BackgroundDeciphering the mechanisms that modulate the inflammatory response induced by microglial activation not only improves our insight into neuroinflammation but also provides avenues for designing novel therapies that could halt inflammation-induced neuronal degeneration. Decreasing glycogen synthase kinase-3β (GSK-3β) activity has therapeutic benefits in inflammatory diseases. However, the exact molecular mechanisms underlying GSK-3β inactivation-mediated suppression of the inflammatory response induced by microglial activation have not been completely clarified. Tumor necrosis factor-α (TNF-α) plays a central role in injury caused by neuroinflammation. We investigated the regulatory effect of GSK-3β on TNF-α production by microglia to discern the molecular mechanisms of this modulation.MethodsLipopolysaccharide (LPS) was used to induce an inflammatory response in cultured primary microglia or murine BV-2 microglial cells. Release of TNF-α was measured by ELISA. Signaling molecules were analyzed by western blotting, and activation of NF-κB and AP-1 was measured by ELISA-based DNA binding analysis and luciferase reporter assay. Protein interaction was examined by coimmunoprecipitation.ResultsInhibition of GSK-3β by selective GSK-3β inhibitors or by RNA interference attenuated LPS-induced TNF-α production in cultured microglia. Exploration of the mechanisms by which GSK-3β positively regulates inflammatory response showed that LPS-induced IκB-α degradation, NF-κBp65 nuclear translocation, and p65 DNA binding activity were not affected by inhibition of GSK-3β activity. However, GSK-3β inactivation inhibited transactivation activity of p65 by deacetylating p65 at lysine 310. Furthermore, we also demonstrated a functional interaction between mixed lineage kinase 3 (MLK3) and GSK-3β during LPS-induced TNF-α production in microglia. The phosphorylated levels of MLK3, MKK4, and JNK were increased upon LPS treatment. Decreasing GSK-3β activity blocked MLK3 signaling cascades through disruption of MLK3 dimerization-induced autophosphorylation, ultimately leading to a decrease in TNF-α secretion.ConclusionThese results suggest that inactivation of GSK-3β might represent a potential strategy to downregulate microglia-mediated inflammatory processes.

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BackgroundThe pathogenesis of several neurodegenerative diseases often involves the microglial activation and associated inflammatory processes. Activated microglia release pro-inflammatory factors that may be neurotoxic. 6-Mercaptopurine (6-MP) is a well-established immunosuppressive drug. Common understanding of their immunosuppressive properties is largely limited to peripheral immune cells. However, the effect of 6-MP in the central nervous system, especially in microglia in the context of neuroinflammation is, as yet, unclear. Tumor necrosis factor-α (TNF-α) is a key cytokine of the immune system that initiates and promotes neuroinflammation. The present study aimed to investigate the effect of 6-MP on TNF-α production by microglia to discern the molecular mechanisms of this modulation.MethodsLipopolysaccharide (LPS) was used to induce an inflammatory response in cultured primary microglia or murine BV-2 microglial cells. Released TNF-α was measured by enzyme-linked immunosorbent assay (ELISA). Gene expression was determined by real-time reverse transcription polymerase chain reaction (RT-PCR). Signaling molecules were analyzed by western blotting, and activation of NF-κB was measured by ELISA-based DNA binding analysis and luciferase reporter assay. Chromatin immunoprecipitation (ChIP) analysis was performed to examine NF-κB p65 and coactivator p300 enrichments and histone modifications at the endogenous TNF-α promoter.ResultsTreatment of LPS-activated microglia with 6-MP significantly attenuated TNF-α production. In 6-MP pretreated microglia, LPS-induced MAPK signaling, IκB-α degradation, NF-κB p65 nuclear translocation, and in vitro p65 DNA binding activity were not impaired. However, 6-MP suppressed transactivation activity of NF-κB and TNF-α promoter by inhibiting phosphorylation and acetylation of p65 on Ser276 and Lys310, respectively. ChIP analyses revealed that 6-MP dampened LPS-induced histone H3 acetylation of chromatin surrounding the TNF-α promoter, ultimately leading to a decrease in p65/coactivator-mediated transcription of TNF-α gene. Furthermore, 6-MP enhanced orphan nuclear receptor Nur77 expression. Using RNA interference approach, we further demonstrated that Nur77 upregulation contribute to 6-MP-mediated inhibitory effect on TNF-α production. Additionally, 6-MP also impeded TNF-α mRNA translation through prevention of LPS-activated PI3K/Akt/mTOR signaling cascades.ConclusionsThese results suggest that 6-MP might have a therapeutic potential in neuroinflammation-related neurodegenerative disorders through downregulation of microglia-mediated inflammatory processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0543-5) contains supplementary material, which is available to authorized users.

