Phospholipid transfer protein (PLTP) deficiency impaired blood–brain barrier integrity by increasing cerebrovascular oxidative stress

Phospholipid transfer protein (PLTP) regulates lipid metabolism and plays an important role in oxidative stress. PLTP is highly expressed in blood–brain barrier (BBB), but the role of PLTP in BBB integrity is not clear. In this study, BBB permeability was detected with in vivo multiphoton imaging and Evans blue assay. We found that PLTP deficient mice exhibited increased BBB permeability, as well as decreased expression of tight junction proteins occludin, zona occludens-1 (ZO-1) and claudin-5 in brain vessels. Cerebrovascular oxidative stress increased in PLTP deficient mice, including increased levels of reactive oxygen species (ROS) and lipid peroxidation marker 4-hydroxy-2-nonenal (HNE) and reduced superoxide dismutase (SOD) activity. Dietary supplementation of antioxidant vitamin E increased BBB integrity and tight junction proteins expression via reducing cerebrovascular oxidative stress. These findings indicated an essential role of PLTP in maintaining BBB integrity, possibly through its ability to transfer vitamin E, and modulate cerebrovascular oxidative stress.     

The role of frataxin in fission yeast iron metabolism: Implications for Friedreich's ataxia

Background

The neurodegenerative disease Friedreich's ataxia is the result of frataxin deficiency. Frataxin is a mitochondrial protein involved in iron–sulfur cluster (Fe–S) cofactor biogenesis, but its functional role in this pathway is debated. This is due to the interconnectivity of iron metabolic and oxidative stress response pathways that make distinguishing primary effects of frataxin deficiency challenging. Since Fe–S cluster assembly is conserved, frataxin overexpression phenotypes in a simple eukaryotic organism will provide additional insight into frataxin function.

Methods

The Schizosaccharomyces pombe frataxin homologue (fxn1) was overexpressed from a plasmid under a thiamine repressible promoter. The S. pombe transformants were characterized at several expression strengths for cellular growth, mitochondrial organization, iron levels, oxidative stress, and activities of Fe–S cluster containing enzymes.

Results

Observed phenotypes were dependent on the amount of Fxn1 overexpression. High Fxn1 overexpression severely inhibited S. pombe growth, impaired mitochondrial membrane integrity and cellular respiration, and led to Fxn1 aggregation. Cellular iron accumulation was observed at moderate Fxn1 overexpression but was most pronounced at high levels of Fxn1. All levels of Fxn1 overexpression up-regulated oxidative stress defense and mitochondrial Fe–S cluster containing enzyme activities.

Conclusions

Despite the presence of oxidative stress and accumulated iron, activation of Fe–S cluster enzymes was common to all levels of Fxn1 overexpression; therefore, Fxn1 may regulate the efficiency of Fe–S cluster biogenesis in S. pombe.

General Significance

We provide evidence that suggests that dysregulated Fe–S cluster biogenesis is a primary effect of both frataxin overexpression and deficiency as in Friedreich's ataxia.     

Metal dyshomeostasis and oxidative stress in Alzheimer’s disease

Alzheimer’s disease is the leading cause of dementia in the elderly and is defined by two pathological hallmarks; the accumulation of aggregated amyloid beta and excessively phosphorylated Tau proteins. The etiology of Alzheimer’s disease progression is still debated, however, increased oxidative stress is an early and sustained event that underlies much of the neurotoxicity and consequent neuronal loss. Amyloid beta is a metal binding protein and copper, zinc and iron promote amyloid beta oligomer formation. Additionally, copper and iron are redox active and can generate reactive oxygen species via Fenton (and Fenton-like chemistry) and the Haber–Weiss reaction. Copper, zinc and iron are naturally abundant in the brain but Alzheimer’s disease brain contains elevated concentrations of these metals in areas of amyloid plaque pathology. Amyloid beta can become pro-oxidant and when complexed to copper or iron it can generate hydrogen peroxide. Accumulating evidence suggests that copper, zinc, and iron homeostasis may become perturbed in Alzheimer’s disease and could underlie an increased oxidative stress burden. In this review we discuss oxidative/nitrosative stress in Alzheimer’s disease with a focus on the role that metals play in this process. Recent studies have started to elucidate molecular links with oxidative/nitrosative stress and Alzheimer’s disease. Finally, we discuss metal binding compounds that are designed to cross the blood brain barrier and restore metal homeostasis as potential Alzheimer’s disease therapeutics.     

