Essential role of ROS - 8-Nitro-cGMP signaling in long-term memory of motor learning and cerebellar synaptic plasticity.

Although reactive oxygen species (ROS) are known to have harmful effects in organisms, recent studies have demonstrated expression of ROS synthases at various parts of the organisms and the controlled ROS generation, suggesting possible involvement of ROS signaling in physiological events of individuals. However, physiological roles of ROS in the CNS, including functional roles in higher brain functions or neuronal activity-dependent ROS production, remain to be elucidated. Here, we demonstrated involvement of ROS - 8-NO2-cGMP signaling in motor learning and synaptic plasticity in the cerebellum. In the presence of inhibitors of ROS signal or ROS synthases, cerebellar motor learning was impaired, and the stimulus inducing long-term depression (LTD), cellular basis for the motor learning, failed to induce LTD but induced long-term potentiation (LTP)-like change at cerebellar synapses. Furthermore, ROS was produced by LTD-inducing stimulus in enzyme-dependent manner, and excess administration of the antioxidant vitamin E impaired cerebellar motor learning, suggesting beneficial roles of endogenous ROS in the learning. As a downstream signal, involvement of 8-NO2-cGMP in motor learning and cerebellar LTD were also revealed. These findings indicate that ROS - 8-NO2-cGMP signal is activated by neuronal activity and is essential for cerebellum-dependent motor learning and synaptic plasticity, demonstrating involvement of the signal in physiological function of brain systems.

Resveratrol Upregulates Senescence Marker Protein 30 by Activating AMPK/Sirt1-Foxo1 Signals and Attenuating H2O2-Induced Damage in FAO Rat Liver Cells.

Resveratrol (RSV) is a polyphenol with numerous biological functions, including anti-inflammatory, antioxidant, and anti-aging activities. The novel senescence marker protein-30 (SMP30) indicates aging, and it suppresses hepatic oxidative stress. However, the effects of RSV on SMP30 expression regulation remain unclear. We observed that RSV positively regulates SMP30 expression in rat hepatoma-derived FAO cells. However, this was abolished by Compound C and EX-527 that specifically inhibit AMP-activated protein kinase (AMPK) and Silent Information Regulator T1 (Sirt1), respectively. We predicted binding sites for AMPK, forkhead box protein O1 (Foxo1), and Sirt1 downstream molecules as possible SMP30 promoters using the JASPAR and UniProtKB databases. We identified a Foxo1 binding site in the promoter region of SMP30. Inhibiting Foxo1 with AS1842527 also decreased the RSV-induced upregulation of SMP30 expression. Moreover, RSV suppressed the substantial downregulation of SMP30 expression caused by oxidative stress and hydrogen peroxide (H2O2) and released accumulated lactate dehydrogenase. These results demonstrate that, as a novel food factor, RSV-induced upregulation of SMP30 by activating AMPK/Sirt1-Foxo1 signaling and may attenuates H2O2-induced oxidative damage. The findings of this study offer new perspectives of the anti-ageing properties of RSV.

Single-cell RNA sequencing to detect age-associated genes that identify senescent cells in the liver of aged mice.

Senescent cells are predicted to occur and increase in animal tissues with aging. However, senescent cells in the tissues of aged animals remain to be identified. We refer to the marker genes to identify senescent cells in tissues as "age-associated genes". In this study, we searched for age-associated genes to identify senescent cells in the livers of aged animals. We performed single-cell RNA sequencing (scRNA-seq) to screen candidates for age-associated genes using young and aged rat primary hepatocytes. To remove animal species specificity, gene expression analyses in mouse livers were performed, confirming age-associated increases in the mRNA expression levels of Glipr1, Clec12a, and Phlda3. Moreover, the mRNA expression levels of Glipr1 and Phlda3 were increased by stress-induced premature senescence using doxorubicin in primary hepatocytes and livers of young mice. Transcriptome data of aged rat hepatocytes suggested that Glipr1, Clec12a, and Phlda3 were expressed in almost identical cells. Fluorescence in situ hybridization (FISH) confirmed the presence of cells with abundant Glipr1, Clec12a, and Phlda3 mRNA in 27-month-old mouse primary hepatocytes, which are considered to be senescent cells. This study is the first to identify Glipr1, Clec12a, and Phlda3 as age-associated genes in the mouse liver.

Release of SMP30 in Extracellular Vesicles under Conditions of Ascorbic Acid Deficiency Is Involved with Acute Phase Response in ODS Rat.

