Yang-Hyeok Jo

α-Synuclein Interacts with Phospholipase D Isozymes and Inhibits Pervanadate-induced Phospholipase D Activation in Human Embryonic Kidney-293 Cells

α-Synuclein has been implicated in the pathogenesis of many neurodegenerative diseases, including Parkinsons disease and Alzheimers disease. Although the function of α-synuclein remains largely unknown, recent studies have demonstrated that this protein can interact with phospholipids. To address the role of α-synuclein in neurodegenerative disease, we have investigated whether it binds phospholipase D (PLD) and affects PLD activity in human embryonic kidney (HEK)-293 cells overexpressing wild type α-synuclein or the mutant forms of α-synuclein (A53T, A30P) associated with Parkinsons disease. Tyrosine phosphorylation of α-synuclein appears to play a modulatory role in the inhibition of PLD, because mutation of Tyr125 to Phe slightly increases inhibitory effect of α-synuclein on PLD activity. Treatment with pervanadate or phorbol myristate acetate inhibits PLD more in HEK 293 cells overexpressing α-synuclein than in control cells. Binding of α-synuclein to PLD requires phox and pleckstrin homology domain of PLD and the amphipathic repeat region and non-Aβ component of α-synuclein. Although biologically important, co-transfection studies indicate that the interaction of α-synuclein with PLD does not influence the tendency of α-synuclein to form pathological inclusions. These results suggest that the association of α-synuclein with PLD, and modulation of PLD activity, is biologically important, but PLD does not appear to play an essential role in the pathophysiology of α-synuclein.

Protective Effects of Epicatechin Against the Toxic Effects of Streptozotocin on Rat Pancreatic Islets : In Vivo and in Vitro

Introduction Green tea catechins have diverse pharmacological effects such as anticarcinogenic and antioxidant activities. Aim To study the protective effects of green tea (−)-epicatechin (EC) against the toxic effects of streptozotocin (STZ), a selective &bgr; cell toxin, on pancreatic islets in vivo and in vitro. Methodology Rats were randomly divided into four groups: control, EC (30 mg/kg)–treated, STZ (60 mg/kg)–treated, and EC plus STZ (same doses; EC+STZ)–treated rats. EC was administered twice a day for 6 days, and a single injection of STZ was used. In EC+STZ–treated rats, EC was administered 6 hours prior to STZ since posttreatment with EC had no beneficial effects on fully developed diabetes in our unpublished study. Insulin and insulin mRNA were detected by immunohistochemical analysis and in situ hybridization, respectively, and physiologic parameters including blood glucose concentration were measured daily. Following isolation of the islets, insulin release, nitrite levels, and islet morphology were observed in the four groups: control, EC (0.8 m M)–treated, STZ (5 m M)–treated, and EC+STZ (same doses)–treated islets. Results In EC+STZ–treated rats, hyperglycemia and weight loss were not observed and islet morphology was well preserved compared with STZ-treated rats. Compared with STZ treatment alone, insulin release was increased and nitrite production was decreased in EC+STZ–treated islets. Conclusion EC appears to be helpful in protecting pancreatic islets against exposure to STZ in both in vivo and in vitro systems.

Transmodulation between Phospholipase D and c-Src Enhances Cell Proliferation

ABSTRACT Phospholipase D (PLD) has been implicated in the signal transduction pathways initiated by several mitogenic protein tyrosine kinases. We demonstrate for the first time that most notably PLD2 and to a lesser extent the PLD1 isoform are tyrosine phosphorylated by c-Src tyrosine kinase via direct association. Moreover, epidermal growth factor induced tyrosine phosphorylation of PLD2 and its interaction with c-Src in A431 cells. Interaction between these proteins is via the pleckstrin homology domain of PLD2 and the catalytic domain of c-Src. Coexpression of PLD1 or PLD2 with c-Src synergistically enhances cellular proliferation compared with expression of either molecule. While PLD activity as a lipid-hydrolyzing enzyme is not affected by c-Src, wild-type PLDs but not catalytically inactive PLD mutants significantly increase c-Src kinase activity, up-regulating c-Src-mediated paxillin phosphorylation and extracellular signal-regulated kinase activity. These results demonstrate the critical role of PLD catalytic activity in the stimulation of Src signaling. In conclusion, we provide the first evidence that c-Src acts as a kinase of PLD and PLD acts as an activator of c-Src. This transmodulation between c-Src and PLD may contribute to the promotion of cellular proliferation via amplification of mitogenic signaling pathways.

