N-acetyl-cysteine blunts 6-hydroxydopamine- and L-buthionine-sulfoximine-induced apoptosis in human mesenchymal stromal cells.

Parkinson disease (PD) is characterized by the loss of dopaminergic (DAergic) neurons linked to environmental toxicants that cause oxidative stress (OS). The aim of this investigation was to establish the molecular response of human mesenchymal stroma cells (MSCs) depleted of glutathione (GSH) by the specific inhibitor L-buthionine-sulfoximine (BSO) to 6-hydroxydopamine (6-OHDA) and/or N-acetylcysteine (NAC) co-treatment. We found that treatment with BSO (10 mM) plus 6-OHDA (200 µM) induced apoptosis in MSCs through an oxidative stress (OS) mechanism involving H2O2, reflected by the detection of dichlorofluorescein-positive (DCF+) cells and oxidation of DJ-1 Cys106-SH into DJ-1 Cys106-SO3; an almost complete reduction in glutathione peroxidase 1 (GPX1) expression; activation of the transcription factor c-JUN, the pro-apoptotic protein BAX and BH-3-only protein PUMA; loss of mitochondrial membrane potential (∆Ψm); activation of the protease caspase-3 (CASP3) and apoptosis-inducing factor (AIF); chromatin condensation; and DNA fragmentation. Strikingly, co-treatment of MSCs with NAC (5 mM) and BSO + 6-OHDA significantly reduced the expression of OS and cell death markers but were unable to restore the expression of GPX1 compared to the expression in untreated or treated cells with NAC only. These findings highlighted the importance of the maintenance of the GSH-dependent (e.g., GPX1, GSH synthesis) and -independent (e.g., ROS scavenger molecules and thiol reducing activity) antioxidant systems (e.g., NAC) in the protection of MSCs from detrimental stress stimuli, thereby increasing the survival of stromal cells.

PARKIN overexpression in human mesenchymal stromal cells from Wharton's jelly suppresses 6-hydroxydopamine-induced apoptosis: Potential therapeutic strategy in Parkinson's disease.

BACKGROUND AIMS: Stem cell transplantation is an excellent option for regenerative or replacement therapy. However, deleterious microenvironmental and endogenous factors (e.g., oxidative stress) compromise ongoing graft survival and longevity. Therefore, (transient or stable) genetically modified cells may be reasonably thought to resist oxidative stress-induced damage. Genetic engineering of mesenchymal stromal cells (MSCs) obtained from Wharton's jelly tissue may offer some therapeutic potential. PARKIN is a multifunctional ubiquitin ligase able to protect dopaminergic cells against stress-related signaling. We, therefore, evaluated the effect of the neurotoxicant 6-hydroxydopamine (6-OHDA) on regulated cell death signaling in MSCs and investigated whether overexpression of PARKIN in MSCs was capable of modulating the effect of 6-OHDA. METHODS: We transiently transfected Wharton's jelly-derived MSCs with an mCherry-PARKIN vector using the Lipofectamine LTX method. Naïve MSCs and MSCs overexpressing PARKIN were exposed to increasing concentrations of 6-OHDA. We used light and fluorescence microscopy, flow cytometry, immunocytochemistry staining, in-cell Western and Western blot analysis. RESULTS: After 12-24 h of 6-OHDA exposure, we detected dichlorofluorescein (DCF)-positive cells (80%) indicative of reactive oxygen species (H2O2) production, reduced cell viability (40-50%), decreased mitochondrial membrane potential (ΔΨm, ~35-45%), DNA fragmentation (18-30%), and G1-arrested cell cycle in the MSCs. 6-OHDA exposure increased the expression of the transcription factor c-JUN, increased the expression of the mitochondria maintenance Phosphatase and tensin homologue-induced putative kinase 1 (PINK1) protein and increased the expression of pro-apoptotic PUMA, caspase-3 and apoptosis-inducing factor (AIF). 6-OHDA exposure also significantly augmented the oxidation of the oxidative stress sensor, DJ-1. Overexpression of PARKIN in MSCs not only significantly reduced the expression of cell death and oxidative stress markers but also significantly reduced DCF-positive cells (~50% reduction). DISCUSSION: 6-OHDA induced apoptosis in MSCs via generation of H2O2, activation of c-JUN and PUMA, mitochondrial depolarization and nuclei fragmentation. Our findings suggest that PARKIN protects MSCs against 6-OHDA toxicity by partly interacting with H2O2, reducing the expression of c-JUN, PUMA, AIF and caspase-3, and maintaining the mitochondrial ΔΨm.

