Glutathione Conjugation at the Blood-CSF Barrier Efficiently Prevents Exposure of the Developing Brain Fluid Environment to Blood-Borne Reactive Electrophilic Substances.

Exposure of the developing brain to toxins, drugs, or deleterious endogenous compounds during the perinatal period can trigger alterations in cell division, migration, differentiation, and synaptogenesis, leading to lifelong neurological impairment. The brain is protected by cellular barriers acting through multiple mechanisms, some of which are still poorly explored. We used a combination of enzymatic assays, live tissue fluorescence microscopy, and an in vitro cellular model of the blood-CSF barrier to investigate an enzymatic detoxification pathway in the developing male and female rat brain. We show that during the early postnatal period the choroid plexus epithelium forming the blood-CSF barrier and the ependymal cell layer bordering the ventricles harbor a high detoxifying capacity that involves glutathione S-transferases. Using a functional knock-down rat model for choroidal glutathione conjugation, we demonstrate that already in neonates, this metabolic pathway efficiently prevents the penetration of blood-borne reactive compounds into CSF. The versatility of the protective mechanism results from the multiplicity of the glutathione S-transferase isoenzymes, which are differently expressed between the choroidal epithelium and the ependyma. The various isoenzymes display differential substrate specificities, which greatly widen the spectrum of molecules that can be inactivated by this pathway. In conclusion, the blood-CSF barrier and the ependyma are identified as key cellular structures in the CNS to protect the brain fluid environment from different chemical classes of potentially toxic compounds during the postnatal period. This metabolic neuroprotective function of brain interfaces ought to compensate for the liver postnatal immaturity.SIGNIFICANCE STATEMENT Brain homeostasis requires a stable and controlled internal environment. Defective brain protection during the perinatal period can lead to lifelong neurological impairment. We demonstrate that the choroid plexus forming the blood-CSF barrier is a key player in the protection of the developing brain. Glutathione-dependent enzymatic metabolism in the choroidal epithelium inactivates a broad spectrum of noxious compounds, efficiently preventing their penetration into the CSF. A second line of detoxification is located in the ependyma separating the CSF from brain tissue. Our study reveals a novel facet of the mechanisms by which the brain is protected at a period of high vulnerability, at a time when the astrocytic network is still immature and liver xenobiotic metabolism is limited.

Characterization of residue-specific glutathionylation of CSF proteins in multiple sclerosis - A MS-based approach.

Reduction of a disulfide linkage between cysteine residues in proteins, a standard step in the preanalytical preparation of samples in conventional proteomics approach, presents a challenge to characterize S-glutathionylation of proteins. S-glutathionylation of proteins has been reported in medical conditions associated with high oxidative stress. In the present study, we attempted to characterize glutathionylation of CSF proteins in patients with multiple sclerosis which is associated with high oxidative stress. Using the nano-LC/ESI-MS platform, we adopted a modified proteomics approach and a targeted database search to investigate glutathionylation at the residue level of CSF proteins. Compared to patients with Intracranial hypertension, the following CSF proteins: Extracellular Superoxide dismutase (ECSOD) at Cys195, α1-antitrypsin (A1AT) at Cys232, Phospholipid transfer protein (PLTP) at Cys318, Alpha-2-HS-glycoprotein at Cys340, Ectonucleotide pyrophosphate (ENPP-2) at Cys773, Gelsolin at Cys304, Interleukin-18 (IL-18) at Cys38 and Ig heavy chain V III region POM at Cys22 were found to be glutathionylated in patients with multiple sclerosis during a relapse. ECSOD, A1AT, and PLTP were observed to be glutathionylated at the functionally important cysteine residues. In conclusion, in the present study using a modified proteomics approach we have identified and characterized glutathionylation of CSF proteins in patients with multiple sclerosis.

3-hydroxykynurenine and other Parkinson's disease biomarkers discovered by metabolomic analysis.

