Sukumal Chongthammakun

Melatonin attenuates methamphetamine‐induced overexpression of pro‐inflammatory cytokines in microglial cell lines

Abstract:  Methamphetamine (METH), the most commonly abused drug, has long been known to induce neurotoxicity. METH causes oxidative stress and inflammation, as well as the overproduction of both reactive oxygen species (ROS) and reactive nitrogen species (RNS). The role of METH‐induced brain inflammation remains unclear. Imbroglio activation contributes to the neuronal damage that accompanies injury, disease and inflammation. METH may activate microglia to produce neuroinflammatory molecules. In highly aggressively proliferating immortalized (HAPI) cells, a rat microglial cell line, METH reduced cell viability in a concentration‐ and time‐dependent manner and initiated the expression of interleukin 1β (IL‐1β), interleukin 6 (IL‐6) and tumor necrosis factor α. METH also induced the production of both ROS and RNS in microglial cells. Pretreatment with melatonin, a major secretory product of the pineal gland, abolished METH‐induced toxicity, suppressed ROS and RNS formation and also had an inhibitory effect on cytotoxic factor gene expression. The expression of cytotoxic factors produced by microglia may contribute to central nervous system degeneration in amphetamine abusers. Melatonin attenuates METH toxicity and inhibits the expression of cytotoxic factor genes associated with ROS and RNS neutralization in HAPI microglia. Thus, melatonin might be one of the neuroprotective agents induced by METH toxicity and/or other immunogens.

Curcumin I protects the dopaminergic cell line SH-SY5Y from 6-hydroxydopamine-induced neurotoxicity through attenuation of p53-mediated apoptosis

Oxidative stress (OS) plays a pivotal role in the pathogenesis of Parkinsons disease (PD). 6-Hydroxydopamine (6-OHDA) is a neurotoxin used to induce oxidative cell death of dopaminergic neurons in experimental models of PD. Curcumin I, or diferuloylmethane is a pure compound isolated from Curcuma longa Linn. that has been reported to have neuroprotective properties. The precise mechanism, however, remains unclear. This study aims to elucidate the mechanisms by which curcumin I exerts its effects, using 6-OHDA-induced neurotoxicity in the human dopaminergic cell line SH-SY5Y. In our experiments, pretreatment with curcumin I improved cell viability, and significantly reduced reactive oxygen species (ROS). Further investigations revealed a reduction of p53 phosphorylation and decrease of the Bax/Bcl-2 ratio, as measured by mRNA expression and protein level. Taken together, these findings indicate that curcumin I protects dopaminergic neurons from 6-OHDA-induced toxicity via the reduction of ROS production, and subsequent attenuation of p53 phosphorylation and reduction of the Bax/Bcl-2 ratio.

Effect of Endogenous Androgens on 17β-Estradiol-Mediated Protection after Spinal Cord Injury in Male Rats

Several groups have recently shown that 17beta-estradiol is protective in spinal cord injury (SCI). Testosterone can be aromatized to 17beta-estradiol and may increase estrogen-mediated protection. Alternatively, testosterone has been shown to increase excitotoxicity in models of central nervous system (CNS) injury. These experiments test the hypothesis that endogenous testosterone in male rats alters 17beta-estradiol-mediated protection by evaluating a delayed administration over a clinically relevant dose range and manipulating testicular-derived testosterone. Adult male Sprague Dawley rats were either gonadectomized or left gonad-intact prior to SCI. SCI was produced by a midthoracic crush injury. At 30 min post SCI, animals received a subcutaneous pellet of 0.0, 0.05, 0.5, or 5.0 mg of 17beta-estradiol, released over 21 days. Hindlimb locomotion was analyzed weekly in the open field. Spinal cords were collected and analyzed for cell death, expression of Bcl-family proteins, and white-matter sparing. Post-SCI administration of the 0.5- or 5.0-mg pellet improved hindlimb locomotion, reduced urinary bladder size, increased neuronal survival, reduced apoptosis, improved the Bax/Bcl-xL protein ratio, and increased white-matter sparing. In the absence of endogenous testicular-derived androgens, SCI induced greater apoptosis, yet 17beta-estradiol administration reduced apoptosis to the same extent in gonadectomized and gonad-intact male rats. These data suggest that delayed post-SCI administration of a clinically relevant dose of 17beta-estradiol is protective in male rats, and endogenous androgens do not alter estrogen-mediated protection. These data suggest that 17beta-estradiol is an effective therapeutic intervention for reducing secondary damage after SCI in males, which could be readily translated to clinical trials.

