5-HT1A receptor agonist 8-OH-DPAT induces serotonergic behaviors in mice via interaction between PKCδ and p47phox.

Serotonin syndrome is an adverse reaction due to increased serotonin (5-hydroxytryptophan: 5-HT) concentrations in the central nervous system (CNS). The full 5-HT1A receptor (5-HT1AR) agonist (±)-8-hydroxy-dipropylaminotetralin (8-OH-DPAT) has been recognized to elicit traditional serotonergic behaviors. Treatment with 8-OH-DPAT selectively increased PKCδ expression out of PKC isoforms and 5-HT turnover rate in the hypothalamus of wild-type mice. Treatment with 8-OH-DPAT resulted in oxidative burdens, co-immunoprecipitation of 5-HT1AR and PKCδ, and phosphorylation and membrane translocation of p47phox. Importantly, p47phox also interacted with 5-HT1AR or PKCδ in the presence of 8-OH-DPAT. Consistently, the interaction and oxidative burdens were attenuated by 5-HT1AR antagonism (i.e., WAY100635), PKCδ inhibition (i.e., rottlerin and genetic depletion of PKCδ), or NADPH oxidase/p47phox inhibition (i.e., apocynin and genetic depletion of p47phox). However, WAY100635, apocynin, or rottlerin did not exhibit any additive effects against the protective effect by inhibition of PKCδ or p47phox. Furthermore, apocynin, rottlerin, or WAY100635 also significantly protected from pro-inflammatory/pro-apoptotic changes induced by 8-OH-DPAT. Therefore, we suggest that 8-OH-DPAT-induced serotonergic behaviors requires oxidative stress, pro-inflammatory, and pro-apoptotic changes, that PKCδ or p47phox mediates the serotonergic behaviors induced by 8-OH-DPAT, and that the inhibition of PKCδ-dependent p47phox activation is critical for protecting against serotonergic behaviors.

PKCδ Knockout Mice Are Protected from Dextromethorphan-Induced Serotonergic Behaviors in Mice: Involvements of Downregulation of 5-HT1A Receptor and Upregulation of Nrf2-Dependent GSH Synthesis.

We investigated whether a specific serotonin (5-HT) receptor-mediated mechanism was involved in dextromethorphan (DM)-induced serotonergic behaviors. We firstly observed that the activation of 5-HT1A receptor, but not 5-HT2A receptor, contributed to DM-induced serotonergic behaviors in mice. We aimed to determine whether the upregulation of 5-HT1A receptor induced by DM facilitates the specific induction of certain PKC isoform, because previous reports suggested that 5-HT1A receptor activates protein kinase C (PKC). A high dose of DM (80 mg/kg, i.p.) induced a selective induction of PKCδ out of PKCα, PKCßI, PKCßII, PKCξ, and PKCδ in the hypothalamus of wild-type (WT) mice. More importantly, 5-HT1A receptor co-immunoprecipitated PKCδ in the presence of DM. Consistently, rottlerin, a pharmacological inhibitor of PKCδ, or PKCδ knockout significantly protected against increases in 5-HT1A receptor gene expression, 5-HT turnover rate, and serotonergic behaviors induced by DM. Treatment with DM resulted in an initial increase in nuclear factor erythroid-2-related factor 2 (Nrf2) nuclear translocation and DNA-binding activity, γ-glutamylcysteine (GCL) mRNA expression, and glutathione (GSH) level. This compensative induction was further potentiated by rottlerin or PKCδ knockout. However, GCL mRNA and GSH/GSSG levels were decreased 6 and 12 h post-DM. These decreases were attenuated by PKCδ inhibition. Our results suggest that interaction between 5-HT1A receptor and PKCδ is critical for inducing DM-induced serotonergic behaviors and that inhibition of PKCδ attenuates the serotonergic behaviors via downregulation of 5-HT1A receptor and upregulation of Nrf2-dependent GSH synthesis.

Ginsenoside Re Protects Trimethyltin-Induced Neurotoxicity via Activation of IL-6-Mediated Phosphoinositol 3-Kinase/Akt Signaling in Mice.

