Orexin receptors exert a neuroprotective effect in Alzheimer's disease (AD) via heterodimerization with GPR103

Orexins are neuropeptides that regulate the sleep-wake cycle and feeding behaviour. QRFP is a newly discovered neuropeptide which exerts similar orexigenic activity, thus playing an important role in energy homeostasis and regulation of appetite. The exact expression and signalling characteristics and physiological actions of QRFP and its receptor GPR103 are poorly understood. Alzheimerâ €™ s disease (AD) patients experience increased nocturnal activity, excessive daytime sleepiness, and weight loss. We hypothesised therefore that orexins and QRFP might be implicated in the pathophysiology of AD. We report that the down-regulation of hippocampal orexin receptors (OXRs) and GPR103 particularly in the cornu ammonis (CA) subfield from AD patients suffering from early onset familial AD (EOFAD) and late onset familial AD (LOAD). Using an in vitro model we demonstrate that this downregulation is due to to Aβ-plaque formation and tau hyper-phosphorylation. Transcriptomics revealed a neuroprotective role for both orexins and QRFP. Finally we provide conclusive evidence using BRET and FRET that OXRs and GPR103 form functional hetero-dimers to exert their effects involving activation of ERK 1/2. Pharmacological intervention directed at the orexigenic system may prove to be an attractive avenue towards the discovery of novel therapeutics for diseases such as AD and improving neuroprotective signalling pathways

Urinary MicroRNA Profiling in the Nephropathy of Type 1 Diabetes

Background: Patients with Type 1 Diabetes (T1D) are particularly vulnerable to development of Diabetic nephropathy (DN) leading to End Stage Renal Disease. Hence a better understanding of the factors affecting kidney disease progression in T1D is urgently needed. In recent years microRNAs have emerged as important post-transcriptional regulators of gene expression in many different health conditions. We hypothesized that urinary microRNA profile of patients will differ in the different stages of diabetic renal disease. Methods and Findings: We studied urine microRNA profiles with qPCR in 40 T1D with >20 year follow up 10 who never developed renal disease (N) matched against 10 patients who went on to develop overt nephropathy (DN), 10 patients with intermittent microalbuminuria (IMA) matched against 10 patients with persistent (PMA) microalbuminuria. A Bayesian procedure was used to normalize and convert raw signals to expression ratios. We applied formal statistical techniques to translate fold changes to profiles of microRNA targets which were then used to make inferences about biological pathways in the Gene Ontology and REACTOME structured vocabularies. A total of 27 microRNAs were found to be present at significantly different levels in different stages of untreated nephropathy. These microRNAs mapped to overlapping pathways pertaining to growth factor signaling and renal fibrosis known to be targeted in diabetic kidney disease. Conclusions: Urinary microRNA profiles differ across the different stages of diabetic nephropathy. Previous work using experimental, clinical chemistry or biopsy samples has demonstrated differential expression of many of these microRNAs in a variety of chronic renal conditions and diabetes. Combining expression ratios of microRNAs with formal inferences about their predicted mRNA targets and associated biological pathways may yield useful markers for early diagnosis and risk stratification of DN in T1D by inferring the alteration of renal molecular processes. © 2013 Argyropoulos et al

Ghrelin Attenuates the Osteoblastic Differentiation of Vascular Smooth Muscle Cells through the ERK Pathway

Vascular calcification results from osteoblastic differentiation of vascular smooth muscle cells (VSMCs) and is a major risk factor for cardiovascular events. Ghrelin is a newly discovered bioactive peptide that acts as a natural endogenous ligand of the growth hormone secretagog receptor (GHSR). Several studies have identified the protective effects of ghrelin on the cardiovascular system, however research on the effects and mechanisms of ghrelin on vascular calcification is still quite rare. In this study, we determined the effect of ghrelin on osteoblastic differentiation of VSMCs and investigated the mechanism involved using the two universally accepted calcifying models of calcifying vascular smooth muscle cells (CVSMCs) and beta-glycerophosphate (beta-GP)-induced VSMCs. Our data demonstrated that ghrelin inhibits osteoblastic differentiation and mineralization of VSMCs due to decreased alkaline phosphatase (ALP) activity, Runx2 expression, bone morphogenetic protein-2 (BMP-2) expression and calcium content. Further study demonstrated that ghrelin exerted this suppression effect via an extracellular signal-related kinase (ERK)-dependent pathway and that the suppression effect of ghrelin was time dependent and dose dependent. Furthermore, inhibition of the growth hormone secretagog receptor (GHSR), the ghrelin receptor, by siRNA significantly reversed the activation of ERK by ghrelin. In conclusion, our study suggests that ghrelin may inhibit osteoblastic differentiation of VSMCs through the GHSR/ERK pathway

