Nitric oxide (NO) plays a key role in neurogenesis as a regulator of cell proliferation and differentiation. NO is synthesized from the amino acid L‐arginine by nitric oxide synthases (NOS1, NOS2, and NOS3), which are encoded by separate genes and display different tissue distributions. We used an in vitro model of RA‐induced neural differentiation of NT2 cells to examine which of the three NO‐synthesizing enzymes is involved in this process. The results revealed a transient induction of NOS3 (known as the constitutively expressed endothelial nitric oxide synthase; eNOS) during the time course of the RA treatment. The peak of gene expression and the nuclear presence of NOS3 protein coincided with cell cycle exit of NT2‐derived neuronal precursors. The subsequent analysis of cytosine methylation and histone H3 acetylation of the human NOS3 5′ regulatory sequences indicated that epigenetic modifications, especially upstream of the proximal promoter (−734 to −989, relative to exon 2 TSS at +1), were also taking place. NOS1 was expressed only in the differentiated neurons (NT2‐N), whereas NOS2 was not expressed at all in this cellular model. Thus, a burst of NO production, possibly required to inhibit neural cell proliferation, was generated by the transient expression of NOS3 . This pattern of gene expression, in turn, required epigenetic remodeling of its regulatory region. Published 2012 Wiley Periodicals, Inc.

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.

Sexual dimorphisms are typically attributed to the hormonal differences arising once sex differentiation has occurred. However, in some sexually dimorphic diseases that differ in frequency but not severity, the differences cannot be logically connected to the sex hormones. Therefore, we asked whether any aspect of sexual dimorphism could be attributed to chromosomal rather than hormonal differences. Cells taken from mice at d 10.5 postconception (PC) before sexual differentiation, at d 17.5 PC after the first embryonic assertion of sexual hormones, and at postnatal day 17 (puberty) were cultured and exposed to 400 microM ethanol or 20 microM camptothecin or to infection with influenza A virus (multiplicity of infection of 5). The results showed that untreated male and female cells of the same age grew at similar rates and manifested similar morphology. However, they responded differently to the applied stressors, even before the production of fetal sex hormones. Furthermore, microarray and qPCR analyses of the whole 10.5 PC embryos also revealed differences in gene expression between male and female tissues. Likewise, the exposure of cells isolated from fetuses and adolescent mice to the stressors and/or sex hormones yielded expression patterns that reflected chromosomal sex, with ethanol feminizing male cells and masculinizing female cells. We conclude that cells differ innately according to sex irrespective of their history of exposure to sex hormones. These differences may have consequences in the course of sexually dimorphic diseases and their therapy.

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. © 2008 Wiley‐Liss, Inc.

BACKGROUND:Transcription 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 (TGACGTCA) 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, TGATGTCA.RESULTS:We 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 TGATGTCA variant CREs are highly conserved in mammals. However, when orthologous sequences differ, canonical CREs in human are most frequently TGATGTCA in the other species and vice-versa. We have identified 207 human CREs showing such differences.CONCLUSION:Our 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 TGATGTCA 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.

Neurogenesis is the process by which new brain cells are produced either during development or in the adult brain. More specifically, it is “the proliferation of neuronal precursor cells to produce neurons.” Both definitions embody a key role for the cell cycle in the process particularly because the brain is an architecturally complex, multicompartmented tissue and the correct numbers of neurons (and glial cells) must be placed into each compartment. The process is made more complicated by the fact that neurons within each compartment are highly specialized, mandating that the new neurons also have the correct phenotype. Therefore, a mechanistic understanding of neurogenesis requires an understanding of several processes—control of the cell cycle to generate neurons in sufficient numbers, spatial mechanisms that ensure the correct number of cells in each compartment, the differentiation process that transforms a progenitor cell into a neuron, and an explanation of how so many neuronal subtypes can be readily created. Equally important is an understanding of the temporal coordination of these four processes, particularly regarding cell cycle exit.

