Chronic neuroinflammation is a common feature of the ageing brain and some neurodegenerative disorders. However, the molecular and cellular mechanisms underlying the regulation of innate immunity in the central nervous system remain elusive. Here we show that the astrocytic dopamine D2 receptor (DRD2) modulates innate immunity through αB-crystallin (CRYAB), which is known to suppress neuroinflammation. We demonstrate that knockout mice lacking Drd2 showed remarkable inflammatory response in multiple central nervous system regions and increased the vulnerability of nigral dopaminergic neurons to neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. Astrocytes null for Drd2 became hyper-responsive to immune stimuli with a marked reduction in the level of CRYAB. Preferential ablation of Drd2 in astrocytes robustly activated astrocytes in the substantia nigra. Gain- or loss-of-function studies showed that CRYAB is critical for DRD2-mediated modulation of innate immune response in astrocytes. Furthermore, treatment of wild-type mice with the selective DRD2 agonist quinpirole increased resistance of the nigral dopaminergic neurons to MPTP through partial suppression of inflammation. Our study indicates that astrocytic DRD2 activation normally suppresses neuroinflammation in the central nervous system through a CRYAB-dependent mechanism, and provides a new strategy for targeting the astrocyte-mediated innate immune response in the central nervous system during ageing and disease.

Caspase-1 is an essential effector of inflammation, pyroptosis, and septic shock. Few caspase-1 substrates have been identified to date, and these substrates do not account for its wide range of actions. To understand the function of caspase-1, we initiated the systematic identification of its cellular substrates. Using the diagonal gel proteomic approach, we identified 41 proteins that are directly cleaved by caspase-1. Among these were chaperones, cytoskeletal and translation machinery proteins, and proteins involved in immunity. A series of unexpected proteins along the glycolysis pathway were also identified, including aldolase, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, ␣-enolase, and pyruvate kinase. With the exception of the latter, the identified glycolysis enzymes were specifically cleaved in vitro by recombinant caspase-1, but not caspase-3. The enzymatic activity of wild-type glyceraldehyde-3-phosphate dehydrogenase, but not a non-cleavable mutant, was dampened by caspase-1 processing. In vivo, stimuli that fully activated caspase-1, including Salmonella typhimurium infection and septic shock, caused a pronounced processing of these proteins in the macrophage and diaphragm muscle, respectively. Notably, these stimuli inhibited glycolysis in wildtype cells compared with caspase-1-deficient cells. The systematic characterization of caspase-1 substrates identifies the glycolysis pathway as a caspase-1 target and provides new insights into its function during pyroptosis and septic shock.Caspases are aspartate-specific cysteine proteases known for their function in regulating programmed cell death and inflammation. Phylogenetically, they are subdivided into the CED3-related enzymes that initiate and execute cell death and the caspase-1-related proteins that process and mature cytokines, viz. IL-1␤, 2 IL-18, and IL-33. Apoptotic caspases execute cell death through the restricted cleavage of key cellular proteins required to maintain cell viability, resulting in the morphological changes observed during apoptosis, including membrane blebbing, nuclear condensation, and cytoskeletal dismantling (1). Caspase-1 is essential during inflammation because of its role in the activation of cytokine signaling pathways. With the exception of its cytokine substrates, very little is known regarding the spectrum of cellular proteins it targets upon full activation. Similar to other caspases, caspase-1 is found in cells as an inactive precursor and is activated in response to inflammatory triggers, including pathogen-derived molecules, as well as danger signals released from infected or dying cells (2). Caspase-1 activation is achieved in a macromolecular complex known as the inflammasome through its recruitment to a scaffolding molecule generally via the adaptor ASC (3). Scaffolding molecules that activate caspase-1 within the inflammasome belong to the cytosolic Nod-like family of pathogen recognition receptors and include Nalp1-3, Ipaf, and Naip5 (4). More recently, a distinct caspase-1 activat...