APOE3 Christchurch modulates tau phosphorylation and ß-catenin/Wnt/Cadherin signaling in induced pluripotent stem cell-derived cerebral organoids from Alzheimer's cases.

Alzheimer's disease (AD) is the most common cause of dementia among older adults. APOE3 Christchurch (R136S, APOE3Ch ) variant homozygosity was reported in an individual with extreme resistance to autosomal dominant AD due to the PSEN1 E280A mutation. This subject had a delayed clinical age at onset and resistance to tauopathy and neurodegeneration despite extremely high amyloid plaque burden. We established induced pluripotent stem (iPS) cell-derived cerebral organoids from this resistant case and from a non-protected kindred control (with PSEN1 E280A and APOE3/3 ). We used CRISPR/Cas9 gene editing to successfully remove the APOE3Ch to wild type in iPS cells from the protected case and to introduce the APOE3Ch as homozygote in iPS cells from the non-protected case to examine causality. We found significant reduction of tau phosphorylation (pTau 202/205 and pTau396) in cerebral organoids with the APOE3Ch variant, consistent with the strikingly reduced tau pathology found in the resistant case. We identified Cadherin and Wnt pathways as signaling mechanisms regulated by the APOE3Ch variant through single cell RNA sequencing in cerebral organoids. We also identified elevated ß-catenin protein, a regulator of tau phosphorylation, as a candidate mediator of APOE3Ch resistance to tauopathy. Our findings show that APOE3Ch is necessary and sufficient to confer resistance to tauopathy in an experimental ex-vivo model establishing a foundation for the development of novel, protected case-inspired therapeutics for tauopathies, including Alzheimer's.

The effects of polyphenols on survival and locomotor activity in Drosophila melanogaster exposed to iron and paraquat.

Parkinson's disease (PD) is a common progressive neurodegenerative disorder, for which at present no causal treatment is available. On the understanding that the causes of PD are mainly oxidative stress and mitochondrial dysfunction, antioxidants and other drugs are expected to be used. In the present study, we demonstrated for the first time that pure polyphenols such as gallic acid, ferulic acid, caffeic acid, coumaric acid, propyl gallate, epicatechin, epigallocatechin, and epigallocatechin gallate protect, rescue and, most importantly, restore the impaired movement activity (i.e., climbing capability) induced by paraquat in Drosophila melanogaster, a valid model of PD. We also showed for the first time that high concentrations of iron (e.g. 15 mM FeSO(4)) are able to diminish fly survival and movement to a similar extent as (20 mM) paraquat treatment. Moreover, paraquat and iron synergistically affect both survival and locomotor function. Remarkably, propyl gallate and epigallocatechin gallate protected and maintained movement abilities in flies co-treated with paraquat and iron. Our findings indicate that pure polyphenols might be potent neuroprotective agents for the treatment of PD against stressful stimuli.

The cannabinoid CP55,940 prolongs survival and improves locomotor activity in Drosophila melanogaster against paraquat: implications in Parkinson's disease.

Cannabinoids have been shown to function as protective agents via receptor-independent and/or receptor-dependent mechanisms against stressful conditions. However, the neuroprotective mechanism of cannabinoids is far from conclusive. Therefore, the genuine antioxidant impact of cannabinoids in vivo is still uncertain. In this study, we demonstrate for the first time that CP55,940, a nonselective CB(1)/CB(2) cannabinoid receptor agonist, significantly protects and rescues Drosophila melanogaster against paraquat (PQ) toxicity via a receptor-independent mechanism. Interestingly, CP55,940 restores the negative geotaxis activity (i.e., climbing capability) of the fly exposed to PQ. Moreover, Drosophila fed with (1-200 microM) SP600125, a specific inhibitor of the stress responsive Jun-N-terminal kinase (JNK) signaling, and 20 mM PQ increased survival percentage and movement function (i.e., climbing capability) when compared to flies only treated with PQ. Taken together our results suggest that exogenous antioxidant cannabinoids can protect against and rescue from locomotor dysfunction in wild type (Canton-S) Drosophila exposed to stress stimuli. Therefore, cannabinoids may offer promising avenues for the design of molecules to prevent, delay, or ameliorate the treatment of population at high risk of suffering Parkinson disease.

