Erythroid Differentiation Regulator 1 as a Regulator of Neuronal GSH Synthesis.

Erythroid differentiation regulator 1 (Erdr1) is a cytokine known to play important roles in cell survival under stressful conditions, maintenance of cellular growth homeostasis, and activation of the immune system. However, the impact of Erdr1 on neurons remains undefined. In this study, we present novel evidence that Erdr1 plays a role in regulating glutathione (GSH) synthesis via glutamate transporter-associated protein 3-18 (GTRAP3-18), an anchor protein in the endoplasmic reticulum that holds excitatory amino acid carrier 1 (EAAC1) in neurons. Both DNA microarray and quantitative real-time PCR analyses revealed an approximately 2-fold increase in Erdr1 levels in the hippocampus of GTRAP3-18-deficient mice compared to those of wild-type mice. Knockdown of Erdr1 in vitro resulted in a decrease in GTRAP3-18 levels, leading to an increase in EAAC1 expression and intracellular GSH levels, and subsequently, cytoprotective effects against oxidative stress. Our findings shed light on the regulatory mechanisms involving Erdr1, GTRAP3-18, EAAC1, and GSH in the context of neuronal defense against oxidative stress. Understanding the intricate interplay among these molecules may pave the way for the development of promising therapeutic strategies for neurodegenerative disorders.

Glutathione-Mediated Neuroprotective Effect of Purine Derivatives.

Numerous basic studies have reported on the neuroprotective properties of several purine derivatives such as caffeine and uric acid (UA). Epidemiological studies have also shown the inverse association of appropriate caffeine intake or serum urate levels with neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson's disease (PD). The well-established neuroprotective mechanisms of caffeine and UA involve adenosine A2A receptor antagonism and antioxidant activity, respectively. Our recent study found that another purine derivative, paraxanthine, has neuroprotective effects similar to those of caffeine and UA. These purine derivatives can promote neuronal cysteine uptake through excitatory amino acid carrier protein 1 (EAAC1) to increase neuronal glutathione (GSH) levels in the brain. This review summarizes the GSH-mediated neuroprotective effects of purine derivatives. Considering the fact that GSH depletion is a manifestation in the brains of AD and PD patients, administration of purine derivatives may be a new therapeutic approach to prevent or delay the onset of these neurodegenerative diseases.

A purine derivative, paraxanthine, promotes cysteine uptake for glutathione synthesis.

Purine derivatives such as caffeine and uric acid have neuroprotective activities and are negatively correlated with the incidence of both Alzheimer's disease and Parkinson's disease. We have reported that an increment of intracellular glutathione (GSH) via cysteine uptake in neuronal cells is one of the mechanisms by which caffeine and uric acid confer neuroprotection. Here, we investigated whether caffeine metabolites such as paraxanthine, theophylline, theobromine, 1,7-dimethyluric acid and monomethylxanthines would increase cysteine uptake in mouse hippocampal slices. The metabolites were administered to hippocampal slices for 30 min at doses of 0, 10, or 100 µM, and then cysteine was added for 30 min. Paraxanthine, a major metabolite of caffeine, increased cysteine content in the slices, whereas the other metabolites did not. In vitro treatment with paraxanthine promoted cysteine uptake and increased GSH in HEK293 cells. The paraxanthine-induced cysteine uptake was inhibited by an excitatory amino-acid carrier-1 (EAAC1) inhibitor, and H2O2-induced cell damage was prevented by the paraxanthine treatment of SH-SY5Y cells. These results suggest that paraxanthine, an active metabolite of caffeine, acts to increase intracellular GSH levels via EAAC1 leading to neuroprotection.

Glutathione Depletion and MicroRNA Dysregulation in Multiple System Atrophy: A Review.