AMP-activated protein kinase (AMPK) serves as an energy sensor and is considered a promising drug target for treatment of type II diabetes and obesity. A previous report has shown that mammalian AMPK ␣1 catalytic subunit including autoinhibitory domain was inactive. To test the hypothesis that small molecules can activate AMPK through antagonizing the autoinhibition in ␣ subunits, we screened a chemical library with inactive human ␣1 394 (␣1, residues 1-394) and found a novel small-molecule activator, PT1, which dose-dependently activated AMPK ␣1 394 , ␣1 335 , ␣2 398 , and even heterotrimer ␣1␤1␥1. Based on PT1-docked AMPK ␣1 subunit structure model and different mutations, we found PT1 might interact with Glu-96 and Lys-156 residues near the autoinhibitory domain and directly relieve autoinhibition. Further studies using L6 myotubes showed that the phosphorylation of AMPK and its downstream substrate, acetyl-CoA carboxylase, were dose-dependently and time-dependently increased by PT1 without an increase in cellular AMP:ATP ratio. Moreover, in HeLa cells deficient in LKB1, PT1 enhanced AMPK phosphorylation, which can be inhibited by the calcium/calmodulin-dependent protein kinase kinases inhibitor STO-609 and AMPK inhibitor compound C. PT1 also lowered hepatic lipid content in a dose-dependent manner through AMPK activation in HepG2 cells, and this effect was diminished by compound C. Taken together, these data indicate that this small-molecule activator may directly activate AMPK via antagonizing the autoinhibition in vitro and in cells. This compound highlights the effort to discover novel AMPK activators and can be a useful tool for elucidating the mechanism responsible for conformational change and autoinhibitory regulation of AMPK.The AMP-activated protein kinase (AMPK) 3 is a highly conserved serine/threonine protein kinase that is widely expressed in higher eukaryotes, yeast, and plants and plays a unique and central role in the responses of cells to metabolic stresses such as nutrient starvation, heat shock, ischemia/hypoxia, and vigorous muscular exercise by depleting cellular ATP and elevating AMP levels (1, 2). Once activated, AMPK prevents depletion of ATP by increasing the rate of ATP generation, triggering changes in the rates of glucose transport, fatty acid oxidation, lipogenesis, sterol synthesis, and gluconeogenesis through direct regulation of key metabolic enzymes and transcriptional control of specific genes (1-4). There is mounting evidence of the involvement of AMPK in human physiological and pathological processes, especially type 2 diabetes and obesity. Previous studies indicate that several of the beneficial effects of rosiglitazone and metformin, two widely used antidiabetic drugs, are mediated by indirect activation of AMPK, suggesting the potential role of the AMPK pathway in the treatment of type 2 diabetes (5, 7). Two adipocyte-derived hormones, leptin and adiponectin, stimulate fatty acid oxidation and glucose uptake in peripheral tissues such as skeletal muscle and liver, whic...

Juvenile hormone (JH) regulates many developmental and physiological events in insects, but its molecular mechanism remains conjectural. Here we report that genetic ablation of the corpus allatum cells of the Drosophila ring gland (the JH source) resulted in JH deficiency, pupal lethality and precocious and enhanced programmed cell death (PCD) of the larval fat body. In the fat body of the JH-deficient animals, Dronc and Drice, two caspase genes that are crucial for PCD induced by the molting hormone 20-hydroxyecdysone (20E), were significantly upregulated. These results demonstrated that JH antagonizes 20E-induced PCD by restricting the mRNA levels of Dronc and Drice. The antagonizing effect of JH on 20E-induced PCD in the fat body was further confirmed in the JH-deficient animals by 20E treatment and RNA interference of the 20E receptor EcR. Moreover, MET and GCE, the bHLH-PAS transcription factors involved in JH action, were shown to induce PCD by upregulating Dronc and Drice. In the Met-and gce-deficient animals, Dronc and Drice were downregulated, whereas in the Met-overexpression fat body, Dronc and Drice were significantly upregulated leading to precocious and enhanced PCD, and this upregulation could be suppressed by application of the JH agonist methoprene. For the first time, we demonstrate that JH counteracts MET and GCE to prevent caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila.

Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary tumor in the central nervous system. One of the most widely used chemotherapeutic drugs for GBM is temozolomide, which is a DNA-alkylating agent and its efficacy is dependent on MGMT methylation status. Little progress in improving the prognosis of GBM patients has been made in the past ten years, urging the development of more effective molecular targeted therapies. Hyper-activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is frequently found in a variety of cancers including GBM, and it plays a central role in the regulation of tumor cell survival, growth, motility, angiogenesis and metabolism. Numerous PI3K inhibitors including pan-PI3K, isoform-selective and dual PI3K/mammalian target of rapamycin (mTOR) inhibitors have exhibited favorable preclinical results and entered clinical trials in a range of hematologic malignancies and solid tumors. Furthermore, combination of inhibitors targeting PI3K and other related pathways may exert synergism on suppressing tumor growth and improving patients’ prognosis. Currently, only a handful of PI3K inhibitors are in phase I/II clinical trials for GBM treatment. In this review, we focus on the importance of PI3K/Akt pathway in GBM, and summarize the current development of PI3K inhibitors alone or in combination with other inhibitors for GBM treatment from preclinical to clinical studies.

TAR DNA-binding protein 43 (TDP-43) inclusions are a pathological hallmark of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), including cases caused by G4C2 repeat expansions in the C9orf72 gene (c9FTD/ALS). Providing mechanistic insight into the link between C9orf72 mutations and TDP-43 pathology, we demonstrated that a glycine-arginine repeat protein [poly(GR)] translated from expanded G4C2 repeats was sufficient to promote aggregation of endogenous TDP-43. In particular, toxic poly(GR) proteins mediated sequestration of full-length TDP-43 in an RNA-independent manner to induce cytoplasmic TDP-43 inclusion formation. Moreover, in GFP-(GR)200 mice, poly(GR) caused the mislocalization of nucleocytoplasmic transport factors and nuclear pore complex proteins. These mislocalization events resulted in the aberrant accumulation of endogenous TDP-43 in the cytoplasm where it co-aggregated with poly(GR). Last, we demonstrated that treating G4C2 repeat–expressing mice with repeat-targeting antisense oligonucleotides lowered poly(GR) burden, which was accompanied by reduced TDP-43 pathology and neurodegeneration, including lowering of plasma neurofilament light (NFL) concentration. These results contribute to clarification of the mechanism by which poly(GR) drives TDP-43 proteinopathy, confirm that G4C2-targeted therapeutics reduce TDP-43 pathology in vivo, and demonstrate that alterations in plasma NFL provide insight into the therapeutic efficacy of disease-modifying treatments.

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Gut-derived lipopolysaccharides (LPS) are critical to the development and progression of chronic low-grade inflammation and metabolic diseases. In this study, the effects of probiotics Lactobacillus and Bifidobacterium on gut-derived lipopolysaccharide and inflammatory cytokine concentrations were evaluated using a human colonic microbiota model. Lactobacillus reuteri, L. rhamnosus, L. plantarum, Bifidobacterium animalis, B. bifidum, B. longum, and B. longum subsp. infantis were identified from the literature for their anti-inflammatory potential. Each bacterial culture was administered daily to a human colonic microbiota model during 14 days. Colonic lipopolysaccharides, and Gram-positive and negative bacteria were quantified. RAW 264.7 macrophage cells were stimulated with supernatant from the human colonic microbiota model. Concentrations of TNF-alpha, IL-1beta, and IL-4 cytokines were measured. Lipopolysaccharide concentrations were significantly reduced with the administration of B. bifidum (-46.45 +/- 5.65%), L. rhamnosus (-30.40 +/- 5.08%), B. longum (-42.50 +/- 1.28%), and B. longum subsp. infantis (-68.85 +/- 5.32%) (p < 0.05). Cell counts of Gram-negative and positive bacteria were distinctly affected by the probiotic administered. There was a probiotic strain-specific effect on immunomodulatory responses of RAW 264.7 macrophage cells. B. longum subsp. infantis demonstrated higher capacities to reduce TNF-alpha concentrations (-69.41 +/- 2.78%; p < 0.05) and to increase IL-4 concentrations (+16.50 +/- 0.59%; p < 0.05). Colonic lipopolysaccharides were significantly correlated with TNF-alpha and IL-1beta concentrations (p < 0.05). These findings suggest that specific probiotic bacteria, such as B. longum subsp. infantis, might decrease colonic lipopolysaccharide concentrations, which might reduce the proinflammatory tone. This study has noteworthy applications in the field of biotherapeutics for the prevention and/or treatment of inflammatory and metabolic diseases.