (TH) disruption in the effects caused by PBDEs. METHODS: We used the two PBDE congeners BDE-47 and BDE-99 (0.1-10 microM), which are most prominent in human tissues. As a model of neural development, we employed primary fetal human neural progenitor cells (hNPCs), which are cultured as neurospheres and mimic basic processes of brain development in vitro: proliferation, migration, and differentiation. RESULTS: PBDEs do not disturb hNPC proliferation but decrease migration distance of hNPCs. Moreover, they cause a reduction of differentiation into neurons and oligodendrocytes. Simultaneous exposure with the TH receptor (THR) agonist triiodothyronine rescues these effects on migration and differentiation, whereas the THR antagonist NH-3 does not exert an additive effect. CONCLUSION: PBDEs disturb development of hNPCs in vitro via endocrine disruption of cellular TH signaling at concentrations that might be of relevance for human exposure.

Human neurospheres as three-dimensional cellular systems for developmental neurotoxicity testing.

BACKGROUND: Developmental neurotoxicity (DNT) of environmental chemicals is a serious threat to human health. Current DNT testing guidelines propose investigations in rodents, which require large numbers of animals. With regard to the "3 Rs" (reduction, replacement, and refinement) of animal testing and the European regulation of chemicals [Registration, Evaluation, and Authorisation of Chemicals (REACH)], alternative testing strategies are needed in order to refine and reduce animal experiments and allow faster and less expensive screening. OBJECTIVES: The goal of this study was to establish a three-dimensional test system for DNT screening based on human fetal brain cells. METHODS: We established assays suitable for detecting disturbances in basic processes of brain development by employing human neural progenitor cells (hNPCs), which grow as neurospheres. Furthermore, we assessed effects of mercury and oxidative stress on these cells. RESULTS: We found that human neurospheres imitate proliferation, differentiation, and migration in vitro. Exposure to the proapoptotic agent staurosporine further suggests that human neurospheres possess functioning apoptosis machinery. The developmental neurotoxicants methylmercury chloride and mercury chloride decreased migration distance and number of neuronal-like cells in differentiated hNPCs. Furthermore, hNPCs undergo caspase-independent apoptosis when exposed toward high amounts of oxidative stress. CONCLUSIONS: Human neurospheres are likely to imitate basic processes of brain development, and these processes can be modulated by developmental neurotoxicants. Thus, this three-dimensional cell system is a promising approach for DNT testing.

ERK-dependent and -independent pathways trigger human neural progenitor cell migration.

Besides differentiation and apoptosis, cell migration is a basic process in brain development in which neural cells migrate several centimeters within the developing brain before reaching their proper positions and forming the right connections. For identifying signaling events that control neural migration and are therefore potential targets of chemicals to disturb normal brain development, we developed a human neurosphere-based migration assay based on normal human neural progenitor (NHNP) cells, in which the distance is measured that cells wander over time. Applying this assay, we investigated the role of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in the regulation of NHNP cell migration. Exposure to model substances like ethanol or phorbol 12-myristate 13-acetate (PMA) revealed a correlation between ERK1/2 activation and cell migration. The participation of phospho-(P-) ERK1/2 was confirmed by exposure of the cells to the MEK inhibitor PD98059, which directly prohibits ERK1/2 phosphorylation and inhibited cell migration. We identified protein kinase C (PKC) and epidermal growth factor receptor (EGFR) as upstream signaling kinases governing ERK1/2 activation, thereby controlling NHNP cell migration. Additionally, treatments with src kinase inhibitors led to a diminished cell migration without affecting ERK1/2 phosphorylation. Based on these results, we postulate that migration of NHNP cells is controlled via ERK1/2-dependent and -independent pathways.

Transcription of potato spindle tuber viroid by RNA polymerase II starts in the left terminal loop.

Viroids are single-stranded, circular RNAs of 250 to 400 bases, that replicate autonomously in their host plants but do not code for a protein. Viroids of the family Pospiviroidae, of which potato spindle tuber viroid (PSTVd) is the type strain, are replicated by the host's DNA-dependent RNA polymerase II in the nucleus. To analyze the initiation site of transcription from the (+)-stranded circles into (-)-stranded replication intermediates, we used a nuclear extract from a non-infected cell culture of the host plant S. tuberosum. The (-)-strands, which were de novo-synthesized in the extract upon addition of circular (+)-PSTVd, were purified by affinity chromatography. This purification avoided contamination by host nucleic acids that had resulted in a misassignment of the start site in an earlier study. Primer-extension analysis of the de novo-synthesized (-)-strands revealed a single start site located in the hairpin loop of the left terminal region in circular PSTVd's secondary structure. This start site is supported further by analysis of the infectivity and replication behavior of site-directed mutants in planta.

Analysis of thermal stress-mediated PSTVd variation and biolistic inoculation of progeny of viroid "thermomutants" to tomato and Brassica species.

Thermal stress of PSTVd-infected Nicotiana benthamiana led to appearance of a broad PSTVd sequence distribution, where most of mutations accumulated in the left half of the viroid's secondary structure including the "pathogenicity" domain. A similar effect had been reported for hop latent viroid [Virology 287 (2001) 349]. The pool of viroid "thermomutants" progenies was transcribed into cDNA and used for biolistic inoculation of Raphanus sativa, where the PSTVd infection was detectable by reverse transcription and polymerase chain reaction (RT-PCR). Newly generated inoculum from R. sativa was used for biolistic transfer to Arabidopsis thaliana wild-type and silencing-deficient mutants bearing one of sde1, sde2, and sde3 locuses. Irrespective to A. thaliana silencing mutants, viroid levels in Brasicaceae species infected with mutated PSTVd variants were of approximately 300 times lower than it is expected for tomato. At the same time, no systemic infection of A. thaliana was achieved with the wild-type PSTVd. In Arabidopsis, a population of PSTVd, consisting of frequent and minor variants, was present and the sequence distribution differed from that of the original viroid "thermomutants"; that is, mutations were not predominantly restricted to the left half of viroid's secondary structure. At least 65% of viroid sequences from Arabidopsis library accumulated mutations in the upper conserved central region (UCCR). In addition, mutants having changes in "hairpin II" domain (C-->A transition at position 229) and in the conserved internal loop element in the left part of viroid structure (single insertion of G at position 39) were detected. All those mutants were inoculated biolistically to tomato and promoted infection especially after prolonged period of plant cultivation (50-80 days pi) when infection reached 70-90%. However, the sequence variants were unstable and reverted to the wild type and to other sequence variants stable in tomato. Our results demonstrate that heat stress-mediated production of viroid quasi-species could be of significance for viroid adaptations.