Multiple system atrophy (MSA) is a rare neurodegenerative disease characterized by parkinsonism, cerebellar impairment, and autonomic failure. Although the causes of MSA onset and progression remain uncertain, its pathogenesis may involve oxidative stress via the generation of excess reactive oxygen species and/or destruction of the antioxidant system. One of the most powerful antioxidants is glutathione, which plays essential roles as an antioxidant enzyme cofactor, cysteine-storage molecule, major redox buffer, and neuromodulator, in addition to being a key antioxidant in the central nervous system. Glutathione levels are known to be reduced in neurodegenerative diseases. In addition, genes regulating redox states have been shown to be post-transcriptionally modified by microRNA (miRNA), one of the most important types of non-coding RNA. miRNAs have been reported to be dysregulated in several diseases, including MSA. In this review, we focused on the relation between glutathione deficiency, miRNA dysregulation and oxidative stress and their close relation with MSA pathology.

Glutathione in the Brain.

Glutathione (GSH) is the most abundant non-protein thiol, and plays crucial roles in the antioxidant defense system and the maintenance of redox homeostasis in neurons. GSH depletion in the brain is a common finding in patients with neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, and can cause neurodegeneration prior to disease onset. Excitatory amino acid carrier 1 (EAAC1), a sodium-dependent glutamate/cysteine transporter that is selectively present in neurons, plays a central role in the regulation of neuronal GSH production. The expression of EAAC1 is posttranslationally controlled by the glutamate transporter-associated protein 3-18 (GTRAP3-18) or miR-96-5p in neurons. The regulatory mechanism of neuronal GSH production mediated by EAAC1 may be a new target in therapeutic strategies for these neurodegenerative diseases. This review describes the regulatory mechanism of neuronal GSH production and its potential therapeutic application in the treatment of neurodegenerative diseases.

Interplay of RNA-Binding Proteins and microRNAs in Neurodegenerative Diseases.

The number of patients with neurodegenerative diseases (NDs) is increasing, along with the growing number of older adults. This escalation threatens to create a medical and social crisis. NDs include a large spectrum of heterogeneous and multifactorial pathologies, such as amyotrophic lateral sclerosis, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease and multiple system atrophy, and the formation of inclusion bodies resulting from protein misfolding and aggregation is a hallmark of these disorders. The proteinaceous components of the pathological inclusions include several RNA-binding proteins (RBPs), which play important roles in splicing, stability, transcription and translation. In addition, RBPs were shown to play a critical role in regulating miRNA biogenesis and metabolism. The dysfunction of both RBPs and miRNAs is often observed in several NDs. Thus, the data about the interplay among RBPs and miRNAs and their cooperation in brain functions would be important to know for better understanding NDs and the development of effective therapeutics. In this review, we focused on the connection between miRNAs, RBPs and neurodegenerative diseases.

The Role of Non-Coding RNAs in the Neuroprotective Effects of Glutathione.

The establishment of antioxidative defense systems might have been mandatory for most living beings with aerobic metabolisms, because oxygen consumption produces adverse byproducts known as reactive oxygen species (ROS). The brain is especially vulnerable to the effect of ROS, since the brain has large amounts of unsaturated fatty acids, which are a target of lipid oxidation, as well as comparably high-energy consumption compared to other organs that results in ROS release from mitochondria. Thus, dysregulation of the synthesis and/or metabolism of antioxidants-particularly glutathione (GSH), which is one of the most important antioxidants in the human body-caused oxidative stress states that resulted in critical diseases, including neurodegenerative diseases in the brain. GSH plays crucial roles not only as an antioxidant but also as an enzyme cofactor, cysteine storage form, the major redox buffer, and a neuromodulator in the central nervous system. The levels of GSH are precisely regulated by uptake systems for GSH precursors as well as GSH biosynthesis and metabolism. The rapid advance of RNA sequencing technologies has contributed to the discovery of numerous non-coding RNAs with a wide range of functions. Recent lines of evidence show that several types of non-coding RNAs, including microRNA, long non-coding RNA and circular RNA, are abundantly expressed in the brain, and their activation or inhibition could contribute to neuroprotection through the regulation of GSH synthesis and/or metabolism. Interestingly, these non-coding RNAs play key roles in gene regulation and growing evidence indicates that non-coding RNAs interact with each other and are co-regulated. In this review, we focus on how the non-coding RNAs modulate the level of GSH and modify the oxidative stress states in the brain.