Senescence marker protein-30 (SMP30) is a senescence marker molecule that exhibits lactonase activity in the ascorbic acid (AsA) biosynthesis pathway, except in primate mammals, including humans. Although numerous studies have shown that hepatic AsA deficiency causes acute-phase responses, details of the relationship between SMP30 expression and acute-phase responses in AsA-deficient conditions remain to be elucidated. Here, we investigated the effects of AsA deficiency on the relationship between SMP30 and acute liver injury in osteogenic disorder Shionogi (ODS) rats, which have a hereditary defect in AsA biosynthesis. Male-ODS rats (4 wk old) were pair-fed an AsA-free diet with distilled or 0.1% AsA-dissolved water for 14 d. Under AsA-deficient conditions, hepatic SMP30 protein level was decreased and liver injury markers, the serum aspartate aminotransferase/alanine transaminase ratio and cytokine-induced neutrophil chemoattractant-1 (CINC-1) concentration, were elevated. In contrast, SMP30 protein level in extracellular vesicles (EVs) was significantly increased in addition to the positive acute proteins haptoglobin and asialoglycoprotein receptor 1 (ASGPR1), hepatic-derived specific markers expression under AsA-deficient conditions. AsA deficiency also activated signal transducer and activator of transcription 3 (STAT3) which is linked to EVs release in the liver. These results suggest that the release of SMP30 in EVs by AsA deficiency is involved with acute-phase responses.

Vitamin C Is Essential for the Maintenance of Skeletal Muscle Functions.

Vitamin C (L-ascorbic acid, VC) is a water-soluble antioxidant essential for collagen polymerization. Previously, we reported that long-term VC deficiency causes muscle atrophy and deterioration in physical ability using female senescence marker protein-30 (SMP30)-deficient mice with a lack of VC synthesis, which is similar to that observed in humans. To determine whether these findings also hold true for male SMP30-deficient mice, two-month-old male SMP30-deficient mice were divided into two groups: the VC-treated group (VC(+)) was administered 1.5 g/L VC, and the VC-untreated group (VC(-)) was supplied water without VC. The VC level at four weeks in the gastrocnemius muscles from the VC(+) and VC(-) groups was 205.7 ± 8.5 nmol/g tissue and 13.1 ± 0.6 nmol/g tissue, respectively. Thus, four weeks was enough to reduce the VC level in the skeletal muscle in the VC-untreated group. On the other hand, muscle weights of the gastrocnemius, soleus, plantaris, tibialis anterior, and extensor digitorum longus in the VC(-) group were significantly reduced by VC deficiency after twelve weeks. The physical endurance of the VC(-) group at eight weeks was markedly lower than that of the VC(+) group. The grasping strength and activity in the cage in the nocturnal phases of the VC(-) group were markedly lower at twelve and sixteen weeks than those of the VC(+) group. Interestingly, muscle atrophy and declined physical ability were completely restored with VC supplementation for twelve weeks after VC deficiency. Thus, VC is essential for maintaining skeletal muscle function in both male and female SMP30-deficient mice with a lack of VC synthesis.

Influence of oestrogen on satellite cells and myonuclear domain size in skeletal muscles following resistance exercise.

BACKGROUND: Oestrogen deficiency reduces skeletal muscle mass and force generation in postmenopausal women. Muscle mass is maintained by satellite cells, which are regulated by oestrogen. Although oestrogen therapy enhances muscle hypertrophy induced by resistance training in postmenopausal women, the molecular mechanism is unclear. METHODS: Adult female rats (10 weeks old) were divided into six groups: sham sedentary (Sham-Sed), sham climbing training (Sham-CT), ovariectomy sedentary (OVX-Sed), ovariectomy climbing training (OVX-CT), ovariectomy plus oestrogen treatment sedentary (OVX+E-Sed), and ovariectomy plus oestrogen treatment climbing training (OVX+E-CT). At 8 weeks after ovariectomy, rats in the training group were trained (one session every 3 days for 8 weeks) to climb a ladder while bearing a load. Oestrogen treatment involved subcutaneous insertion of a 17ß-oestradiol pellet. After 8 weeks, the flexor hallucis longus muscle was collected and analysed. RESULTS: Following climbing training, the flexor hallucis longus muscle mass and muscle-to-body weight ratios were dramatically increased by training (main effect of training, P < 0.01); the OVX+E-CT group showed the highest values (main effect of group, P < 0.01). The cross-sectional area of all muscle fibre types was increased by training (main effect of training, P < 0.01). Particularly, the cross-sectional area of MHC IIa in the OVX+E-CT group was significantly larger than that in the Sham-CT and OVX-CT groups. Satellite cell numbers were increased in all training groups (main effect of training, P < 0.05), and the myonuclear number was increased by training (main effect of training, P < 0.01), but there was no main group effect. The myonuclear domain size of all muscle fibre types and MHC IIa was increased in all training groups (main effect of training, P < 0.01) and showed a main group effect (P < 0.01). The myonuclear domain sizes of all muscle fibre types and MHC IIa in the OVX+E-CT group were significantly larger than those in the Sham-CT and OVX-CT groups. The total RNA contents revealed main effects of training and the group (P < 0.01); the OVX+E-CT group showed the highest contents (main effect of group, P < 0.01). The mRNA and protein levels of rpS6 were increased in the OVX+E-Sed and CT groups (main effects of group, P < 0.05). Particularly, the 28S ribosomal RNA content in OVX+E-Sed group was significantly higher than that in the OVX-Sed group. CONCLUSIONS: Oestrogen enhanced the resistance training-induced increase in myonuclear domain size but did not affect satellite cells and ribosome biogenesis.