The ubiquitin Carboxyl-terminal Hydrolase-L1 gene (UCHL1) is a key enzyme in the protein degradation pathway; however, its precise role in protecting cells under stress conditions is unclear. In the present study we investigated the activity of this gene in human NT2/D1 embryonal carcinoma cells subjected to oxygen-glucose deprivation (OGD) and reoxygenation. OGD/reoxygenation cause global metabolic changes due to energy withdrawal and the subsequent generation of reactive oxygen species which initiates either a stress-adaptation-survival response or cell death, depending on the severity of the insult. A bi-phasic change in UCHL1 expression was observed by Q-PCR, Western blotting and flow cytometry. Down regulation of UCHL1 was detected immediately after OGD treatment and its expression was subsequently restored and increased 6 h after OGD treatment as well as during reoxygenation. Furthermore, flow cytometry analysis detected a lower level of UCHL1 only in apoptotic cells that had severe loss of mitochondrial membrane potential. Accordingly, down-regulation of endogenous UCHL1 by antisense cDNA in mouse N2a neuroblastoma cells increased the cell's sensitivity to OGD treatment. This down-regulation of endogenous UCHL1 led to the accumulation of p27, suggesting that UCHL1 is an essential gene to maintain cell homeostasis under normal growth and oxidative stress conditions.

International Journal of Developmental NeuroscienceVolume 24, Issue 8 p. 560-560 Abstract [P149]: Differential DNA methylation and histone acetylation of Sox2 SRR1 and SRR2 enhancers control its expression in neurons and astrocytes M. Sikorska, Corresponding Author M. Sikorska n/[email protected] Institute for Biological Sciences, National Research Council of Canada, CanadaSearch for more papers by this authorA. Jezierski, A. Jezierski Institute for Biological Sciences, National Research Council of Canada, CanadaSearch for more papers by this authorJ.K. Sandhu, J.K. Sandhu Institute for Biological Sciences, National Research Council of Canada, CanadaSearch for more papers by this authorP. Deb-Rinker, P. Deb-Rinker Institute for Biological Sciences, National Research Council of Canada, CanadaSearch for more papers by this authorC. Charlebois, C. Charlebois Institute for Biological Sciences, National Research Council of Canada, CanadaSearch for more papers by this authorM. Ribecco-Lutkiewicz, M. Ribecco-Lutkiewicz Institute for Biological Sciences, National Research Council of Canada, CanadaSearch for more papers by this authorM. Bani-Yaghoub, M. Bani-Yaghoub Institute for Biological Sciences, National Research Council of Canada, CanadaSearch for more papers by this authorP.R. Walker, P.R. Walker Institute for Biological Sciences, National Research Council of Canada, CanadaSearch for more papers by this author M. Sikorska, Corresponding Author M. Sikorska n/[email protected] Institute for Biological Sciences, National Research Council of Canada, CanadaSearch for more papers by this authorA. Jezierski, A. Jezierski Institute for Biological Sciences, National Research Council of Canada, CanadaSearch for more papers by this authorJ.K. Sandhu, J.K. Sandhu Institute for Biological Sciences, National Research Council of Canada, CanadaSearch for more papers by this authorP. Deb-Rinker, P. Deb-Rinker Institute for Biological Sciences, National Research Council of Canada, CanadaSearch for more papers by this authorC. Charlebois, C. Charlebois Institute for Biological Sciences, National Research Council of Canada, CanadaSearch for more papers by this authorM. Ribecco-Lutkiewicz, M. Ribecco-Lutkiewicz Institute for Biological Sciences, National Research Council of Canada, CanadaSearch for more papers by this authorM. Bani-Yaghoub, M. Bani-Yaghoub Institute for Biological Sciences, National Research Council of Canada, CanadaSearch for more papers by this authorP.R. Walker, P.R. Walker Institute for Biological Sciences, National Research Council of Canada, CanadaSearch for more papers by this author First published: 16 November 2006 https://doi.org/10.1016/j.ijdevneu.2006.09.210Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume24, Issue8ABSTRACTS TO THE 16TH BIENNIAL MEETING OF THE INTERNATIONAL SOCIETY FOR DEVELOPMENTAL NEUROSCIENCE, 24‐28 AUGUST 2006, BANFF, CANADADecember 2006Pages 560-560 RelatedInformation

