Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription

Background: Glycogen synthase kinase-3 (GSK-8) is a serine/threonine protein kinase, the activity of which is inhibited by a variety of extracellular stimuli including insulin, growth factors, cell specification factors and cell adhesion. Consequently, inhibition of GSK-3 activity has been proposed to play a role in the regulation of numerous signalling pathways that elicit pleiotropic cellular responses. This report describes the identification and characterisation of potent and selective small molecule inhibitors of GSK-3.

Inhibition of Secretion of Interleukin (IL)-12/IL-23 Family Cytokines by 4-Trifluoromethyl-celecoxib Is Coupled to Degradation via the Endoplasmic Reticulum Stress Protein HERP

Interleukin-12 (IL-12), p80, and IL-23 are structurally related cytokines sharing a p40 subunit. We have recently demonstrated that celecoxib and its COX-2-independent analogue 4-trifluoromethyl-celecoxib (TFM-C) inhibit secretion but not transcription of IL-12 (p35/p40) and p80 (p40/p40). This is associated with a mechanism involving altered cytokine-chaperone interaction in the endoplasmic reticulum (ER). In the present study, we found that celecoxib and TFM-C also block secretion of IL-23 (p40/p19 heterodimers). Given the putative ER-centric mode of these compounds, we performed a comprehensive RTPCR analysis of 23 ER-resident chaperones/foldases and associated co-factors. This revealed that TFM-C induced 1.5-3-fold transcriptional up-regulation of calreticulin, GRP78, GRP94, GRP170, ERp72, ERp57, ERdj4, and ERp29. However, more significantly, a 7-fold up-regulation of homocysteine-inducible ER protein (HERP) was observed. HERP is part of a high molecular mass protein complex involved in ER-associated protein degradation (ERAD). Using co-immunoprecipitation assays, we show that TFM-C induces protein interaction of p80 and IL-23 with HERP. Both HERP siRNA knockdown and HERP overexpression coupled to cycloheximide chase assays revealed that HERP is necessary for degradation of intracellularly retained p80 by TFM-C. Thus, our data suggest that targeting cytokine folding in the ER by small molecule drugs could be therapeutically exploited to alleviate in appropriate inflammation in autoimmune conditions.

Hyperglycaemia-induced pro-inflammatory responses by retinal Müller glia are regulated by the receptor for advanced glycation end-products (RAGE)

Aims/hypothesis: Up-regulation of the receptor for AGEs (RAGE) and its ligands in diabetes has been observed in various tissues. Here, we sought to determine levels of RAGE and one of its most important ligands, S100B, in diabetic retina, and to investigate the regulatory role of S100B and RAGE in Müller glia.

Methods: Streptozotocin-diabetes was induced in Sprague-Dawley rats. RAGE, S100B and glial fibrillary acidic protein (GFAP) were detected in retinal cryosections. In parallel, the human retinal Müller cell line, MIO-M1, was maintained in normal glucose (5.5 mmol/l) or high glucose (25 mmol/l). RAGE knockdown was achieved using small interfering RNA (siRNA), while soluble RAGE was used as a competitive inhibitor of RAGE ligand binding. RAGE, S100B and cytokines were detected using quantitative RT-PCR, western blotting, cytokine protein arrays or ELISA. Activation of mitogen-activated protein kinase (MAPK) by RAGE was determined by western blotting.

Results: Compared with non-diabetic controls, RAGE and S100B were significantly elevated in the diabetic retina with apparent localisation in the Müller glia, occurring concomitantly with upregulation of GFAP. Exposure of MIO-M1 cells to high glucose induced increased production of RAGE and S100B. RAGE signalling via MAPK pathway was linked to cytokine production. Blockade of RAGE prevented cytokine responses induced by high glucose and S100B in Müller glia.

Conclusions/interpretation: Hyperglycaemia in vivo and in vitro exposure to high glucose induce upregulation of RAGE and its ligands, leading to RAGE signalling, which links to pro-inflammatory responses by retinal Müller glia. These data shed light on the potential clinical application of RAGE blockade to inhibit the progression of diabetic retinopathy.

Genetic Evidence Implicates the Immune System and Cholesterol Metabolism in the Aetiology of Alzheimer's Disease

Background: Late Onset Alzheimer's disease (LOAD) is the leading cause of dementia. Recent large genome-wide association studies (GWAS) identified the first strongly supported LOAD susceptibility genes since the discovery of the involvement of APOE in the early 1990s. We have now exploited these GWAS datasets to uncover key LOAD pathophysiological processes. Methodology: We applied a recently developed tool for mining GWAS data for biologically meaningful information to a LOAD GWAS dataset. The principal findings were then tested in an independent GWAS dataset.

Persistent Inflammation Subverts Thrombospondin-1–Induced Regulation of Retinal Angiogenesis and Is Driven by CCR2 Ligation

Neovascular retinal disease is a leading cause of blindness orchestrated by inflammatory responses. Although noninfectious uveoretinitis is mediated by CD4(+) T cells, in the persistent phase of disease, angiogenic responses are observed, along with degeneration of the retina. Full clinical manifestation relies on myeloid-derived cells, which are phenotypically distinct from, but potentially sharing common effector responses to age-related macular degeneration. To interrogate inflammation-mediated angiogenesis, we investigated experimental autoimmune uveoretinitis, an animal model for human uveitis. After the initial acute phase of severe inflammation, the retina sustains a persistent low-grade inflammation with tissue-infiltrating leukocytes for over 4 months. During this persistent phase, angiogenesis is observed as retinal neovascular membranes that arise from inflamed venules and postcapillary venules, increase in size as the disease progresses, and are associated with infiltrating arginase-1(+) macrophages. In the absence of thrombospondin-1, retinal neovascular membranes are markedly increased and are associated with arginase-1(-) CD68(+) macrophages, whereas deletion of the chemokine receptor CCR2 resulted in reduced retinal neovascular membranes in association with a predominant neutrophil infiltrate. CCR2 is important for macrophage recruitment to the retina in experimental autoimmune uveoretinitis and promotes chronicity in the form of a persistent angiogenesis response, which in turn is regulated by constitutive expression of angiogenic inhibitors like thrombospondin-1. This model offers a new platform to dissect the molecular and cellular pathology of inflammation-induced ocular angiogenesis.

