Jun Kim

DJ-1 facilitates the interaction between STAT1 and its phosphatase, SHP-1, in brain microglia and astrocytes: A novel anti-inflammatory function of DJ-1.

Parkinsons disease (PD) is a progressive neurodegenerative movement disorder caused by the death of dopaminergic neurons in the substantia nigra. Importantly, altered astrocyte and microglial functions could contribute to neuronal death in PD. In this study, we demonstrate a novel mechanism by which DJ-1 (PARK7), an early onset autosomal-recessive PD gene, negatively regulates inflammatory responses of astrocytes and microglia by facilitating the interaction between STAT1 and its phosphatase, SHP-1 (Src-homology 2-domain containing protein tyrosine phosphatase-1). Astrocytes and microglia cultured from DJ-1-knockout (KO) mice exhibited increased expression of inflammatory mediators and phosphorylation levels of STAT1 (p-STAT1) in response to interferon-gamma (IFN-γ) compared to cells from wild-type (WT) mice. DJ-1 deficiency also attenuated IFN-γ-induced interactions of SHP-1 with p-STAT1 and STAT1, measured 1 and 12h after IFN-γ treatment, respectively. Subsequent experiments showed that DJ-1 directly interacts with SHP-1, p-STAT1, and STAT1. Notably, DJ-1 bound to SHP-1 independently of IFN-γ, whereas the interactions of DJ-1 with p-STAT1 and STAT1 were dependent on IFN-γ. Similar results were obtained in brain slice cultures, where IFN-γ induced much stronger STAT1 phosphorylation and inflammatory responses in KO slices than in WT slices. Moreover, IFN-γ treatment induced neuronal damage in KO slices. Collectively, these findings suggest that DJ-1 may function as a scaffold protein that facilitates SHP-1 interactions with p-STAT1 and STAT1, thereby preventing extensive and prolonged STAT1 activation. Thus, the loss of DJ-1 function may increase the risk of PD by enhancing brain inflammation.

Lobule-specific membrane excitability of cerebellar Purkinje cells

Non‐technical summaryu2002 Cerebellar vermis consists of 10 lobules, and each lobule receives different sensory information. Afferent inputs are integrated in cerebellar Purkinje cells (PCs) which are the sole output of the cerebellar cortex. We show that intrinsic membrane properties are widely different between PCs in the spinocerebellum (lobules III–V) and vestibulocerebellum (lobule X).

A Role of Canonical Transient Receptor Potential 5 Channel in Neuronal Differentiation from A2B5 Neural Progenitor Cells

Store-operated Ca2+ entry (SOCE) channels are the main pathway of Ca2+ entry in non-excitable cells such as neural progenitor cells (NPCs). However, the role of SOCE channels has not been defined in the neuronal differentiation from NPCs. Here, we show that canonical transient receptor potential channel (TRPC) as SOCE channel influences the induction of the neuronal differentiation of A2B5+ NPCs isolated from postnatal-12-day rat cerebrums. The amplitudes of SOCE were significantly higher in neural cells differentiated from proliferating A2B5+ NPCs and applications of SOCE blockers, 2-aminoethoxy-diphenylborane (2-APB), and ruthenium red (RR), inhibited their rise of SOCE. Among TRPC subtypes (TRPC1-7), marked expression of TRPC5 and TRPC6 with turned-off TRPC1 expression was observed in neuronal cells differentiated from proliferating A2B5+ NPCs. TRPC5 small interfering RNA (siRNA) blocked the neuronal differentiation from A2B5+ NPCs and reduced the rise of SOCE. In contrast, TRPC6 siRNA had no significant effect on the neuronal differentiation from A2B5+ NPCs. These results indicate that calcium regulation by TRPC5 would play a key role as a switch between proliferation and neuronal differentiation from NPCs.

Amyotrophic lateral sclerosis : Serum factors enhance spontaneous and evoked transmitter release at the neuromuscular junction

Sera from 30 patients with sporadic amyotrophic lateral sclerosis (ALS) were tested to determine their effects at the neuromuscular junction. Spontaneous transmitter release was significantly increased, as evidenced by a 151% increase in MEPP frequency, by sera from 16 ALS patients. In addition, 16 patients sera elevated EEP quantal content by an average of 89%. Eleven sera produced both effects. There was no consistent change in MEPP amplitude or time course, indicating the absence of a humoral effect on postjunctional ACh receptors or endplate membrane function. These results suggest that a portion of the sporadic ALS patient population possess serum factors that can alter presynaptic function of the motor nerve terminal. Evidence from the present experiments indicates that alterations at the neuromuscular junction are a result of a combination of increased Ca2+ influx into the cell and an independent increase in intracellular calcium concentration.