Genetic vitamin E deficiency does not affect MPTP susceptibility in the mouse brain

Oxidative stress is involved in the degeneration of the nigrostriatal dopaminergic system in Parkinson's disease (PD). Vitamin E (alpha-tocopherol) is a potent antioxidant in the cell membrane that can trap free radicals and prohibit lipid peroxidation. The retention and secretion of vitamin E are regulated by alpha-tocopherol transfer protein (TTP) in the brain and liver. Dysfunction of TTP results in systemic deficiency of vitamin E in humans and mice, and increased oxidative stress in mouse brain. In this study, we investigated the effect of vitamin E deficiency in PD development by generating an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD using TTP knockout (TTP-/-) mice. Vitamin E concentration in the brains of TTP+/- mice was half that in TTP+/+ mice, and in TTP-/- mice, was undetectable. MPTP treatment tended to decrease striatal dopamine, but the effect was comparable and not significant in any of the three genotypes. Furthermore, the extent of loss of dopaminergic cell bodies in the substantia nigra did not differ among the groups. One the other hand, oral administration of vitamin E resulted in the partial protection of striatal dopaminergic terminals against MPTP toxicity. Our results suggest that vitamin E does not play a major protective role in MPTP-induced nigrostriatal dopaminergic neurodegeneration in the brain.     

High glucose-induced mitochondrial respiration and reactive oxygen species in mouse cerebral pericytes is reversed by pharmacological inhibition of mitochondrial carbonic anhydrases: Implications for cerebral microvascular disease in diabetes

Hyperglycemia-induced oxidative stress leads to diabetes-associated damage to the microvasculature of the brain. Pericytes in close proximity to endothelial cells in the brain microvessels are vital to the integrity of the blood–brain barrier and are especially susceptible to oxidative stress. According to our recently published results, streptozotocin-diabetic mouse brain exhibits oxidative stress and loose pericytes by twelve weeks of diabetes, and cerebral pericytes cultured in high glucose media suffer intracellular oxidative stress and apoptosis. Oxidative stress in diabetes is hypothesized to be caused by reactive oxygen species (ROS) produced during hyperglycemia-induced enhanced oxidative metabolism of glucose (respiration). To test this hypothesis, we investigated the effect of high glucose on respiration rate and ROS production in mouse cerebral pericytes. Previously, we showed that pharmacological inhibition of mitochondrial carbonic anhydrases protects the brain from oxidative stress and pericyte loss. The high glucose-induced intracellular oxidative stress and apoptosis of pericytes in culture were also reversed by inhibition of mitochondrial carbonic anhydrases. Therefore, we extended our current study to determine the effect of these inhibitors on high glucose-induced increases in pericyte respiration and ROS. We now report that both the respiration and ROS are significantly increased in pericytes challenged with high glucose. Furthermore, inhibition of mitochondrial carbonic anhydrases significantly slowed down both the rate of respiration and ROS production. These data provide new evidence that pharmacological inhibitors of mitochondrial carbonic anhydrases, already in clinical use, may prove beneficial in protecting the brain from oxidative stress caused by ROS produced as a consequence of hyperglycemia-induced enhanced respiration.     

