Wolf-Dieter Rausch

Transition metals, ferritin, glutathione, and ascorbic acid in parkinsonian brains

Abstract: The regional distributions of iron, copper, zinc, magnesium, and calcium in parkinsonian brains were compared with those of matched controls. In mild Parkinsons disease (PD), there were no significant differences in the content of total iron between the two groups, whereas there was a significant increase in total iron and iron (III) in substantia nigra of severely affected patients. Although marked regional distributions of iron, magnesium, and calcium were present, there were no changes in magnesium, calcium, and copper in various brain areas of PD. The most notable finding was a shift in the iron (II)/iron (III) ratio in favor of iron (III) in substantia nigra and a significant increase in the iron (III)‐binding protein, ferritin. A significantly lower glutathione content was present in pooled samples of putamen, globus pallidus, substantia nigra, nucleus basalis of Meynert, amygdaloid nucleus, and frontal cortex of PD brains with severe damage to substantia nigra, whereas no significant changes were observed in clinicopathologically mild forms of PD. In all these regions, except the amygdaloid nucleus, ascorbic acid was not decreased. Reduced glutathione and the shift of the iron (II)/iron (III) ratio in favor of iron (III) suggest that these changes might contribute to pathophysiological processes underlying PD.

Ginsenosides Rb1 and Rg1 effects on mesencephalic dopaminergic cells stressed with glutamate.

Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), is a well known and popular herbal medicine used worldwide. Among more than 30 ginsenosides, the active ingredients of ginseng, ginsenosides Rb1 and Rg1 are regarded as the main compounds responsible for many pharmaceutical actions of ginseng. In our study, primary cultures from embryonic mouse mesencephala were exposed to neurotoxic glutamate concentration and potential protective effects of these two ginsenosides on survival and neuritic growth of dopaminergic cells were tested. Treatment of primary mesencephalic culture with 500 microM glutamate for 15 min on the 10th day in vitro (DIV) increased the release of lactate dehydrogenase (LDH) into the culture medium, the propidium iodide (PI) uptake by cultured cells and the total number of nuclei with condensed and fragmented chromatin (apoptotic features) as evaluated with Hoechst 33342. Moreover, it extensively decreased the number of tyrosine hydroxylase immunopositive (TH+) cells and adversely affected the length and number of their neuronal processes. The toxic effect of glutamate was primarily mediated by over-activation of N-methyl-D-aspartate receptor (NMDA) as treatment of cultured cells with (+)-MK 801, an NMDA receptor antagonist, nearly abolished dopaminergic cells loss and LDH release induced by glutamate. When either ginsenoside was added alone for six consecutive days (at final concentrations 0.1, 1, 10, 20 microM), ginsenoside Rb1 (at 10 microM) significantly enhanced the survival of dopaminergic neurons compared to untreated controls. In these cultures, neurite lengths and numbers were not affected by both ginsenosides. Against glutamate exposure, ginsenosides Rb1 and Rg1 could not prevent cell death. However when pre-treating for 4 days or post-treating for 2 days following glutamate exposure, they significantly increased the numbers and lengths of neurites of surviving dopaminergic cells. Thus our study indicates that ginsenosides Rb1 and Rg1 have a partial neurotrophic and neuroprotective role in dopaminergic cell culture.

Iron accumulation in the substantia nigra in rats visualized by ultrasound

In recent studies, we have found a marked increase in substantia nigra (SN) echogenicity in patients with Parkinsons disease (PD) using transcranial ultrasound. Because a substantial body of evidence has accumulated indicating a selective elevation of iron in the SN from patients with PD, we set out to test the hypothesis that trace metals like iron could lead to the observed increase of SN echogenicity in PD. Rat brains were scanned after stereotactic injection of iron in different concentrations into the SN and after injecting ferritin, zinc and 6-OHDA alone, and after the addition of desferrioxamine. The amount of iron in the SN was measured spectroscopically. For iron, and partly for 6-OHDA, in different concentrations, a dose-dependent increase of SN echogenicity could be visualized, corresponding to an increase of iron measured by spectroscopy. No increase of echogenicity was visualized after the injection of ferritin and the addition of desferrioxamine to 6-OHDA, though an increase of iron was measured by spectroscopy. Therefore, we conclude that iron not bound to these proteins may lead to an increase of echogenicity of the SN.