Thimerosal decreases TRPV1 activity by oxidation of extracellular sulfhydryl residues

TRPV1, a receptor for capsaicin, plays a key role in mediating thermal and inflammatory pain. Because the modulation of ion channels by the cellular redox state is a significant determinant of channel function, we investigated the effects of sulfhydryl modification on the activity of TRPV1. Thimerosal, which oxidizes sulfhydryls, blocked the capsaicin-activated inward current (I(cap)) in cultured sensory neurons, in a reversible and dose-dependent manner, which was prevented by the co-application of the reducing agent, dithiothreitol. Among the three cysteine residues of TRPV1 that are exposed to the extracellular space, the oxidation-induced effect of thimerosal on I(cap) was blocked only by a point mutation at Cys621. These results suggest that the modification of an extracellular thiol group can alter the activity of TRPV1. Consequently, we propose that such a modulation of the redox state might regulate the physiological activity of TRPV1.

Activation of the cGMP/Protein Kinase G Pathway by Nitric Oxide Can Decrease TRPV1 Activity in Cultured Rat Dorsal Root Ganglion Neurons

Recent studies have demonstrated that nitric oxide (NO) activates transient receptor potential vanilloid subtype 1 (TRPV1) via S-nitrosylation of the channel protein. NO also modulates various cellular functions via activation of the soluble guanylyl cyclase (sGC)/protein kinase G (PKG) pathway and the direct modification of proteins. Thus, in the present study, we investigated whether NO could indirectly modulate the activity of TRPV1 via a cGMP/PKG-dependent pathway in cultured rat dorsal root ganglion (DRG) neurons. NO donors, sodium nitroprusside (SNP) and S-nitro-N-acetylpenicillamine (SNAP), decreased capsaicin-evoked currents (Icap). NO scavengers, hemoglobin and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO), prevented the inhibitory effect of SNP on Icap. Membrane-permeable cGMP analogs, 8-bromoguanosine 3, 5-cyclic monophosphate (8bromo-cGMP) and 8-(4chlorophenylthio)-guanosine 3,5-cyclic monophosphate (8-pCPT-cGMP), and the guanylyl cyclase stimulator YC-1 mimicked the effect of SNP on Icap. The PKG inhibitor KT5823 prevented the inhibition of Icap by SNP. These results suggest that NO can downregulate the function of TRPV1 through activation of the cGMP/PKG pathway in peripheral sensory neurons.

Endothelial progenitor cells functionally express inward rectifier potassium channels

Since the first isolation of endothelial progenitor cells (EPCs) from human peripheral blood in 1997, many researchers have conducted studies to understand the characteristics and therapeutic effects of EPCs in vascular disease models. Nevertheless, the electrophysiological properties of EPCs have yet to be clearly elucidated. The inward rectifier potassium channel (Kir) performs a major role in controlling the membrane potential and cellular events. Here, via the whole cell patch-clamp technique, we found inwardly rectifying currents in EPCs and that these currents were inhibited by Ba(2+) (100 μM) and Cs(+) (1 mM), known as Kir blockers, in a dose-dependent manner (Ba(2+), 91.2 ± 1.4% at -140 mV and Cs(+), 76.1 ± 6.9% at -140 mV, respectively). Next, using DiBAC(3), a fluorescence indicator of membrane potential, we verified that Ba(2+) induced an increase of fluorescence in EPCs (10 μM, 123 ± 2.8%), implying the depolarization of EPCs. At the mRNA and protein levels, we confirmed the existence of several Kir subtypes, including Kir2.x, 3.x, 4.x, and 6.x. In a functional experiment, we observed that, in the presence of Ba(2+), the number of tubes on Matrigel formed by EPCs was dose-dependently reduced (10 μM, 62.3 ± 6.5%). In addition, the proliferation of EPCs was increased in a dose-dependent fashion (10 μM, 157.9 ± 17.4%), and specific inhibition of Kir2.1 by small interfering RNA also increased the proliferation of EPCs (116.2 ± 2.5%). Our results demonstrate that EPCs express several types of Kir which may modulate the endothelial function and proliferation of EPCs.

Transient Upregulation of Postsynaptic IP3-Gated Ca Release Underlies Short-Term Potentiation of Metabotropic Glutamate Receptor 1 Signaling in Cerebellar Purkinje Cells

