Tet3 mediates stable glucocorticoid-induced alterations in DNA methylation and Dnmt3a/Dkk1 expression in neural progenitors

Developmental exposure to excess glucocorticoids (GCs) has harmful neurodevelopmental effects, which include persistent alterations in the differentiation potential of embryonic neural stem cells (NSCs). The mechanisms, however, are largely unknown. Here, we investigated the effects of dexamethasone (Dex, a synthetic GC analog) by MeDIP-like genome-wide analysis of differentially methylated DNA regions (DMRs) in NSCs isolated from embryonic rat cortices. We found that Dex-induced genome-wide DNA hypomethylation in the NSCs in vitro. Similarly, in utero exposure to Dex resulted in global DNA hypomethylation in the cerebral cortex of 3-day-old mouse pups. Dex-exposed NSCs displayed stable changes in the expression of the DNA methyltransferase Dnmt3a, and Dkk1, an essential factor for neuronal differentiation. These alterations were dependent on Tet3 upregulation. In conclusion, we propose that GCs elicit strong and persistent effects on DNA methylation in NSCs with Tet3 playing an essential role in the regulation of Dnmt3a and Dkk1. Noteworthy is the occurrence of similar changes in Dnmt3a and Dkk1 gene expression after exposure to excess GC in vivo.Glucocorticoid (GC) hormones are critical for the terminal maturation of organs, but fetal exposure to high levels of GCs have detrimental effects on the development of the nervous system, including impaired neurogenesis, alterations of the hypothalamic-pituitary-adrenal axis, and behavioral changes.^{1, 2, 3, 4, 5, 6, 7, 8} The fetus is protected from surges of GC by placental enzymes (namely 11bHSD2) that convert circulating GC into inactive, water soluble metabolites.⁹ Conditions that are associated with high fetal GC levels include severe maternal stress, placental failure, and exogenous administration of GC agonists in cases of high risk of premature delivery (reviewed in Harris and Seckl¹⁰). We have previously shown that neurons and neural stem cells (NSCs) of rats prenatally exposed to high levels of the synthetic GC dexamethasone (Dex) exhibit a long-lasting increased susceptibility to oxidative stress.^{11, 12} Dex treatment in vitro decreases NSC proliferation, neuronal differentiation, and modifies the expression of genes associated with cellular senescence and mitochondrial functions in a GC receptor (GR)-mediated manner.¹³ The phenotypical alterations are associated with a decrease in total DNA methylation and the expression of DNA methyltransferases (DNMTs), and notably these global changes persists in ''daughter'' NSCs never directly exposed to Dex, suggesting a bona fide epigenetic mechanism.¹³DNA methylation is catalyzed and maintained by DNMTs (Dnmt1, Dnmt3a, and Dnmt3b).¹⁴ Dnmt1 and Dnmt3a are required for proper proliferation as well as neuronal and glial differentiation of NSCs.^{15, 16, 17} Genetic deletion of Dnmt3a leads to premature glial differentiation,^{16, 18, 19} and conditional knockout mice exhibit decreased adult neurogenesis.^{17, 20, 21} The understanding of the dynamic regulation of DNA methylation has increased significantly with the discovery of the ten-eleven translocation (Tet) family of methylcytosine dioxygenases (Tet1, Tet2, and Tet3). Tets catalyze the oxidation of 5-methylcytosine (5-mC) and generate 5-mC derivatives, including 5-hydroxymethylcytosine (5-hmC). Recent reports have demonstrated that deficiency of Tet1 is associated with impaired embryonic and adult neurogenesis^{22, 23} whereas overexpression of Tet1 impairs memory formation in mice.²⁴ Tet3 is required for normal survival, proliferation, and differentiation of neural progenitor cells, but the mechanisms involved are not clarified.^{25, 26} Hence, a fine tuning of both Dnmts and Tets appears to be critical for the correct development and function of the brain.In this study, we aimed at elucidating the mechanisms underlying the programming effects of the GC agonist Dex on the epigenome in cortical NSCs. By analyzing genome-wide DNA methylation with a MeDIP-like approach, we found a dramatic decrease in DNA methylation and identified numerous differentially methylated regions (DMRs) in Dex-exposed proliferating NSCs. The genome-wide Dex-induced changes in methylation were associated with a downregulation of Dnmt3a and an upregulation of Tet3 in both parent (P) NSCs and daughter (D) cells, which were never directly exposed to Dex. Interestingly, we found a similar global DNA hypomethylation along with Dnmt3a downregulation and Tet1–3 upregulation in the cerebral cortex of pups exposed to Dex in utero. We have previously identified Dickkopf 1 (Dkk1) as a direct target of Dex acting via GR binding to the Dkk1 promoter.²⁷ Proteins of the Dkk family inhibit the canonical Wnt signaling and are essential for brain development.^{28, 29} We now show that the Dex-induced Dkk1 upregulation is heritable and dependent on Tet3 expression. Our results show that transient exposure to excess GC have dramatic and long-lasting effects on the epigenome of NSCs and specifically point to a critical Tet3-mediated dysregulation of Dnmt3a and Dkk1, both essential factors for proper forebrain development.     

A voltage-sensitive dye-based assay for the identification of differentiated neurons derived from embryonic neural stem cell cultures

Background

Pluripotent and multipotent stem cells hold great therapeutical promise for the replacement of degenerated tissue in neurological diseases. To fulfill that promise we have to understand the mechanisms underlying the differentiation of multipotent cells into specific types of neurons. Embryonic stem cell (ESC) and embryonic neural stem cell (NSC) cultures provide a valuable tool to study the processes of neural differentiation, which can be assessed using immunohistochemistry, gene expression, Ca²⁺-imaging or electrophysiology. However, indirect methods such as protein and gene analysis cannot provide direct evidence of neuronal functionality. In contrast, direct methods such as electrophysiological techniques are well suited to produce direct evidence of neural functionality but are limited to the study of a few cells on a culture plate.

