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.

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.

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.

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.

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.