Cliona O'Driscoll

Hydroquinone Increases 5-Hydroxymethylcytosine Formation through Ten Eleven Translocation 1 (TET1) 5-Methylcytosine Dioxygenase

Background: Hydroquinone is a benzene metabolite shown to lead to decreased DNA methylation. Results: Hydroquinone exposure increases Ten Eleven Translocation 1 methylcytosine dioxygenase activity and 5-hydroxymethylcytosine levels and decreases DNA methylation. Conclusion: Hydroquinone leads to DNA demethylation through a Ten Eleven Translocation 1-dependent mechanism. Significance: This mechanism may explain observations of decreased DNA methylation and cytotoxicity following exposure to benzene and hydroquinone. DNA methylation regulates gene expression throughout development and in a wide range of pathologies such as cancer and neurological disorders. Pathways controlling the dynamic levels and targets of methylation are known to be disrupted by chemicals and are therefore of great interest in both prevention and clinical contexts. Benzene and its metabolite hydroquinone have been shown to lead to decreased levels of DNA methylation, although the mechanism is not known. This study employs a cell culture model to investigate the mechanism of hydroquinone-mediated changes in DNA methylation. Exposures that do not affect HEK293 cell viability led to genomic and methylated reporter DNA demethylation. Hydroquinone caused reactivation of a methylated reporter plasmid that was prevented by the addition of N-acetylcysteine. Hydroquinone also caused an increase in Ten Eleven Translocation 1 activity and global levels of 5-hydroxymethylcytosine. 5-Hydroxymethylcytosine was found enriched at LINE-1 prior to a decrease in both 5-hydroxymethylcytosine and 5-methylcytosine. Ten Eleven Translocation-1 knockdown decreased 5-hydroxymethylcytosine formation following hydroquinone exposure as well as the induction of glutamate-cysteine ligase catalytic subunit and 14-3-3σ. Finally, Ten Eleven Translocation 1 knockdown decreased the percentage of cells accumulating in G2+M following hydroquinone exposure, indicating that it may have a role in cell cycle changes in response to toxicants. This work demonstrates that hydroquinone exposure leads to active and functional DNA demethylation in HEK293 cells in a mechanism involving reactive oxygen species and Ten Eleven Translocation 1 5-methylcytosine dioxygenase.

Toward a 3D model of human brain development for studying gene/environment interactions

This project aims to establish and characterize an in vitro model of the developing human brain for the purpose of testing drugs and chemicals. To accurately assess risk, a model needs to recapitulate the complex interactions between different types of glial cells and neurons in a three-dimensional platform. Moreover, human cells are preferred over cells from rodents to eliminate cross-species differences in sensitivity to chemicals. Previously, we established conditions to culture rat primary cells as three-dimensional aggregates, which will be humanized and evaluated here with induced pluripotent stem cells (iPSCs). The use of iPSCs allows us to address gene/environment interactions as well as the potential of chemicals to interfere with epigenetic mechanisms. Additionally, iPSCs afford us the opportunity to study the effect of chemicals during very early stages of brain development. It is well recognized that assays for testing toxicity in the developing brain must consider differences in sensitivity and susceptibility that arise depending on the time of exposure. This model will reflect critical developmental processes such as proliferation, differentiation, lineage specification, migration, axonal growth, dendritic arborization and synaptogenesis, which will probably display differences in sensitivity to different types of chemicals. Functional endpoints will evaluate the complex cell-to-cell interactions that are affected in neurodevelopment through chemical perturbation, and the efficacy of drug intervention to prevent or reverse phenotypes. The model described is designed to assess developmental neurotoxicity effects on unique processes occurring during human brain development by leveraging human iPSCs from diverse genetic backgrounds, which can be differentiated into different cell types of the central nervous system. Our goal is to demonstrate the feasibility of the personalized model using iPSCs derived from individuals with neurodevelopmental disorders caused by known mutations and chromosomal aberrations. Notably, such a human brain model will be a versatile tool for more complex testing platforms and strategies as well as research into central nervous system physiology and pathology.

Small molecule glutaminase inhibitors block glutamate release from stimulated microglia

Glutaminase plays a critical role in the generation of glutamate, a key excitatory neurotransmitter in the CNS. Excess glutamate release from activated macrophages and microglia correlates with upregulated glutaminase suggesting a pathogenic role for glutaminase. Both glutaminase siRNA and small molecule inhibitors have been shown to decrease excess glutamate and provide neuroprotection in multiple models of disease, including HIV-associated dementia (HAD), multiple sclerosis and ischemia. Consequently, inhibition of glutaminase could be of interest for treatment of these diseases. Bis-2-(5-phenylacetimido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and 6-diazo-5-oxo-l-norleucine (DON), two most commonly used glutaminase inhibitors, are either poorly soluble or non-specific. Recently, several new BPTES analogs with improved physicochemical properties were reported. To evaluate these new inhibitors, we established a cell-based microglial activation assay measuring glutamate release. Microglia-mediated glutamate levels were significantly augmented by tumor necrosis factor (TNF)-α, phorbol 12-myristate 13-acetate (PMA) and Toll-like receptor (TLR) ligands coincident with increased glutaminase activity. While several potent glutaminase inhibitors abrogated the increase in glutamate, a structurally related analog devoid of glutaminase activity was unable to block the increase. In the absence of glutamine, glutamate levels were significantly attenuated. These data suggest that the in vitro microglia assay may be a useful tool in developing glutaminase inhibitors of therapeutic interest.