Upregulation of phospholipase D in astrocytes in response to transient forebrain ischemia

Previous in vitro studies using cell cultures or brain slices have demonstrated that phospholipase D (PLD) in the nervous system is involved in the signaling mechanism in response to a variety of agonists. However, little is known about the pathophysiological role of PLD‐mediated signaling in the adult brain. We examined the changes in the expression of a PLD isozyme, PLD1, in the adult rat hippocampus, using immunological approaches and an assay for PLD activity after transient forebrain ischemia (four‐vessel occlusion model) that results in the selective delayed death of CA1 pyramidal cells and induces reactive astrocytes in the CA1 subfield. In the control hippocampus, PLD1 the level of immunoreactivity was very low. After ischemia, in parallel with the results of Western blot analysis and the PLD activity assay, immunohistochemical analysis of PLD1 demonstrated that the immunoreactive proteins peaked at 7–14 days and were most prominent in the CA1 and the dentate hilar region. The temporal and spatial patterns of immunoreactivity of both PLD1 and glial fibrillary acidic protein (GFAP) were very similar, indicating that reactive astrocytes express PLD1, confirmed by double staining for PLD1 and GFAP. These results demonstrate that reactive astrocytes upregulate PLD in vivo after injury in the adult rat hippocampus. GLIA 30:311–317, 2000.

Expression and regulation of phospholipase D during neuronal differentiation of PC12 cells.

To assess a possible role for phospholipase D (PLD) in PC12 cell signal transduction and differentiation, we have investigated the expression of PLD in PC12 cells and found that the differentiation factor, nerve growth factor (NGF) increased PLD1 protein expression and phorbol 12-myristate 13 acetate (PMA)-induced PLD activity. During neuronal differentiation, this effect showed correlation to the protein expression levels of classical protein kinase C (PKC) isozymes, PKC-alpha and -beta II, but there was no significant increase in the protein level of RhoA, another regulatory factor for PLD activation. Interestingly, PLD1 was associated with PKC-alpha or beta II, and its association gradually increased as NGF-induced neuronal differentiation progressed. PKC inhibitor, Ro-31-8220, caused a significant inhibition of neurite outgrowth and PLD activity. Furthermore, PLD1 was constitutively associated with the Shc adaptor molecule, the overexpression of which is known to induce PLD activity and to induce neurite outgrowth. Taken together, the data in this study suggests that PLD1 is closely implicated in neuronal differentiation of PC12 cells.

Inhibition by fluoxetine of voltage-activated ion channels in rat PC12 cells.

The effects of fluoxetine (Prozac) on voltage-activated K+, Ca2+ and Na+ channels were examined using the whole-cell configuration of the patch clamp technique in rat pheochromocytoma (PC12) cells. When applied to the external bath solution, fluoxetine (1, 10, 100 microM) decreased the peak amplitude of K+ currents. The K+ current inhibition by fluoxetine (10 microM) was voltage-independent and the fraction of current inhibition was 39.7-51.3% at all voltages tested (0 to +50 mV). Neither the activation and inactivation curves nor the reversal potential for K+ currents was significantly changed by fluoxetine. The inhibition by fluoxetine of K+ currents was use- and concentration-dependent with an IC50 of 16.0 microM. The inhibition was partially reversible upon washout of fluoxetine. The action of fluoxetine was independent of the protein kinases, because the protein kinase C or A inhibitors (H-7, staurosporine, Rp-cAMPS) did not prevent the inhibition by fluoxetine. Intracellular infusion with GDPbetaS or pretreatment with pertussis toxin did not block the inhibitory effects of fluoxetine. The inhibitory action of fluoxetine was not specific to K+ currents because it also inhibited both Ca2+ (IC50 = 13.4 microM) and Na+ (IC50 = 25.6 microM) currents in a concentration-dependent manner. Our data indicate that when applied to the external side of cells, fluoxetine inhibited voltage-activated K+, Ca2+ and Na+ currents in PC12 cells and its action on K+ currents does not appear to be mediated through protein kinases or G proteins.