Iron uncouples oxidative phosphorylation in brain mitochondria isolated from vitamin E-deficient rats

Few, if any, studies have examined the effect of vitamin E deficiency on brain mitochondrial oxidative phosphorylation. The latter was studied using brain mitochondria isolated from control and vitamin E-deficient rats (13 months of deficiency) after exposure to iron, an inducer of oxidative stress. Mitochondria were treated with iron (2 to 50 microM) added as ferrous ammonium sulfate. Rates of state 3 and state 4 respiration, respiratory control ratios, and ADP/O ratios were not affected by vitamin E deficiency alone. However, iron uncoupled oxidative phosphorylation in vitamin E-deficient mitochondria, but not in controls. In vitamin E-deficient mitochondria, iron decreased ADP/O ratios and markedly stimulated state 4 respiration; iron had only a modest effect on these parameters in control mitochondria. Thus, vitamin E may have an important role in sustaining oxidative phosphorylation. Low concentrations of iron (2 to 5 microM) oxidized mitochondrial tocopherol that exists in two pools. The release of iron in brain may impair oxidative phosphorylation, which would be exacerbated by vitamin E deficiency. The results are important for understanding the pathogenesis of human brain disorders known to be associated with abnormalities in mitochondrial function as well as iron homeostasis (e.g., Parkinson's disease).     

AIF,reactive oxygen species,and neurodegeneration: A “complex” problem

Apoptosis-inducing factor (AIF) is a flavin-binding mitochondrial intermembrane space protein that is implicated in diverse but intertwined processes that include maintenance of electron transport chain function, reactive oxygen species regulation, cell death, and neurodegeneration. In acute brain injury, AIF acquires a pro-death role upon translocation from the mitochondria to the nucleus, where it initiates chromatin condensation and large-scale DNA fragmentation. Although harlequin mice exhibiting an 80–90% global reduction in AIF protein are resistant to numerous forms of acute brain injury, they paradoxically undergo slow, progressive neurodegeneration beginning at three months of age. Brain deterioration, accompanied by markers of oxidative stress, is most pronounced in the cerebellum and retina, although it also occurs in the cortex, striatum, and thalamus. Loss of an AIF pro-survival function linked to assembly or stabilization of electron transport chain complex I underlies chronic neurodegeneration. To date, most studies of neurodegeneration have failed to adequately separate the relative importance of the mitochondrial and nuclear functions of AIF in determining the extent of injury, or whether oxidative stress plays a causative role. This review explores the complicated relationship among AIF, complex I, and the regulation of mitochondrial reactive oxygen species levels. It also discusses the controversial role of complex I deficiency in Parkinson’s disease, and what can be learned from the AIF- and complex I-depleted harlequin mouse.     

Astrocyte functions in the copper homeostasis of the brain

Copper is an essential element that is required for a variety of important cellular functions. Since not only copper deficiency but also excess of copper can seriously affect cellular functions, the cellular copper metabolism is tightly regulated. In brain, astrocytes appear to play a pivotal role in the copper metabolism. With their strategically important localization between capillary endothelial cells and neuronal structures they are ideally positioned to transport copper from the blood–brain barrier to parenchymal brain cells. Accordingly, astrocytes have the capacity to efficiently take up, store and to export copper. Cultured astrocytes appear to be remarkably resistant against copper-induced toxicity. However, copper exposure can lead to profound alterations in the metabolism of these cells. This article will summarize the current knowledge on the copper metabolism of astrocytes, will describe copper-induced alterations in the glucose and glutathione metabolism of astrocytes and will address the potential role of astrocytes in the copper metabolism of the brain in diseases that have been connected with disturbances in brain copper homeostasis.     

Homocysteine contribution to DNA damage in cystathionine β-synthase-deficient patients

High blood levels of homocysteine (Hcy) are found in patients affected by homocystinuria, a genetic disorder caused by deficiency of cystathionine β-synthase (CBS) activity, as well as in nutritional deficiencies (vitamin B₁₂ or folate) and in abnormal renal function. We previously demonstrated that lipid and protein oxidative damage is increased and the antioxidant defenses diminished in plasma of CBS-deficient patients, indicating that oxidative stress is involved in the pathophysiology of this disease. In the present work, we extended these investigations by evaluating DNA damage through the comet assay in peripheral leukocytes from CBS-deficient patients, as well as by analyzing of the in vitro effect of Hcy on DNA damage in white blood cells. We verified that DNA damage was significantly higher in the CBS-deficient patients under treatment based on a protein-restricted diet and pyridoxine, folic acid, betaine and vitamin B₁₂ supplementation, when compared to controls. Furthermore, the in vitro study showed a concentration-dependent effect of Hcy inducing DNA damage. Taken together, the present data indicate that DNA damage occurs in treated CBS-deficient patients, possibly due to high Hcy levels.     

