NRF2 activation protects against valproic acid-induced disruption of neurogenesis in P19 cells.

Valproic acid (VPA) is a commonly prescribed antiepileptic drug that causes fetal valproate syndrome (FVS) in developing embryos exposed to it. Symptoms of FVS include neural tube defects (NTDs), musculoskeletal abnormalities, and neurodevelopmental difficulties. One proposed mechanism of VPA-induced developmental toxicity is via oxidative stress, defined as the disruption of redox-sensitive cell signaling. We propose that redox imbalances caused by VPA exposure result in improper cellular differentiation that may contribute to FVS. In undifferentiated P19 mouse embryonal carcinoma cells treated with VPA, glutathione disulfide (GSSG) concentrations were higher and the glutathione (GSH)/GSSG redox potential (Eh) was more oxidizing compared to vehicle-treated control cells, both of which are indications of potential intracellular oxidative stress. Interestingly, VPA had no effect on GSH or GSSG levels in differentiated P19 neurons. Undifferentiated cells pretreated with 3H-1,2-dithiole-3-thione (D3T), an inducer of the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant response that combats cellular redox disruption, were protected from VPA-induced alterations to the GSH/GSSG system. To assess differential periods of susceptibility, P19 cells were exposed to VPA at various time points during their neuronal differentiation. Cells exposed to VPA early in the differentiation process did not undergo normal neurogenesis as measured by POU domain, class 5, transcription factor 1 (OCT4) and tubulin beta-3 chain (ßIII-tubulin), markers of cell stemness and neuronal differentiation, respectively. Neurogenesis was improved with D3T pretreatments prior to VPA exposure. Furthermore, differentiating P19 cells treated with VPA exhibited increased protein oxidation that was diminished with D3T pretreatment. These findings demonstrate that VPA inhibits neurogenesis and propose NRF2-mediated redox homeostasis as a means to promote normal neuronal differentiation, thereby potentially decreasing the prevalence of FVS outcomes.

Ceramide: a novel inducer for neural tube defects.

BACKGROUND: Circulating plasma ceramides, a class of bioactive sphingolipids, are elevated in metabolic disorders, including obesity. Infants of women with these disorders are at 2- to 3-fold greater risk for developing a neural tube defect (NTD). This study aimed to test the effects of embryonic exposure to C2-ceramides (C2) during neural tube closure. Preliminary data shows an increase in NTDs in chick embryos after C2 exposure, and addresses potential mechanisms. RESULTS: Cell and embryo models were used to examine redox shifts after ceramide exposure. While undifferentiated P19 cells were resistant to ceramide exposure, neuronally differentiated P19 cells exhibited an oxidizing shift. Consistent with these observations, GSH E h curves revealed a shift to a more oxidized state in C2 treated embryos without increasing apoptosis or changing Pax3 expression, however cell proliferation was lower. Neural tube defects were observed in 45% of chick embryos exposed to C2, compared to 12% in control embryos. CONCLUSIONS: C2 exposure during critical developmental stages increased the frequency of NTDs in the avian model. Increased ROS generation in cell culture, along with the more oxidative GSH E h profiles of C2 exposed cells and embryos, support a model wherein ceramide affects neural tube closure via altered tissue redox environments.

Comparative analysis of lipid-mediated CRISPR-Cas9 genome editing techniques.

CRISPR-Cas technology has revolutionized genome engineering. While Cas9 was not the first programmable endonuclease identified, its simplicity of use has driven widespread adoption in a short period of time. While CRISPR-Cas genome editing holds enormous potential for clinical applications, its use in laboratory settings for genotype-phenotype studies and genome-wide screens has led to breakthroughs in the understanding of many molecular pathways. Numerous protocols have been described for introducing CRISPR-Cas components into cells, and here we sought to simplify and optimize a protocol for genome editing using readily available and inexpensive tools. We compared plasmid, ribonucleoprotein (RNP), and RNA transfection to determine which was method was most optimal for editing cells in a laboratory setting. We limited our comparison to lipofection-mediated introduction because the reagents are widely available. To facilitate optimization, we developed a novel reporter assay to measure gene disruption and the introduction of a variety of exogenous DNA tags. Each method efficiently disrupted endogenous genes and was able to stimulate the introduction of foreign DNA at specific sites, albeit to varying efficiencies. RNP transfection produced the highest level of gene disruption and was the most rapid and efficient method overall. Finally, we show that very short homology arms of 30 base pairs can mediate site-specific editing. The methods described here should broaden the accessibility of RNP-mediated lipofection for laboratory genome-editing experiments.

Valproic Acid Causes Redox-Regulated Post-Translational Protein Modifications That Are Dependent upon P19 Cellular Differentiation States.