Parkinson's disease (PD) biomarkers are needed to enhance therapeutics research and to understand PD pathogenesis. Methods that simultaneously measure hundreds of small molecular-weight compounds-metabolomic analysis-"fingerprint" disease-specific alterations in individual compounds or metabolic pathways. Beyond a nontargeted search for PD biomarkers, we hypothesized that PD cerebrospinal fluid would show increased formation of the excitotoxin 3-hydroxykynurenine and diminished concentration of the antioxidant glutathione. Cerebrospinal fluid was collected at <4 hours postmortem from 48 pathologically-verified PD subjects and 57 comparably-aged controls. Assays involved ultra-high-performance liquid and gas chromatography linked to mass spectrometry. We used univariate techniques to determine fold-changes in concentrations of biochemicals; false-discovery rates were calculated to exclude spurious findings. Data was modeled using a Support Vector Machine for analyzing compounds selected by Welch's t test. Classification accuracy was determined by cross-validation. Of 243 structurally-identified biochemicals,19 compounds differentiated PD from controls at a 20% false-discovery level. In PD, mean 3-hydroxykynurenine concentration was increased by one-third, and mean oxidized glutathione was decreased by 40% (for each, P < .01). Four of the 19 compounds differentiating PD from controls were N-acetylated amino acids, suggesting a generalized alteration in N-acetylation activity. The Support Vector Machine classification model distinguished between groups at 83% sensitivity and 91% specificity for the learning data, and at 65% and 79% from cross-validation. In this study, the first for metabolomic profiling of PD cerebrospinal fluid, we found several novel biomarkers and offer new directions for recognizing disease-specific biochemical indicators. The findings support involvement of excitotoxicity and oxidative stress in the pathogenesis of PD.

Reduced but not oxidized cerebrospinal fluid glutathione levels are lowered in Lewy body diseases.

Reduced (GSH(R)) but not oxidized glutathione (GSSG) has been shown to be dramatically altered in the substantia nigra (SN) of Lewy body disease (LBD) patients post mortem; but up to now, there is no convincing evidence that these changes can be monitored in vivo. We investigated GSH(R) and GSSG in rapidly processed cerebrospinal fluid (CSF) and plasma samples of 80 LBD and 35 control subjects and detected reduced CSF GSH(R) levels in LBD subjects. The reduction was negatively associated with age but not with disease-associated parameters. Plasma GSH(R), CSF GSSG, and plasma GSSG levels did not significantly differ between the groups. Our findings confirm the results from neuropathologic studies, which demonstrated an alteration of the glutathione system in LBD. We hypothesize that alterations of the glutathione system occur in a very early stage of the disease or may even represent a risk marker for LBD.

Oxidative stress in mice infected with Angiostrongylus cantonensis coincides with enhanced glutathione-dependent enzymes activity.

This study aimed to estimate reactive oxygen species (ROS) production, antioxidants activity, and biomarkers level of oxidative damage to protein and DNA in the cerebrospinal fluid (CSF) of C57BL/6 mice infected with Angiostrongylus cantonensis. The mean ROS concentration in the CSF of infected mice increased gradually, and the increase in ROS in CSF became statistical significance at days 12-30 post-infection compared to that before infection (P<0.001), and then ROS returned to normal level at day 45 after infection. In parallel with the increase in ROS in the CSF, infected mice showed similar of changes in reduced glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST) as that in ROS in the CSF. GSH, GR, GPx, and GST in the CSF of infected mice were all significantly higher than they were before infection during days 12-30 post-infection. However, protein carbonyl content and 8-hydroxy-2'-deoxyguanosine, biomarkers of oxidative damage to protein and DNA, respectively, were also significantly higher in the CSF of infected mice during this period. These results suggest that oxidative stress occur in the cells of central nervous system of mice infected with A. cantonensis during days 12-30 after infection due to ROS overproduction in CSF despite the increase in antioxidants during this period.

DNA-nanohydrogel self-assembled gold nanoparticles: co-profiling of multiple small molecule reductants in rat brain.