Functional expression of carnitine/organic cation transporter OCTN1 in mouse brain neurons: Possible involvement in neuronal differentiation

The aim of the present study is to clarify the functional expression and physiological role in brain neurons of carnitine/organic cation transporter OCTN1/SLC22A4, which accepts the naturally occurring antioxidant ergothioneine (ERGO) as a substrate in vivo. After intracerebroventricular administration, the distribution of [³H]ERGO in several brain regions of octn1^−/− mice was much lower than that in wild-type mice, whereas extracellular marker [¹⁴C]mannitol exhibited similar distribution in the two strains. The [³H]ERGO distribution in wild-type mice was well correlated with the amount of ERGO derived from food intake and the OCTN1 mRNA level in each brain region. Immunohistochemical analysis revealed colocalization of OCTN1 with neuronal cell markers microtubule-associated protein 2 (MAP2) and βIII-tubulin in mouse brain and primary cultured cortical neurons, respectively. Moreover, cultured cortical neurons exhibited time-dependent and saturable uptake of [³H]ERGO. These results demonstrate that OCTN1 is functionally expressed in brain neurons. The addition of ERGO simultaneously with serum to culture medium of cortical neurons attenuated mRNA and protein expressions of MAP2, βIII-tubulin and synapse formation marker synapsin I, and induced those of sex determining region Y-box 2 (Sox2), which is required to maintain the properties of undifferentiated neural stem cells. In neuronal model Neuro2a cells, knockdown of OCTN1 by siRNA reduced the uptake of [³H]ERGO with concomitant up-regulation of oxidative stress marker HO-1 and Sox2, and down-regulation of neurite outgrowth marker GAP43. Interestingly, the siRNA knockdown decreased the number of differentiated Neuro2a cells showing long neurites, but increased the total number of cells. Thus, OCTN1 is involved in cellular differentiation, but inhibits their proliferation, possibly via the regulation of cellular oxidative stress. This is the first evidence that OCTN1 plays a role in neuronal differentiation and proliferation, which are required for brain development.     

Expression of SOD1 G93A or wild-type SOD1 in primary cultures of astrocytes down-regulates the glutamate transporter GLT-1: lack of involvement of oxidative stress

Glutamate excitotoxicity is implicated in the aetiology of amyotrophic lateral sclerosis (ALS) with impairment of glutamate transport into astrocytes a possible cause of glutamate-induced injury to motor neurons. It is possible that mutations of Cu/Zn superoxide dismutase (SOD1), responsible for about 20% of familial ALS, down-regulates glutamate transporters via oxidative stress. We transfected primary mouse astrocytes to investigate the effect of the FALS-linked mutant hSOD1(G93A) and wild-type SOD1 (hSOD1wt) on the glutamate uptake system. Using western blotting, immunocytochemistry and RT-PCR it was shown that expression of either hSOD1(G93A) or hSOD1wt in astrocytes produced down-regulation of the levels of a glutamate transporter GLT-1, without alterations in its mRNA level. hSOD1(G93A) or hSOD1wt expression caused a decrease of the monomeric form of GLT-1 without increasing oxidative multimers of GLT-1. The effects were selective to GLT-1, since another glutamate transporter GLAST protein and mRNA levels were not altered. Reflecting the decrease in GLT-1 protein, [3H]d-aspartate uptake was reduced in cultures expressing hSOD1(G93A) or hSOD1wt. The hSOD1-induced decline in GLT-1 protein and [3H]d-aspartate uptake was not blocked by the antioxidant Trolox nor potentiated by antioxidant depletion using catalase and glutathione peroxidase inhibitors. Measurement of 2',7'-dichlorofluorescein (DCF)-induced fluorescence revealed that expression of hSOD1(G93A) or hSOD1wt in astrocytes does not lead to detectable increase of intracellular reactive oxygen species. This study suggests that levels of GLT-1 protein in astrocytes are reduced rapidly by overexpression of hSOD1, and is due to a property shared between the wild-type and G93A mutant form, but does not involve the production of intracellular oxidative stress.     

