ying Li

Ascorbate transport by primary cultured neurons and its role in neuronal function and protection against excitotoxicity

Neurons maintain relatively high intracellular concentrations of ascorbic acid, which is achieved primarily by the activity of the sodium‐dependent vitamin C transporter SVCT2. In this work, we studied the mechanisms by which neuronal cells in culture transport and maintain ascorbate as well as whether this system contributes to maturation of neuronal function and cellular defense against oxidative stress and excitotoxic injury. We found that the SVCT2 helps to maintain high intracellular ascorbate levels, normal ascorbate transport kinetics, and activity‐dependent ascorbate recycling. Immunocytochemistry studies revealed that SVCT2 is expressed primarily in the axons of mature hippocampal neurons in culture. In the absence of SVCT2, hippocampal neurons exhibited stunted neurite outgrowth, less glutamate receptor clustering, and reduced spontaneous neuronal activity. Finally, hippocampal cultures from SVCT2‐deficient mice showed increased susceptibility to oxidative damage and N‐methyl‐D‐aspartate‐induced excitotoxicity. Our results revealed that maintenance of intracellular ascorbate as a result of SVCT2 activity is crucial for neuronal development, functional maturation, and antioxidant responses.

Elevated oxidative stress and sensorimotor deficits but normal cognition in mice that cannot synthesize ascorbic acid

Oxidative stress is implicated in the cognitive deterioration associated with normal aging as well as neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. We investigated the effect of ascorbic acid (vitamin C) on oxidative stress, cognition, and motor abilities in mice null for gulono‐γ‐lactone oxidase (Gulo). Gulo−/− mice are unable to synthesize ascorbic acid and depend on dietary ascorbic acid for survival. Gulo−/− mice were given supplements that provided them either with ascorbic acid levels equal to‐ or slightly higher than wild‐type mice (Gulo‐sufficient), or lower than physiological levels (Gulo‐low) that were just enough to prevent scurvy. Ascorbic acid is a major anti‐oxidant in mice and any reduction in ascorbic acid level is therefore likely to result in increased oxidative stress. Ascorbic acid levels in the brain and liver were higher in Gulo‐sufficient mice than in Gulo‐low mice. F4‐neuroprostanes were elevated in cortex and cerebellum in Gulo‐low mice and in the cortex of Gulo‐sufficient mice. All Gulo−/− mice were cognitively normal but had a strength and agility deficit that was worse in Gulo‐low mice. This suggests that low levels of ascorbic acid and elevated oxidative stress as measured by F4‐neuroprostanes alone are insufficient to impair memory in the knockouts but may be responsible for the exacerbated motor deficits in Gulo‐low mice, and ascorbic acid may have a vital role in maintaining motor abilities.

Low vitamin C and increased oxidative stress and cell death in mice that lack the sodium-dependent vitamin C transporter SVCT2.

The sodium-dependent vitamin C transporter (SVCT2) is responsible for the transport of vitamin C into cells in multiple organs, from either the blood or the cerebrospinal fluid. Mice null for SVCT2 (SVCT2(-/-)) do not survive past birth but the cause of death has not yet been ascertained. After mating of SVCT2(+/-) males and SVCT2(+/-) females, fewer SVCT2(-/-) and SVCT2(+/-) progeny were observed than would be expected according to Mendelian ratios. Vitamin C levels in SVCT2(-/-), SVCT2(+/-), and SVCT2(+/+) were genotype-dependent. SVCT2(-/-) fetuses had significantly lower vitamin C levels than littermates in placenta, cortex, and lung, but not in liver (the site of vitamin C synthesis). Low vitamin C levels in placenta and cortex were associated with elevations in several markers of oxidative stress: malondialdehyde, isoketals, F(2)-isoprostanes, and F(4)-neuroprostanes. Oxidative stress was not elevated in fetal SVCT2(-/-) lung tissue despite low vitamin C levels. In addition to the expected severe hemorrhage in cortex, we also found hemorrhage in the brain stem, which was accompanied by cell loss. We found evidence of increased apoptosis in SVCT2(-/-) mice and disruption of the basement membrane in fetal brain. Together these data show that SVCT2 is critical for maintaining vitamin C levels in fetal and placental tissues and that the lack of SVCT2, and the resulting low vitamin C levels, results in fetal death and, in SVCT2(-/-) mice that survive the gestation period, in oxidative stress and cell death.

Antioxidants and cognitive training interact to affect oxidative stress and memory in APP/PSEN1 mice

Abstract The present study investigated the relationships among oxidative stress, β-amyloid and cognitive abilities in the APP/PSEN1 double-transgenic mouse model of Alzheimers disease. In two experiments, long-term dietary supplements were given to aged APP/PSEN1 mice containing vitamin C alone (1 g/kg diet; Experiment 1) or in combination with a high (750 IU/kg diet, Experiments 1 and 2) or lower (400 IU/kg diet, Experiment 2) dose of vitamin E. Oxidative stress, measured by F4-neuroprostanes or malondialdehyde, was elevated in cortex of control-fed APP/PSEN1 mice and reduced to wild-type levels by vitamin supplementation. High-dose vitamin E with C was less effective at reducing oxidative stress than vitamin C alone or the low vitamin E+C diet combination. The high-dose combination also impaired water maze performance in mice of both genotypes. In Experiment 2, the lower vitamin E+C treatment attenuated spatial memory deficits in APP/PSEN1 mice and improved performance in wild-type mice in the water maze. Amyloid deposition was not reduced by antioxidant supplementation in either experiment.

Ascorbic acid uptake and regulation of type I collagen synthesis in cultured vascular smooth muscle cells.