DNA nanohydrogel assembled AuNPs were proposed as a high-throughput multidimensional sensing strategy for small molecule reductant profiling in rat brain. The equilibrium among AuNPs, DNA nanohydrogel and targets produced a unique fingerprint-like pattern for differentiating the reducing capacity.

A comparative study of nitric oxide, glutathione, and glutathione peroxidase activities in cerebrospinal fluid from children with convulsive diseases/children with aseptic meningitis.

It has been reported that active oxygen and/or free radicals are produced in the central nervous system (CNS) compartment in patients with bacterial meningitis, so it is supposed that the levels of endogenous antioxidative scavengers in the cerebrospinal fluid (CSF) are elevated as an adaptive reaction to bacterial meningitis, which exerts severe stress on the human body. We assumed that they are also elevated in patients with convulsive diseases. Nitric oxide (NO) and endogenous antioxidative scavengers (glutathione (GSH), glutathione peroxidase (GPX), (total) superoxide dismutase (T-SOD), manganese superoxide dismutase (Mn-SOD), and catalase) were measured in CSF from a group of child patients with various neurological diseases and a control group. NO, GSH, and GPX activities in CSF from the patients with convulsive diseases were significantly higher than in those with aseptic meningitis or in the controls. Furthermore, all parameters in CSF from patients with bacterial meningitis were significantly higher than in any other group. The present study suggests that oxidative stress may be associated with the pathophysiology of convulsion and that its clinical attenuation will lead to improvement in the prognosis for convulsive diseases.

Cerebrospinal fluid markers of disturbed brain cell metabolism.

Reversibly and irreversibly disturbed brain cell metabolism may be monitored in an indirect way by the analyses of enzymes in the CSF according to the hypothesis of cell swelling induced by energy shortage. Adenylate kinase fulfils the criteria for an ideal CSF marker with the exception that it is not organspecific, which necessitates precautions to avoid influence of AK in erythrocytes and serum. When taking such limitating factors into account, AK determinations may be diagnostically useful in combination with radiological and clinical observations. Besides, it is possible that a combination of AK analyses and clinical signs are useful in the prognostication in individual patients suffering from global cerebral ischemia and cerebral infarction.

Neuronal Glutathione Content and Antioxidant Capacity can be Normalized In Situ by N-acetyl Cysteine Concentrations Attained in Human Cerebrospinal Fluid.

N-acetyl cysteine (NAC) supports the synthesis of glutathione (GSH), an essential substrate for fast, enzymatically catalyzed oxidant scavenging and protein repair processes. NAC is entering clinical trials for adrenoleukodystrophy, Parkinson's disease, schizophrenia, and other disorders in which oxidative stress may contribute to disease progression. However, these trials are hampered by uncertainty about the dose of NAC required to achieve biological effects in human brain. Here we describe an approach to this issue in which mice are used to establish the levels of NAC in cerebrospinal fluid (CSF) required to affect brain neurons. NAC dosing in humans can then be calibrated to achieve these NAC levels in human CSF. The mice were treated with NAC over a range of doses, followed by assessments of neuronal GSH levels and neuronal antioxidant capacity in ex vivo brain slices. Neuronal GSH levels and antioxidant capacity were augmented at NAC doses that produced peak CSF NAC concentrations of ≥50 nM. Oral NAC administration to humans produced CSF concentrations of up to 10 µM, thus demonstrating that oral NAC administration can surpass the levels required for biological activity in brain. Variations of this approach may similarly facilitate and rationalize drug dosing for other agents targeting central nervous system disorders.

Effects of WR1065 on 6-hydroxydopamine-induced motor imbalance: Possible involvement of oxidative stress and inflammatory cytokines.