Rotenone induces cell death in primary dopaminergic culture by increasing ROS production and inhibiting mitochondrial respiration

Although the definite etiology of Parkinsons disease is still unclear, increasing evidence has suggested an important role for environmental factors such as exposure to pesticides in increasing the risk of developing Parkinsons disease. In the present study, primary cultures prepared from embryonic mouse mesencephala were applied to investigate the toxic effects and underlying mechanisms of rotenone-induced neuronal cell death relevant to Parkinsons disease. Results revealed that rotenone destroyed dopaminergic neurons in a dose- and time-dependent manner. Consistent with the cytotoxic effect of rotenone as evidenced by dopaminergic cell loss, it significantly increased the release of lactate dehydrogenase into the culture medium, the number of necrotic cells in the culture and the number of nuclei showing apoptotic features. Rotenone exerted toxicity by decreasing the mitochondrial membrane potential, increasing reactive oxygen species production and shifting respiration to a more anaerobic state.

The flavone hispidulin, a benzodiazepine receptor ligand with positive allosteric properties, traverses the blood–brain barrier and exhibits anticonvulsive effects

The functional characterization of hispidulin (4′,5,7‐trihydroxy‐6‐methoxyflavone), a potent benzodiazepine (BZD) receptor ligand, was initiated to determine its potential as a modulator of central nervous system activity. After chemical synthesis, hispidulin was investigated at recombinant GABAA/BZD receptors expressed by Xenopus laevis oocytes. Concentrations of 50 nM and higher stimulated the GABA‐induced chloride currents at tested receptor subtypes (α1−3,5,6β2γ2S) indicating positive allosteric properties. Maximal stimulation at α1β2γ2S was observed with 10 μM hispidulin. In contrast to diazepam, hispidulin modulated the α6β2γ2S‐GABAA receptor subtype. When fed to seizure‐prone Mongolian gerbils (Meriones unguiculatus) in a model of epilepsy, hispidulin (10 mg kg−1 body weight (BW) per day) and diazepam (2 mg kg−1 BW per day) markedly reduced the number of animals suffering from seizures after 7 days of treatment (30 and 25% of animals in the respective treatment groups, vs 80% in the vehicle group). Permeability across the blood–brain barrier for the chemically synthesized, 14C‐labelled hispidulin was confirmed by a rat in situ perfusion model. With an uptake rate (Kin) of 1.14 ml min−1 g−1, measurements approached the values obtained with highly penetrating compounds such as diazepam. Experiments with Caco‐2 cells predict that orally administered hispidulin enters circulation in its intact form. At a concentration of 30 μM, the flavone crossed the monolayer without degradation as verified by the absence of glucuronidated metabolites.

On the mode of action of L-deprenyl in the human central nervous system.

It has been shown that 5 or 10 mg (-)deprenyl after i.v. application inhibited platelet MAO within 30 min. This effect correlated with the improvement of parkinsonian patients disability. Platelet MAO is purely of type B, thus resembling the human brain enzyme, which is 80% of type B. In other organs of the human MAO-A is of higher activity, thus it can oxidatively deaminate 5-HT, noradrenaline and tyramine in the periphery. The rather low peripheral side effects of (-)deprenyl can be explained by this fact. In vitro studies demonstrated that (-)deprenyl in comparison to d, l-tranylcypromine, clorgyline and harmaline is by far the most potent inhibitor of human brain MAO. Post-mortem studies in different human brain areas showed that there are differences in the behaviour of (-)deprenyl (10 mg) between short- and long-term treatment. Both show sufficient inhibition of DA-sensitive MAO (85–90%). However, when 5-HT is used as a substrate short-term treatment inhibits by about 40–50% whereas long-term treatment inhibits by about 65% which is higher than that mentioned before but not sufficient to increase brain 5-HT or decrease 5-HIAA. Therefore, long-term treatment with more than 1 mg/10 kg body weight could result in an accumulation of (-)deprenyl in the brain. Evidence for this derives from one parkinsonian patient, who was treated with 100 mg (-)deprenyl in which case both forms of the enzyme were inhibited sufficiently to increase DA and 5-HT in several brain regions.

