1-Methyl-4-phenylpyridinium-induced down-regulation of dopamine transporter function correlates with a reduction in dopamine transporter cell surface expression

The mechanisms whereby 1-methyl-4-phenylpyridinium (MPP(+)) mediates cell death and Parkinsonism are still unclear. We have shown that dopamine transporter (DAT) is required for MPP(+)-mediated cytotoxicity in HEK-293 cells stably transfected with human DAT. Furthermore, MPP(+) produced a concentration- and time-dependent reduction in the uptake of [3H]dopamine. We observed a significant decrease in [3H]WIN 35428 binding in the intact cells with MPP(+). The saturation analysis of the [3H]WIN 35428 binding obtained from total membrane fractions revealed a decrease in the transporter density (B(max)) with an increase in the dissociation equilibrium constant (K(d)) after MPP(+) treatment. Furthermore, biotinylation assays confirmed that MPP(+) reduced both plasma membrane and intracellular DAT immunoreactivity. Taken together, these findings suggest that the reduction in cell surface DAT protein expression in response to MPP(+) may be a contributory factor in the down-regulation of DAT function while enhanced lysosomal degradation of DAT may signal events leading to cellular toxicity.     

Platelet serotonin transporter in schizophrenic patients with and without neuroleptic treatment

Among various hypotheses put forth to account for the etiology of schizophrenia, the abnormal function of serotonergic system has recently gained marked interest. Our previous study showed that drug-free schizophrenic patients had a significant increase in maximum numbers (B(max)) of platelet 5-HT(2A) receptors that declined to normal level after treatment with different neuroleptic drugs. To elucidate the role of the serotonin system in schizophrenia, the serotonin transporters on human platelets were examined in this study. Platelet serotonin transporters obtained from normal control subjects and schizophrenic patients were identified by using [(3)H]imipramine as the radioligand and fluoxetine to define the non-specific binding. The data showed that the mean B(max) of serotonin transporter sites for schizophrenic patients without neuroleptic therapy was significantly higher than in normal controls. The B(max) values for schizophrenic patients on phenothiazine, butyrophenone, thioxanthene and serotonin-dopamine antagonist (SDA) therapies were significantly lower than the B(max) values obtained from schizophrenic patients without neuroleptic therapy, and were comparable to those found in normal control subjects. The dissociation equilibrium constant (K(d)) values in all subject groups remained unchanged. The effect of various medication periods on platelet serotonin transporters was also studied. We found that, B(max) values of 1-4 weeks, 1-4 months, 4-12 months and >1 year of neuroleptic therapies were significantly decreased when compared with the unmedicated group. Significant reduction of brief psychiatric rating scale (BPRS) occurred in all types of neuroleptics and every period of drug treatments compared with the unmedicated group. The present results indicate that alteration of platelet serotonin transporters is associated with schizophrenia. Treatment with various types of neuroleptics suppresses the hypersensitivity of platelet serotonin transporters. The mechanisms of how neuroleptics achieve their therapeutic effects, whether they act via or modulate serotonin system in certain brain area, still need to be further evaluated.     

BIOGENIC AMINE-MEDIATED ALTERATION OF PYRIDOXAL PHOSPHATE FORMATION IN RAT BRAIN

Abstract— DOPA, dopamine, norepinephrine, tyramine, serotonin, histamine and GABA inhibited pyridoxal kinase; whereas, tyrosine, 5-hydroxytryptophan, histidine, glutamic acid, hypotaurine and taurine were without inhibitory effects. Tetrahydroisoquinoline derivatives formed from Pictet-Spengler condensation between DOPA, dopamine and norepinephrine with pyridoxal and pyridoxal phosphate did not inhibit pyridoxal kinase. These results are interpreted to indicate that interaction of biogenic amines and pyridoxal kinase may alter the formation of pyridoxal phosphate which in turn may influence the activity of numerous pyridoxal phosphate dependent enzymatic reactions in brain.     

L-(3H) glutamate binding to a membrane preparation from the optic lobe of the giant freshwater prawn Macrobrachium rosenbergii de Man.

Membrane preparation from the optic lobe of the giant freshwater prawn, Macrobrachium rosenbergii de Man, was examined for the presence of specific L-(3H) glutamate binding. The optic lobes were isolated from live animals. The tissue was homogenized and the membrane fraction isolated by differential centrifugation. The membrane suspension was incubated with 10-1,000 nM of L-(3H) glutamate at 37 degrees C for 60 min. Nonspecific binding was determined by incubating the mixture with 100 microM L-glutamate. L-(3H) glutamate specifically bound to the membrane fraction with a dissociation equilibrium constant (Kd) of 205 nM and maximum number of binding sites (Bmax) of 2.04 n mol/mg protein. By using LIGAND computerized program, the saturation isotherm binding pattern indicates a single type of binding. To determine the type of glutamate receptors, competitive inhibition and IC50 of several glutamate agonists and antagonists were determined. The study reveals a metabotropic type of binding site.     