Cardiac oxidative stress in a mouse model of neutral lipid storage disease

Cardiac oxidative stress has been implicated in the pathogenesis of hypertrophy, cardiomyopathy and heart failure. Systemic deletion of the gene encoding adipose triglyceride lipase (ATGL), the enzyme that catalyzes the rate-limiting step of triglyceride lipolysis, results in a phenotype characterized by severe steatotic cardiac dysfunction. The objective of the present study was to investigate a potential role of oxidative stress in cardiac ATGL deficiency. Hearts of mice with global ATGL knockout were compared to those of mice with cardiomyocyte-restricted overexpression of ATGL and to those of wildtype littermates. Our results demonstrate that oxidative stress, measured as lucigenin chemiluminescence, was increased ~ 6-fold in ATGL-deficient hearts. In parallel, cytosolic NADPH oxidase subunits p67phox and p47phox were upregulated 4–5-fold at the protein level. Moreover, a prominent upregulation of different inflammatory markers (tumor necrosis factor α, monocyte chemotactant protein-1, interleukin 6, and galectin-3) was observed in those hearts. Both the oxidative and inflammatory responses were abolished upon cardiomyocyte-restricted overexpression of ATGL. Investigating the effect of oxidative and inflammatory stress on nitric oxide/cGMP signal transduction we observed a ~ 2.5-fold upregulation of soluble guanylate cyclase activity and a ~ 2-fold increase in cardiac tetrahydrobiopterin levels. Systemic treatment of ATGL-deficient mice with the superoxide dismutase mimetic Mn(III)tetrakis (4-benzoic acid) porphyrin did not ameliorate but rather aggravated cardiac oxidative stress. Our data suggest that oxidative and inflammatory stress seems involved in lipotoxic heart disease. Upregulation of soluble guanylate cyclase and cardiac tetrahydrobiopterin might be regarded as counterregulatory mechanisms in cardiac ATGL deficiency.     

Raised calcium and oxidative stress cooperatively promote alpha-synuclein aggregate formation

Cell loss in Parkinson’s and Parkinson’s-plus diseases is linked to abnormal, aggregated forms of the cytoplasmic protein, α-synuclein (α-syn). The factors causing α-syn aggregation may include oxidative stress, changes in protein turnover and dysregulation of calcium homeostasis, resulting in cytotoxic aggregated α-syn species. Recently, we showed that raised calcium can promote α-syn aggregation. We have now investigated the effects of raised calcium combined with oxidation/oxidative stress on α-syn aggregation both in vitro and in vivo. We treated monomeric α-syn with calcium, hydrogen peroxide or calcium plus hydrogen peroxide in vitro and used size exclusion chromatography, fluorescence correlation spectroscopy, atomic force microscopy and scanning electron microscopy to investigate protein aggregation. Our in vitro data is consistent with a cooperative interaction between calcium and oxidation resulting in α-syn oligomers. In cell culture experiments, we used thapsigargin or ionophore A23187 to induce transient increases of intracellular free calcium in human 1321N1 cells expressing an α-syn-GFP construct both with and without co-treatment with hydrogen peroxide and observed α-syn aggregation by fluorescence microscopy. Our in vivo cell culture data shows that either transient increase in intracellular free calcium or hydrogen peroxide treatment individually were able to induce significantly (P = 0.01) increased 1–4 μm cytoplasmic α-syn aggregates after 12 h in cells transiently transfected with α-syn-GFP. There was a greater proportion of cells positive for aggregates when both raised calcium and oxidative stress were combined, with a significantly increased proportion (P = 0.001) of cells with multiple (3 or more) discrete α-syn focal accumulations per cell in the combined treatment compared to raised calcium only. Our data indicates that calcium and oxidation/oxidative stress can cooperatively promote α-syn aggregation both in vitro and in vivo and suggests that oxidative stress may play an important role in the calcium-dependent aggregation mechanism.     

