The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes1–4. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context5. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance2,4,6, as well as in development, physiology and disease3,7. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX)8. The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.

Significance Brain ischemia is a major cause of death and disability worldwide, but the cellular mechanisms of delayed neuronal loss and brain atrophy after cerebral ischemia are poorly understood and thus currently untreatable. Surprisingly, we find that after cerebral ischemia, brain macrophages phagocytose viable and functional neurons, causing brain atrophy and motor dysfunction. Our data show that delayed neuronal death and functional impairment after cerebral ischemia can be prevented by blocking specific phagocytic pathways, and therefore highlight new therapeutic targets for stroke and dementia.

In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the molecular mechanisms and therapeutic potential of class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) is impaired by the lack of potent and selective chemical probes. Here we report the discovery of inhibitors that fill this void with an unprecedented metal-binding group, trifluoromethyloxadiazole (TFMO), which circumvents the selectivity and pharmacologic liabilities of hydroxamates. We confirm direct metal binding of the TFMO through crystallographic approaches and use chemoproteomics to demonstrate the superior selectivity of the TFMO series relative to a hydroxamate-substituted analog. We further apply these tool compounds to reveal gene regulation dependent on the catalytic active site of class IIa HDACs. The discovery of these inhibitors challenges the design process for targeting metalloenzymes through a chelating metal-binding group and suggests therapeutic potential for class IIa HDAC enzyme blockers distinct in mechanism and application compared to current HDAC inhibitors.

It has been postulated that the G protein-coupled receptor, GPR55, is a third cannabinoid receptor. Given that the ligands at the CB(1) and CB(2) receptors are effective analgesic and anti-inflammatory agents, the role of GPR55 in hyperalgesia associated with inflammatory and neuropathic pain has been investigated. As there are no well-validated GPR55 tool compounds, a GPR55 knockout (GPR55(-/-)) mouse line was generated and fully backcrossed onto the C57BL/6 strain. General phenotypic analysis of GPR55(-/-) mice revealed no obvious primary differences, compared with wild-type (GPR55(+/+)) littermates. GPR55(-/-) mice were then tested in the models of adjuvant-induced inflammation and partial nerve ligation. Following intraplantar administration of Freund's complete adjuvant (FCA), inflammatory mechanical hyperalgesia was completely absent in GPR55(-/-) mice up to 14 days post-injection. Cytokine profiling experiments showed that at 14 days post-FCA injection there were increased levels of IL-4, IL-10, IFN gamma and GM-CSF in paws from the FCA-injected GPR55(-/-) mice when compared with the FCA-injected GPR55(+/+) mice. This suggests that GPR55 signalling can influence the regulation of certain cytokines and this may contribute to the lack of inflammatory mechanical hyperalgesia in the GPR55(-/-) mice. In the model of neuropathic hypersensitivity, GPR55(-/-) mice also failed to develop mechanical hyperalgesia up to 28 days post-ligation. These data clearly suggest that the manipulation of GPR55 may have therapeutic potential in the treatment of both inflammatory and neuropathic pain.

No abstract

Microglia are resident brain macrophages that become activated and proliferate following brain damage or stimulation by immune mediators, such as IL-1β or TNF-α. We investigated the mechanisms by which microglial proliferation is regulated in primary cultures of rat glia. We found that basal proliferation of microglia was stimulated by proinflammatory cytokines IL-1β or TNF-α, and this proliferation was completely inhibited by catalase, implicating hydrogen peroxide as a mediator of proliferation. In addition, inhibitors of NADPH oxidase (diphenylene iodonium or apocynin) also prevented microglia proliferation, suggesting that this may be the source of hydrogen peroxide. IL-1β and TNF-α rapidly stimulated the rate of hydrogen peroxide produced by isolated microglia, and this was inhibited by diphenylene iodonium, implying that the cytokines were acting directly on microglia to stimulate the NADPH oxidase. Low concentrations of PMA or arachidonic acid (known activators of NADPH oxidase) or xanthine/xanthine oxidase or glucose oxidase (generating hydrogen peroxide) also increased microglia proliferation and this was blocked by catalase, showing that NADPH oxidase activation or hydrogen peroxide was sufficient to stimulate microglia proliferation. In contrast to microglia, the proliferation of astrocytes was unaffected by the presence of catalase. In conclusion, these findings indicate that microglial proliferation in response to IL-1β or TNF-α is mediated by hydrogen peroxide from NADPH oxidase.

Background: Inflammation-activated glia are seen in many CNS pathologies and may kill neurons through the release of cytotoxic mediators, such as nitric oxide from inducible NO synthase (iNOS), and possibly superoxide from NADPH oxidase (NOX). We set out to determine the relative role of these species in inducing neuronal death, and to test the dual-key hypothesis that the production of both species simultaneously is required for significant neuronal death.

Inflammatory-activated glia are seen in numerous central nervous system (CNS) pathologies and can kill nearby neurons through the release of cytotoxic mediators. Glia, when activated, can express the inducible isoform of nitric oxide synthase (iNOS) producing high levels of nitric oxide (NO), which can kill neurons in certain conditions. We show, however, that inflammatory activation of glia in a mature culture of cerebellar granule neurons and glia causes little or no neuronal death under normal (21%) oxygen conditions. Similarly, hypoxia (2% oxygen) or low levels of an NO donor (100 microM DETA/NO) caused little or no neuronal death in nonactivated cultures. If inflammatory activation of glia or addition of NO donor was combined with hypoxia, however, extensive neuronal death occurred. Death in both cases was prevented by the N-methyl-D-aspartate (NMDA) receptor blocker MK-801, implying that death was mediated by the glutamate receptor. Low levels of NO were found to increase the apparent K(M) of cellular oxygen consumption for oxygen, probably due to NO-induced inhibition of mitochondrial respiration, in competition with oxygen, at cytochrome oxidase. Necrotic death, induced by hypoxia plus DETA/NO, was increased further by deoxyglucose, an inhibitor of glycolysis, suggesting that necrosis was mediated by energy depletion. Hypoxia was found to be a potent stimulator of microglia proliferation, but this proliferation was not significant in inflammatory-activated cultures. These results suggest that low levels of NO can induce neuronal death under hypoxic conditions, mediated by glutamate after NO inhibition of respiration in competition with oxygen. Brain inflammation can thus sensitize to hypoxia-induced death, which may be important in pathologies such as stroke, neurodegeneration, and brain aging.