Mun-Yong Lee

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.

Expression of vascular endothelial growth factor mRNA following transient forebrain ischemia in rats

Using a reproducible two-vessel occlusion model for forebrain ischemia in rats, we investigated the temporal and spatial changes of vascular endothelial growth factor (VEGF) expression after transient forebrain ischemia with Northern blot analysis and in situ hybridization. Northern blot analysis revealed that VEGF mRNA of the hippocampus was increased from 12 h after reperfusion, with a peak at 1 day. In situ hybridization and double labeling for VEGF mRNA and glial fibrillary acidic protein showed a transient induction of VEGF mRNA in the neurons of the hippocampus from 12 h of reperfusion with a peak at 1 day, and in the astrocytes of the hippocampus, caudoputamen, thalamus and cortical regions at 1 day. After 3 days, no more VEGF signal was detected. Our results demonstrate that astrocytes and neurons each upregulate VEGF mRNA in different temporal and spatial patterns after transient forebrain ischemia in the rat, and these patterns appear to be different from those in transient focal cerebral ischemia.

The immunocytochemical localization of connexin 36 at rod and cone gap junctions in the guinea pig retina

Connexin 36 (Cx36) is a channel‐forming protein found in the membranes of apposed cells, forming the hexameric hemichannels of intercellular gap junction channels. It localizes to certain neurons in various regions of the brain including the retina. We characterized the expression pattern of neuronal Cx36 in the guinea pig retina by immunocytochemistry using specific antisera against Cx36 and green/red cone opsin or recoverin. Strong Cx36 immunoreactivity was visible in the ON sublamina of the inner plexiform layer and in the outer plexiform layer, as punctate labelling patterns. Double‐labelling experiments with antibody directed against Cx36 and green/red cone opsin or recoverin showed that strong clustered Cx36 immunoreactivity localized to the axon terminals of cone or close to rod photoreceptors. By electron microscopy, Cx36 immunoreactivity was visible in the gap junctions as well as in the cytoplasmic matrices of both sides of cone photoreceptors. In the gap junctions between cone and rod photoreceptors, Cx36 immunoreactivity was only visible in the cytoplasmic matrices of cone photoreceptors. These results clearly indicate that Cx36 forms homologous gap junctions between neighbouring cone photoreceptors, and forms heterologous gap junctions between cone and rod photoreceptors in guinea pig retina. This focal location of Cx36 at the terminals of the photoreceptor suggests that rod photoreceptors can transmit rod signals to the pedicle of a neighbouring cone photoreceptor via Cx36, and that the cone in turn signals to corresponding ganglion cells via ON and OFF cone bipolar cells.

Upregulation of ciliary neurotrophic factor in reactive Müller cells in the rat retina following optic nerve transection.

We have investigated the expression and cellular localization of ciliary neurotrophic factor (CNTF) in the rat retina following optic nerve transection. In the normal retina, CNTF immunoreactivity was restricted to profiles in the ganglion cell layer. Following optic nerve transection, immunoreactivity appeared in Müller cell somata and processes and its intensity increased between three and seven days post-lesion. Quantitative evaluation by immunoblotting confirmed that CNTF expression increased continuously up to 7 days after optic nerve transection (to 430% of control levels), but decreased again to 250% of controls at 4 weeks post-lesion. Our findings suggest that CNTF supplied by Müller cells may play a protective role for axotomized ganglion cells in the rat retina.

Upregulation of gp130 and STAT3 activation in the rat hippocampus following transient forebrain ischemia

To determine whether the pathophysiological processes after transient forebrain ischemia are mediated via a signal pathway involving gp130 (a signal transducer for the interleukin‐6 family), we analyzed changes in the expression of gp130 and its downstream transcription factor, signal transducer and activator of transcription factor 3 (STAT3), in the rat hippocampus of a four‐vessel occlusive ischemia model. Expression of gp130 mRNA was restricted to neurons of the pyramidal cell and granule cell layers in control animals. Four hours after ischemic injury, astrocytes expressed gp130 mRNA. Expression of gp130 increased preferentially in the CA1 and dentate hilar regions, and was maintained for at least 2 weeks. Increase in gp130 expression was accompanied by the activation of STAT3 following ischemic injury. Four hours after injury, STAT3 and phosphorylated STAT3 (pSTAT3) were observed in the nuclei of the dentate hilar region, and sequentially in the CA1 region at day 1. By day 3, STAT3 immunoreactivity markedly increased in these areas, where small cells with the morphology of astrocytes showed nuclear and cytoplasmic STAT3 and nuclear pSTAT3 immunoreactivities. These patterns were especially maintained in the CA1 area until 14 days of reperfusion. Double‐labeling experiments revealed that the cells expressing STAT3 and pSTAT3 were glial fibrillary acidic protein‐expressing reactive astrocytes. These results show a coordinated and long‐lasting upregulation of gp130 mRNA and STAT3 activation in reactive astrocytes of the postischemic hippocampus, indicating that they may be involved in the astrocytic response to an ischemic insult. GLIA 41:237–246, 2003.

