Objective: To define how the catabolic cytokines (Interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF alpha)) affect the circadian clock mechanism and the expression of clock-controlled catabolic genes within cartilage, and to identify the downstream pathways linking the cytokines to the molecular clock within chondrocytes.Methods: Ex vivo cartilage explants were isolated from the Cry1-luc or PER2::LUC clock reporter mice. Clock gene dynamics were monitored in real-time by bioluminescence photon counting. Gene expression changes were studied by qRT-PCR. Functional luc assays were used to study the function of the core Clock/BMAL1 complex in SW-1353 cells. NF kappa B pathway inhibitor and fluorescence live-imaging of cartilage were performed to study the underlying mechanisms.Results: Exposure to IL-1 beta severely disrupted circadian gene expression rhythms in cartilage. This effect was reversed by an anti-inflammatory drug dexamethasone, but not by other clock synchronizing agents. Circadian disruption mediated by IL-1 beta was accompanied by disregulated expression of endogenous clock genes and clock-controlled catabolic pathways. Mechanistically, NF kappa B signalling was involved in the effect of IL-1 beta on the cartilage clock in part through functional interference with the core Clock/BMAL1 complex. In contrast, TNF alpha had little impact on the circadian rhythm and clock gene expression in cartilage.Conclusion: In our experimental system (young healthy mouse cartilage), we demonstrate that IL-1 beta (but not TNF alpha) abolishes circadian rhythms in Cry1-luc and PER2::LUC gene expression. These data implicate disruption of the chondrocyte clock as a novel aspect of the catabolic responses of cartilage to proinflammatory cytokines, and provide an additional mechanism for how chronic joint inflammation may contribute to osteoarthritis (OA). (C) 2015 The Authors. Published by Elsevier Ltd and Osteoarthritis Research Society International.

Stimulus generalization occurs when stimuli with characteristics similar to a previously conditioned stimulus (CS) become able to evoke a previously conditioned response. Experimental data (Lissek et al., 2005) indicate that patients with post-traumatic stress disorder (PTSD), more often show stimulus generalization following fear conditioning when tested under laboratory conditions. Factors surrounding this observation may contribute to two common features of PTSD: 1) hyper-responsiveness to sensory stimuli reminiscent of those associated with the original trauma, and 2) resistance of PTSD to extinction-based therapies. Adverse early experience is considered a risk factor for the later development of PTSD and in the present experiments we hypothesized that stimulus generalization would occur in an animal model of adverse early experience, the prenatally stressed (PS) rat. Adult PS and control (CON) rats underwent extensive pre-habituation to a conditioning chamber followed by conventional auditory fear conditioning. The next day both groups began an extinction regimen where a series of quieter (attenuated), CSs were administered prior to the full 75 dB training CS. When tested in this manner, PS rats froze at significantly lower tone amplitudes than did CON offspring on the first day of extinction training. This suggests that the PS rats had stimulus-generalized the CS to lower decibel tones. In addition to this finding, we also observed that PS rats froze more often and longer during three ensuing days of extinction training to attenuated tones. Group differences vanished when PS and CON rats were extinguished under conventional conditions. Thus, it appears that the two extinction regimens differed in their aversive cue saliency for the PS vs. CON rats. Follow-up prefrontal cortex transcriptome probing suggests that cholinergic and dopaminergic alterations may be involved.

Astrocytic Toll-like receptor 3 (TLR3) plays an important role not only in antiviral response but also in regeneration/healing of the CNS. The present study was undertaken to determine whether the neuroprotective effects of TLR3 signaling also include antioxidative protection. TLR3 ligation in human astrocytes induced protracted resistance of the cells to H2O2 toxicity. Similar resistance was induced by conditioned medium from TLR3-ligated astrocytes indicating the involvement of paracrine signaling mechanisms. Out of 13 major antioxidative genes only the gene encoding superoxide dismutase 2 (SOD2) was postligationally upregulated suggesting that SOD2 is the major enzyme responsible for this protection.