The production of midbrain dopaminergic (mDA) neurons requires precise extrinsic inductive signals and intrinsic transcriptional cascade at a specific time point in development. Urocortin (UCN) is a peptide of the corticotropin-releasing hormone family that mediates various responses to stress. UCN was first cloned from adult rat midbrain. However, the contribution of UCN to the development of mDA neurons is poorly understood. Here, we show that UCN is endogenously expressed in the developing ventral midbrain (VM) and its receptors are exhibited in Nurr1 1 postmitotic mDA precursors and TH 1 neurons, suggesting possible roles in regulating their terminal differentiation. UCN treatment increased DA cell numbers in rat VM precursor cultures by promoting the conversion of Nurr1 1 precursors into DA neurons. Furthermore, neutralization of secreted UCN with anti-UCN antibody resulted in a reduction in the number of DA neurons. UCN induced an abundance of acetylated histone H3 and enhanced late DA regulator Nurr1, Foxa2, and Pitx3 expressions. Using pharmacological and RNA interference approaches, we further demonstrated that histone deacetylase (HDAC) inhibition and late transcriptional factors upregulation contribute to UCN-mediated DA neuron differentiation. Chromatin immunoprecipitation analyses revealed that UCN promoted histone acetylation of chromatin surrounding the TH promoter by directly inhibiting HDAC and releasing of methyl CpG binding protein 2-CoREST-HDAC1 repressor complex from the promoter, ultimately leading to an increase in Nurr1/coactivators-mediated transcription of TH gene. Moreover, UCN treatment in vivo also resulted in increased DA neuron differentiation. These findings suggest that UCN might contribute to regulate late mDA neuron differentiation during VM development. STEM CELLS

Peroxiredoxins (Prxs) are a family of thioredoxin peroxidases. Accumulating evidence suggests that changes in the expression of Prxs may be involved in neurodegenerative diseases pathology. However, the expression and function of Prxs in Parkinson's disease (PD) remains unclear.Here, we showed that Prx5 was the most downregulated of the six Prx subtypes in dopaminergic (DA) neurons in rotenone-induced cellular and rat models of PD, suggesting possible roles in regulating their survival. Depletion of Prx5 sensitized SH-SY5Y DA neuronal cells to rotenone-induced apoptosis. The extent of mitochondrial membrane potential collapse, cytochrome c release, and caspase activation was increased by Prx5 loss. Furthermore, Prx5 knockdown enhanced the induction of PUMA by rotenone through a p53-dependent mechanism. Using RNA interference approaches, we demonstrated that the p53/PUMA signaling was essential for Prx5 silencing-exacerbated mitochondria-driven apoptosis. Additionally, downregulation of Prx5 augmented rotenone-induced DNA damage manifested as induction of phosphorylated histone H2AX (γ-H2AX) and activation of ataxia telangiectasia mutated (ATM) kinase. The pharmacological inactivation of ATM revealed that ATM was integral to p53 activation by DNA damage. These findings provided a novel link between Prx5 and DNA damage-triggered ATM/p53/PUMA signaling in a rotenone-induced PD model. Thus, Prx5 might play an important role in protection against rotenone-induced DA neurodegeneration.Cells 2020, 9, 22 2 of 25 ubiquitin-proteasome system, and autophagy-lysosome pathway may be involved in the pathogenesis of PD [3,4]. Recently, DNA damage is hypothesized to constitute a unifying component across different neurodegenerative diseases [5]. Increased DNA strand breaks and oxidative DNA damages have been found in the SN and peripheral blood of PD patients [6][7][8], suggesting that DNA damage may compromise genomic integrity leading to the onset of PD [9].Peroxiredoxins (Prxs) represent a superfamily of thiol-dependent antioxidant enzymes that participate directly in eliminating H 2 O 2 and regulate the transduction of various cellular signals by modulating redox signaling [10,11]. In mammals, six isoforms of Prxs have been identified, comprising typical 2-Cys Prxs (Prx1-4), atypical 2-Cys Prxs (Prx5), and 1-Cys Prxs (Prx6). Proposed functions of Prxs include regulation of cellular proliferation, differentiation, immune responses, and apoptosis [12]. Prx5 is broadly localized in the cytosol, mitochondria, peroxisome, and nucleus, and performs specific functions according to its subcellular localization [13]. Initially, Prx5 was described as a DNA-binding protein able to suppress RNA polymerase III-dependent transcription of Alu retroposons in vitro [14]. The cytoprotective antioxidant function of Prx5 has been investigated after human Prx5 was characterized as a peroxidase but also because of the existence of this enzyme in peroxisomes and mitochondria, the two major places of reactive oxygen species (ROS)/reactive nitrogen ...