Over production of reactive oxygen species (ROS) is postulated to be the main contributor in degeneration of nigrostriatal dopaminergic neurons. In this study we investigated the effects of WR1065, a free radical scavenger, on motor imbalance, oxidative stress parameters and inflammatory cytokines in CSF and brain of hemi-parkinsonian rats. Lesion of dopaminergic neurons was done by unilateral infusion of 6-hydroxydopamine into the central region of the substentia nigra pars compacta (SNc) to induce hemi-parkinsonism and motor imbalance in rats. WR1065 (20, 40 and 80µg/2µl/rat) was administered three days before 6-OHDA administration. After three weeks behavioral study was performed and then brain and CSF samples were collected to assess tumor necrosis factor (TNFα), interlukin (IL-1ß), reduced glutathione (GSH), and malondialdehyde (MDA). WR1065 pre-treatment in rats before receiving 6-OHDA, improved significantly motor impairment and caused reduction of MDA and inflammatory cytokines TNFα and IL-1ß levels, while GSH level significantly increased when compared with lesioned rats. Our study indicated that WR1065 could improve 6-OHDA-induced motor imbalance. Furthermore, it decreased lipid peroxidation and inflammatory cytokines and restored the level of GSH up to normal range. We suggest that WR1065 can be proposed as a potential neuroprotective agent in motor impairments of PD. However to prove this hypothesis more clinical trial studies should be done.

Oxidative stress in brain during experimental bacterial meningitis: differential effects of alpha-phenyl-tert-butyl nitrone and N-acetylcysteine treatment.

Antioxidant treatment has previously been shown to be neuroprotective in experimental bacterial meningitis. To obtain quantitative evidence for oxidative stress in this disease, we measured the major brain antioxidants ascorbate and reduced glutathione, and the lipid peroxidation endproduct malondialdehyde in the cortex of infant rats infected with Streptococcus pneumoniae. Cortical levels of the two antioxidants were markedly decreased 22 h after infection, when animals were severely ill. Total pyridine nucleotide levels in the cortex were unaltered, suggesting that the loss of the two antioxidants was not due to cell necrosis. Bacterial meningitis was accompanied by a moderate, significant increase in cortical malondialdehyde. While treatment with either of the antioxidants alpha-phenyl-tert-butyl nitrone or N-acetylcysteine significantly inhibited this increase, only the former attenuated the loss of endogenous antioxidants. Cerebrospinal fluid bacterial titer, nitrite and nitrate levels, and myeloperoxidase activity at 18 h after infection were unaffected by antioxidant treatment, suggesting that they acted by mechanisms other than modulation of inflammation. The results demonstrate that bacterial meningitis is accompanied by oxidative stress in the brain parenchyma. Furthermore, increased cortical lipid peroxidation does not appear to be the result of parenchymal oxidative stress, because it was prevented by NAC, which had no effect on the loss of brain antioxidants.

Cerebrospinal fluid S-adenosylmethionine (SAMe) and glutathione concentrations in HIV infection: effect of parenteral treatment with SAMe.

The methylation and transsulfuration pathways are intimately linked and have been implicated in the progression of neurologic damage and immune cell depletion caused by human immunodeficiency virus (HIV) infection. We studied the following metabolites related to these pathways: S-adenosylmethionine (SAMe), homocysteine, cysteine, cysteinyl-glycine, and glutathione (GSH) in blood and CSF of 16 HIV-infected patients with neurologic complications and 20 HIV-negative control patients undergoing lumbar punctures for other medical reasons. We confirmed recent studies of decreased CSF SAMe concentrations in HIV infection and demonstrated that diastereomers of SAMe are present in CSF but not in plasma or erythrocytes from both HIV-infected and HIV-negative patients. In HIV-infected patients, CSF GSH and cysteinyl-glycine, but not homocysteine or cysteine, were significantly reduced. This is the first report of decreased CSF GSH induced by HIV infection. GSH has a regulatory effect on the synthesis of SAMe in hepatic tissue, and the same mechanism may also apply in the CNS. Administration of SAMe-butanedisulphonate, 800 mg/d intravenously for 14 days, was associated with significant increases in CSF SAMe and GSH. These findings have potentially important therapeutic implications for the use of SAMe in protecting against SAMe and GSH deficiency in the CNS of HIV-infected patients.

The pharmacokinetics and pharmaco-dynamics of Procysteine in amyotrophic lateral sclerosis.