Fast transdifferentiation of human Wharton's jelly mesenchymal stem cells into neurospheres and nerve-like cells.

BACKGROUND: The human mesenchymal stem cells derived from Wharton's jelly tissue (hWJ-MSCs) represent a tool for cell-based therapies and regenerative medicine. hWJ-MSCs form neurospheres (NSs) within 3-7 days. No data is available to establish the neuro-phenotypic markers and time of formation of nerve-like (NLCs) and glial cells from NSs derived from hWJ-MSCs. NEW METHOD: hWJ-MSCs were incubated with Fast-N-Spheres medium for 24 and 72h. The new formed NSs were in turn incubated with forskolin in neurogenic NeuroForsk medium for 1-7days. RESULTS: hWJ-MSCs cultured with Fast-N-Spheres medium trans-differentiated into NSs in just 24h compared to 72h for hWJ-MSCs cultured with classic growth factor medium. The NSs generated from the Fast-N-Spheres medium expressed reduced levels SOX2, OCT4 and NANOG, as markers of pluripotency compared to undifferentiated hWJ-MSCs. The formed NSs exposed to NeuroForsk medium differentiated into NLCs in 4days as evidenced by high levels of protein expression of the neuronal markers, and no expression of the glial marker GFAP. COMPARISON WITH EXISTING METHOD(S): Currently, the formation and harvest of NSs is expensive and time consuming. Published protocols require 3-7days to form NSs from whole human umbilical cord MSCs. We report for the first time, to our knowledge, the differentiation of NSs-derived from hWJ-MSCs into NLCs. CONCLUSIONS: The fastest method to obtain NSs and NLCs from hWJ-MSCs takes only five days using the two-step incubation media Fast-N-Spheres and NeuroForsk.

A new method of standartization of health-promoting pomegranate fruit (Punica granatum) extract.

This study analyzes the major phenolic constituents of pomegranate fruit juice and pericarp feedstock, and dry extracts thereof, using high-performance liquid chromatography. Pomegranate pericarp was extracted with water (WE) and alcohol (AE) as solvents, and liquid extracts were subsequently freeze-dried. The results indicate that ellagitannins punicalagin A and punicalagin B are the major constituents in the primary pomegranate feedstock and in both types of extracts. Ellagic acid, a common botanical constituent that is currently used to standardize pomegranate extracts, as well as ellagitannin punicalin, were found to be only minor constituents. Total punicalagins (the sum of punicalagins A+B and punicalin) and ellagic acid content in the pomegranate fruit pericarp feedstock WE were 7,6+/-0,3% and 0,2+/-0,1% by dry weight, respectively, and in the AE feedstock 7,0+/-0,2 and 0,4+/-0,1%, respectively. Total phenolic content (the sum of punicalagins and ellagic acid) in the pomegranate WE and AE were 45,8+/-1,2% and 42,3+/-1,1%, respectively. The concentrations of ellagic acid in the pomegranate WE and AE were 0,8+/-0,2% and 3,9+/-0,2%, respectively. Total phenolics in fresh single-strength pomegranate whole fruit juice contained 2,216+/-70 mg/L (95% punicalagins), whereas commercial pomegranate juice that was purchased from local stores was 317+/-13 mg/L (70% punicalagins). Our results strongly suggest that the commercially produced pomegranate extracts should be standardized to the content of total punicalagins as well as ellagic acid. The current standard uses only ellagic acid, which is unreliable, potentially misleading and vulnerable to commercial adulteration.

Efficiency of pharmacologically-active antioxidant phytomedicine Radical Fruits in treatment hypercholesteremia at men.