Melatonin inhibits amphetamine-induced nitric oxide synthase mRNA overexpression in microglial cell lines

Amphetamine (AMPH) derivatives are the most commonly abused drugs. Chronic or intermittent AMPH abuse may create temporary or permanent disturbances in the dopaminergic system of the brain that may predispose individuals to Parkinsonism. AMPH induces a massive release of dopamine from synaptic vesicles and then generates reactive oxygen species (ROS). Furthermore, nitric oxide (NO), produced in the central nervous system (CNS) mediated by the activation of microglia, appears to play a critical role in stress-induced brain damage. In the present study, we examined the involvement of NO in the neurotoxic effects of AMPH, to investigate the hypothesis that altered nitric oxide synthase (NOS) function was involved. AMPH at a concentration of 0.4-3.2mM has a cytotoxic effect on highly aggressively proliferating immortalized (HAPI) cells, a rat microglial cell line. The effect of AMPH on increasing inducible NOS (iNOS) mRNA in HAPI microglial cells is concentration-dependent. Pretreatment with either S-methylisothiourea (S-MT), a selective iNOS inhibitor, or melatonin, a major secretory product of pineal gland, counteracted the over expression of iNOS induced by AMPH in a concentration-dependent manner. The induction of iNOS by AMPH in microglial cells could be an important source of NO in CNS inflammatory disorders associated with the death of neurons and oligodendrocytes. Administration of exogenous melatonin will be beneficial, as it reduces iNOS mRNA expression, and may, therefore, be able to be used as a neuroprotective agent in toxicity induced by AMPH or other immunogens.

Activation of group I metabotropic glutamate receptors leads to brain-derived neurotrophic factor expression in rat C6 cells

Brain-derived neurotrophic factor (BDNF), which mediates neuronal growth, neuroprotection and synaptic modulation, is expressed in neurons and glial cells. The present study investigated the expression of BDNF in response to the activation of group I metabotropic glutamate receptors (mGluRs) by (S)-3,5-Dihydroxyphenylglycine (DHPG) in rat C6 glioma cells. The increase in BDNF mRNA in DHPG-stimulated cells, which peaked by 12h after DHPG exposure, was attenuated by the mGluR5 inhibitor MPEP, but not by the mGluR1 inhibitor CPCCOEt. DHPG-induced BDNF mRNA expression reduced in cultures pretreated with protein kinase C (PKC) inhibitor, GFX, but not with calcium/calmodulin kinase II (CaMKII) inhibitor, KN-93. Immunostaining revealed high BDNF expression in cytoplasm of C6 cells after 48h of incubation with 1muM DHPG, but this was lower in MPEP-pretreated cells. These results indicate that activation of group I mGluRs induces BDNF mRNA and protein expression via mGluR5 subtype and PKC-dependent signaling pathway in C6 glioma cells.

A priming role of local estrogen on exogenous estrogen-mediated synaptic plasticity and neuroprotection

The localization of estrogen (E2) has been clearly shown in hippocampus, called local hippocampal E2. It enhanced neuronal synaptic plasticity and protected neuron form cerebral ischemia, similar to those effects of exogenous E2. However, the interactive function of hippocampal and exogenous E2 on synaptic plasticity activation and neuroprotection is still elusive. By using hippocampal H19-7 cells, we demonstrated the local hippocampal E2 that totally suppressed by aromatase inhibitor anastrozole. Anastrozole also suppressed estrogen receptor (ER)β, but not ERα, expression. Specific agonist of ERα (PPT) and ERβ (DPN) restored ERβ expression in anastrozole-treated cells. In combinatorial treatment with anastrozole and phosphoinositide kinase-3 (PI-3K) signaling inhibitor wortmannin, PPT could not improve hippocampal ERβ expression. On the other hand, DPN induced basal ERβ translocalization into nucleus of anastrozole-treated cells. Exogenous E2 increased synaptic plasticity markers expression in H19-7 cells. However, exogenous E2 could not enhance synaptic plasticity in anastrozole-treated group. Exogenous E2 also increased cell viability and B-cell lymphoma 2 (Bcl2) expression in H2O2-treated cells. In combined treatment of anastrozole and H2O2, exogenous E2 failed to enhance cell viability and Bcl2 expression in hippocampal H19-7 cells. Our results provided the evidence of the priming role of local hippocampal E2 on exogenous E2-enhanced synaptic plasticity and viability of hippocampal neurons.