In 13 subjects with ALS, we studied the safety and pharmacokinetic properties of Procysteine, a cysteine prodrug that increases levels of intracellular glutathione. We found that oral administration of Procysteine was safe. Procysteine enters CSF after both IV and oral dosing and accumulates to significant levels in CSF. We also observed that CSF levels of glutathione fall dramatically with aging.

Abnormal cobalamin-dependent transmethylation in AIDS-associated myelopathy.

BACKGROUND: White matter vacuolization of the spinal cord is common in patients with AIDS and may lead to clinical manifestations of myelopathy. The pathogenesis of AIDS-associated myelopathy (AM) is unknown and may be related to metabolic abnormalities rather than to direct HIV infection. The striking pathologic similarity between AM and the vacuolar myelopathy associated with vitamin B(12) deficiency suggests that abnormal metabolism of the B(12)-dependent transmethylation pathway may be important in the pathogenesis of AM. METHODS: The authors compared S-adenosyl-methionine (SAM), methionine, homocysteine, and glutathione in serum and CSF of 15 patients with AM vs. 13 HIV-infected controls without myelopathy (HWM). They also compared the results with a non-HIV--infected reference population (NC). All patients had normal B(12), folate, and methylmalonic acid levels. RESULTS: There was a decrease in CSF SAM in the AM group compared with the HWM group (p < 0.0001) and the NC group (p < 0.0001). CSF SAM in the HWM group was also lower than that in the NC group (p = 0.015). Serum methionine was also reduced in serum of the myelopathic group compared with the NC group (p = 0.006). CONCLUSIONS: AM is associated with an abnormality of the vitamin B(12)-dependent transmethylation pathway.

Dopamine-induced oxidative stress in neurons with glutathione deficit: implication for schizophrenia.

Glutathione (GSH) is the main non-protein antioxidant and plays a critical role in protecting cells from damage by reactive oxygen species (ROS) generated by dopamine (DA) metabolism. We reported a decrease of GSH levels ([GSH]) in CSF and in prefrontal cortex in vivo in schizophrenics [Eur. J. Neurosci. 12 (2000) 3721]. A GSH deficit may lead to membrane peroxidation and microlesions around dopaminergic terminals, resulting in loss of connectivity. To test this hypothesis, we studied the effect of DA in cultured cortical neurons with low [GSH]. DA alone decreased [GSH] by 40%. This effect appears to result from direct conjugation of DA semiquinone/quinone with GSH. Ethacrynic acid (EA) decreased [GSH] in a concentration-dependent manner. When added to EA, DA further lowers [GSH]. As this additional decrease is blocked by superoxide dismutase (SOD) or D(1)/D(2) receptor antagonists, it likely involves the generation of superoxide via activation of DA receptors. It also reduces the mitochondrial membrane potential. Most interestingly, a significant decrease in number of neuronal processes (spines analogous) was induced by 24-h application of DA only in low [GSH]. These data, compatible with our hypothesis, is consistent with the dendritic spines reduction reported in schizophrenia and could be related to abnormalities in synaptic connectivity.

Reduced and oxidized forms of glutathione and alpha-tocopherol in the cerebrospinal fluid of parkinsonian patients: comparison between before and after L-dopa treatment.

In the cerebrospinal fluid of untreated patients with Parkinson's disease (PD) the concentrations of reduced glutathione (GSH) and alpha-tocopherol (alpha-TOH) were unaltered but the concentration of oxidized glutathione (glutathione disulfide, GSSG) (P < 0.001), the GSSG/GSH ratio (P < 0.06), alpha-tocopherol quinone (alpha-TQ) (P < 0.001), and the alpha-TQ/alpha-TOH ratio (P < 0.01) were reduced significantly. In L-dopa-treated patients, the concentrations of GSH, GSSG, and the alpha-TQ concentration and the alpha-TQ/alpha-TOH ratio (P < 0.05) increased compared with untreated PD patients. These results suggest that oxidation of GSH and alpha-TOH is decreased in untreated PD patients, but is activated to a control level or more after L-dopa treatment.