Phospholipase C, Protein Kinase C, Ca2+/Calmodulin-Dependent Protein Kinase II, and Tyrosine Phosphorylation Are Involved in Carbachol-Induced Phospholipase D Activation in Chinese Hamster Ovary Cells Expressing Muscarinic Acetylcholine Receptor of Caenorhabditis elegans

Abstract : Recently, we have isolated a cDNA encoding a muscarinic acetylcholine receptor (mAChR) from Caenorhabditis elegans. To investigate the regulation of phospholipase D (PLD) signaling via a muscarinic receptor, we generated stable transfected Chinese hamster ovary (CHO) cells that overexpress the mAChR of C. elegans (CHO‐GAR‐3). Carbachol (CCh) induced inositol phosphate formation and a significantly higher Ca2+ elevation and stimulated PLD activity through the mAChR ; this was insensitive to pertussis toxin, but its activity was abolished by the phospholipase C (PLC) inhibitor U73122. Western blot analysis revealed several apparent tyrosine‐phosphorylated protein bands after CCh treatment. The CCh‐induced PLD activation and tyrosine phosphorylation were significantly reduced by the protein kinase C (PKC) inhibitor calphostin C and down‐regulation of PKC and the tyrosine kinase inhibitor genistein. Moreover, the Ca2+‐calmodulin‐dependent protein kinase II (CaM kinase II) inhibitor KN62, in addition to chelation of extracellular or intracellular Ca2+ by EGTA and BAPTA/AM, abolished CCh‐induced PLD activation and protein tyrosine phosphorylation. Taken together, these results suggest that the PLC/PKC‐PLD pathway and the CaM kinase II/tyrosine kinase‐PLD pathway are involved in the activation of PLD through mAChRs of C. elegans.

Effects of norfluoxetine, the major metabolite of fluoxetine, on the cloned neuronal potassium channel Kv3.1

The effects of fluoxetine and its major metabolite, norfluoxetine, were studied using the patch-clamp technique on the cloned neuronal rat K(+) channel Kv3.1, expressed in Chinese hamster ovary cells. In whole-cell recordings, fluoxetine and norfluoxetine inhibited Kv3.1 currents in a reversible concentration-dependent manner, with an IC(50) value and a Hill coefficient of 13.11+/-0.91 microM and 1.33+/-0.08 for fluoxetine and 0.80+/-0.06 microM and 1.65+/-0.08 for norfluoxetine at +40 mV, respectively. In inside-out patches, norfluoxetine applied to the cytoplasmic surface inhibited Kv3.1 with an IC(50) value of 0.19+/-0.01 microM. The inhibition of Kv3.1 currents by both drugs was characterized by an acceleration in the apparent rate of current decay, without modification of the activation time course and with relatively fewer effects on peak amplitude. The degree of inhibition of Kv3.1 by norfluoxetine was voltage-dependent. The inhibition increased steeply between 0 and +30 mV, which corresponded with the voltage range for channel opening. In the voltage range positive to +30 mV, inhibition displayed a weak voltage dependence, consistent with an electrical distance delta of 0.31+/-0.05. The association (k(+1)) and dissociation (k(-1)) rate constants for norfluoxetine-induced inhibition of Kv3.1 were 21.70+/-3.39 microM(-1) s(-1) and 14.68+/-3.94 s(-1), respectively. The theoretical K(D) value derived by k(-1)/k(+1) yielded 0.68 microM. Norfluoxetine did not affect the ion selectivity of Kv3.1. The reversal potential under control conditions was about -85 mV and was not affected by norfluoxetine. Norfluoxetine slowed the deactivation time course, resulting in a tail crossover phenomenon when the tail currents, recorded in the presence and absence of norfluoxetine, were superimposed. The voltage dependence of steady-state inactivation was not changed by the drug. Norfluoxetine produced use-dependent inhibition of Kv3.1 at a frequency of 1 Hz and slowed the recovery from inactivation. It is concluded that at clinically relevant concentrations, both fluoxetine and its major metabolite norfluoxetine inhibit Kv3.1, and that norfluoxetine directly inhibits Kv3.1 as an open channel blocker.

