P. Roy Walker

Sequential DNA methylation of the Nanog and Oct-4 upstream regions in human NT2 cells during neuronal differentiation.

Human NT2 cells, which differentiate into neurons and astrocytes, initially express and then permanently down-regulate Nanog and Oct-4 (POU5F1). We investigated the relationship between the expression of these genes and the methylation state of their 5′-flanking regions. Gene expression and DNA methylation were assayed with quantitative polymerase chain reaction and bisulfite genomic sequencing, respectively. Retinoic acid-induced differentiation of NT2 cells to neurons is accompanied by a sequential decrease in the expression of both genes, paralleled by sequential epigenetic modification of their upstream regions. This is the first report demonstrating changes in DNA methylation in the promoter regions of Nanog and Oct-4 in a human cell line.

Molecular mechanisms of glutamate neurotoxicity in mixed cultures of NT2-derived neurons and astrocytes: protective effects of coenzyme Q10.

Although glutamate excitotoxicity has long been implicated in neuronal cell death associated with a variety of neurological disorders, the molecular mechanisms underlying this process are not yet fully understood. In part, this is due to the lack of relevant experimental cell systems recapitulating the in vivo neuronal environment, mainly neuronal–glial interactions. To explore these mechanisms, we have analyzed the cytotoxic effects of glutamate on mixed cultures of NT2/N neurons and NT2/A astrocytes derived from human NT2/D1 cells. In these cultures, the neurons were resistant to glutamate alone (up to 2 mM for 24–48 hr), but they responded to a simultaneous exposure to 0.5 mM glutamate and 6 hr of hypoxia. Neuronal cell death occurred during subsequent periods of reoxygenation (>30% within 24 hr). This was associated with a marked decrease of intracellular ATP, a significant increase in reactive oxygen species (ROS) and downregulation of glutamate uptake by astrocytes. Thus, under energy failure and high levels of ROS production, only the neurons from these mixed cultures succumbed to glutamate neurotoxicity; the astrocytic cells remained unaffected by the treatment. Taken together, our data suggested that glutamate excitotoxicity might be due to the energy failure and oxidative stress affecting the properties of the NMDA glutamate receptors and causing impairment of glutamate transporters. Cells pretreated for 72 hr with 10 μg/ml of coenzyme Q10 (functions both as a ROS scavenger and co‐factor of mitochondrial electron transport), were protected, suggesting a useful role for coenzyme Q10 in treatments of neurological diseases associated with glutamate excitotoxicity. A model of the complex interactions between neurons and astrocytes in regulating glutamate metabolism is presented.

Calcium, cyclic AMP and protein kinase C — partners in mitogenesis

SummaryEvidence is steadily mounting that the proto-oncogenes, whose products organize and start the programs that drive normal eukaryotic cells through their chromosome replication/mitosis cycles, are transiently stimulated by sequential signals from a multi-purpose, receptor-operated mechanism (consisting of internal surges of Ca2+ and bursts of protein kinase C activity resulting from phosphatidylinositol 4,5-bisphosphate breakdown and the opening of membrane Ca2+ channels induced by receptor-associated tyrosine-protein kinase activity) and bursts of cyclic AMP-dependent kinase activity. The bypassing or subversion of the receptor-operated Ca2+/phospholipid breakdown/protein kinase C signalling mechanism is probably the basis of the freeing of cell proliferation from external controls that characterizes all neoplastic transformations.

Characterization of astrocytes derived from human NTera‐2/D1 embryonal carcinoma cells

Astrocytes are the predominant cell type in the vicinity of glutamatergic synapses, where they monitor and maintain low levels of glutamate. Synaptic homeostasis of glutamate involves its removal from the synaptic cleft via high‐affinity glutamate transporters, glutamate transporter‐1 (GLT‐1)/excitatory amino acid transporters (EAAT)2 and glutamate and aspartate transporter (GLAST)/EAAT1, and glutamate‐catabolizing enzyme, glutamine synthase. Glutamate transporters have been mostly characterized in rodent astrocytes, due to the lack of a convenient human cell system. We report here that NTera‐2 (NT2/D1, a cell line derived from a human teratocarcinoma and known to differentiate into neurons) can also be differentiated by a 4‐week treatment with retinoic acid into functional astrocytes (NT2/A). Differentiation was accompanied by decreased cell proliferation and cell‐cycle arrest, as measured by flow cytometry, immunostaining for Ki67 and incorporation of 5‐bromo‐2′deoxyuridine (BrdU). Immunocytochemistry and Western blot analysis showed that NT2/A expressed glial fibrillary acidic protein, vimentin and S100β. Reverse transcription polymerase chain reaction (PCR) detected mRNA encoding glutamate transporters GLT‐1/EAAT2 and GLAST/EAAT1. The expression level of GLAST/EAAT1 was higher than that of GLT‐1/EAAT2, which is a typical expression pattern for primary astrocytes. Functionality of the transporters was demonstrated by the uptake of 3H‐glutamate. NT2/A also expressed active glutamine synthase, and treatment with glutamate (up to 1 mM for 24 hr) was non‐toxic, suggesting that these cells were capable of converting it to non‐toxic metabolites. NT2/A and NT2‐derived neurons could be grown as mixed cultures and this may prove to be a useful experimental model to study molecular mechanisms underlying glutamate excitotoxicity.