The purpose of this randomized, double-blind, placebo controlled intervention clinical trial was to investigate the effect of orally administered dietary supplement Radical Fruits (www.gardenoflife.com) consisting of highly concentrated edible fruits and berries on the concentration of plasma cholesterol, urinary 8-epi-prostaglandin F2alpha (8-epi-PGF2alpha) and 11-dehydrothromboxane B2 (11-dehydro-TXB2) in none-obese, non-smoking, non-diabetic hypercholesteremic male volunteers. Forty four (n=44) none-obese, non-smoking, non-diabetic male volunteers with an average age of 40+/-12 years, average body weight of 77,4+/-5,0 kg, average body mass index (BMI) of 22,2+/-2,7 kg/m2 and average total plasma cholesterol level 280+/-22 mg/dL were recruited to take part in a 4 week double-blind, randomized, placebo-controlled clinical trial. After evaluating 12 different antioxidant supplements for their phytochemical compositions and bioavailability, a dietary supplement Radical Fruits was chosen for this clinical trial. Radical Fruits contains standardized extracts and concentrates of prune, pomegranate, apple, grape, raspberry, blueberry, white cherry and strawberry. Subjects were randomly assigned to the treatment group and the placebo group using Simple Randomization Procedure. Subjects in the treatment group (n=22) were directed to take 900 mg of Radical Fruits supplement three times a day before meals. Subjects in the control group (n=22) were directed to take placebo according to the same schedule. Food record analysis, body composition, blood and urine samples were assessed on admission and then once a week. The duration of the clinical trial was 4 weeks. Administration of Radical Fruits(TM) for 4 weeks resulted in statistically significant reduction of total plasma cholesterol from 280+/-23 to 250+/-11 mg/dL, (p<0,001). Total plasma cholesterol changes in the placebo group were not statistically significant. The average plasma LDL was reduced from 195+/-23 to 169+/-21 mg/dL (p<0,001) in the Radical Fruits(TM) group, while in the placebo group there were no statistically significant changes. Plasma HDL increased by 3,2=/-0,6% in the Radical Fruits treated group (p<0,001). No significant changes in the HDL levels were observed in the placebo group. Urinary 8-epi-PGF2alpha level decreased from 450+/-170 to 330+/-159 pg/mg creatinine (p<0,001); urinary 11-dehydro-TXB2 level decreased from 1,200+/-420 to 790+/-320 pg/mg creatinine (p<0,001) with no changes in the placebo group. Administration of pharmacologically active antioxidant supplement Radical Fruit(TM) in hypercholesteremic men significantly increased plasma HDL and reduced total cholesterol and LDL, and urinary oxidative and inflammatory isoprostanes and thromboxane.

Effect of Blueberin on fasting glucose, C-reactive protein and plasma aminotransferases, in female volunteers with diabetes type 2: double-blind, placebo controlled clinical study.

In a 4-week randomized placebo-controlled clinical trial we investigated the effect of 300 mg Blueberin, a phytomedicine containing 250 mg Blueberry leaves (Vaccinium arctostaphylos L, Ericaceae) extract providing minimum 50 mg 3,4-caffeoylquinic (chlorogenic) acid, and 50 mg myricetin, on fasting plasma glucose, alanine aminotransferases (ALT), aspartate aminotransferases (AST), glutamyltransferase (GGT) enzymes levels, and serum inflammatory C-Reactive proteins (CRP) in forty-two volunteer subjects (46+/-15 year of age, BMI 25+/-3 kgs/(m2)) diagnosed with Type 2 diabetes. During the 4-week trial, the Blueberin supplement was administered three times per day, 15-30 minutes prior to a meal along with 100 ml of water. Results of this trial revealed that the supplementation of Blueberin reduced fasting plasma glucose from 143+/-5,2mg/L to 104+/-5,7 mg/L (p<0,001), whereas there was no statistically significant changes in the Placebo group from 138+/-4,8 mg/L to 126+/-5,1mg/L (p>0,05). The reduction of fasting glucose was correlated with the reduction of serum CRP and in the Blueberin group from 5,18+/-1,4 mg/l to 2,14+/-1,8 mg/L (p<0,05), whereas in the Placebo group CRP levels were not significantly reduced from 5,11+/-1,7 mg/l to 4,94+/-1,1mg/L (p>0,05). Furthermore, the Blueberin also significantly reduced the levels of plasma enzymes ALT, AST and GGT, indicating that, in addition to anti-diabetes effects, the Blueberin also possess pharmacologically relevant anti-inflammatory properties.