Reactive oxygen species production and MAPK activation are implicated in tetrahydrobiopterin-induced SH-SY5Y cell death

Tetrahydrobiopterin (BH4), an obligatory cofactor for dopamine (DA) synthesis, has been shown to produce reactive oxygen species (ROS) upon its autoxidation and induce selective dopaminergic cell death in many in vivo and in vitro models of Parkinsons disease (PD). The precise molecular mechanisms underlying neuronal death upon BH4 exposure, however, have not yet been well elucidated. The present study aims to examine the intracellular ROS production and the signal transduction pathways underlying the toxic effects of BH4 on human dopaminergic SH-SY5Y cells. The results show that BH4 treatment at concentrations ranging from 50microM to 400microM induces neuronal death in a dose-dependent manner. In concomitant with the elevation of intracellular ROS formation, BH4-induced activation of MAPK, p38 and ERK1/2 in SH-SY5Y cells is attenuated by pretreatment with MAPK inhibitors, SB203580 or PD98059. These data indicate that MAPK activation and oxidative stress are involved in BH4-induced dopaminergic cell death, possibly through the autoxidation of BH4 and subsequent ROS production.

Transcriptional regulation of iNOS and COX-2 by a novel compound from Curcuma comosa in lipopolysaccharide-induced microglial activation

Overproduction of pro-inflammatory mediators resulting from chronic activation of microglia has been implicated in many neurodegenerative disorders, such as Parkinsons disease and Alzheimers disease. In this study, we investigated the effects of (3R) 1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol, or compound 049 on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-treated microglia. Compound 049 is a pure compound fractionated from the hexane extract of Curcuma comosa, an indigenous plant of Thailand traditionally used as an anti-inflammatory agent for the treatment of uterine inflammation. It was found that pretreatment of the highly aggressively proliferating immortalized (HAPI), rat microglial cell line, with compound 049, at the concentrations of 0.1, 0.5 and 1microM significantly decreased LPS-induced NO and PGE(2) production in a concentration-dependent manner. Parallel to the decreases in NO and PGE(2) production was a reduction in the expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2) as measured by mRNA and protein levels. These results indicate that compound 049 possesses an anti-inflammatory activity and may have a therapeutic potential for the treatment of neurodegenerative diseases related to microglial activation.

Platelet-activating factor enhancement of calcium influx and interleukin-6 expression, but not production, in human microglia

Calcium-sensitive fluorescence microscopy and molecular biology analysis have been used to study the effects of platelet-activating factor (PAF) on intracellular calcium [Ca2+]i and IL-6 expression in human microglia. PAF (applied acutely at 100 nM) elicited a biphasic response in [Ca2+]i consisting of an initial rapid increase of [Ca2+]i due to release from internal stores, followed by a sustained influx. The latter phase of the [Ca2+]i increase was blocked by SKF96365, a non-selective store-operated channel (SOC) inhibitor. RT-PCR analysis showed PAF treatment of microglia induced expression of the pro-inflammatory cytokine IL-6 in a time-dependent manner which was blocked in the presence of SKF96365. However, ELISA assay showed no production of IL-6 was elicited at any time point (1–24 h) for microglial exposures to PAF. These findings suggest that PAF stimulation of human microglia induces expression, but not production, of IL-6 and that SOC-mediated [Ca2+]i influx contributes to the enhanced expression of the cytokine.

Estrogen stimulates activity-regulated cytoskeleton associated protein (Arc) expression via the MAPK-and PI-3K-dependent pathways in SH-SY5Y cells

Activity-regulated cytoskeleton associated protein (Arc) is known to be induced by synaptic plasticity following memory consolidation. Since estrogen has been shown to play an important role in synaptogenesis, a key aspect of the synaptic plasticity, we aimed to study the effects of estrogen on Arc expression in SH-SY5Y human neuroblastoma cells. Using quantitative real-time PCR, Western blot, and confocal immunocytochemistry techniques we found that estrogen markedly increased Arc mRNA and protein expression in SH-SY5Y cells. Estrogen-activated Arc expression was mediated via mitogen-activated protein kinase (MAPK) and phosphoinositide-3 kinase (PI-3K), but not protein kinase C (PKC) and Rho-associated kinase (ROCK), and in the estrogen receptor (ER)-dependent manner. Estrogen also significantly upregulated the dendritic spine scaffolding protein, postsynaptic density-95 (PSD-95), as well as expression of the presynaptic vesicle protein, synaptophysin. Our findings demonstrate the possible mechanisms of estrogen-induced synaptic plasticity, as well as memory consolidation.