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Macrophage migration inhibitory factor (MIF) is a pluripotent pro-inflammatory cytokine and is related to acute and chronic inflammatory responses, immune disorders, tumors, and other diseases. In this study, an integrated virtual screening strategy and bioassays were used to search for potent MIF inhibitors. Twelve compounds with better bioactivity than the prototypical MIF-inhibitor ISO-1 (IC50= 14.41 μM) were identified by an in vitro enzymatic activity assay. Structural analysis revealed that these inhibitors have novel structural scaffolds. Compound 11was then chosen for further characterization in vitro, and it exhibited marked anti-inflammatory efficacy in LPS-activated BV-2 microglial cells by suppressing the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs). Our findings suggest that MIF may be involved in the regulation of microglial inflammatory activation and that small-molecule MIF inhibitors may serve as promising therapeutic agents for neuroinflammatory diseases.

Parkinson’s disease (PD) is a common neurodegenerative disease characterized by motor impairment and progressive loss of dopamine (DA) neurons. At present, the acute application of neurotoxic drugs such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) are commonly used to simulate the pathology of PD; however, it is difficult to induce the progressive pathogenesis of PD with these models. In this study, we employed DAT promoter-mediated Cre transgenic mice to establish tamoxifen-inducible Dicer conditional knockout (cKO) mice in an effort to mimic the progressive loss of DA neurons and the development of PD-like behavioral phenotypes. The results showed that Dicer cKO mice exhibited progressive loss of DA neurons in the substantia nigra (SN) following tamoxifen administration. Significant DA loss was observed 6 weeks after tamoxifen administration; accordingly, progressive motor function impairment was also observed. We also found that a significant neuroinflammatory response, as evidenced by microglial proliferation, another hallmark of PD pathogenesis, accompanied the loss of DA neurons. The acute application of levo-DOPA (l-DOPA) relieved the PD-like motor impairments in Dicer cKO mice to exert its antiparkinsonian action, indicating that the model can be used to evaluate the antiparkinsonian efficacy of PD drugs. To further elucidate the potential application of this novel PD animal model for PD drug development, we employed the powerful neuroprotective agent dihydromyricetin (DHM) (10 mg/kg) and the selective sigma-1 receptor agonist PRE-084 (1 mg/kg), both of which were previously shown to produce antiparkinsonian effects. The results indicated that the chronic administration of either DHM or PRE-084 attenuated the Dicer cKO-induced loss of DA neurons and motor impairments, although the two drugs acted through different mechanisms. These data indicate that the Dicer cKO mouse model may be a useful model for investigating the pathological development of PD and intervention-mediated changes. In conclusion, this transgenic mouse model appears to simulate the progressive pathogenesis of PD and may be a potentially useful model for PD drug discovery.

Microglia, the brain-resident macrophage, is known as the innate immune cell type in the central nervous system. Microglia is also the major cellular component of tumor mass of gliomas that plays a key role in glioma development. Mutations of isocitrate dehydrogenases 1 and 2 (IDH1/2) frequently occur in gliomas, which leads to accumulation of oncometabolic product 2-hydroxyglutarate (2HG). Moreover, IDH1/2 mutations were found to correlate with better prognosis in glioma patients. In the present study, we investigated the effects of the 2HG on microglial inflammatory activation. We showed that the conditioned media (CM) from GL261 glioma cells stimulated the activation of BV-2 microglia cells, evidenced by markedly increased expression of interleukin-6 (IL-6), IL-1β, tumor necrosis factor-α (TNF-α), CCL2 (C-C motif chemokine ligand 2) and CXCL10 (C-X-C motif chemokine 10). CM-induced expression of proinflammatory genes was significantly suppressed by pretreatment with a synthetic cell-permeable 2HG (1 mM) or a nuclear factor-κB (NF-κB) inhibitor BAY11-7082 (10 μM). In lipopolysaccharide (LPS)- or TNF-α-stimulated BV-2 microglia cells and primary microglia, pretreatment with 2HG (0.25–1 mM) dose-dependently suppressed the expression of proinflammatory genes. We further demonstrated that 2HG significantly suppressed LPS-induced phosphorylation of IκB kinase α/β (IKKα/β), IκBα and p65, IκB degradation, and nuclear translocation of p65 subunit of NF-κB, as well as NF-κB transcriptional activity. Similarly, ectopic expression of mutant isocitrate dehydrogenase 1 (IDH1) (R132H) significantly decreased TNF-α-induced activation of NF-κB signaling pathway. Finally, we revealed that activation of adenosine 5′-monophosphate-activated protein kinase (AMPK) and subsequent inhibition of mammalian target of rapamycin (mTOR) signaling contributed to the inhibitory effect of 2HG on NF-κB signaling pathway in BV-2 cells. Taken together, these results, for the first time, show that oncometabolite 2HG inhibits microglial activation through affecting AMPK/mTOR/NF-κB signaling pathway and provide evidence that oncometabolite 2HG may regulate glioma development via modulating microglial activation in tumor microenvironment.

Polo-like kinase 2 (PLK2) is a serine/threonine protein kinase associated with the regulation of synaptic plasticity and centriole duplication. We identify PLK2 as a crucial early response gene in lipopolysaccharide (LPS)-stimulated microglial cells. Knockdown or inhibition of PLK2 remarkably attenuates LPS-induced expression of pro-inflammatory factors in microglial cells by suppressing the IKKβ-NF-κB signaling pathway. We identify heat shock protein 90 alpha (HSP90α), a regulator of IKKβ activity, as a novel PLK2 substrate. Knockdown or pharmacological inhibition of HSP90α abolishes PLK2-mediated activation of NF-κB transcriptional activity and microglial inflammatory activation. Furthermore, phosphoproteomic analysis pinpoints Ser252 and Ser263 on HSP90α as novel phosphorylation targets of PLK2. Lastly, conditional knockout of PLK2 in microglial cells dramatically ameliorates neuroinflammation and subsequent dopaminergic neuron loss in an intracranial LPS-induced mouse PD model. The present study reveals that PLK2 promotes microglial activation through the phosphorylation of HSP90α and subsequent activation of the IKKβ-NF-κB signaling pathway.