Redox cycling activity of monoamine-serotonin binding protein conjugates.

It has been shown recently that the covalent binding of labelled dopamine and serotonin to serotonin binding proteins (SBP) from bovine frontal cortex is potently inhibited by their related neurotoxins. The present study reveals that the monoamine-SBP conjugates of serotonin, dopamine, and related toxins are able to catalyse redox cycling reactions. Using an improved method to detect quinoproteins in SDS-PAGE gels, we were also able to demonstrate that the redox cycling activity corresponded to two major protein components with molecular weights of 45 and 56 kDa. The covalent monoamine-SBP conjugates may be referred to as "artificial quinoproteins."

Interaction of serotonin- and dopamine-related neurotoxins with "serotonin binding proteins" in bovine frontal cortex.

Binding of [3H]serotonin and [3H]dopamine to serotonin-binding proteins (SBP) from soluble extracts of bovine frontal cortex is increased by Fe2+. This group recently attributed this effect of Fe2+ to its ability to enhance the oxidation of [3H]serotonin and [3H]dopamine in the presence of dissolved molecular oxygen, and to the ability of the formed oxidation products to bind covalently to cysteine residues of SBP. In this study it is shown that the binding of both ligands is potently inhibited by dopamine as well as by several catecholamine-and serotonin-related neurotoxins: adrenochrome, 5,6-dihydroxytryptamine, 5,7-dihydroxytryptamine, 6-hydroxydopamine and 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline. In contrast, serotonin can only potently inhibit part (36%) of the [3H]dopamine binding, while 1,2,3,4-tetrahydroisoquinoline is only a weak competitor for both ligands. Potent inhibition by the toxins is associated with the presence of electrophilic centres at the aromatic ring, either of the products themselves (adrenochrome) or of their oxidation products (all other competitors). These findings suggest that "SBP" represent an important target for the Fe(2+)-mediated binding of [3H]-serotonin, [3H]dopamine and related neurotoxins.

Manganese and copper promote the binding of dopamine to "serotonin binding proteins" in bovine frontal cortex.

The authors previously reported that Fe2+ is capable of increasing the binding of dopamine and of serotonin to "serotonin binding proteins" which are present in soluble extracts from calf brain. In this study, it is shown that Mn2+ and Cu2+ are also capable of increasing the binding, but for dopamine only. As for Fe2+, Mn2+ and Cu2+ are likely to promote the binding by virtue of their ability to enhance the oxidation of dopamine into dopamine-O-quinone, a derivative which is known to undergo covalent association with sulfhydryl groups of proteins. Data such as the irreversible nature of the majority of the binding, the inhibitory action of reducing agents (sodium ascorbate) and of reagents which contain, or modify sulfhydryl groups (reduced glutathione) are compatible with such a mechanism. The three metal ions are also capable of inactivating part of the binding sites on SBP directly; this effect is more pronounced for Cu2+ than for Fe2+ and it is only weak for Mn2+. The Fe(2+)-mediated binding of dopamine is inhibited by the superoxide dismutase enzyme, and it was therefore suggested that Fe2+ enhances the oxidation of dopamine by virtue of its ability to produce superoxide radicals out of dissolved molecular oxygen. Such a mechanism does not appear to take place in the case of Mn2+ and Cu2+. Instead, it is likely that Cu2+ and dopamine form a complex which is highly susceptible towards oxidation by dissolved molecular oxygen. Mn2+, on the other hand, can easily be oxidized into Mn3+, which is capable to oxidize dopamine by itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Soluble serotonin and catecholamine binding proteins in the bovine adrenal medulla.