Data mining of gene expression changes in Alzheimer brain

Genome-wide transcription profiling is a powerful technique for studying the enormous complexity of cellular states. Moreover, when applied to disease tissue it may reveal quantitative and qualitative alterations in gene expression that give information on the context or underlying basis for the disease and may provide a new diagnostic approach. However, the data obtained from high-density microarrays is highly complex and poses considerable challenges in data mining. The data requires care in both pre-processing and the application of data mining techniques. This paper addresses the problem of dealing with microarray data that come from two known classes (Alzheimer and normal). We have applied three separate techniques to discover genes associated with Alzheimer disease (AD). The 67 genes identified in this study included a total of 17 genes that are already known to be associated with Alzheimers or other neurological diseases. This is higher than any of the previously published Alzheimers studies. Twenty known genes, not previously associated with the disease, have been identified as well as 30 uncharacterized expressed sequence tags (ESTs). Given the success in identifying genes already associated with AD, we can have some confidence in the involvement of the latter genes and ESTs. From these studies we can attempt to define therapeutic strategies that would prevent the loss of specific components of neuronal function in susceptible patients or be in a position to stimulate the replacement of lost cellular function in damaged neurons. Although our study is based on a relatively small number of patients (four AD and five normal), we think our approach sets the stage for a major step in using gene expression data for disease modeling (i.e. classification and diagnosis). It can also contribute to the future of gene function identification, pathology, toxicogenomics, and pharmacogenomics.

Large-Scale Expression Analysis Reveals Distinct MicroRNA Profiles at Different Stages of Human Neurodevelopment

Background MicroRNAs (miRNAs) are short non-coding RNAs predicted to regulate one third of protein coding genes via mRNA targeting. In conjunction with key transcription factors, such as the repressor REST (RE1 silencing transcription factor), miRNAs play crucial roles in neurogenesis, which requires a highly orchestrated program of gene expression to ensure the appropriate development and function of diverse neural cell types. Whilst previous studies have highlighted select groups of miRNAs during neural development, there remains a need for amenable models in which miRNA expression and function can be analyzed over the duration of neurogenesis. Principal Findings We performed large-scale expression profiling of miRNAs in human NTera2/D1 (NT2) cells during retinoic acid (RA)-induced transition from progenitors to fully differentiated neural phenotypes. Our results revealed dynamic changes of miRNA patterns, resulting in distinct miRNA subsets that could be linked to specific neurodevelopmental stages. Moreover, the cell-type specific miRNA subsets were very similar in NT2-derived differentiated cells and human primary neurons and astrocytes. Further analysis identified miRNAs as putative regulators of REST, as well as candidate miRNAs targeted by REST. Finally, we confirmed the existence of two predicted miRNAs; pred-MIR191 and pred-MIR222 associated with SLAIN1 and FOXP2, respectively, and provided some evidence of their potential co-regulation. Conclusions In the present study, we demonstrate that regulation of miRNAs occurs in precise patterns indicative of their roles in cell fate commitment, progenitor expansion and differentiation into neurons and glia. Furthermore, the similarity between our NT2 system and primary human cells suggests their roles in molecular pathways critical for human in vivo neurogenesis.

Epigenetic Modifications of SOX2 Enhancers, SRR1 and SRR2, Correlate With In Vitro Neural Differentiation

SOX2 is a key neurodevelopmental gene involved in maintaining the pluripotency of stem cells and proliferation of neural progenitors and astroglia. Two evolutionally conserved enhancers, SRR1 and SRR2, are involved in controlling SOX2 expression during neurodevelopment; however, the molecular mechanisms regulating their activity are not known. We have examined DNA methylation and histone H3 acetylation at both enhancers in NT2‐D1 progenitors, neurons and astrocytes, to establish the role of epigenetic mechanisms in cell‐type‐specific SOX2 expression. This study showed that 1) unmethylated DNA and acetylated histones at both enhancers correlated with a high level of SOX2 expression in proliferating neural progenitors and 2) reversible modifications of the SRR1 element were observed during gene reexpression in astrocytes, whereas permanent epigenetic marks on the SRR2 enhancer were seen in neurons where the gene was silenced. Taken together, these results are clear illustrations of cell‐type‐specific epigenomes and suggest mechanisms by which they may be created and maintained.