Synaptic plasticity lasting ∼100 s has been suggested to function as a temporary buffer for neural information. One example of this was reported by Batchelor and Garthwaite (1997), who found that a slow metabotropic glutamate receptor 1 (mGluR1)-evoked EPSP produced by burst stimulation of cerebellar parallel fiber–Purkinje cell synapses could be potentiated by a conditioning stimulus consisting of prior activation of climbing fiber synapses (or injection of depolarizing current) with a delay of up to 90 s. What is the molecular basis of the signal that spans this temporal gap? Here, we show that mGluR1-evoked slow EPSCs evoked by parallel fiber burst test stimuli show a similar form of short-term potentiation (mGluR1-STP) and that this phenomenon is also observed when parallel fiber bursts are replaced by pressure pulses of an exogenous mGluR1 agonist. Ca imaging experiments revealed that cytosolic Ca levels returned to baseline within several seconds after conditioning depolarization, indicating that this cannot underlie mGluR1-STP. To test the hypothesis that transient upregulation of inositol-1,4,5-trisphosphate (IP3)-gated Ca release underlies this phenomenon, we used local photolytic uncaging of IP3 to deplete IP3-gated Ca stores. IP3 uncaging in the interval between conditioning depolarization and the test pulse produced a complete blockade of mGluR1-STP, as did blockade of IP3 receptors with heparin. When Ca transients evoked by IP3 uncaging were used as a test stimulus, conditioning depolarization produced a large STP of Ca response amplitudes. These data suggest that transient upregulation of postsynaptic IP3-gated Ca signaling constitutes a novel form of short-term synaptic plasticity.

GABAB receptor-mediated presynaptic inhibition of glycinergic transmission onto substantia gelatinosa neurons in the rat spinal cord

&NA; The GABAB receptor‐mediated presynaptic inhibition of glycinergic transmission was studied from young rat substantia gelatinosa (SG) neurons using a conventional whole‐cell patch clamp technique. Action potential‐dependent glycinergic inhibitory postsynaptic currents (IPSCs) were recorded from SG neurons in the presence of 3 mM kynurenic acid and 10 μM SR95531. In these conditions, baclofen (30 μM), a selective GABAB receptor agonist, greatly reduced the amplitude of glycinergic IPSCs and increased the paired‐pulse ratio. Such effects were completely blocked by 3 μM CGP55845, a selective GABAB receptor antagonist, indicating that the activation of presynaptic GABAB receptors decreases glycinergic synaptic transmission. Glycinergic IPSCs were largely dependent on Ca2+ influxes passing through presynaptic N‐ and P/Q‐type Ca2+ channels, and these channels contributed equally to the baclofen‐induced inhibition of glycinergic IPSCs. However, the baclofen‐induced inhibition of glycinergic IPSCs was not affected by either 100 μM SQ22536, an adenylyl cyclase inhibitor, or 1 mM Ba2+, a G‐protein coupled inwardly rectifying K+ channel blocker. During the train stimulation (10 pulses at 20 Hz), which caused a marked synaptic depression of glycinergic IPSCs, baclofen at a 30 μM concentration completely blocked glycinergic synaptic depression, but at a 3 μM concentration it largely preserved glycinergic synaptic depression. Such GABAB receptor‐mediated dynamic changes in short‐term synaptic plasticity of glycinergic transmission onto SG neurons might contribute to the central processing of sensory signals.

Modulation of synaptic transmission from primary afferents to spinal substantia gelatinosa neurons by group III mGluRs in GAD65-EGFP transgenic mice.

Group III metabotropic glutamate receptors (mGluRs) are involved in nociceptive transmission in the spinal cord. However, the cellular mechanism underlying the modulation of synaptic transmission from nociceptive primary afferents to dorsal horn neurons by group III mGluRs has yet to be explored. In this study, we used transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of the glutamate decarboxylase (GAD) 65 promoter to identify specific subpopulations of GABAergic inhibitory interneurons. By GABA immunolabeling, we confirmed the majority of GAD65-EGFP-expressing neurons were GABAergic. Because GAD65-EGFP-expressing neurons have not been examined in detail before, we first investigated the physiological properties of GAD65-EGFP- and non-EGFP-expressing neurons in substantia gelatinosa (SG) of the spinal dorsal horn. Membrane properties, such as the resting membrane potential, membrane capacitance, action potential threshold, and action potential height, differed significantly between these two groups of neurons. Most EGFP-expressing neurons displayed a tonic firing pattern (73% of recorded neurons) and received monosynaptic Aδ and/or C primary afferent inputs (85% of recorded neurons). In contrast, we observed a delayed firing pattern in 53% of non-EGFP-expressing neurons. After identifying the physiological properties of EGFP-expressing neurons, we tested the effects of group III mGluRs on synaptic transmission pharmacologically. A group III mGluR agonist, L-AP4, attenuated Aδ fiber-evoked synaptic transmission but did not affect C fiber-evoked synaptic transmission to EGFP-expressing neurons. Similar primary afferent-specific inhibition by L-AP4 was also observed in non-EGFP-expressing neurons. Moreover, Aδ fiber-evoked synaptic transmission was suppressed by a selective mGluR7 agonist, AMN082. These results suggest that modulation of the synaptic transmission from primary afferents to SG neurons by group III mGluR agonist is specific to the type of nociceptive primary afferents but not to the type of target neurons.