Abstract Flavin-dependent histone demethylases play an important role in epigenetic gene regulation. The most studied enzyme, LSD1 (KDM1), catalyzes the oxidative demethylation of methylated Lys4 of histone H3. Known LSD1 inhibitors also inhibit other flavin-dependent enzymes, in particular monoamine oxidase (MAO). Therefore, selective inhibition of LSD1 is a significant drug discovery target. We designed, synthesized and evaluated in vitro a series of eleven N-substituted tetrahydropyridines as inhibitors of flavin-dependent enzymes. Complete chemical structures of the compounds used will be disclosed at the time of presentation. The inhibition of LSD1 was measured in rat glioma C6 cells. Briefly, cells grown in serum-free medium were treated with the inhibitors for 24 h, and the level of Lys4 dimethylated form of histone H3 was measured by immunoblot. MAO A activity was determined using human recombinant enzyme with kynuramine as substrate. The most selective among the inhibitors showed low nanomolar potency toward LSD1 and weak inhibition of MAO A. The observed high potency and substrate selectivity justifies further evaluation of the drug discovery potential of the tetrahydropyridine scaffold. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3255. doi:10.1158/1538-7445.AM2011-3255

Previously, we have shown that peripheral challenge of mice with double stranded RNA (dsRNA), a viral mimic, evokes global upregulation of cerebral inflammatory genes and, particularly, genes encoding chemokines. Because chemokine networks are potent modulators of brain function, the present study was undertaken to comprehensively characterize the cerebral response of chemokine ligand and receptor genes to peripheral immune system stimulation. Briefly, C57BL/6 mice were intraperitoneally injected with 12 mg/kg of polyinosinic-polycytidylic acid (PIC) and the expression of 39 mouse chemokine ligand and 20 receptor genes was monitored in the cerebellum by real time quantitative RT-PCR within 24 h. Almost half of the ligand genes featured either transient or sustained upregulation from several- to several thousand-fold. Five CXC type genes, i.e., Cxcl9, Cxcl11, Cxcl10, Cxcl2 and Cxcl1, were the most robustly upregulated, and were followed by six CC type genes, i.e., Ccl2, Ccl7, Ccl5, Ccl12, Ccl4 and Ccl11. Seven genes showed moderate upregulation, whereas the remaining genes were unresponsive. Six receptor genes, i.e., Cxcr2, Ccr7, Cxcr5, Ccr6, Ccr1 and Ccr5, featured a several-fold upregulation. Similar chemokine gene response was observed in the forebrain and brainstem. This upregulation of chemokine genes could be induced in naïve mice by transfer of blood plasma from PIC-challenged mice. Employing oligodeoxynucleotide-labeled PIC we further showed that intraperitoneally injected PIC was not transferred to the blood. In conclusion, peripheral PIC challenge elicits a broad upregulation of cerebral chemokine genes, and this upregulation is mediated by blood-borne agents.

Clinical evidence implicates peripheral inflammatory diseases as comorbid factors in epilepsy. The present study was designed to determine the effect of the acute phase of antiviral response on seizure susceptibility. Young adult mice were intraperitoneally injected with 12 mg/kg of a viral mimic, polyinosinic:polycytidylic acid (PIC). After 48 h, seizures were induced by subcutaneous injection of kainic acid (KA). PIC-pretreatment profoundly enhances vulnerability to excitotoxic insult as evidenced by increased seizure intensity and extended duration of status epilepticus. These results support the notion that peripheral viral infections may alter brain function resulting in enhanced predilection to seizures.

It is well established that mediators of peripheral inflammation are relayed to the brain and elicit sickness behavior via neuroinflammatory agents that target neuronal substrates. In the present study, we used double‐stranded RNA (dsRNA), a viral replication intermediate, to mimic the acute phase of viral infection. C57BL/6 mice were injected intraperitoneally with 12 mg/kg of synthetic dsRNA, i.e., polyinosinic‐polycytidylic acid (PIC). The treatment induced severe sickness behavior in the animals as revealed by the burrowing test performed 6 hr postinjection. PIC challenge also induced up‐regulation of mRNA for several cytokines in the brain as determined by real‐time quantitative RT‐PCR. In all brain regions, i.e., the forebrain, brainstem, and cerebellum, the gene encoding the CXCL2 chemokine featured the most robust up‐regulation over the basal level (saline‐injected animals), followed by the genes encoding the CCL2 chemokine, interferon‐β (IFNβ), interleukin‐6 (IL‐6), tumor necrosis factor‐α (TNFα), and interleukin‐1β (IL‐1β). The forebrain featured the highest extent of up‐regulation of the Ifnb gene, whereas the other genes attained the highest expression in the cerebellum. Most of the genes featured transient up‐regulation, with peaks occurring 3–6 hr after PIC challenge. The TNFα, CCL2, CXCL2, IFNβ, and IL‐1β messages remained profoundly up‐regulated even at 24 hr. The expression of genes encoding inducible and neuronal nitric oxide synthase (NOS) in the brain was not affected by the peripheral PIC challenge. However, the endothelial NOS message was initially down‐regulated and subsequently up‐regulated, indicating stimulation of cerebral vasculature. © 2008 Wiley‐Liss, Inc.