Expression of CNTF in Müller cells of the rat retina after pressure-induced ischemia.

We have investigated the expression and cellular localization of ciliary neurotrophic factor (CNTF) in the rat retina following ischemia induced by transiently increasing the intraocular pressure. In the normal retina, CNTF immunoreactivity was restricted to profiles in the ganglion cell layer. Following ischemia and reperfusion, immunoreactivity appeared in Müller cell somata and processes and its intensity increased between 1 day and 2 weeks post-lesion. Quantitative evaluation by immunoblotting confirmed that CNTF expression continuously increased up to 2 weeks after ischemic injury (to 600% of control levels), but had declined again to 250% of controls at 4 weeks post-lesion. Our findings suggest that CNTF supplied by Müller cells has a protective function for lesioned neurons following transient ischemia.

Auranofin blocks interleukin‐6 signalling by inhibiting phosphorylation of JAK1 and STAT3

Auranofin (AF) is a sulphur‐containing gold compound. Because of its anti‐inflammatory and immunosuppressive activities, AF has been widely used for the therapeutic treatment of rheumatoid arthritis. However, little is known about its mechanism of action. To elucidate the molecular mechanism underlying the anti‐inflammatory effect of AF, we studied the effects of AF on cellular responses to interleukin‐6 (IL‐6). In HepG2 human hepatoma cells, AF markedly inhibited IL‐6‐induced phosphorylation of janus kinase 1 (JAK1) and signal transducer and activator of transcription 3 (STAT3) and STAT3 translocation into the nucleus. Consistent with this, AF diminished IL‐6‐induced production of the acute‐phase proteins, haptoglobin, fibrinogen, C3 complement and α1‐acid glycoprotein, and gene expression of vascular endothelial growth factor, all of whose transcriptional activities are regulated by STAT3. The inhibitory activity of AF on STAT3 phosphorylation was also demonstrated in primary cells, i.e. fibroblast‐like synoviocytes from rheumatoid arthritis patients, human umbilical vein endothelial cells and rat astrocytes. Auranofin‐mediated inhibition of STAT3 phosphorylation was recovered by pretreatment with antioxidants containing thiol groups. These findings suggest that the anti‐inflammatory action of AF is associated with a blockade of JAK1/STAT3 signalling. Thiol‐group‐reactive proteins may be involved in AF‐induced suppression of JAK1/STAT3 phosphorylation.

Induction of Neuronal Vascular Endothelial Growth Factor Expression by cAMP in the Dentate Gyrus of the Hippocampus Is Required for Antidepressant-Like Behaviors

The cAMP cascade and vascular endothelial growth factor (VEGF) are critical modulators of depression. Here we have tested whether the antidepressive effect of the cAMP cascade is mediated by VEGF in the adult hippocampus. We used a conditional genetic system in which the Aplysia octopamine receptor (Ap oa1), a Gs-coupled receptor, is transgenically expressed in the forebrain neurons of mice. Chronic activation of the heterologous Ap oa1 by its natural ligand evoked antidepressant-like behaviors, accompanied by enhanced phosphorylation of cAMP response element-binding protein and transcription of VEGF in hippocampal dentate gyrus (DG) neurons. Selective knockdown of VEGF in these cells during the period of cAMP elevation inhibited the antidepressant-like behaviors. These findings reveal a molecular interaction between the cAMP cascade and VEGF expression, and the pronounced behavioral consequences of this interaction shed light on the mechanism underlying neuronal VEGF functions in antidepression.

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.

Transient upregulation of osteopontin mRNA in hippocampus and striatum following global forebrain ischemia in rats

We investigated the spatial and temporal expression of osteopontin (OPN) mRNA following transient forebrain ischemia in rats. Experiments were carried out using a four-vessel occlusion model for forebrain ischemia. The transient induction of OPN mRNA after global ischemia occurred earlier in the striatum than in the hippocampus. It was pronounced in the dorsomedial striatum close to the lateral ventricle and in the CA1 subfield and the subiculum of the hippocampus before microglial cells became more reactive. It also could be detected in the dentate hilus and to a marginal extent in the CA3. Our results suggest that the hippocampus and the striatum following global forebrain ischemia upregulate OPN mRNA in different spatiotemporal profiles.