Inhibition of miR-96-5p in the mouse brain increases glutathione levels by altering NOVA1 expression.

Glutathione (GSH) is an important antioxidant that plays a critical role in neuroprotection. GSH depletion in neurons induces oxidative stress and thereby promotes neuronal damage, which in turn is regarded as a hallmark of the early stage of neurodegenerative diseases. The neuronal GSH level is mainly regulated by cysteine transporter EAAC1 and its inhibitor, GTRAP3-18. In this study, we found that the GTRAP3-18 level was increased by up-regulation of the microRNA miR-96-5p, which was found to decrease EAAC1 levels in our previous study. Since the 3'-UTR region of GTRAP3-18 lacks the consensus sequence for miR-96-5p, an unidentified protein should be responsible for the intermediate regulation of GTRAP3-18 expression by miR-96-5p. Here, we discovered that RNA-binding protein NOVA1 functions as an intermediate protein for GTRAP3-18 expression via miR-96-5p. Moreover, we show that intra-arterial injection of a miR-96-5p-inhibiting nucleic acid to living mice by a drug delivery system using microbubbles and ultrasound decreased the level of GTRAP3-18 via NOVA1 and increased the levels of EAAC1 and GSH in the dentate gyrus of the hippocampus. These findings suggest that the delivery of a miR-96-5p inhibitor to the brain would efficiently increase the neuroprotective activity by increasing GSH levels via EAAC1, GTRAP3-18 and NOVA1.

[Mechanism of glutathione production in neurons].

Glutathione (GSH) is a tripeptide consisting of glutamate, cysteine, and glycine that acts as an important neuroprotective molecule in the central nervous system. In neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, GSH levels in the brain would be decreased before the onset, and GSH dysregulation is considered to be involved in the development of these neurodegenerative diseases. Cysteine uptake into neurons is the rate-limiting step for GSH synthesis. Excitatory amino acid carrier 1 (EAAC1), which is a glutamate/cysteine cotransporter, is responsible for the neuronal cysteine uptake, and EAAC1 dysfunction reduces GSH levels in the brain and has a significant influence on the process of neurodegeneration. Since miR-96-5p, which is one of microRNAs, suppresses EAAC1 expression, it is conceivable that miR-96-5p inhibitor suppresses the onset or slows the progression of neurodegenerative diseases by increasing EAAC1 levels leading to promoting neuronal GSH production.

Novel TLR2xTLR4 Bispecific Antibody Inhibits Bacterial Sepsis.

Toll-like receptors (TLRs) sense microbial infection through recognition of pathogen-associated molecular patterns. For example, TLR4 responds to the lipopolysaccharide of gram-negative bacteria, whereas TLR2 recognizes a broad range of microbial ligands. Both receptors are, therefore, compelling targets for treating sepsis. Here, we developed a TLR2xTLR4 tetravalent bispecific antibody designated ICU-1, which inhibits both receptors. The inhibitory activity of ICU-1 was comparable to that of the parental antibodies in neutralization assays using a human monocyte cell line. Moreover, ICU-1 completely blocked stimulation of human peripheral blood mononuclear cells by clinically relevant bacterial species. These findings provide convincing evidence that ICU-1 offers a novel approach for treating bacterial sepsis.

Generation and Characterization of a Novel Anti-Rat TLR4/MD2 Antibody with Potent Neutralizing Activity In Vivo.

Toll-like receptor 4 (TLR4) plays a critical role in the innate immune system and is involved in the pathogenesis of multiple diseases. Here, we report the antagonistic and ratized antibody, 52-1H4 e2 (e2), which completely inhibited lipopolysaccharide-induced interleukin-6 secretion in vitro. The average serum drug concentration was above 10 µg/mL for 28 days in rats injected with e2. The novel anti-rat TLR4/myeloid differentiation factor 2 antibody, e2, may be a useful tool for investigating the role of TLR4 in rat disease models.