Cyclic AMP is part of an endogenous mechanism that downregulates inflammatory response, and its intracellular concentration is regulated chiefly by cyclic nucleotide phosphodiesterases type 4. The goal of the present study was to determine whether phosphodiesterases 4 are involved in the inflammatory response of astrocytes mediated by Toll-like receptors. Astrocyte cultures established from newborn rat brain were challenged with lipoteichoic acid, a ligand of Toll-like receptor 2, polyinosinic-polycytidylic acid, a ligand of Toll-like receptor 3, or lipopolysaccharide, a ligand of Toll-like receptor 4. After 24 h the expression of genes encoding phosphodiesterase 4A, phosphodiesterase 4B and phosphodiesterase 4D was determined by real time reverse transcription polymerase chain reaction. The challenge of astrocytes with the ligands profoundly up-regulated expression of the phosphodiesterase 4B mRNA, while the phosphodiesterase 4A and 4D mRNA was either unaffected or downregulated. Moreover, Toll-like receptor ligation specifically up-regulated expression of the phosphodiesterase 4B2 transcriptional variant. Thus, polyinosinic-polycytidylic acid, lipopolysaccharide and lipoteichoic acid induced approximately 7-, 5- and 4-fold up-regulation of the message, respectively. Toll-like receptor ligation also led to an over 2-fold increase in the protein level of phosphodiesterase 4B2 as revealed by immunoblot analysis. The inactivation of Rho proteins by pretreatment with toxin B form C. difficile enhanced ligation-induced up-regulation of the phosphodiesterase 4B2 message by 4-9-fold. However, in spite of this increase in the message abundance, there was no increase in the protein level compared to cells challenged with the ligands alone. These results demonstrate that the phosphodiesterase 4B2 gene is an effector of Toll-like receptor signaling in astrocytes, and that its up-regulation at the protein level is controlled by complex mechanisms.

The family of Toll‐like receptors (TLRs) expressed by innate immune cells recognizes a spectrum of microbial components as well as molecules released from injured tissues. TLR ligation activates intracellular signaling cascades that culminate in the up‐regulation of proinflammatory genes. We have recently demonstrated that the up‐regulation of inflammatory cytokines mediated by TLR4 in astrocytes is negatively controlled by the monomeric GTPases of Rho subfamily. The present study was undertaken to examine further the involvement of Rho proteins in the inflammatory response of astrocytes elicited by the ligation of three TLRs that use divergent signaling pathways. Astrocyte cultures established from newborn rat brains were challenged with ligands of TLR2, TLR3, and TLR4. The expression of genes encoding interleukin (IL)‐1β, IL‐6, tumor necrosis factor‐α (TNFα), interferon‐β (IFNβ), and inducible nitric oxide synthase (NOS2) was up‐regulated 24 hr after the challenge as determined by real‐time RT‐PCR. Pretreatment of the cells with toxin B, which specifically inactivates Rho proteins, enhanced the up‐regulation of gene expression. The extent of this enhancement was both receptor and gene dependent. The enhancing effect of Rho protein inactivation was also evident at the protein level of IL‐6 and NOS2 as reveled by ELISA and immunoblot analyses, respectively. These results suggest that Rho proteins control TLR‐mediated up‐regulation of inflammatory genes in astrocytes by interfering with multiple events along the signaling pathways. © 2008 Wiley‐Liss, Inc.