Serotonin inhibits the induction of NMDA receptor-dependent long-term potentiation in the rat primary visual cortex.

An increase in serotonin [5-hydroxytryptamine (5-HT)] levels in the rat visual cortex is correlated with the developmental decrease in long-term potentiation (LTP), and 5-HT may play an important role in the closure of the critical period by regulating LTP. The effect of 5-HT on the induction of N-methyl-D-aspartate receptor (NMDAR)-dependent and metabotropic glutamate receptor (mGluR)-dependent LTP in visual cortex slices from young rats was investigated. The field potential in layer II/III was recorded by stimulating the underlying layer IV. NMDAR-dependent LTP was induced in slices from 3-week-old rats by theta-burst stimulation (TBS) but not in slices from 5-week-old rats. However, LTP was induced in 5-HT-depleted slices from 5-week-old rats by incubation with para-chloroamphetamine (10 microM, 2 h), a 5-HT-depleting agent. The reinstated LTP in 5-HT-depleted slices was inhibited by the application of D-aminopentanoate, an NMDAR antagonist (50 microM) and 5-HT (10 and 30 microM). In contrast, the induction of mGluR-dependent LTP by weak TBS in disinhibited slices with picrotoxin (1 microM) in the bath was not affected by 5-HT application. The coapplication of 5-HT1A and 5-HT2 receptor agonists inhibited the induction of NMDAR-dependent LTP in 5-HT-depleted slices. 5-HT levels in the visual cortex increased with age. Based on these findings, we conclude that NMDAR-dependent LTP is specifically inhibited by coactivation of 5-HT1A and 5-HT2 receptors with the increase in 5-HT levels in the rat visual cortex at the end of the critical period.

Fluoxetine inhibits A-type potassium currents in primary cultured rat hippocampal neurons

The effects of fluoxetine (Prozac) on the transient A-currents (IA) in primary cultured hippocampal neurons were examined using the whole-cell patch clamp technique. Fluoxetine did not significantly decrease the peak amplitude of whole-cell K+ currents, but it accelerated the decay rate of inactivation, and thus decreased the current amplitude at the end of the pulse. For further analysis, IA and delayed rectifier K+ currents (IDR) were isolated from total K+ currents. Fluoxetine decreased IA (the integral of the outward current) in a concentration-dependent manner with an IC50 of 5.54 microM. Norfluoxetine, the major active metabolite of fluoxetine, was a more potent inhibitor of IA than was fluoxetine, with an IC50 of 0.90 microM. Fluoxetine (3 microM) inhibited IA in a voltage-dependent manner over the whole range of membrane potentials tested. Analysis of the time dependence of inhibition gave estimates of 34.72 microM(-1) s(-1) and 116.39 s(-1) for the rate constants of association and dissociation, respectively. The resulting apparent Kd was 3.35 microM, similar to the IC50 value obtained from the concentration-response curve. In current clamp configuration, fluoxetine (3 microM) induced depolarization of resting membrane potential and reduced the rate of action potential. Our results indicate that fluoxetine produces a concentration- and voltage-dependent inhibition of IA, and that this effect could affect the excitability of hippocampal neurons.