MicroRNA: A Key Player for the Interplay of Circadian Rhythm Abnormalities, Sleep Disorders and Neurodegenerative Diseases.

Circadian rhythms are endogenous 24-h oscillators that regulate the sleep/wake cycles and the timing of biological systems to optimize physiology and behavior for the environmental day/night cycles. The systems are basically generated by transcription-translation feedback loops combined with post-transcriptional and post-translational modification. Recently, evidence is emerging that additional non-coding RNA-based mechanisms are also required to maintain proper clock function. MicroRNA is an especially important factor that plays critical roles in regulating circadian rhythm as well as many other physiological functions. Circadian misalignment not only disturbs the sleep/wake cycle and rhythmic physiological activity but also contributes to the development of various diseases, such as sleep disorders and neurodegenerative diseases. The patient with neurodegenerative diseases often experiences profound disruptions in their circadian rhythms and/or sleep/wake cycles. In addition, a growing body of recent evidence implicates sleep disorders as an early symptom of neurodegenerative diseases, and also suggests that abnormalities in the circadian system lead to the onset and expression of neurodegenerative diseases. The genetic mutations which cause the pathogenesis of familial neurodegenerative diseases have been well studied; however, with the exception of Huntington's disease, the majority of neurodegenerative diseases are sporadic. Interestingly, the dysfunction of microRNA is increasingly recognized as a cause of sporadic neurodegenerative diseases through the deregulated genes related to the pathogenesis of neurodegenerative disease, some of which are the causative genes of familial neurodegenerative diseases. Here we review the interplay of circadian rhythm disruption, sleep disorders and neurodegenerative disease, and its relation to microRNA, a key regulator of cellular processes.

Development of a simultaneous quantification method for ten trichothecenes including deoxynivalenol-3-glucoside in feed.

An analytical method for the simultaneous quantitation of ten trichothecenes of type A (HT-2 toxin, T-2 toxin, diacetoxyscirpenol, and neosolaniol) and type B (3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, deoxynivalenol, deoxynivalenol-3-glucoside, nivalenol, and fusarenon-X) in feed has been developed using liquid chromatography with tandem mass spectrometry. Mycotoxins extracted twice from samples using aqueous acetonitrile were purified using a multifunctional clean-up column, followed by a phospholipid removal column. Trichothecenes were analysed using liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry. The extraction efficiency of the mycotoxins and the repeatability of some were improved by repeated extractions. Ionization enhancement (signal enhancement) of some mycotoxins was improved by using the phospholipid removal column at the clean-up step. Spike and recovery tests of trichothecenes were conducted on maize, barley, soybean meal, rapeseed meal, and formula feeds (for starting broiler chicks, suckling pigs, and beef cattle). The mean recovery values were 70.6-119% with relative standard deviations < 17%. The limit of quantification and the limit of detection of our method were 20 and 6 µg/kg, respectively, for 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol; 10 and 3 µg/kg, respectively, for T-2 toxin, deoxynivalenol, and fusarenon-X; and 5 and 2 µg/kg, respectively, for nivalenol and the remaining mycotoxins.

GTRAP3-18 regulates food intake and body weight by interacting with pro-opiomelanocortin.