Methodology/Principal Findings

In this study we describe a novel method for the detection of action potential-capable neurons differentiated from embryonic NSC cultures using fast voltage-sensitive dyes (VSD). We found that the use of extracellularly applied VSD resulted in a more detailed labeling of cellular processes compared to calcium indicators. In addition, VSD changes in fluorescence translated precisely to action potential kinetics as assessed by the injection of simulated slow and fast sodium currents using the dynamic clamp technique. We further demonstrate the use of a finite element model of the NSC culture cover slip for optimizing electrical stimulation parameters.

Conclusions/Significance

Our method allows for a repeatable fast and accurate stimulation of neurons derived from stem cell cultures to assess their differentiation state, which is capable of monitoring large amounts of cells without harming the overall culture.     

Nicotine self-medication of cognitive-attentional processing

This article selectively reviews research concerning nicotine's effects on cognition, including the neurobiological mechanism for these effects, task and experimental features that may be important for elucidating these effects, and why these effects may have amplified motivational significance among smokers with cognitive deficit. Nicotine has effects on various cognitive processes, though most studies in humans have focused on the amelioration of cognitive deficits experienced during drug withdrawal. The direct cognitive-enhancing effect of nicotine remains a controversial topic. The relationship between attentional and non-attentional cognitive effects of nicotine is discussed in the context of cognitive self-medication. Further research should include theory-driven examination of cognitive effects of nicotine, and develop targeted smoking cessation programs based on an improved understanding of the role of cognitive self-medication in high-risk individuals.     

Contractile behavior of the forelimb digital flexors during steady-state locomotion in horses (Equus caballus): an initial test of muscle architectural hypotheses about in vivo function

The forelimb digital flexors of the horse display remarkable diversity in muscle architecture despite each muscle-tendon unit having a similar mechanical advantage across the fetlock joint. We focus on two distinct muscles of the digital flexor system: short compartment deep digital flexor (DDF(sc)) and the superficial digital flexor (SDF). The objectives were to investigate force-length behavior and work performance of these two muscles in vivo during locomotion, and to determine how muscle architecture contributes to in vivo function in this system. We directly recorded muscle force (via tendon strain gauges) and muscle fascicle length (via sonomicrometry crystals) as horses walked (1.7 m s(-1)), trotted (4.1 m s(-1)) and cantered (7.0 m s(-1)) on a motorized treadmill. Over the range of gaits and speeds, DDF(sc) fascicles shortened while producing relatively low force, generating modest positive net work. In contrast, SDF fascicles initially shortened, then lengthened while producing high force, resulting in substantial negative net work. These findings suggest the long fibered, unipennate DDF(sc) supplements mechanical work during running, whereas the short fibered, multipennate SDF is specialized for economical high force and enhanced elastic energy storage. Apparent in vivo functions match well with the distinct architectural features of each muscle.     

Insights into the evolutionary origins of clostridial neurotoxins from analysis of the <Emphasis Type="Italic">Clostridium botulinum</Emphasis> strain A neurotoxin gene cluster

Background  

Clostridial neurotoxins (CNTs) are the most deadly toxins known and causal agents of botulism and tetanus neuroparalytic diseases. Despite considerable progress in understanding CNT structure and function, the evolutionary origins of CNTs remain a mystery as they are unique to Clostridium and possess a sequence and structural architecture distinct from other protein families. Uncovering the origins of CNTs would be a significant contribution to our understanding of how pathogens evolve and generate novel toxin families.     

Studies on the actin-binding protein HS1 in platelets

Background  

The platelet cytoskeleton mediates the dramatic change in platelet morphology that takes place upon activation and stabilizes thrombus formation. The Arp2/3 complex plays a vital role in these processes, providing the protrusive force for lamellipodia formation. The Arp2/3 complex is highly regulated by a number of actin-binding proteins including the haematopoietic-specific protein HS1 and its homologue cortactin. The present study investigates the role of HS1 in platelets using HS1^-/- mice.     

Utilization of MHC class I transgenic mice for development of minigene DNA vaccines encoding multiple HLA-restricted CTL epitopes

We engineered a multiepitope DNA minigene encoding nine dominant HLA-A2.1- and A11-restricted epitopes from the polymerase, envelope, and core proteins of hepatitis B virus and HIV, together with the PADRE (pan-DR epitope) universal Th cell epitope and an endoplasmic reticulum-translocating signal sequence. Immunization of HLA transgenic mice with this construct resulted in: 1) simultaneous CTL induction against all nine CTL epitopes despite their varying MHC binding affinities; 2) CTL responses that were equivalent in magnitude to those induced against a lipopeptide known be immunogenic in humans; 3) induction of memory CTLs up to 4 mo after a single DNA injection; 4) higher epitope-specific CTL responses than immunization with DNA encoding whole protein; and 5) a correlation between the immunogenicity of DNA-encoded epitopes in vivo and the in vitro responses of specific CTL lines against minigene DNA-transfected target cells. Examination of potential variables in minigene construct design revealed that removal of the PADRE Th cell epitope or the signal sequence, and changing the position of selected epitopes, affected the magnitude and frequency of CTL responses. Our results demonstrate the simultaneous induction of broad CTL responses in vivo against multiple dominant HLA-restricted epitopes using a minigene DNA vaccine and underline the utility of HLA transgenic mice in development and optimization of vaccine constructs for human use.