l-carnitine supplementation as a potential antioxidant therapy for inherited neurometabolic disorders

In recent years increasing evidence has emerged suggesting that oxidative stress is involved in the pathophysiology of a number of inherited metabolic disorders. However the clinical use of classical antioxidants in these diseases has been poorly evaluated and so far no benefit has been demonstrated. l-Carnitine is an endogenous substance that acts as a carrier for fatty acids across the inner mitochondrial membrane necessary for subsequent beta-oxidation and ATP production. Besides its important role in the metabolism of lipids, l-carnitine is also a potent antioxidant (free radical scavenger) and thus may protect tissues from oxidative damage. This review addresses recent findings obtained from patients with some inherited neurometabolic diseases showing that l-carnitine may be involved in the reduction of oxidative damage observed in these disorders. For some of these diseases, reduced concentrations of l-carnitine may occur due to the combination of this compound to the accumulating toxic metabolites, especially organic acids, or as a result of protein restricted diets. Thus, l-carnitine supplementation may be useful not only to prevent tissue deficiency of this element, but also to avoid oxidative damage secondary to increased production of reactive species in these diseases. Considering the ability of l-carnitine to easily cross the blood–brain barrier, l-carnitine supplementation may also be beneficial in preventing neurological damage derived from oxidative injury. However further studies are required to better explore this potential.     

Vitamin E as a novel enhancer of macroautophagy in rat hepatocytes and H4-II-E cells

Autophagy is an intracellular bulk degradation process induced by nutrient starvation, and contributes to macromolecular turnover and rejuvenation of cellular organelles. We demonstrated that vitamin E was a novel nutritional enhancer of autophagy in freshly isolated rat hepatocytes and rat hepatoma H4-II-E cells. Supplementation of fresh hepatocytes with vitamin E (up to 100 μM) increased proteolysis significantly in the presence or absence of amino acids in a dose-dependent manner. The cytosolic LC3 ratio, a newly established index of autophagic flux, was significantly increased by vitamin E, strongly suggesting that the possible site of action is the LC3 conversion step, an early step in autophagosome formation. A typical antioxidant, α-lipoic acid, exerted autophagy suppression, while H₂O₂ stimulated autophagy. It is conceivable that autophagy was stimulated by oxidative stress and this stimulation was cancelled by cellular antioxidative effects. However, in our studies, vitamin E could have enhanced autophagy over-stimulation by H₂O₂, rather than suppress it. From these results, using a new cytosolic LC3 ratio, vitamin E increases autophagy by accelerating LC3 conversion through a new signaling pathway, emerging as a novel enhancer of autophagy.     

综述:金属离子代谢平衡失调与阿尔茨海默病早期发病机制

研究表明,脑内金属离子代谢失衡与阿尔茨海默病(AD)有关,但其机理尚需深入探讨．结合本实验室研究结果,作者对金属离子代谢紊乱与氧化应激,金属离子代谢紊乱与β-淀粉样蛋白、转铁蛋白和转铁蛋白受体、铁调节蛋白、二价金属离子转运体以及天然抗氧化剂通过调节金属离子代谢平衡缓解β-淀粉样蛋白的毒性和保护细胞的作用进行探讨．提出：铁、铜等金属离子缺乏可能主要与AD早期关系密切,而铁、铜等金属离子过载可能主要与AD后期损伤关系密切的学术观点．