Pro-opiomelanocortin (POMC)-expressing neurons provide α-melanocyte-stimulating hormone (α-MSH), which stimulates melanocortin 4 receptor to induce hypophagia by AMPK inhibition in the hypothalamus. α-MSH is produced by POMC cleavage in secretory granules and released. However, it is not known yet whether any posttranscriptional regulatory mechanism of POMC signaling exists upstream of the secretory granules in neurons. Here we show that glutamate transporter-associated protein 3-18 (GTRAP3-18), an anchor protein that retains interacting proteins in the endoplasmic reticulum, is a critical regulator of food intake and body weight by interacting with POMC. GTRAP3-18-deficient mice showed hypophagia, lean bodies, and lower blood glucose, insulin, and leptin levels with increased serum and brain α-MSH levels, leading to AMPK inhibition. Intraperitoneal glucose tolerance tests revealed significantly decreased blood glucose levels and areas under the curve in GTRAP3-18-deficient mice compared to wild-type mice. An intracerebroventricular infusion of a selective melanocortin 4 receptor antagonist to GTRAP3-18-deficient mice significantly increased their food intake and body weight. A fluorescence resonance energy transfer study showed an interaction between GTRAP3-18 and POMC in vitro These findings suggest that activation of the melanocortin pathway by modulating GTRAP3-18/POMC interaction could be an alternative strategy for obesity and/or type 2 diabetes.-Aoyama, K., Bhadhprasit, W., Watabe, M., Wang, F., Matsumura, N., Nakaki, T. GTRAP3-18 regulates food intake and body weight by interacting with pro-opiomelanocortin.

Neuroprotection afforded by circadian regulation of intracellular glutathione levels: A key role for miRNAs.

Circadian rhythms are approximately 24-h oscillations of physiological and behavioral processes that allow us to adapt to daily environmental cycles. Like many other biological functions, cellular redox status and antioxidative defense systems display circadian rhythmicity. In the central nervous system (CNS), glutathione (GSH) is a critical antioxidant because the CNS is extremely vulnerable to oxidative stress; oxidative stress, in turn, causes several fatal diseases, including neurodegenerative diseases. It has long been known that GSH level shows circadian rhythm, although the mechanism underlying GSH rhythm production has not been well-studied. Several lines of recent evidence indicate that the expression of antioxidant genes involved in GSH homeostasis as well as circadian clock genes are regulated by post-transcriptional regulator microRNA (miRNA), indicating that miRNA plays a key role in generating GSH rhythm. Interestingly, several reports have shown that alterations of miRNA expression as well as circadian rhythm have been known to link with various diseases related to oxidative stress. A growing body of evidence implicates a strong correlation between antioxidative defense, circadian rhythm and miRNA function, therefore, their dysfunctions could cause numerous diseases. It is hoped that continued elucidation of the antioxidative defense systems controlled by novel miRNA regulation under circadian control will advance the development of therapeutics for the diseases caused by oxidative stress.

Sterigmatocystin and aflatoxin B1 contamination of corn, soybean meal, and formula feed in Japan.

Sterigmatocystin (STC) and aflatoxin B1 (AFB1) were analyzed in 246 corn samples, 126 soybean meal samples, and 861 formula feed samples from the Japanese market between April 2010 and March 2015. The detection rate, the highest concentration, and the mean concentration of STC were respectively 14%, 6.4 µg/kg, and 1.2 µg/kg for corn; 14%, 1.1 µg/kg, and 0.63 µg/kg for soybean meal; and 43%, 9.1 µg/kg, and 0.97 µg/kg for formula feed. The detection rate, the highest concentration, and the mean concentration of AFB1 were respectively 46%, 24 µg/kg, and 3.9 µg/kg for corn; 30%, 6.7 µg/kg, and 1.1 µg/kg for soybean meal; and 47%, 20 µg/kg, and 1.6 µg/kg for formula feed. A weak negative correlation between the STC and AFB1 concentrations was observed: there was a high concentration of AFB1 in samples that contained a lower concentration of STC and vice versa. Spearman's rank correlation coefficient showed a weak negative correlation of - 0.30 (p < 0.001, n = 128) for corn and - 0.23 (p < 0.001, n = 575) for formula feed. In conclusion, no correlation was observed between the mean concentrations of STC contamination in formula feed (0.97 µg/kg) and in corn (1.2 µg/kg) and the blending rate (approximately 50%). The rate of STC contamination in the formula feed (43%) was higher than that in corn (14%). Therefore, it is likely that ingredients other than corn contribute to the contamination of formula feed with STC. In this study, regarding STC, problematic samples were not found.