Determination of tissue-specific interaction between vitamin C and vitamin E in vivo using senescence marker protein-30 knockout mice as a vitamin C synthesis deficiency model.

Vitamin E (α-tocopherol; VE) is known to be regenerated from VE radicals by vitamin C (L-ascorbic acid; VC) in vitro. However, their in vivo interaction in various tissues is still unclear. Therefore, we alternatively examined the in vivo interaction of VC and VE by measurement of their concentrations in various tissues of senescence marker protein-30 (SMP30) knockout (KO) mice as a VC synthesis deficiency model. Male SMP30-KO mice were divided into four groups (VC+/VE+, VC+/VE-, VC-/VE+ and VC-/VE-), fed diets with or without 500 mg/kg VE and given water with or without 1·5 g/l VC ad libitum. Then, VC and VE concentrations in the plasma and various tissues were determined. Further, gene expression levels of transporters associated with VC and VE, such as α-tocopherol transfer protein (α-TTP) and sodium-dependent vitamin C transporters (SVCTs), were examined. These results showed that the VE levels in the VC-depleted (VC-/VE+) group were significantly lower than those in the VC+/VE+ group in the liver and heart; the VC levels in the VE-depleted (VC+/VE-) group were significantly lower than those in the VC+/VE+ group in the kidneys. The α-TTP gene expression in the liver and kidneys was decreased by VC and/or VE depletion. Moreover, SVCT1 gene expression in the liver was decreased by both VC and VE depletion. In conclusion, these results indicate that VC spares VE mainly in the liver and heart and that VE spares VC in the kidneys of SMP30-KO mice. Thus, interaction between VC and VE is likely to be tissue specific.

Reduced Plasma Ascorbate and Increased Proportion of Dehydroascorbic Acid Levels in Patients Undergoing Hemodialysis.

Ascorbate functions as an electron donor and scavenges free radicals. Dehydroascorbic acid (DHA), the oxidized form of ascorbate, is generated as a result of these reactions. While low plasma ascorbate levels have been reported in hemodialysis patients worldwide, no studies have measured DHA because it is not generalized. In this study, we aimed to clarify whether plasma ascorbate levels are low in dialysis patients and whether plasma ascorbate levels fluctuate before and after dialysis. Moreover, we applied our previously established method to measure the plasma ascorbate and DHA levels in chronic kidney disease (CKD) stage G3-G5 non-hemodialysis-dependent patients, and pre- and post-dialysis plasma ascorbate and DHA levels in CKD stage G5D hemodialysis patients. The sample size was calculated using G-power software. The pre-dialysis plasma total ascorbate levels, including DHA, were significantly (56%) lower in hemodialysis patients than in non-hemodialysis-dependent CKD patients. After dialysis, there was a 40% reduction in the plasma total ascorbate levels. Hemodialysis increased the post-dialysis plasma proportions of DHA from 37% to 55%. The study results demonstrated lower plasma total ascorbate levels in hemodialysis patients compared with in non-hemodialysis-dependent CKD patients; these low levels in hemodialysis patients were further reduced by hemodialysis and increased DHA proportion.

Radiation-induced gastrointestinal syndrome is exacerbated in vitamin C-insufficient SMP30/GNL knockout mice.

OBJECTIVES: Accidental exposure to high-dose radiation causes life-threatening acute radiation syndrome, features that include gastrointestinal syndrome (GIS) and hematopoietic syndrome (HS). Administration of vitamin C (VC), a free radical scavenger, has been reported to increase survival of mice in GIS and HS models. The effect of nutritional VC status on radiation injury remains unknown because, unlike humans, mice can synthesize VC. The aim of this study was to investigate the effect of VC insufficiency on acute radiation syndrome using senescence marker protein 30 (SMP30)/gluconolactonase knockout (SMP30-KO) mice. METHODS: SMP30-KO mice, which cannot synthesize VC, were given water with or without sufficient VC supplementation, and were analyzed in GIS and HS models. RESULTS: In the GIS model, in which bone marrow failure is rescued by bone marrow transplantation, VC-insufficient mice had a lower survival rate than VC-sufficient mice. The intestine of VC-insufficient GIS mice showed epithelial cell atrophy, inflammatory cell infiltration, and decreased crypt cell proliferation. We observed rapid VC oxidation after total body irradiation in the intestine of mice supplemented with VC-sufficient water. In the HS model, which was not combined with bone marrow transplantation, there was no difference in survival between VC-insufficient and -sufficient mice. CONCLUSION: The results of this study demonstrated that nutritionally sufficient VC exerts a radioprotective effect against radiation-induced GIS.