Valproic acid (VPA) is a common anti-epileptic drug and known neurodevelopmental toxicant. Although the exact mechanism of VPA toxicity remains unknown, recent findings show that VPA disrupts redox signaling in undifferentiated cells but has little effect on fully differentiated neurons. Redox imbalances often alter oxidative post-translational protein modifications and could affect embryogenesis if developmentally critical proteins are targeted. We hypothesize that VPA causes redox-sensitive post-translational protein modifications that are dependent upon cellular differentiation states. Undifferentiated P19 cells and P19-derived neurons were treated with VPA alone or pretreated with D3T, an inducer of the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant pathway, prior to VPA exposure. Undifferentiated cells treated with VPA alone exhibited an oxidized glutathione redox couple and increased overall protein oxidation, whereas differentiated neurons were protected from protein oxidation via increased S-glutathionylation. Pretreatment with D3T prevented the effects of VPA exposure in undifferentiated cells. Taken together, our findings support redox-sensitive post-translational protein alterations in undifferentiated cells as a mechanism of VPA-induced developmental toxicity and propose NRF2 activation as a means to preserve proper neurogenesis.

NRF2 activation inhibits valproic acid-induced neural tube defects in mice.

Valproic acid (VPA) is a widely prescribed medication that has traditionally been used to treat epilepsy, yet embryonic exposure to VPA increases the risk of the fetus developing neural tube defects (NTDs). While the mechanism by which VPA causes NTDs is unknown, we hypothesize that VPA causes dysmorphogenesis through the disruption of redox-sensitive signaling pathways that are critical for proper embryonic development, and that protection from the redox disruption may decrease the prevalence of NTDs. Time-bred CD-1 mice were treated with 3H-1,2-dithiole-3-thione (D3T), an inducer of nuclear factor erythroid 2-related factor 2 (NRF2)-a transcription factor that activates the intracellular antioxidant response to prevent redox disruptions. Embryos were then collected for whole embryo culture and subsequently treated with VPA in vitro. The glutathione (GSH)/glutathione disulfide (GSSG) redox potential (Eh), a measure of the intracellular redox environment, was measured in the developing mouse embryos. Embryos treated with VPA exhibited a transiently oxidizing GSH/GSSG Eh, while those that received D3T pretreatment prior to VPA exposure showed no differences compared to controls. Moving to an in utero mouse model, time-bred C57BL/6 J dams were pretreated with or without D3T and then exposed to VPA, after which all embryos were collected for morphological analyses. The prevalence of open neural tubes in embryos treated with VPA significantly decreased with D3T pretreatment, as did the severity of the observed defects evaluated by a morphological assessment. These data show that NRF2 induction via D3T pretreatment protects against VPA-induced redox dysregulation and decreases the prevalence of NTDs in developing mouse embryos.

Spatiotemporal evaluation of the mouse embryonic redox environment and histiotrophic nutrition following treatment with valproic acid and 1,2-dithiole-3-thione during early organogenesis.

Redox regulation during metazoan development ensures that coordinated metabolic reprogramming and developmental signaling are orchestrated with high fidelity in the hypoxic embryonic environment. Valproic acid (VPA), an anti-seizure medication, is known to increase markers of oxidation and also increase the risk of neural tube defects (NTDs) when taken during pregnancy. It is unknown, however, whether oxidation plays a direct role in failed neural tube closure (NTC). Spatial and temporal fluctuations in total glutathione (GSH) and total cysteine (Cys) redox steady states were seen during a 24 h period of CD-1 mouse organogenesis in untreated conceptuses and following exposure to VPA and the Nrf2 antioxidant pathway inducer, 1,2-dithiole-3-thione (D3T). Glutathione, glutathione disulfide (GSSG), and Cys, cystine (CySS) concentrations, measured in conceptal tissues (embryo/visceral yolk sac) and fluids (yolk sac fluid/amniotic fluid) showed that VPA did not cause extensive and prolonged oxidation during the period of NTC, but instead produced transient periods of oxidation, as assessed by GSH:GSSG redox potentials, which revealed oxidation in all four conceptal compartments at 4, 10, and 14 h, corresponding to the period of heartbeat activation and NTC. Other changes were tissue and time specific. VPA treatment also reduced total FITC-Ab clearance from the medium over 3 h, indicating potential disruption of nutritive amino acid supply. Overall, these results indicated that VPA's ability to affect cellular redox status may be limited to tissue-specific windows of sensitivity during the period of NTC. The safety evaluation of drugs used during pregnancy should consider time and tissue specific redox factors.

Valproic acid promotes SOD2 acetylation: a potential mechanism of valproic acid-induced oxidative stress in developing systems.