Generation and characterisation of Friedreich ataxia YG8R mouse fibroblast and neural stem cell models

This article has been made available through the Brunel Open Access Publishing Fund.Background: Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by GAA repeat expansion in the first intron of the FXN gene, which encodes frataxin, an essential mitochondrial protein. To further characterise the molecular abnormalities associated with FRDA pathogenesis and to hasten drug screening, the development and use of animal and cellular models is considered essential. Studies of lower organisms have already contributed to understanding FRDA disease pathology, but mammalian cells are more related to FRDA patient cells in physiological terms. Methodology/Principal Findings: We have generated fibroblast cells and neural stem cells (NSCs) from control Y47R mice (9 GAA repeats) and GAA repeat expansion YG8R mice (190+120 GAA repeats). We then differentiated the NSCs in to neurons, oligodendrocytes and astrocytes as confirmed by immunocytochemical analysis of cell specific markers. The three YG8R mouse cell types (fibroblasts, NSCs and differentiated NSCs) exhibit GAA repeat stability, together with reduced expression of frataxin and reduced aconitase activity compared to control Y47R cells. Furthermore, YG8R cells also show increased sensitivity to oxidative stress and downregulation of Pgc-1α and antioxidant gene expression levels, especially Sod2. We also analysed various DNA mismatch repair (MMR) gene expression levels and found that YG8R cells displayed significant reduction in expression of several MMR genes, which may contribute to the GAA repeat stability. Conclusions/Significance: We describe the first fibroblast and NSC models from YG8R FRDA mice and we confirm that the NSCs can be differentiated into neurons and glia. These novel FRDA mouse cell models, which exhibit a FRDA-like cellular and molecular phenotype, will be valuable resources to further study FRDA molecular pathogenesis. They will also provide very useful tools for preclinical testing of frataxin-increasing compounds for FRDA drug therapy, for gene therapy, and as a source of cells for cell therapy testing in FRDA mice. © 2014 Sandi et al

Response of the Gypsy Moth, Lymantria dispar to Transgenic Poplar, Populus simonii x P. nigra, Expressing Fusion Protein Gene of the Spider Insecticidal Peptide and Bt-toxin C-peptide

The response of the Asian gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae) to a fusion gene consisting of the spider, Atrax robustus Simon (Araneae: Hexanthelidae) ω?-ACTX-Ar1 sequence coding for an ω?-atracotoxin and a sequence coding for the Bt-toxin C-peptide, expressed in transgenic poplar Populus simonii x P. nigra L. (Malphigiales: Salicaceae) was investigated. Individual performance, feeding selection, midgut proteinase activity and nutrition utilization were monitored. The growth and development of L. dispar were significantly affected by continually feeding on the transgenic poplar, with the larval instars displaying significantly shorter developmental times than those fed on nontransgenic poplar, but pupation was delayed. Mortality was higher in populations fed transgenic poplar leaves, than for larvae fed nontransgenic poplar leaves. The cumulative mortality during all stages of larvae fed transgenic leaves was 92% compared to 16.7% of larvae on nontransgenic leaves. The highest mortality observed was 71.7% in the last larval instar stage. A two-choice test showed that fifth-instar larvae preferred to feed on nontransgenic leaves at a ratio of 1:1.4. Feeding on transgenic leaves had highly significant negative effects on relative growth of larvae, and the efficiency of conversion of ingested and digested food. Activity of major midgut proteinases was measured using substrates TAME and BTEE showed significant increases in tryptase and chymotrypsinlike activity (9.2- and 9.0-fold, respectively) in fifth-instar larvae fed on transgenic leaves over control. These results suggest transgenic poplar is resistant to L. dispar, and the mature L. dispar may be weakened by the transgenic plants due to Bt protoxins activated by elevated major midgut proteinase activity. The new transgenic poplar expressing fusion protein genes of Bt and a new spider insecticidal peptide are good candidates for managing gypsy moth