Acerola (Malpighia emarginata DC.) Promotes Ascorbic Acid Uptake into Human Intestinal Caco-2 Cells via Enhancing the Gene Expression of Sodium-Dependent Vitamin C Transporter 1.

Acerola (Malpighia emarginata DC.) is a fruit containing abundant ascorbic acid (AsA) and numerous functional phytochemicals. We previously reported that the intake of acerola juice increased the absorption of AsA in plasma of healthy Japanese subjects. The functional phytochemicals in acerola may influence the intestinal epithelial cells to increase the cellular uptake of AsA. Therefore, in this study, we compared the AsA uptake into Caco-2 cells between AsA alone and that in acerola juice at the same concentration using a human intestinal model. Caco-2 cells were incubated with 3 mM AsA and 3 mM AsA in acerola juice. Intracellular AsA contents gradually increased until 24 h upon incubation with both AsA alone and AsA in acerola juice; however, these contents when incubated with AsA in acerola juice, were significantly higher than those incubated with AsA alone at 2, 3, 4, 8, and 24 h. Furthermore, the mRNA expression level of the sodium-dependent vitamin C transporter (SVCT) 1 was significantly higher in the cells incubated with AsA in acerola juice than those incubated with AsA alone. Moreover, polyphenols such as cyanidin-3-glucoside chloride and quercetin enhanced the SVCT1 gene expression in Caco-2 cells. Collectively, these results suggest that acerola polyphenols enhances the SVCT1 gene expression in Caco-2 cells and promotes AsA uptake.

High-Dose Vitamin C Preadministration Reduces Vancomycin-Associated Nephrotoxicity in Mice.

Vancomycin is recommended for treating severe infections caused by Gram-positive cocci, including methicillin-resistant Staphylococcus aureus. However, renal damage often occurs as a side effect because vancomycin is mainly excreted via the kidneys. The mechanism of vancomycin-associated nephrotoxicity is thought to involve the elevation of oxidative stress in the kidneys. Vitamin C (VC) has strong antioxidant properties; therefore, we evaluated the effect of high-dose VC preadministration on vancomycin-associated nephrotoxicity. Vancomycin was intraperitoneally injected into mice once daily for 7 d. Additionally, high-dose VC was intraperitoneally injected into mice at 30 min before vancomycin administration for 7 d. The plasma creatinine and urea nitrogen levels were increased by vancomycin treatment; however, high-dose VC preadministration suppressed the increase in these levels. Histological examination also revealed that high-dose VC preadministration reduced the characteristics of vancomycin-associated nephrotoxicity, such as dilated renal tubules with casts, the dilation of renal proximal tubules, and tubular epithelial desquamation. Furthermore, high-dose VC preadministration reduced the appearance of apoptotic cells presumably derived from the epithelial cells in the dilated proximal tubules. Thus, intraperitoneally injected high-dose VC preadministration reduced vancomycin-associated nephrotoxicity in mice. These novel findings may indicate that vancomycin-associated nephrotoxicity in humans may be reduced by high-dose VC preadministration.

Reduced aqueous humour ascorbic-acid concentration in women with smaller anterior chamber depth.

Short anterior chamber depth (ACD) is considered a risk factor of endothelial-cell loss after phacoemulsification. However, whether it is an independent risk factor or not remains controversial. We investigated the relationship between ascorbic acid (AA) concentrations in the aqueous humour (AqH) and ACD. We analysed 165 AqH samples of 97 patients (42 men and 55 women) who underwent small incision cataract surgery. AqH and plasma AA concentrations were measured using a high-performance liquid chromatography - electrochemical detection method. Patient characteristics were compared between and within the sexes. As a result, age and ACD were significantly correlated with AqH AA concentrations (r = -0.206, P = 0.045; r = 0.339, P < 0.001) only in women. Moreover, plasma AA concentrations were significantly correlated with AqH AA concentrations (r = 0.420, P < 0.001; r = 0.316, P = 0.002) both in men and women. After adjusting for confounding factors (age and plasma AA concentrations), ACD was significantly and positively correlated with AqH AA concentrations (partial.r = 0.275, P = 0.009) only in women. In conclusion, AqH AA concentrations were reduced in women with smaller ACD. This may suggest that women with short ACD could be more susceptible to oxidative damage.