Inhibition of triclabendazole metabolism in vitro by ketoconazole increases disruption to the tegument of a triclabendazole-resistant isolate of Fasciola hepatica

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of drug metabolism. The cytochrome P450 (CYP 450) enzyme pathway was inhibited using ketoconazole (KTZ) to see whether a TCBZ-resistant isolate could be made more sensitive to TCBZ action. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP 450 system was inhibited by a 2-h pre-incubation in ketoconazole (40 mu M), then incubated for a further 22 h in NCTC medium containing either KTZ, KTZ+nicotinamide adenine dinucleotide phosphate (NADPH) (1 nM), KTZ+NADPH+TCBZ (15 mu g/ml), or KTZ+NADPH+triclabendazole sulphoxide (TCBZ. SO; 15 mu g/ml). Changes to fluke ultrastructure following drug treatment and metabolic inhibition were assessed using transmission electron microscopy. After treatment with either TCBZ or TCBZ. SO on their own, there was greater disruption to the TCBZ-susceptible than TCBZ-resistant isolate. However, co-incubation with KTZ+TCBZ, but more particularly KTZ+TCBZ. SO, led to more severe changes to the TCBZ-resistant isolate than with each drug on its own: in the syncytium, for example, there was severe swelling of the basal infolds and their associated mucopolysaccharide masses, accompanied by an accumulation of secretory bodies just below the apex. Golgi complexes were greatly reduced or absent in the tegumental cells and the synthesis, production, and transport of secretory bodies were badly disrupted. With the TCBZ-susceptible Cullompton isolate, there was limited potentiation of drug action. The results support the concept of altered drug metabolism in TCBZ-resistant flukes and this process may play a role in the development of drug resistance.

Novel Analysis of Oceanic Surface Water Metagenomes Suggests Importance of Polyphosphate Metabolism in Oligotrophic Environments

Polyphosphate is a ubiquitous linear homopolymer of phosphate residues linked by high-energy bonds similar to those found in ATP. It has been associated with many processes including pathogenicity, DNA uptake and multiple stress responses across all domains. Bacteria have also been shown to use polyphosphate as a way to store phosphate when transferred from phosphate-limited to phosphate-rich media - a process exploited in wastewater treatment and other environmental contaminant remediation. Despite this, there has, to date, been little research into the role of polyphosphate in the survival of marine bacterioplankton in oligotrophic environments. The three main proteins involved in polyphosphate metabolism, Ppk1, Ppk2 and Ppx are multi-domain and have differential inter-domain and inter-gene conservation, making unbiased analysis of relative abundance in metagenomic datasets difficult. This paper describes the development of a novel Isofunctional Homolog Annotation Tool (IHAT) to detect homologs of genes with a broad range of conservation without bias of traditional expect-value cutoffs. IHAT analysis of the Global Ocean Sampling (GOS) dataset revealed that genes associated with polyphosphate metabolism are more abundant in environments where available phosphate is limited, suggesting an important role for polyphosphate metabolism in marine oligotrophs.

Intervention With an Erythropoietin-Derived Peptide Protects Against Neuroglial and Vascular Degeneration During Diabetic Retinopathy.

OBJECTIVE:
Erythropoietin (EPO) may be protective for early stage diabetic retinopathy, although there are concerns that it could exacerbate retinal angiogenesis and thrombosis. A peptide based on the EPO helix-B domain (helix B-surface peptide [pHBSP]) is nonerythrogenic but retains tissue-protective properties, and this study evaluates its therapeutic potential in diabetic retinopathy.
RESEARCH DESIGN AND METHODS:
After 6 months of streptozotocin-induced diabetes, rats (n = 12) and age-matched nondiabetic controls (n = 12) were evenly split into pHBSP and scrambled peptide groups and injected daily (10 µg/kg per day) for 1 month. The retina was investigated for glial dysfunction, microglial activation, and neuronal DNA damage. The vasculature was dual stained with isolectin and collagen IV. Retinal cytokine expression was quantified using real-time RT-PCR. In parallel, oxygen-induced retinopathy (OIR) was used to evaluate the effects of pHBSP on retinal ischemia and neovascularization (1-30 µg/kg pHBSP or control peptide).
RESULTS:
pHBSP or scrambled peptide treatment did not alter hematocrit. In the diabetic retina, Müller glial expression of glial fibrillary acidic protein was increased when compared with nondiabetic controls, but pHBSP significantly reduced this stress-related response (P < 0.001). CD11b+ microglia and proinflammatory cytokines were elevated in diabetic retina responses, and some of these responses were attenuated by pHBSP (P < 0.01-0.001). pHBSP significantly reduced diabetes-linked DNA damage as determined by 8-hydroxydeoxyguanosine and transferase-mediated dUTP nick-end labeling positivity and also prevented acellular capillary formation (P < 0.05). In OIR, pHBSP had no effect on preretinal neovascularization at any dose.
CONCLUSIONS:
Treatment with an EPO-derived peptide after diabetes is fully established can significantly protect against neuroglial and vascular degenerative pathology without altering hematocrit or exacerbating neovascularization. These findings have therapeutic implications for disorders such as diabetic retinopathy.