Ginseng (Panax ginseng), an herbal medicine, has been used to prevent neurodegenerative disorders. Ginsenosides (e.g., Re, Rb1, or Rg1) were obtained from Korean mountain cultivated ginseng. The anticonvulsant activity of ginsenoside Re (20 mg/kg/day × 3) against trimethyltin (TMT) insult was the most pronounced out of ginsenosides (e.g., Re, Rb1, and Rg1). Re itself did not significantly alter tumor necrosis factor-α (TNF-α), interferon-ϒ (IFN-ϒ), and interleukin-1ß (IL-1ß) expression, however, it significantly increases the interleukin-6 (IL-6) expression. In addition, Re attenuated the TMT-induced decreases in IL-6 protein level. Therefore, IL-6 knockout (-/-) mice were employed to investigate whether Re requires IL-6-dependent neuroprotective activity against TMT toxicity. Re significantly attenuated TMT-induced lipid peroxidation, protein peroxidation, and reactive oxygen species in the hippocampus. Re-mediated antioxidant effects were more pronounced in IL-6 (-/-) mice than in WT mice. Consistently, TMT-induced increase in c-Fos-immunoreactivity (c-Fos-IR), TUNEL-positive cells, and nuclear chromatin clumping in the dentate gyrus of the hippocampus were significantly attenuated by Re. Furthermore, Re attenuated TMT-induced proapoptotic changes. Protective potentials by Re were comparable to those by recombinant IL-6 protein (rIL-6) against TMT-insult in IL-6 (-/-) mice. Moreover, treatment with a phosphoinositol 3-kinase (PI3K) inhibitor, LY294002 (1.6 µg, i.c.v) counteracted the protective potential mediated by Re or rIL-6 against TMT insult. The results suggest that ginsenoside Re requires IL-6-dependent PI3K/Akt signaling for its protective potential against TMT-induced neurotoxicity.

Treatment with Mountain-Cultivated Ginseng Alleviates Trimethyltin-Induced Cognitive Impairments in Mice via IL-6-Dependent JAK2/STAT3/ERK Signaling.

Panax ginseng is the most widely used herbal medicine for improving cognitive functions. The pharmacological activity and underlying mechanisms of mountain-cultivated ginseng, however, have yet to be clearly elucidated, in particular, against trimethyltin-induced cognitive dysfunction. We previously reported that interleukin-6 plays a protective role against trimethyltin-induced cognitive dysfunction. Because of this, we have implemented a study system that uses interleukin-6 null (-/-) and wild-type mice. Interestingly, mountain-cultivated ginseng significantly upregulated interleukin-6 expression. With this study, we sought to determine whether the interleukin-6-dependent modulation of the Janus kinase 2/signal transducer activator of transcription 3 and extracellular signal-regulated kinase signaling network is also associated with the pharmacological activity of mountain-cultivated ginseng against trimethyltin-induced cognitive dysfunction. Trimethyltin treatment (2.4 mg/kg, intraperitoneal) causes the downregulation of Janus kinase 2/signal transducer activator of transcription 3, extracellular signal-regulated kinase signaling, and impairment of the cholinergic system. We found that mountain-cultivated ginseng treatment (50 mg/kg, intraperitoneal) significantly attenuated cognitive impairment normally induced by trimethyltin by upregulating p-Janus kinase 2/signal transducer activator of transcription 3, p-extracellular signal-regulated kinase signaling, and the cholinergic system. Trimethyltin-induced cognitive impairments were more pronounced in interleukin-6 (-/-) mice than wild-type mice, and they were markedly reduced by treatment with either mountain-cultivated ginseng or recombinant interleukin-6 protein (6 ng, intracerebroventricular). Additionally, treatment with either AG490 (20 mg/kg, intraperitoneal), a Janus kinase 2/signal transducer activator of transcription 3 inhibitor, or U0126 (2 µg/head, intracerebroventricular), an extracellular signal-regulated kinase inhibitor, reversed the effects of mountain-cultivated ginseng treatment. The effects of mountain-cultivated ginseng treatment were comparable to those of recombinant interleukin-6 protein in interleukin-6 (-/-) mice. Our results, therefore, suggest that mountain-cultivated ginseng acts through interleukin-6-dependent activation of Janus kinase 2/signal transducer activator of transcription 3/extracellular signal-regulated kinase signaling in order to reverse cognitive impairment caused by trimethyltin treatment.