Ginsenosides Rb1 and Rg1 effects on survival and neurite growth of MPP+-affected mesencephalic dopaminergic cells

Summary.Ginsenosides Rb1 and Rg1 are the main active ingredients of Panax ginseng C.A. Meyer (Araliaceae). They appear to exert protection against ischaemia and anoxic damage in animal models, suggesting an antioxidative and cytoprotective role. In our study, primary cultures from embryonic mouse mesencephalon are applied to examine the effects of these two ginsenosides on neuritic growth of dopaminergic cells and their survival affected by 1-methyl-4-phenylpyridinium-iodide (MPP+). Ginsenoside Rb1 (at 10 µM) enhanced the survival of dopaminergic neurons by 19% compared to untreated control. MPP+ (at 1 µM) significantly reduced the number of dopaminergic neurons and severely affected neuronal processes. Both ginsenosides counteracted these degenerations and significantly protected lengths and numbers of neurites of TH+ cells. Both compounds however could not prevent the cell loss caused by MPP+. Our study thus indicates partial neurotrophic and neuroprotective actions of ginsenosides Rb1 and Rg1 in dopaminergic cell culture.

Tyrosine Hydroxylase Activity in Caudate Nucleus from Parkinson's Disease: Effects of Iron and Phosphorylating Agents

Abstract: Tyrosine hydroxylase (TH) activity of human postmortem brain tissues from controls and patients with Parkinsons disease (PD) was examined in the presence of Fe2+ and phosphorylation agents, such as cyclic AMP, exogenous protein kinase, calcium plus calmodulin (Ca2+‐CaM), and ATP. TH activity from parkinsonian tissue was increased by 48% with statistical significance in the presence of exogenous protein kinase. Cyclic AMP alone had no effect, whereas Ca2+‐CaM increased the activity by only 10%. The presence of acetylcholine resulted in a slight decrease in enzyme activity. Human TH was stimulated 13.17‐fold in the presence of 1 mM Fe2+. For iron dependence, no significant differences could be shown for the Km values of TH in striata of PD, while the activity of TH was half of that of controls. Here stimulation with 1 mM Fe2+ raised the activity of TH 11‐fold. Stimulation of rat, gerbil, pig, and human caudate nucleus TH with Fe2+ shows remarkable species differences. In particular, the sensitivity of human TH to stimulating processes is noteworthy. H2O2 decreases TH activity only at high concentrations. Species differences are noted for the combined incubation of Fe2+ and H2O2. In the gerbil caudate nucleus, H2O2 does not prevent the stimulating properties of Fe2+, while the pig shows a dose‐dependent decline of TH activity. In conclusion, there are no significant changes in the stimulating properties of human caudate nucleus TH activity with Fe2+ in PD, while such differences are noted by using exogenous protein kinase. Furthermore, experimental evidence shows that TH activity declines at high concentration of H2O2 only. Potentiation of this effect by Fe2+ seems to be species‐dependent.

Oxidative Stress to Dopaminergic Neurons as Models of Parkinson's Disease

Abstract: The effects of exogenous toxins (MPP+, rotenone) and potentially neurotoxic properties of levodopa (L‐DOPA) on the survival rate of dopaminergic neurons in dissociated primary culture are presented. Dopamine agonists show a capacity to counteract MPP+‐toxicity. Moreover, a preserving potential of the antioxidant and bioenergetic coenzyme Q10 (CoQ10) on the activities of tyrosine hydroxylase (TH), complexes I and II of the respiratory chain, and hexokinase activity in striatal slice cultures against MPP+ is demonstrated.

Ginsenosides and their CNS targets.

Ginsenosides are a special group of triterpenoid saponins attributed to medical effects of ginseng. Therefore, they have been research targets over the last three decades to explain ginseng actions and a wealth of literature has been presented reporting on ginsenosides’ effects on the human body. Recently, there is increasing evidence on beneficial effects of ginsenosides to the central nervous system (CNS). Using a wide range of in vitro and in vivo models, researchers have attributed these effects to specific pharmacological actions of ginsenosides on cerebral metabolism, oxidative stress and radical formation, neurotransmitter imbalance and membrane stabilizing effects, and even antiapoptotic effects. Modulating these particular mechanisms by ginsenosides has thus been reported to exert either general stimulatory effects on the brain functions or protecting the CNS against various disease conditions. In this review, we try to address the recently reported ginsenosides’ actions on different CNS targets particularly those supporting possible therapeutic efficacies in CNS disorders and neurodegenerative diseases.