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.

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.

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.

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.

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.

Rhythmic oscillations of the microRNA miR-96-5p play a neuroprotective role by indirectly regulating glutathione levels.

Glutathione (GSH) is a key antioxidant that plays an important neuroprotective role in the brain. Decreased GSH levels are associated with neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. Here we show that a diurnal fluctuation of GSH levels is correlated with neuroprotective activity against oxidative stress in dopaminergic cells. In addition, we found that the cysteine transporter excitatory amino acid carrier 1 (EAAC1), which is involved in neuronal GSH synthesis, is negatively regulated by the microRNA miR-96-5p, which exhibits a diurnal rhythm. Blocking miR-96-5p by intracerebroventricular administration of an inhibitor increased the level of EAAC1 as well as that of GSH and had a neuroprotective effect against oxidative stress in the mouse substantia nigra. Our results suggest that the diurnal rhythm of miR-96-5p may play a role in neuroprotection by regulating neuronal GSH levels via EAAC1.

Impaired glutathione synthesis in neurodegeneration.

Glutathione (GSH) was discovered in yeast cells in 1888. Studies of GSH in mammalian cells before the 1980s focused exclusively on its function for the detoxication of xenobiotics or for drug metabolism in the liver, in which GSH is present at its highest concentration in the body. Increasing evidence has demonstrated other important roles of GSH in the brain, not only for the detoxication of xenobiotics but also for antioxidant defense and the regulation of intracellular redox homeostasis. GSH also regulates cell signaling, protein function, gene expression, and cell differentiation/proliferation in the brain. Clinically, inborn errors in GSH-related enzymes are very rare, but disorders of GSH metabolism are common in major neurodegenerative diseases showing GSH depletion and increased levels of oxidative stress in the brain. GSH depletion would precipitate oxidative damage in the brain, leading to neurodegenerative diseases. This review focuses on the significance of GSH function, the synthesis of GSH and its metabolism, and clinical disorders of GSH metabolism. A potential approach to increase brain GSH levels against neurodegeneration is also discussed.

Neuroprotective properties of the excitatory amino acid carrier 1 (EAAC1).

Extracellular glutamate should be maintained at low levels to conserve optimal neurotransmission and prevent glutamate neurotoxicity in the brain. Excitatory amino acid transporters (EAATs) play a pivotal role in removing extracellular glutamate in the central nervous system (CNS). Excitatory amino acid carrier 1 (EAAC1) is a high-affinity Na⁺-dependent neuronal EAAT that is ubiquitously expressed in the brain. However, most glutamate released in the synapses is cleared by glial EAATs, but not by EAAC1 in vivo. In the CNS, EAAC1 is widely distributed in somata and dendrites but not in synaptic terminals. The contribution of EAAC1 to the control of extracellular glutamate levels seems to be negligible in the brain. However, EAAC1 can transport not only extracellular glutamate but also cysteine into the neurons. Cysteine is an important substrate for glutathione (GSH) synthesis in the brain. GSH has a variety of neuroprotective functions, while its depletion induces neurodegeneration. Therefore, EAAC1 might exert a critical role for neuroprotection in neuronal GSH metabolism rather than glutamatergic neurotransmission, while EAAC1 dysfunction would cause neurodegeneration. Despite the potential importance of EAAC1 in the brain, previous studies have mainly focused on the glutamate neurotoxicity induced by glial EAAT dysfunction. In recent years, however, several studies have revealed regulatory mechanisms of EAAC1 functions in the brain. This review will summarize the latest information on the EAAC1-regulated neuroprotective functions in the CNS.

Inhibition of GTRAP3-18 may increase neuroprotective glutathione (GSH) synthesis.

Glutathione (GSH) is a tripeptide consisting of glutamate, cysteine, and glycine; it has a variety of functions in the central nervous system. Brain GSH depletion is considered a preclinical sign in age-related neurodegenerative diseases, and it promotes the subsequent processes toward neurotoxicity. A neuroprotective mechanism accomplished by increasing GSH synthesis could be a promising approach in the treatment of neurodegenerative diseases. In neurons, cysteine is the rate-limiting substrate for GSH synthesis. Excitatory amino acid carrier 1 (EAAC1) is a neuronal cysteine/glutamate transporter in the brain. EAAC1 translocation to the plasma membrane promotes cysteine uptake, leading to GSH synthesis, while being negatively regulated by glutamate transport associated protein 3-18 (GTRAP3-18). Our recent studies have suggested GTRAP3-18 as an inhibitory factor for neuronal GSH synthesis. Inhibiting GTRAP3-18 function is an endogenous mechanism to increase neuron-specific GSH synthesis in the brain. This review gives an overview of EAAC1-mediated GSH synthesis, and its regulatory mechanisms by GTRAP3-18 in the brain, and a potential approach against neurodegeneration.

Timeline for development of autologous bone marrow infusion (ABMi) therapy and perspective for future stem cell therapy.

Liver cirrhosis patients generally progress to liver failure. To cure this progressive disease, we developed a novel cell therapy using bone marrow cells; autologous bone marrow cell infusion (ABMi) therapy. We previously described the possible action mechanism of ABMi therapy in the cirrhotic liver, and showed the timeline and results of clinical studies of ABMi therapy. We have also carried out other clinical studies using bone marrow cells and granulocyte colony-stimulating factor. Here, we report a new randomized clinical trial to evaluate the effects of ABMi therapy. However, ABMi therapy may not be possible in patients who are unable to undergo general anesthesia; therefore, we have started to develop a next-generation stem cell therapy using cultured mesenchymal stem cells.