Formation Ratios of Zearalanone, Zearalenols, and Zearalanols versus Zearalenone during Incubation of Fusarium semitectum on Sorghum and Ratios in Naturally Contaminated Sorghum.

We incubated Fusarium semitectum on sorghum and measured the production of zearalenone (ZEN) and ZEN-related compounds (zearalanone (ZAN), α-zearalenol (α-ZEL), ß-zearalenol (ß-ZEL), α-zearalanol (α-ZAL) and ß-zearalanol (ß-ZAL)) in the culture by LC-MS. Of the five ZEN-related compounds, ZAN and ß-ZEL were mainly detected. The concentrations of ZEN and the five ZEN-related compounds increased until 9 days after incubation and then increased slightly or stayed constant between days 9 and 15. The ratios of α-ZEL, ß-ZEL, α-ZAL and ß-ZAL to ZEN decreased in a similar manner after 7 days, whereas the ratio of ZAN to ZEN remained constant after 5 days. Analysis of naturally contaminated sorghum by LC-MS/MS revealed that the production ratio of α-ZEL to ZEN was inconsistent with that of our in vitro incubation analysis. The results indicate that ZAN might not be suitable for use as an internal standard.

Glutathione in Cellular Redox Homeostasis: Association with the Excitatory Amino Acid Carrier 1 (EAAC1).

Reactive oxygen species (ROS) are by-products of the cellular metabolism of oxygen consumption, produced mainly in the mitochondria. ROS are known to be highly reactive ions or free radicals containing oxygen that impair redox homeostasis and cellular functions, leading to cell death. Under physiological conditions, a variety of antioxidant systems scavenge ROS to maintain the intracellular redox homeostasis and normal cellular functions. This review focuses on the antioxidant system's roles in maintaining redox homeostasis. Especially, glutathione (GSH) is the most important thiol-containing molecule, as it functions as a redox buffer, antioxidant, and enzyme cofactor against oxidative stress. In the brain, dysfunction of GSH synthesis leading to GSH depletion exacerbates oxidative stress, which is linked to a pathogenesis of aging-related neurodegenerative diseases. Excitatory amino acid carrier 1 (EAAC1) plays a pivotal role in neuronal GSH synthesis. The regulatory mechanism of EAAC1 is also discussed.

Occurrence of four Fusarium mycotoxins, deoxynivalenol, zearalenone, T-2 toxin, and HT-2 toxin, in wheat, barley, and Japanese retail food.

A survey of the contamination of wheat, barley, and Japanese retail food by four Fusarium mycotoxins, deoxynivalenol (DON), zearalenone (ZEN), T-2 toxin (T-2), and HT-2 toxin (HT-2), was performed between 2010 and 2012. A method for the simultaneous determination of the four mycotoxins by liquid chromatography-tandem mass spectrometry was validated by a small-scale interlaboratory study using two spiked wheat samples (DON was spiked at 20 and 100 µg/kg and ZEN, T-2, and HT-2 at 6 and 20 µg/kg in the respective samples). The recovery of the four mycotoxins ranged from 77.3 to 107.2%. A total of 557 samples of 10 different commodities were analyzed over 3 years by this validated method. Both T-2 and HT-2 were detected in wheat, wheat flour, barley, Job's tears products, beer, corn grits, azuki beans, soybeans, and rice with mixed grains. Only T-2 toxin was detected in sesame seeds. The highest concentrations of T-2 toxin (48.4 µg/kg) and HT-2 toxin (85.0 µg/kg) were present in azuki beans and wheat, respectively. DON was frequently detected in wheat, wheat flour, beer, and corn grits. The contamination level of wheat was below the provisional standard in Japan (1,100 µg/kg). The maximum contamination level of DON was present in a sample of a Job's tears product (1,093 µg/kg). ZEN was frequently detected in Job's tears products, corn grits, azuki beans, rice with mixed grains, and sesame seeds. A sample of a Job's tears product presented the highest ZEN contamination (153 µg/kg). These results indicate that continuous monitoring by multiple laboratories is effective and necessary due to the percentage of positive samples detected.