Temporal and Regional Alterations in NMDA Receptor Expression in Mecp2-Null Mice

Our previous postmortem study of girls with Rett Syndrome (RTT), a development disorder caused by MECP2 mutations, found increases in the density of N‐Methyl‐D‐aspartate (NMDA) receptors in the prefrontal cortex of 2–8‐year‐old girls, whereas girls older than 10 years had reductions in NMDA receptors compared with age‐matched controls (Blue et al., Ann Neurol 1999b;45:541–545). Using [3H]‐CGP to label NMDA‐type glutamate receptors in 2‐ and 7‐week old wild‐type (WT), Mecp2‐null, and Mecp2‐heterozygous (HET) mice (Bird model), we found that frontal areas of the brain also exhibited a bimodal pattern in NMDA expression, with increased densities of NMDA receptors in Mecp2‐null mice at 2 weeks of age but decreased densities at 7 weeks of age. Visual cortex showed a similar pattern, while other cortical regions only exhibited changes in NMDA receptor densities at 2 weeks (retrosplenial granular) or 7 weeks (somatosensory). In thalamus of null mice, NMDA receptors were increased at 2 and 7 weeks. No significant differences in density were found between HET and WT mice at both ages. Western blots for NMDAR1 expression in frontal brain showed higher levels of expression in Mecp2‐null mice at 2 weeks of age but not at 1 or 7 weeks of age. Our mouse data support the notion that deficient MeCP2 function is the primary cause of the NMDA receptor changes we observed in RTT. Furthermore, the findings of regional and temporal differences in NMDA expression illustrate the importance of age and brain region in evaluating different genotypes of mice. Anat Rec, 2011.

Evaluation of the Interactions Between Multiwalled Carbon Nanotubes and Caco-2 Cells

The aim of this study was to determine whether multiwalled carbon nanotubes (MWNCT) are taken up by and are toxic to human intestinal enterocytes using the Caco-2 cell model. Caco-2 cells were exposed to 50 μg/ml MWCNT (oxidized or pristine) for 24 h, and experiments were repeated in the presence of 2.5 mg/L natural organic matter. Cells displayed many of the properties that characterize enterocytes, such as apical microvilli, basolateral basement membrane, and glycogen. The cell monolayers also displayed tight junctions and electrical resistance. Exposure to pristine and oxidized MWCNT, with or without natural organic matter, did not markedly affect viability, which was assessed by measuring activity of released lactate dehydrogenase (LDH) and staining with propidium iodide. Ultrastructural analysis revealed some damage to microvilli colocalized with the MWCNT; however, neither type of MWCNT was taken up by Caco-2 cells. In contrast, pristine and oxidized MWCNT were taken up by the macrophage RAW 264.7 line. Our study suggests that intestinal enterocytes cells do not take up MWCNT.

Methyl CpG binding protein 2 deficiency enhances expression of inflammatory cytokines by sustaining NF-κB signaling in myeloid derived cells

Knocking down methyl CpG binding protein 2 (MeCP2) enhances NF-κB activation in human peripheral blood mononuclear cells (PBMC). In this study, we examined whether this caused the expression of cytokines to be elevated. Increased levels of TNFα, IL-6, and IL-3 mRNAs were observed in human PBMC made MeCP2 deficient with a lentiviral shRNA MeCP2 vector and in splenocytes from MeCP2-null mice. TNFα neutralizing antibody attenuated expression of IL-6 and TNFα but did not affect expression of IL-3. Lipopolysaccharide-mediated increases in TNFα, IL-6, and IL-3 mRNAs were also enhanced in MeCP2-deficient PBMC. Two inhibitors of NF-κB blocked the increased levels of IL-6, TNFα, and IL-3 in MeCP2-deficient PBMC treated with lipopolysaccharide. MeCP2 deficiency also enhanced expression of IL-6 and TNFα mRNAs in the THP1 human monocyte cell line, which were also attenuated by the NF-κB inhibitors. In chromatin immunoprecipitation assays, the binding of the NF-κB family member p65 and acetylated H3 to the TNFα promoter was greater after treatment with LPS in MeCP2-deficient THP1 cells. MeCP2 did not bind to the TNFα promoter. In summary, the data indicates that MeCP2 deficiency increases expression of TNFα and other inflammatory cytokines by enhancing NF-κB signaling.