Identification of functional dopamine receptors in human teratocarcinoma NT2 cells

In search of a cellular model suitable for studying molecular events contributing to brain disorders, we have characterised the expression and functionality of dopamine receptors in human teratocarcinoma NT2 cells. The cells were differentiated by a 4-week retinoic acid treatment, followed by a 3-week mitotic inhibitor treatment in the absence of retinoic acid. The messages of two D(2)-like family members, D(2L) and D(3), were expressed in undifferentiated NT2 cells. The retinoic acid treatment resulted in increased expression of both spliced variants of the D(2) receptor, D(2L) and D(2S) isoforms and a significant induction of D(1) and D(5) gene transcripts. The same treatment turned off expression of the D(3) gene. Further induction of the D(5) gene was observed in the post-mitotic NT2N neurons. The NT2N cells stained positively for D(2) and D(5) receptor proteins, and the intracellular cyclic AMP level increased in response to forskolin, dopamine and the D(1)-receptor agonist SKF-81297. Furthermore, dopamine was ineffective in the presence of the D(2) receptor agonist PPHT and the D(1) receptor antagonist cis-(z)-flupenthixol. These results indicated that upon ligand/agonist/antagonist binding, the receptors could be coupled to the adenylyl cyclase system, hence were functional. To our knowledge, NT2 is the only human immortalized cell line expressing functional dopamine receptors of both families.

Evolution of motif variants and positional bias of the cyclic-AMP response element

BackgroundTranscription factors regulate gene expression by interacting with their specific DNA binding sites. Some transcription factors, particularly those involved in transcription initiation, always bind close to transcription start sites (TSS). Others have no such preference and are functional on sites even tens of thousands of base pairs (bp) away from the TSS.The Cyclic-AMP response element (CRE) binding protein (CREB) binds preferentially to a palindromic sequence (TGACGTCA), known as the canonical CRE, and also to other CRE variants. CREB can activate transcription at CREs thousands of bp away from the TSS, but in mammals CREs are found far more frequently within 1 to 150 bp upstream of the TSS than in any other region. This property is termed positional bias.The strength of CREB binding to DNA is dependent on the sequence of the CRE motif. The central CpG dinucleotide in the canonical CRE (TGACG TCA) is critical for strong binding of CREB dimers. Methylation of the cytosine in the CpG can inhibit binding of CREB. Deamination of the methylated cytosines causes a C to T transition, resulting in a functional, but lower affinity CRE variant, TGAT GTCA.ResultsWe performed genome-wide surveys of CREs in a number of species (from worm to human) and showed that only vertebrates exhibited a CRE positional bias. We performed pair-wise comparisons of human CREs with orthologous sequences in mouse, rat and dog genomes and found that canonical and TGAT GTCA variant CREs are highly conserved in mammals. However, when orthologous sequences differ, canonical CREs in human are most frequently TGAT GTCA in the other species and vice-versa. We have identified 207 human CREs showing such differences.ConclusionOur data suggest that the positional bias of CREs likely evolved after the separation of urochordata and vertebrata. Although many canonical CREs are conserved among mammals, there are a number of orthologous genes that have canonical CREs in one species but the TGAT GTCA variant in another. These differences are likely due to deamination of the methylated cytosines in the CpG and may contribute to differential transcriptional regulation among orthologous genes.

Neither Caspase-3 nor DNA Fragmentation Factor Is Required for High Molecular Weight DNA Degradation in Apoptosis

In this paper, we show that there is a two‐step process of DNA fragmentation in apoptosis; DNA is first cleaved to large fragments of 50–300 kb that are subsequently cleaved to smaller oligonucleosomes in some, but not all cells. Significantly, only the first stage is considered essential for cell death since some cells, for example human MCF7 breast carcinoma cells and human NT2 neuronal cells, do not show this behavior but still display normal nuclear morphological apoptotic changes. In cells that usually produce small fragments blocking the second (internucleosomal) stage of DNA fragmentation prevents neither nuclear condensation nor apoptosis. We are beginning to understand why the extent of DNA fragmentation during apoptosis varies enormously and why it appears to be a function of the cell type not the inducer. Presumably, this reflects the content of not only endonuclease activit(ies) but also on the ability of the cells to activate caspases, particularly caspase‐3, and other proteases that may be involved in endonuclease activation. Since NT2 cells activate caspase‐3, but do not correctly process DFF45, b other factors must also impinge on the inevitability of that process.