Increase in oxidized NO products and reduction in oxidized glutathione in cerebrospinal fluid from patients with sporadic form of amyotrophic lateral sclerosis.

To determine the role of free radical mechanisms in the pathogenesis of amyotrophic lateral sclerosis (ALS), cerebrospinal fluid concentrations of oxidized nitric oxide (NO) products (nitrite and nitrate) and reduced and oxidized forms of glutathione (GSH and GSSG, respectively) were compared between patients with the sporadic form of ALS (SALS) and controls. In the SALS patients, the nitrate levels were significantly higher (by 73%) in contrast to remarkably lower GSSG/GSH ratio, approximately 3-fold, compared to controls. These results suggest that NO production or oxidation is activated in SALS patients, leading to a decrease in superoxide radicals to oxidize GSH. The subsequent generation of a highly reactive anion, peroxynitrite, may play a causal role in the pathogenesis of SALS.

Measurement of free radical scavengers in the spinal cord of rats with experimental autoimmune encephalomyelitis.

Antioxidants (ascorbic acid, glutathione, cysteine, alpha-tocopherol) and uric acid were measured using two high-pressure liquid chromatographic methods in 3 regions (cervical, thoracic, lumbar) of the spinal cord and in blood of Lewis rats during the attack and recovery of experimental autoimmune encephalomyelitis (EAE). Uric acid, which is thought to be a marker of free radical release, was greatly increased and glutathione correspondingly decreased in lumbar and thoracic regions. Cysteine and ascorbic acid were practically unchanged, whereas alpha-tocopherol was significantly increased during attack and recovery. Results, which could have therapeutic implications, generally support the hypothesis that free radicals are released during EAE.

Normal cerebrospinal fluid glutathione concentrations in Parkinson's disease, Alzheimer's disease and multiple system atrophy.

We measured total glutathione concentrations in the cerebrospinal fluid (CSF) of non-demented Parkinson's disease patients (PD; n=71), demented PD patients (PDD; n=13), multiple system atrophy patients (MSA; n=10), Alzheimer's disease patients (AD; n=17) and age-matched controls (n=21). No statistically significant differences in the mean total CSF glutathione concentrations were found between groups and dopaminomimetic treatment was not found to have any effect on total CSF glutathione levels. Our main conclusion is that total glutathione is not useful as a CSF marker for assumed oxidative stress in patients with PD, MSA or AD.

Do multidrug resistance-associated protein-1 and -2 play any role in the elimination of estradiol-17 beta-glucuronide and 2,4-dinitrophenyl-S-glutathione across the blood-cerebrospinal fluid barrier?

The purpose of this study was to examine the role of multidrug resistance-associated protein-1 and -2 (Mrp1 and Mrp2) in the efflux transport of organic anions across the blood-cerebrospinal fluid (CSF) barrier. The CSF concentration of estradiol-17beta-glucuronide (E(2)17betaG) and 2,4-dinitrophenyl-S-glutathione (DNP-SG) in the CSF after intracerebroventricular and intravenous injection were compared between wild-type and Mrp1 gene knockout mice. There was no significant difference in the apparent CSF elimination rate constants of E(2)17betaG (0.158 and 0.145 min(-1)) and DNP-SG (0.116 and 0.0779 min(-1)) between wild-type and Mrp1 knockout mice, respectively. After intravenous administration of E(2)17betaG, its brain-to-serum and CSF-to-serum concentration ratios in Mrp1 knockout mice were not significantly different from those in the wild-type. Results from in vivo and in vitro studies using Eisai hyperbilirubinemic rats, in which Mrp2 is hereditarily deficient, were similar to those using normal rats. Quantitative polymerase chain reaction (PCR) showed that the expression level of Mrp4 and Mrp5 was several times higher than that of Mrp1, whereas the expression levels of Mrp2, Mrp3, and Mrp6 were negligible or low. Therefore, Mrp4 and Mrp5 may contribute to the efflux transport of E(2)17betaG and DNP-SG from the CSF.