WTH3 is a direct target of the p53 protein

Previous results showed that overexpression of the WTH3 gene in multidrug resistance (MDR) cells reduced MDR1 gene expression and converted their resistance to sensitivity to various anticancer drugs. The WTH3 gene promoter was found to be differentially regulated in paired MDR vs non-MDR MCF7 cells owing to epigenetic modifications and transcription factor modulations. To understand further the mechanisms that govern WTH3's differential expression, we uncovered a p53-binding site in its promoter, which indicated that WTH3 could be regulated by the p53 gene. This hypothesis was then tested by different strategies. The resulting data revealed that (1) the WTH3 promoter was upregulated by the p53 transgene in diverse host cells; (2) there was a correlation between WTH3 expression levels and p53 gene status in a cell line panel; (3) a WTH3 promoter region was directly targeted by the p53 protein in vitro and in vivo. In addition, overexpression of the WTH3 gene promoted the apoptotic phenotype in host cells. On the basis of these findings, we believe that the negative role played by the WTH3 gene in MDR development is through its proapoptotic potential that is regulated by multiple mechanisms at the transcription level, and one of these mechanisms is linked to the p53 gene

Methylation of WTH3, a possible drug resistant gene, inhibits p53 regulated expression

<p>Abstract</p> <p>Background</p> <p>Previous results showed that over-expression of the <it>WTH3 </it>gene in MDR cells reduced <it>MDR1 </it>gene expression and converted their resistance to sensitivity to various anticancer drugs. In addition, the <it>WTH3 </it>gene promoter was hypermethylated in the MCF7/AdrR cell line and primary drug resistant breast cancer epithelial cells. <it>WTH3 </it>was also found to be directly targeted and up regulated by the <it>p53 </it>gene. Furthermore, over expression of the <it>WTH3 </it>gene promoted the apoptotic phenotype in various host cells.</p> <p>Methods</p> <p>To further confirm <it>WTH3</it>'s drug resistant related characteristics, we recently employed the small hairpin RNA (shRNA) strategy to knockdown its expression in HEK293 cells. In addition, since the <it>WTH3 </it>promoter's p53-binding site was located in a CpG island that was targeted by methylation, we were interested in testing the possible effect this epigenetic modification had on the <it>p53 </it>transcription factor relative to <it>WTH3 </it>expression. To do so, the <it>in vitro </it>methylation method was utilized to examine the <it>p53 </it>transgene's influence on either the methylated or non-methylated <it>WTH3 </it>promoter.</p> <p>Results</p> <p>The results generated from the gene knockdown strategy showed that reduction of <it>WTH3 </it>expression increased <it>MDR1 </it>expression and elevated resistance to Doxorubicin as compared to the original control cells. Data produced from the methylation studies demonstrated that DNA methylation adversely affected the positive impact of <it>p53 </it>on <it>WTH3 </it>promoter activity.</p> <p>Conclusion</p> <p>Taken together, our studies provided further evidence that <it>WTH3 </it>played an important role in MDR development and revealed one of its transcription regulatory mechanisms, DNA methylation, which antagonized <it>p53</it>'s positive impact on <it>WTH3 </it>expression.</p

Transepithelial Transport and Enzymatic Detoxification of Gluten in Gluten-Sensitive Rhesus Macaques

In a previous report, we characterized a condition of gluten sensitivity in juvenile rhesus macaques that is similar in many respects to the human condition of gluten sensitivity, celiac disease. This animal model of gluten sensitivity may therefore be useful toward studying both the pathogenesis and the treatment of celiac disease. Here, we perform two pilot experiments to demonstrate the potential utility of this model for studying intestinal permeability toward an immunotoxic gluten peptide and pharmacological detoxification of gluten in vivo by an oral enzyme drug candidate.Intestinal permeability was investigated in age-matched gluten-sensitive and control macaques by using mass spectrometry to detect and quantify an orally dosed, isotope labeled 33-mer gluten peptide delivered across the intestinal epithelium to the plasma. The protective effect of a therapeutically promising oral protease, EP-B2, was evaluated in a gluten-sensitive macaque by administering a daily gluten challenge with or without EP-B2 supplementation. ELISA-based antibody assays and blinded clinical evaluations of this macaque and of an age-matched control were conducted to assess responses to gluten.Labeled 33-mer peptide was detected in the plasma of a gluten-sensitive macaque, both in remission and during active disease, but not in the plasma of healthy controls. Administration of EP-B2, but not vehicle, prevented clinical relapse in response to a dietary gluten challenge. Unexpectedly, a marked increase in anti-gliadin (IgG and IgA) and anti-transglutaminase (IgG) antibodies was observed during the EP-B2 treatment phase.Gluten-sensitive rhesus macaques may be an attractive resource for investigating important aspects of celiac disease, including enhanced intestinal permeability and pharmacology of oral enzyme drug candidates. Orally dosed EP-B2 exerts a protective effect against ingested gluten. Limited data suggest that enhanced permeability of short gluten peptides generated by gastrically active glutenases may trigger an elevated antibody response, but that these antibodies are not necessarily causative of clinical illness