The soluble serotonin-binding proteins (SBP) present in the adrenal medulla and in chromaffin cells, are very similar to those reported for the bovine brain and retina. Binding of [3H]serotonin and [3H]dopamine to these SBP is increased by Fe2+ but not by Fe3+. At an optimal concentration of Fe2+ (0.1 mM) these proteins behave as a single class of non-cooperative sites for [3H]serotonin (Bmax = 124 +/- 28 pmol/mg protein, KD = 0.51 +/- 0.13 microM) and [3H]dopamine (Bmax = 685 +/- 118 pmol/mg protein, KD = 0.46 +/- 0.06 microM). Binding of [3H]dopamine is also increased by Cu2+ and Mn2+, but to a lesser extent than by Fe2+. Catecholamines are good competitors for [3H]serotonin binding (Ki = 0.31 microM for dopamine, 0.6 microM for adrenaline and 0.9 microM for noradrenaline). The serotonin binding proteins from adrenal medulla elute in the void volume of a Sephacryl 100 HR gel filtration column, reflecting aggregation, and migrate mainly with an apparent molecular weight of 45 kDa in native polyacrylamide gel electrophoresis experiments. Subcellular localization studies and release experiments suggest that SBP are not present in chromaffin granules, but in the cytosol of purified chromaffin cells. The present data suggest that these proteins must have other functions than storing monoamines in synaptic vesicles.

Serotonin binding proteins in bovine retina: binding of serotonin and catecholamines.

Serotonin binding proteins (SBP) are present in the soluble fraction of bovine retina homogenates. These proteins can be precipitated with 30% ammonium sulphate and their binding and physicochemical characteristics are very similar to those of SBP in bovine and rat brain. Binding of [3H]serotonin to bovine retina SBP requires Fe2+ but not Fe3+. In the presence of an optimal concentration of Fe2+ (0.1 mM), these proteins behave as a single class of non-cooperative sites for [3H]serotonin (Bmax = 242 +/- 10 pmol/mg protein, KD = 0.22 +/- 0.44 microM). Competition binding studies reveal that serotonin analogs possessing an hydroxyl group on the indole ring and catecholamine analogs possessing an intact catechol moiety are potent competitors (K1 from 0.12 to 0.3 microM). In both cases, the affinity is strongly decreased if aromatic hydroxyl groups are methoxylated. Catecholamine SBP interactions can also be demonstrated directly by binding experiments with [3H]dopamine. Binding of this catecholamine is greatly enhanced by Fe2+, to a lesser extent by Cu2+ and Mn2+, but not by Fe3+. The Fe(2+)-dependent binding component is saturable (Bmax = 505 +/- 30 pmol/mg protein. KD = 0.34 +/- 0.04 microM). The SBP from bovine retina show the same physicochemical properties as SBP from bovine and rat brain: they elute immediately after the void volume on a Sephacryl S100 HR (1.6 x 140 cm) gel filtration column (reflecting aggregation) and they migrate with apparent molecular weights of respectively 43 kDa and 57 kDa on native polyacrylamide gel electrophoresis. The serotonin-storing role of SBP in serotonergic neurones has already been well documented.(ABSTRACT TRUNCATED AT 250 WORDS)

Fe(2+)-mediated binding of serotonin and dopamine to skeletal muscle actin: resemblance to serotonin binding proteins.

Fe2+ stimulates the binding of [3H]serotonin and [3H]dopamine to rabbit skeletal muscle actin. This binding is inhibited by reducing agents (sodium ascorbate, vitamin E), by superoxide dismutase and by sulfhydryl group-modifying reagents (N-ethyl-maleimide, 2,2'-dinitro-5,5'-dithiobenzoic acid). The effect of Fe2+ is mimicked by oxidants (sodium periodate, potassium nitroso-disulfonate) and by superoxide radicals. Once formed, the binding cannot be decreased by a large excess of monoamine. It is proposed that Fe2+ catalyses the autoxidation of the monoamines by generating oxygen free radicals, and the oxidation products are likely to bind covalently to exposed cysteine residues of actin. Digestion of [3H]dopamine-labelled actin by cyanogen bromide and then by V8 protease (EC3.4.21.19) yields two labelled peptides whose apparent molecular weights (4.1 and 1.2 kDa) are compatible with the labelling of cysteine-10 and -374. Fe2+ also inactivates some of the binding sites on actin. This inactivation, and the covalent nature of the binding precludes the interpretation of monoamine saturation and competition binding data in terms of reversible bimolecular interactions.