The mammalian neocortex is established from neural stem and progenitor cells that utilize specific transcriptional and environmental factors to create functional neurons and astrocytes. Here, we examined the mechanism of Sox2 action during neocortical neurogenesis and gliogenesis. We established a robust Sox2 expression in neural stem and progenitor cells within the ventricular zone, which persisted until the cells exited the cell cycle. Overexpression of constitutively active Sox2 in neural progenitors resulted in upregulation of Notch1, recombination signal-sequence binding protein-J (RBP-J) and hairy enhancer of split 5 (Hes5) transcripts and the Sox2 high mobility group (HMG) domain seemed sufficient to confer these effects. While Sox2 overexpression permitted the differentiation of progenitors into astroglia, it inhibited neurogenesis, unless the Notch pathway was blocked. Moreover, neuronal precursors engaged a serine protease(s) to eliminate the overexpressed Sox2 protein and relieve the repression of neurogenesis. Glial precursors and differentiated astrocytes, on the other hand, maintained Sox2 expression until they reached a quiescent state. Sox2 expression was re-activated by signals that triggered astrocytic proliferation (i.e., injury, mitogenic and gliogenic factors). Taken together, Sox2 appears to act upstream of the Notch signaling pathway to maintain the cell proliferative potential and to ensure the generation of sufficient cell numbers and phenotypes in the developing neocortex.

BACKGROUND:Alzheimer's disease (AD) is a complex disorder that involves multiple biological processes. Many genes implicated in these processes may be present in low abundance in the human brain. DNA microarray analysis identifies changed genes that are expressed at high or moderate levels. Complementary to this approach, we described here a novel technology designed specifically to isolate rare and novel genes previously undetectable by other methods. We have used this method to identify differentially expressed genes in brains affected by AD. Our method, termed Subtractive Transcription-based Amplification of mRNA (STAR), is a combination of subtractive RNA/DNA hybridization and RNA amplification, which allows the removal of non-differentially expressed transcripts and the linear amplification of the differentially expressed genes.RESULTS:Using the STAR technology we have identified over 800 differentially expressed sequences in AD brains, both up- and down- regulated, compared to age-matched controls. Over 55% of the sequences represent genes of unknown function and roughly half of them were novel and rare discoveries in the human brain. The expression changes of nearly 80 unique genes were further confirmed by qRT-PCR and the association of additional genes with AD and/or neurodegeneration was established using an in-house literature mining tool (LitMiner).CONCLUSION:The STAR process significantly amplifies unique and rare sequences relative to abundant housekeeping genes and, as a consequence, identifies genes not previously linked to AD. This method also offers new opportunities to study the subtle changes in gene expression that potentially contribute to the development and/or progression of AD.

We isolated a fragment of the fukutin gene promoter from differentiated human NT2 cells using chromatin immunoprecipitation technique with an anti-CREB antibody. This fragment contained a CRE-like sequence and here we describe its functional validation. The results showed that the element was functional in vitro and in vivo and that CREB in neurons was involved in the transcriptional regulation of the fukutin gene. Moreover, its expression in neurons was regulated by cAMP and calcium ions, known triggers of CREB phosphorylation. To our knowledge, this is the first report on the regulation of fukutin gene by transcription factor CREB in response to the signals generated by synaptic activity. The true biological function of fukutin, the gene responsible for Fukuyama-type congenital muscular dystrophy and mental retardation, is at present not known. However, it has been suggested that it might possess glycosyltransferase activity and its intracellular localization within the Golgi structures is consistent with this function. As such, fukutin might play a significant role in post-translational modification of synaptic proteins in neuronal cells.

DNA fragmentation in apoptosis, especially in lymphocytic cells, is initiated at scaffold/matrix attachment regions (S/ MARs) and is preceded by the degradation of nuclear proteins. The present study was performed to establish whether the same mechanism occurred in human NT2 cells subjected to oxygen and glucose deprivation (OGD). We analyzed the integrity of c-myc S/ MAR containing a base-unpairing region ( BUR)-like element, which we established to be a binding site of the transcription factor Sox2. An accumulation of DNA breaks in close proximity to this element and a degradation of Sox2 were observed early in the OGD-induced apoptotic response. Identification of Sox2 as a novel c-myc BUR-binding protein was achieved through yeast one-hybrid screening and the Sox2/DNA interaction was confirmed by electrophoretic mobility shift assay and immunoprecipitation with Sox2 antibody. Our data support the notion that early proteolysis of unique BUR-binding proteins might represent a universal mechanism that renders these DNA sites vulnerable to endonucleolysis.

Human NT2 cells, which differentiate into neurons and astrocytes, initially express and then permanently down-regulate Nanog and Oct-4 (POU51F1). 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.