Modified Collis-Nissen procedure for long gap pure esophageal atresia.

BACKGROUND/PURPOSE: Esophageal reconstruction in long gap esophageal atresia (EA) is technically challenging, and several procedures have been described. The purpose of this study is to review our experience with the modified Collis-Nissen procedure in the repair of long gap pure EA. METHODS: Six patients with pure EA were treated at our institution from 1985 to 2008. Patients' demographics, surgical technique, timing of repair, early and late complications, and long-term functional outcomes were retrospectively reviewed. RESULTS: Five primary cases and 1 redo case were included. The mean gap length was 5.3 vertebral bodies (range, 4-6). Modified Collis-Nissen procedure was performed at a mean age of 11.6 months (range, 9-14 months) in primary cases. There was 1 anastomotic leak in the redo case, which healed spontaneously. Two patients had anastomotic strictures requiring balloon dilatations. Patients were weaned from tube feeding at a mean duration of 4 months (range, 1-6 months) postoperatively. All patients have normal oral intake at the last follow-up visit. Two adult patients had normal growth and development and no digestive symptoms. Endoscopic examination and pH monitoring showed no signs of significant gastroesophageal reflux. CONCLUSIONS: Modified Collis-Nissen procedure is a good option to consider in patients with long gap pure EA and is associated with an acceptable complication rate and promising short- and long-term results.

Increased neuronal glutathione and neuroprotection in GTRAP3-18-deficient mice.

Glutathione (GSH) is an important neuroprotective molecule in the brain. The strategy to increase neuronal GSH level is a promising approach to the treatment of neurodegenerative diseases. However, the regulatory mechanism by which neuron-specific GSH synthesis is facilitated remains elusive. Glutamate transporter-associated protein 3-18 (GTRAP3-18) is an endoplasmic reticulum protein interacting with excitatory amino acid carrier 1 (EAAC1), which is a neuronal glutamate/cysteine transporter. To investigate the potential regulatory mechanism to increase neuronal GSH level in vivo, we generated GTRAP3-18-deficient (GTRAP3-18(-/-)) mice using a gene-targeting approach. Disruption of the GTRAP3-18 gene resulted in increased EAAC1 expression in the plasma membrane, increased neuronal GSH content and neuroprotection against oxidative stress. In addition, GTRAP3-18(-/-) mice performed better in motor/spatial learning and memory tests than wild-type mice. Therefore, the suppression of GTRAP3-18 increases neuronal resistance to oxidative stress by increasing GSH content and also facilitates cognitive function. The present results may provide a molecular basis for the development of treatments for neurodegenerative diseases.

Modulation of neuronal glutathione synthesis by EAAC1 and its interacting protein GTRAP3-18.

Glutathione (GSH) plays essential roles in different processes such as antioxidant defenses, cell signaling, cell proliferation, and apoptosis in the central nervous system. GSH is a tripeptide composed of glutamate, cysteine, and glycine. The concentration of cysteine in neurons is much lower than that of glutamate or glycine, so that cysteine is the rate-limiting substrate for neuronal GSH synthesis. Most neuronal cysteine uptake is mediated through the neuronal sodium-dependent glutamate transporter, known as excitatory amino acid carrier 1 (EAAC1). Glutamate transporters are vulnerable to oxidative stress and EAAC1 dysfunction impairs neuronal GSH synthesis by reducing cysteine uptake. This may start a vicious circle leading to neurodegeneration. Intracellular signaling molecules functionally regulate EAAC1. Glutamate transporter-associated protein 3-18 (GTRAP3-18) activation down-regulates EAAC1 function. Here, we focused on the interaction between EAAC1 and GTRAP3-18 at the plasma membrane to investigate their effects on neuronal GSH synthesis. Increased level of GTRAP3-18 protein induced a decrease in GSH level and, thereby, increased the vulnerability to oxidative stress, while decreased level of GTRAP3-18 protein induced an increase in GSH level in vitro. We also confirmed these results in vivo. Our studies demonstrate that GTRAP3-18 regulates neuronal GSH level by controlling the EAAC1-mediated uptake of cysteine.

Undifferentiated carcinoma of the common bile duct with intraductal tumor thrombi: report of a case.

We report a case of undifferentiated carcinoma of the common bile duct with intraductal tumor thrombi. A 73-year-old man presented with general malaise. Abdominal computed tomography and magnetic resonance imaging revealed a mass in the distal common bile duct, accompanied by dilatation of the intra- and extrahepatic bile ducts. The patient underwent pancreaticoduodenectomy with regional lymphadenectomy. Gross examination revealed that the distal common bile duct was obstructed by an elastic hard mass, 3.2 × 2.6 cm, accompanied by intraductal tumor thrombi. Microscopically, the nodule was well defined and composed of atypical large tumor cells with bizarre nuclei and little cytoplasm. Immunohistochemically, the tumor cells were diffusely positive for cytokeratin-7 and CAM5.2, but negative for CD56, chromogranin A, and synaptophysin. Thus, a histological diagnosis of undifferentiated carcinoma of the common bile duct was made. The patient recovered uneventfully and has remained free of any signs of recurrence for 18 months since the operation. Undifferentiated carcinomas of the extrahepatic bile duct can be detected early, with the chance of a good prognosis; however, because their biologic growth behavior is still considered aggressive, careful observation after surgery and the initiation of multidisciplinary treatment against recurrence are necessary.