Binding of serotonin and dopamine to 'serotonin binding proteins' in bovine frontal cortex: evidence for iron-induced oxidative mechanisms.

Binding of [3H]serotonin and of [3H]dopamine to serotonin binding proteins (SBP) from soluble extracts of bovine frontal cortex is increased by Fe2+ but not by Fe3+. It was generally believed that Fe2+ first binds to sulfhydryl groups of SBP and that the monoamines form coordination bonds with the trapped iron. We report two series of findings that are incompatible with this mechanism. First, the binding of both radioligands is an irreversible process since it is not diminished when a large excess (1 mM) of serotonin or dopamine is added to a pre-equilibrated mixture of SBP, 0.1 mM Fe2+ and 0.2 microM radioligand. Once formed, binding is not impaired by chelating agents such as ethyleneglycoltetraacetic acid and desferal. Second, the Fe(2+)-stimulated binding is inhibited by reducing agents (sodium ascorbate, vitamin E, sodium metabisulfite) and by agents which deplete superoxide radicals (superoxide dismutase and hydrogen peroxide). Moreover, the effect of Fe2+ can be mimicked by oxidants (sodium periodate, potassium superoxide) and by the generation of superoxide radicals by the xanthine oxidase-catalysed oxidation of xanthine. To integrate these findings, we formulate the hypothesis that Fe2+ reacts with dissolved molecular oxygen to produce superoxide radicals, that these radicals oxidise [3H]serotonin and [3H]dopamine, and that the formed oxidation products bind covalently to cysteine residues of SBP. This alternative mechanism is also based on the ability of reagents which contain or modify sulfhydryl groups to decrease the binding and on the inability of hydroxyl radical scavengers (dimethyl sulfoxide, mannitol, ethanol and thiourea) to do so. Fe2+ is also able to irreversibly inactivate part of the binding sites on SBP (81% of the specific binding of [3H]serotonin, and 61% for [3H]dopamine). This Fe(2+)-mediated inactivation, as well as the covalent nature of the binding, preclude the interpretation of saturation and competition binding data in terms of reversible bimolecular interactions. Yet, such experiments indicate that, at the same concentration, [3H]dopamine binds to 2 to 3 times more sites than [3H]serotonin. Unlabelled dopamine acts also as a potent competitor at all the [3H]serotonin binding sites, whereas unlabelled serotonin only acts as a potent competitor at part (30%) of the [3H]dopamine binding sites. SBP were initially proposed to be involved in the storage, protection and/or transport of serotonin, and recently also of catecholamines. However, these potential functions of SBP can hardly be reconciled with the molecular mechanism of the binding. Moreover, it is conceivable that this binding actually represents an in vitro model for neurodegeneration.

Serotonin-binding proteins in the bovine cerebral cortex: interaction with serotonin and catecholamines.

The soluble serotonin-binding proteins (SBP) present in bovine frontal cortex are very similar to those reported in rat brain. Binding of [3H]serotonin to SBP, present in ammonium sulphate-precipitated proteins from bovine cortex, requires Fe2+ but not Fe3+. In the presence of an optimal concentration of Fe2+ (0.1 mM), bovine SBP behave as a single class of non-cooperative sites for [3H]serotonin binding (Bmax = 120 +/- 12 pmol/mg protein, KD = 0.12 +/- 0.04 microM, n = 3). Binding of [3H]serotonin is decreased by nucleotides and by reagents which modify sulfhydryl groups and reduce disulfide bonds and by metal ion chelators. Serotonin analogs possessing an hydroxyl group on the indole ring and catecholamine analogs possessing an intact catechol moiety are effective competitors (Ki from 0.1 to 0.3 microM). In both cases, the aliphatic amino group does not contribute to the binding, but the affinity is strongly decreased if aromatic hydroxyl groups are methoxylated. Catecholamine-SBP interactions can also be demonstrated directly by binding experiments. Binding of [3H]dopamine is greatly enhanced by Fe2+, Cu2+ and Mn2+, but not by Fe3+. The Fe(2+)-dependent binding component of [3H]dopamine is saturable (Bmax = 279 +/- 64 pmol/mg protein, KD = 0.19 +/- 0.02 microM, n = 3), and possesses the same physicochemical properties as SBP: it elutes immediately after the void volume on a Sephacryl S100 HR (1.6 x 140 cm) gel filtration column (reflecting aggregation) and it migrates with an apparent molecular weight of 57-58 kDa on native polyacrylamide gel electrophoresis. Whereas the serotonin-storing role of SBP in serotonergic neurons has already been well documented, the present data advocate that these proteins may also possess catecholamine-storing properties.