DNaseY, a Ca(2+)- and Mg(2+)-dependent endonuclease, has been implicated in apoptotic DNA degradation; however, the molecular mechanisms controlling its involvement in this process have not been fully elucidated. We have obtained evidence from yeast two-hybrid screening and coimmunoprecipitation experiments that DNaseY interacted physically with actinin-alpha4 and this interaction significantly enhanced its endonuclease activity. Accordingly, simultaneous overexpression of both proteins in PC12 cells dramatically increased the rate of apoptosis in response to teniposide' VM26. However, overexpression of DNaseY alone neither triggered apoptosis nor facilitated cell death in response to VM26 or serum deprivation. Instead, the overexpression of DNaseY increased the production of single-strand DNA breaks and evoked a profound upregulation of DNA repair pathways. Taken together, our results point to a novel regulatory mechanism of DNaseY activity and offer an explanation for why cells must first cleave key DNA repair and replication proteins before the successful execution of apoptosis.

Cis-regulatory motifs are often overrepresented in promoters and may exhibit frequency biases in subpromoter regions (SPRs). Many probabilistic algorithms have been used to predict such motifs, but they tend to generate many false positives. We devised a novel algorithm, MotifFilter, that computes representation indices (RIs) for putative motifs. MotifFilter's RI is a ratio of the actual over expected frequency of a motif in promoters, SPRs or random genomic DNA that takes into account of the nucleotide probability distributions in these regions. This approach was applied to a genome-wide survey of putative cAMP-response elements (CREs) for motifs generated by a profile hidden Markov model. Twenty of 144 putative CRE motifs found in the survey were retained by the MotifFilter.

The purpose of this study was to develop a method for identifying useful patterns in gene expression time-series data. We have developed a novel data mining approach that identifies interesting patterns. The method consists of a combination of data pre-processing as well as unsupervised and supervised learning techniques. To evaluate our approach, we have analyzed three time series data sets which investigate the temporal transcriptome changes that occur during: 1) the cell cycle of budding yeast (S. cerevisiae) [3], 2) the epithelial to mesenchymal transition induced by Transforming Growth Factor-β1 in mouse mammary epithelial BRI-JM01 cells, and 3) the program of differentiation induced by retinoic acid in human embryonal teratocarcinoma NT-2 cells. We present the results from all of our experiments, discuss the patterns discovered through the use of our approach and briefly explain future plans and directions for improving our method.

We have produced a family of novel carriers enabling water solubilization of highly lipophilic molecules. The compound carriers were synthesized by conjugating polyethylene glycol to alpha-tocopherol, tocotrienols, beta-sitosterol or cholesterol via an alkanedioyl linker. These PEG- conjugates were amphiphilic and formed stable non-covalent complexes (nanomicelles) with a wide range of molecules including vitamins, carotenoids, ubiquinones, poly-unsaturated fatty acids and polyene macrolide antibiotics. The resulting formulations were water-soluble, non-toxic and had excellent stability. This solubilization method represents a major advance in the delivery of lipophilic molecules and could be used to reformulate drugs with near term patent expiry or those that have failed clinical trials due to low solubility. Furthermore, the technology could also be applied for delivery of active ingredients for dietary supplement, functional food, cosmetic and animal health industries.

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 Alzheimer's or other neurological diseases. This is higher than any of the previously published Alzheimer's 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.

The level of glutathione (GSH) is often reduced in brains that are affected by neurodegeneration. It is not known, however,whether this is a cause or a consequence of the disease. Here we have examined the effects of GSH depletion on the viability of human neurons cultured in either the presence or the absence of astrocytes, both derived from NT2/D1 cells. We established that the endogenous concentration of GSH is 10 times lower in neurons than in astrocytes (1.42 versus 18.9 pmol microg protein(-1)) and that pure neuronal cultures begin to die by apoptosis within 24 h of GSH depletion. By contrast, neurons that are co-cultured with astrocytes remain viable for several days, even with a profoundly decreased GSH content. However, they die rapidly when challenged additionally with nitrative stress. In addition, astrocytes survive for prolonged periods of time (>12 days) under severely reduced GSH concentrations. Our study shows clear differences in the content and sensitivity to depletion of GSH in neurons and astrocytes and establishes the significance of neuronal-glial interactions for the maintenance of neuronal viability under reduced GSH content. However, with chronic GSH depletion, these interactions might not be sufficient to protect neurons from other injurious factors (i.e. reactive oxygen and nitrogen species), which indicates that defective GSH metabolism might facilitate the progression of neurodegeneration.