Valproic acid (VPA) is an antiepileptic, bipolar, and migraine medication, which is associated with embryonic dysmorphology, more specifically neural tube defects (NTDs), if taken while pregnant. One mechanism by which VPA may cause NTDs is through oxidative stress that cause disruption of cell signaling. However, mechanisms of VPA-induced oxidative stress are not fully understood. Since VPA is a deacetylase inhibitor, we propose that VPA promotes mitochondrial superoxide dismutase-2 (SOD2) acetylation, decreasing SOD2 activity and increasing oxidant levels. Using the pluripotent embryonal carcinoma cell line, P19, VPA effects were evaluated in undifferentiated and neurodifferentiated cells. VPA treatments increased oxidant levels, oxidized the glutathione (GSH)/glutathione disulfide (GSSG) redox couple, and decreased total SOD and SOD2 activity in undifferentiated P19 cells but not in differentiated P19 cells. VPA caused a specific increase in mitochondrial oxidants in undifferentiated P19 cells, VPA did not alter respirometry measurements. Immunoblot analyses demonstrated that VPA increased acetylation of SOD2 at lysine68 (AcK68 SOD2) in undifferentiated P19 cells but not in differentiated P19 cells. Pretreatments with the Nrf2 inducer, dithiol-3-thione (D3T), in undifferentiated P19 cells prevented increased oxidant levels, GSH/GSSG redox oxidation and restored total SOD and SOD2 activity, correlating with a decrease in AcK68 SOD2 levels. In embryos, VPA decreased total SOD and SOD2 activity and increased levels of AcK68 SOD2, and D3T pretreatments prevented VPA effects, increasing total SOD and SOD2 activity and lowering levels of AcK68 SOD2. These data demonstrate a potential, contributing oxidizing mechanism by which VPA incites teratogenesis in developing systems. Moreover, these data also suggest that Nrf2 interventions may serve as a means to protect developmental signaling and inhibit VPA-induced malformations.

Lack of skeletal muscle liver kinase B1 alters gene expression, mitochondrial content, inflammation and oxidative stress without affecting high-fat diet-induced obesity or insulin resistance.

Ad libitum high-fat diet (HFD) induces obesity and skeletal muscle metabolic dysfunction. Liver kinase B1 (LKB1) regulates skeletal muscle metabolism by controlling the AMP-activated protein kinase family, but its importance in regulating muscle gene expression and glucose tolerance in obese mice has not been established. The purpose of this study was to determine how the lack of LKB1 in skeletal muscle (KO) affects gene expression and glucose tolerance in HFD-fed, obese mice. KO and littermate control wild-type (WT) mice were fed a standard diet or HFD for 14 weeks. RNA sequencing, and subsequent analysis were performed to assess mitochondrial content and respiration, inflammatory status, glucose and insulin tolerance, and muscle anabolic signaling. KO did not affect body weight gain on HFD, but heavily impacted mitochondria-, oxidative stress-, and inflammation-related gene expression. Accordingly, mitochondrial protein content and respiration were suppressed while inflammatory signaling and markers of oxidative stress were elevated in obese KO muscles. KO did not affect glucose or insulin tolerance. However, fasting serum insulin and skeletal muscle insulin signaling were higher in the KO mice. Furthermore, decreased muscle fiber size in skmLKB1-KO mice was associated with increased general protein ubiquitination and increased expression of several ubiquitin ligases, but not muscle ring finger 1 or atrogin-1. Taken together, these data suggest that the lack of LKB1 in skeletal muscle does not exacerbate obesity or insulin resistance in mice on a HFD, despite impaired mitochondrial content and function and elevated inflammatory signaling and oxidative stress.

Exposure to Static Magnetic and Electric Fields Treats Type 2 Diabetes.

Aberrant redox signaling underlies the pathophysiology of many chronic metabolic diseases, including type 2 diabetes (T2D). Methodologies aimed at rebalancing systemic redox homeostasis have had limited success. A noninvasive, sustained approach would enable the long-term control of redox signaling for the treatment of T2D. We report that static magnetic and electric fields (sBE) noninvasively modulate the systemic GSH-to-GSSG redox couple to promote a healthier systemic redox environment that is reducing. Strikingly, when applied to mouse models of T2D, sBE rapidly ameliorates insulin resistance and glucose intolerance in as few as 3 days with no observed adverse effects. Scavenging paramagnetic byproducts of oxygen metabolism with SOD2 in hepatic mitochondria fully abolishes these insulin sensitizing effects, demonstrating that mitochondrial superoxide mediates induction of these therapeutic changes. Our findings introduce a remarkable redox-modulating phenomenon that exploits endogenous electromagneto-receptive mechanisms for the noninvasive treatment of T2D, and potentially other redox-related diseases.

Use of Primary Mouse Embryonic Fibroblasts in Developmental Toxicity Assessments.

Mouse embryonic fibroblasts (MEFs) are commonly collected as a means to maintain the culture and growth of embryonic stem cells (ESCs). However, their utility can extend well beyond their use exclusively in ESC culture. With collection from various transgenic mouse models, use of MEFs may serve as a more simplistic means to reconstitute in vivo/in utero toxicological assessments in an in vitro format for evaluation of function of specific proteins during toxic insults. The ease of collection, rapid growth kinetics, and large-scale expansion to perform multiple, high-throughput experiments are just some of the advantages of MEF use. Here, we describe procedures for successful MEF isolation and culture. As an example of MEF utility, we use MEFs collected form wild-type (WT) and Nrf2 knockout mice. After collection, MEFs were pretreated with the Nrf2 activator, dithiol-3-thione (D3T; 10 µM) for 12 h, and then treated with either hydrogen peroxide (0-2000 µM) or mercury (0-100 µM) for another 24 h. Viability was measured via MTT assay after 24 h of treatment.