Molecular mechanism of monoamine toxicity in Parkinson's disease: hypothetical cell death model.

Although there have been experimental approaches to understanding the etiology of Parkinson's disease, the cause of cell degeneration in this neurological disorder remains a mystery. Herein, a hypothetical model is proposed to explain the mechanism leading neurons to die. The model is based on recent experimental evidence and it attempts to dissect the actions of dopamine and metal ions as potential triggers for the activation of an ordered cascade of events of the cell death machinery.

[Apoptosis in neurodegenerative diseases: facts and controversies]. / La apoptosis en las enfermedades neurodegenerativas: evidencias y controversias.

OBJECTIVES: In this article, the authors analyzed critically the morphological and biochemical evidences of cell death by apoptosis from postmortem studies in Alzheimer s, Parkinson s, Huntington s and Wilson s diseases. DEVELOPMENT: During the last few years, apoptosis has been postulated as a type of neuronal death responsible for the neurodegenerative process in those heterogeneous, chronic and progressive neurological disorders, which are characterized by a selective and a symmetric loss of neurons in motor, sensory or cognitive systems. With regard to neuronal death mechanism and the contribution of the mutated or metabolic altered proteins such as betaA, P-tau, alpha-synuclein, Parkin, Huntingtin, ATP78B, proteins in the pathogenesis of those disorders are still unknown. CONCLUSIONS: We consider that the morphological (e.g. DNA fragmentation without showing classical apoptotic morphology) and biochemical evidences are still insufficient and contradictory to formally indict apoptosis as the mechanism of neuronal cell death in those neurological disorders. The establishment of the molecular mechanisms leading neurons to cell death (by apoptosis?) could provide significant information for the design of therapeutic strategies to retard or prevent the development of such neurodegenerative diseases in affected individuals.

[Neuronal DNA damage correlates with a positive detection of c-Jun, nuclear factor kB, p53 and Par-4 transcription factors in Alzheimer's disease]. / El daño nuclear neuronal se correlaciona con la detección in situ de los factores de transcripción c-Jun, factor nuclear kB, p53 y Par-4 en la enfermedad de Alzheimer.

INTRODUCTION AND OBJECTIVES: Alzheimer s disease is a neurodegenerative disorder characterized neuropathologically by beta amyloid plaques, neurofibrillary tangles, gliosis and neuronal loss. Recently, we have elucidated a molecular cascade of cell death induced by A beta 25 35 involving the activation of nuclear factor kappa B (NF kB), p53, and c Jun transcription factors in vitro. At present, no comparative reports have been published to establish a similar cell death signalization pathway in in vitro and in in vivo. The aim of this investigation was to determine if AD brains might activate NF kB, p53, c Jun, Par 4 transcription factors and to establish whether there exist a relationship between neuronal DNA damage and transcription factors activation. PATIENTS AND METHODS: We investigated Ab plaques, neurofibrillary tangles and NF kB, p53, and c Jun transcription factor activation in five cerebral regions from 3 normal subjects and from six demented patient with sporadic AD and one patient with AD familiar according to CERAD criteria. Using TUNEL we determine neuronal damage. RESULTS: We demonstrated neuronal damage in 17 out of 50 regions evaluated as TUNEL positive, and their distribution was heterogeneous in all brain regions evaluated; and the activation of NF kB, p53, c Jun and Par 4 transcription factors from case # 24 and #22, corresponding to TUNEL positive. CONCLUSIONS: We found a correlation between severity of DNA damage and nuclear activation of the transcription factors. These findings suggest that the AD brain may induce cell death by a molecular signalization similar to a non neuronal model by Ab exposure. This in situ study might validate previous Ab induced cell death observations in vitro.