Background/Aims: Vascular smooth muscle cells contribute both to the structure and function of arteries, but are also involved in pathologic changes that accompany inflammatory diseases such as atherosclerosis. Since inflammation is associated with oxidant stress, we examined the uptake and cellular effects of the antioxidant vitamin ascorbic acid in cultured A10 vascular smooth muscle cells. Methods/Results: A10 cells concentrated ascorbate against a gradient in a sodium-dependent manner, most likely on the sodium-dependent vitamin C transporter type 2 (SVCT2) ascorbate transporter, which was present in immunoblots of cell extracts. Although ascorbate did not affect A10 cell proliferation, it stimulated radiolabeled proline incorporation and type I collagen synthesis. The latter was evident in the cells as increases in proα1(I) collagen and conversion of proα1(I) and proα2(I) collagen to mature forms that were released from the cells and deposited as extracellular matrix. Intracellular type I procollagen maturation was optimal at intracellular ascorbate concentrations of 200 μM and below, which were readily achieved by culture of the cells at plasma physiologic ascorbate concentrations. Conclusion: These results show that the SVCT2 facilitates ascorbate uptake by vascular smooth muscle cells, which in turn increases both the synthesis and maturation of type I collagen.

Combined Vitamin C and Vitamin E Deficiency Worsens Early Atherosclerosis in Apolipoprotein E–Deficient Mice

Objective—To assess the role of combined deficiencies of vitamins C and E on the earliest stages of atherosclerosis (an inflammatory condition associated with oxidative stress), 4 combinations of vitamin supplementation (low C/low E, low C/high E, high C/low E, and high C/high E) were studied in atherosclerosis-prone apolipoprotein E–deficient mice also unable to synthesize their own vitamin C (gulonolactone oxidase−/−); and to evaluate the effect of a more severe depletion of vitamin C alone in a second experiment using gulonolactone oxidase−/− mice carrying the hemizygous deletion of SVCT2 (the vitamin C transporter). Methods and Results—After 8 weeks of a high-fat diet (16% lard and 0.2% cholesterol), atherosclerosis developed in the aortic sinus areas of mice in all diet groups. Each vitamin-deficient diet significantly decreased liver and brain contents of the corresponding vitamin. Combined deficiency of both vitamins increased lipid peroxidation, doubled plaque size, and increased plaque macrophage content by 2- to 3-fold in male mice, although only plaque macrophage content was increased in female mice. A more severe deficiency of vitamin C in gulonolactone oxidase−/− mice with defective cellular uptake of vitamin C increased both oxidative stress and atherosclerosis in apolipoprotein E−/− mice compared with littermates receiving a diet replete in vitamin C, again most clearly in males. Conclusion—Combined deficiencies of vitamins E and C are required to worsen early atherosclerosis in an apolipoprotein E–deficient mouse model. However, a more severe cellular deficiency of vitamin C alone promotes atherosclerosis when vitamin E is replete.

Selective macrophage ascorbate deficiency suppresses early atherosclerosis.

To test whether severe ascorbic acid deficiency in macrophages affects progression of early atherosclerosis, we used fetal liver cell transplantation to generate atherosclerosis-prone apolipoprotein E-deficient (apoE(-/-)) mice that selectively lacked the ascorbate transporter (SVCT2) in hematopoietic cells, including macrophages. After 13 weeks of chow diet, apoE(-/-) mice lacking the SVCT2 in macrophages had surprisingly less aortic atherosclerosis, decreased lesion macrophage numbers, and increased macrophage apoptosis compared to control-transplanted mice. Serum lipid levels were similar in both groups. Peritoneal macrophages lacking the SVCT2 had undetectable ascorbate; increased susceptibility to H(2)O(2)-induced mitochondrial dysfunction and apoptosis; decreased expression of genes for COX-2, IL1β, and IL6; and decreased lipopolysaccharide-stimulated NF-κB and antiapoptotic gene expression. These changes were associated with decreased expression of both the receptor for advanced glycation end products and HIF-1α, either or both of which could have been the proximal cause of decreased macrophage activation and apoptosis in ascorbate-deficient macrophages.

Cobalt‐induced oxidant stress in cultured endothelial cells: Prevention by ascorbate in relation to HIF‐1α

Endothelial cells respond to hypoxia by decreased degradation of hypoxia‐inducible factor 1α (HIF‐1α), accumulation of which leads to increased transcription of numerous proteins involved in cell growth and survival. Ascorbic acid prevents HIF‐1α stabilization in many cell types, but the physiologic relevance of such effects is uncertain. Given their relevance for angiogenesis, endothelial cells in culture were used to evaluate the effects of ascorbate on HIF‐1α expression induced by hypoxia and the hypoxia mimic cobalt. Although EA.hy926 cells in culture under oxygenated conditions did not contain ascorbate, HIF‐1α expression was very low, showing that the vitamin is not necessary to suppress HIF‐1α. On the other hand, hypoxia‐ or cobalt‐induced HIF‐1α expression/stabilization was almost completely suppressed by what are likely physiologic intracellular ascorbate concentrations. Increased HIF‐1α expression was not associated with significant changes in expression of the SVCT2, the major transporter for ascorbate in these cells. Cobalt at concentrations sufficient to stabilize HIF‐1α both oxidized intracellular ascorbate and induced an oxidant stress in the cells that was prevented by ascorbate. Whereas the interaction of ascorbate and cobalt is complex, the presence of physiologic low millimolar concentrations of ascorbate in endothelial cells effectively decreases HIF‐1α expression and protects against cobalt‐induced oxidant stress.