Induction of a trophoblast-like phenotype by hydralazine in the p19 embryonic carcinoma cell line

Chemicals that affect cellular differentiation through epigenetic mechanisms have potential utility in treating a wide range of diseases. Hydralazine decreases DNA methylation in some cell types but its effect on differentiation has not been well explored. After five days of exposure to hydralazine, P19 embryocarcinoma cells displayed a giant cell morphology and were binucleate, indicative of a trophoblast-like morphology. Other trophoblast-like properties included the intermediary filament Troma-1/cytokeratin 8 and the transcription factor Tead4. A decrease in CpG methylation at three sites in the TEAD4 promoter and the B1 repeated sequence was observed. Knocking down expression of Tead4 with siRNA blocked the increase in Troma-1/cytokeratin 8 and over expression of Tead4 induced the expression of Troma-1/cytokeratin 8. Cells treated for 5days with hydralazine were no longer capable of undergoing retinoic acid-mediated neuronal differentiation. An irreversible loss of the pluripotent transcription factor Oct-4 was observed following hydralazine exposure. In summary, hydralazine induces P19 cells to assume a trophoblast-like phenotype by upregulating Tead4 expression through a mechanism involving DNA demethylation.

Twenty-four hour quantitative-EEG and in-vivo glutamate biosensor detects activity and circadian rhythm dependent biomarkers of pathogenesis in Mecp2 null mice

Mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (Mecp2) cause most cases of Rett syndrome (RTT). Currently there is no cure for RTT. Abnormal EEGs are found in 100% of RTT cases and are associated with severe sleep dysfunction, the cause of which is not well understood. Mice deficient in MeCP2 protein have been studied and characterized for their neuropathological and behavioral deficits to better understand RTT. With the goal to study the non-ictal EEG correlates in symptomatic Mecp2 KO mice (Mecp2tm1.1Bird/y), and determine novel EEG biomarkers of their reported progressive neurodegeneration, we used 24 h video-EEG/EMG with synchronous in-vivo cortical glutamate biosensor in the frontal cortex. We scored the EEG for activity states and spectral analysis was performed to evaluate correlations to the synchronous extracellular glutamate fluctuations underlying Mecp2 inactivation as compared to WT. Significant alterations in sleep structure due to dark cycle-specific long wake states and poor quality of slow-wave sleep were associated with a significant increase in glutamate loads per activity cycle. The dynamics of the activity-state-dependent physiological rise and fall of glutamate indicative of glutamate homeostasis were significantly altered in the KO mice. Colorimetric quantitation of absolute glutamate levels in frontal cortex also indicated the presence of significantly higher levels in KO. This study for the first time found evidence of uncompensated sleep deprivation-like EEG biomarkers that were associated with glutamate homeostatic dysfunction in the Mecp2 KO mice.

Relationship between Mecp2 and NFκb signaling during neural differentiation of P19 cells

The pluripotent P19 embryo carcinoma cell line was studied to determine a signaling pathway regulating MeCP2 expression. P19 cells were induced to differentiate into neurons by RA and express β-III tubulin at one day after induction and synaptophysin by 7 days. MeCP2 was first observed after β-III tubulin expression was detected and continued to rise over the course of differentiation. Both Mecp2 e1 and e2 mRNA forms progressively increased in differentiating cells. MeCP2 expression was increased by tumor necrosis factor (TNF) in early differentiating cells, which was blocked by NFκB inhibitors. TNF did not increase MeCP2 expression in naïve cells. Moreover, TNF did not increase NFκB reporter gene activity in naïve cells even though increases were observed in early differentiating cells. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) increased MeCP2 expression in naïve P19 cells, which was also blocked by NFκB inhibitors. Interestingly, PMA increased NFκB reporter gene activity in naïve cells. Finally, PMA, but not TNF, induced IκBα degradation in naïve P19 cells. Taken together, our data indicates that MeCP2 expression is regulated in part by signaling pathways involving NFκB.

Assessing Blood–Brain Barrier Function Using In Vitro Assays

The impermeability of the blood-brain barrier (BBB) is due to a number of properties including tight junctions on adjoining endothelial cells, absence of pinocytic vesicles, and expression of multidrug transporters. Although the permeability of many chemicals can be predicted by their polarity, or oil/water partition coefficient, many lipophilic chemicals are not permeable because of multidrug transporters at the luminal and abluminal membranes. In contrast, many nutrients, which are usually polar, cross the BBB more readily than predicted by their oil/water partition coefficients due to the expression of specific nutrient transporters. In vitro models are being developed because rodent models are of low input and relatively expensive. Isolated brain microvessels and cell culture models each offers certain advantages and disadvantages. Isolated brain microvessels are useful in measuring multidrug drug transporters and tight junction integrity, whereas cell culture models allow the investigator to measure directional transport and can be genetically manipulated. In this chapter, we describe how to isolate large batches of brain microvessels from freshly slaughtered cows. The different steps in the isolation procedure include density gradient centrifugations and filtering. Purity is determined microscopically and by marker enzymes. Permeability is assessed by measuring the uptake of fluorescein-labeled dextran in an assay that has been optimized to have a large dynamic range and low inter-day variability. We also describe how to evaluate transendothelial cell electrical resistance and paracellular transport in cell culture models.