Mechanism of 1-Methyl-4-Phenylpyridinium-Induced Dopamine Release from PC12 Cells

The molecular mechanism of 1-methyl-4-phenylpyridinium (MPP⁺), a Parkinsonism-inducing neurotoxin, has been studied in PC12 cells. The cells treated with MPP⁺ (100 μM) induced a rapid increase in phosphorylation of tyrosine residues of several proteins, including synaptophysin, a major 38 kDa synaptic vesicle protein implicated in exocytosis. An accelerated release of dopamine by MPP⁺ correlated with phosphorylation of synaptophysin. Exposing the cells to MPP⁺ triggered reactive oxygen species (ROS) generation within 60 min of treatment and the said effect was blocked by mazindol, a dopamine uptake blocker. In addition, pretreatment with 50–100 μM of selegiline, a selective MAO-B inhibitor, significantly suppressed MPP⁺-mediated ROS generation. These effects of MPP⁺ result in the generation of ROS, which may be involved in neuronal degeneration seen in Parkinson’s disease.     

Neuroprotective effects of quercetin, rutin and okra (Abelmoschus esculentus Linn.) in dexamethasone-treated mice

The administration of dexamethasone, a synthetic glucocorticoid receptor agonist, causes neuronal death in the CA3 layer of the hippocampus, which has been associated with learning and memory impairments. This study aimed to examine the ability of okra (Abelmoschus esculentus Linn.) extract and its derivatives (quercetin and rutin) to protect neuronal function and improve learning and memory deficits in mice subjected to dexamethasone treatment. Learning and memory functions in mice were examined using the Morris water maze test. The results showed that the mice treated with dexamethasone had prolonged water maze performance latencies and shorter time spent in the target quadrant while mice pretreated with quercetin, rutin or okra extract prior to dexamethasone treatment showed shorter latencies and longer time spent in target quadrant. Morphological changes in pyramidal neurons were observed in the dexamethasone treated group. The number of CA3 hippocampal neurons was significantly lower while pretreated with quercetin, rutin or okra attenuated this change. Prolonged treatment with dexamethasone altered NMDA receptor expression in the hippocampus. Pretreatment with quercetin, rutin or okra extract prevented the reduction in NMDA receptor expression. Dentate gyrus (DG) cell proliferation was examined using the 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry technique. The number of BrdU-immunopositive cells was significantly reduced in dexamethasone-treated mice compared to control mice. Pretreatment with okra extract, either quercetin or rutin was found to restore BrdU-immunoreactivity in the dentate gyrus. These findings suggest that quercetin, rutin and okra extract treatments reversed cognitive deficits, including impaired dentate gyrus (DG) cell proliferation, and protected against morphological changes in the CA3 region in dexamethasone-treated mice. The precise mechanism of the neuroprotective effect of these plant extracts should be further investigated.     

Salsolinol,an Endogenous Neurotoxin,Activates JNK and NF-κB Signaling Pathways in Human Neuroblastoma Cells

Salsolinol, an endogenous neurotoxin, is known to be involved in the pathogenesis of Parkinson’s disease (PD). In the present study, we have investigated the effects of salsolinol on the activation of two different signaling pathways that involve c-Jun N-terminal kinase (JNK), and nuclear factor-κB, (NF-κB) in human dopaminergic neuroblastoma SH-SY5Y cells. Salsolinol treatment caused upregulation in the levels of c-Jun and phosphorylated c-Jun. It also caused degradation of IκBα and translocated the active NF-κB into the nucleus. The binding activity of NF-κB to DNA was enhanced by salsolinol in a concentration dependent manner. Furthermore, salsolinol decreased the levels of the anti-apoptotic protein Bcl-2, and increased pro-apoptotic protein Bax, while enhancing the release of cytochrome-c from mitochondria. Mitochondrial complex-I activity was significantly decreased and reactive oxygen species (ROS) were increased in salsolinol treated cells. These results partly suggest that salsolinol-induced JNK and NF-κB signaling pathways may be involved in induction of apoptosis in human dopaminergic neurons, as seen in Parkinson’s disease.