Gx‐50 reduces β‐amyloid‐induced TNF‐α, IL‐1β, NO,and PGE2 expression and inhibits NF‐κB signaling in a mouse model of Alzheimer's disease

Chronic inflammation, which is regulated by overactivated microglia in the brain, accelerates the occurrence and development of Alzheimer's disease (AD). Gx‐50 has been investigated as a novel drug for the treatment of AD in our previous studies. Here, we investigated whether gx‐50 possesses anti‐inflammatory effects in primary rat microglia and a mouse model of AD, amyloid precursor protein (APP) Tg mice. The expression of TNF‐α, IL‐1β, NO, prostaglandin E2, and the expression of iNOS and COX2 were inhibited by gx‐50 in amyloid β (Aβ) treated rat microglia; additionally, microglial activation and the expression of IL‐1β, iNOS, and COX2 were also significantly suppressed by gx‐50 in APP⁺ transgenic mice. Furthermore, gx‐50 inhibited the activation of NF‐κB and MAPK cascades in vitro and in vivo in APP‐Tg mice. Moreover, the expression of TLR4 and its downstream signaling proteins MyD88 and tumor necrosis factor receptor associated factor 6 (TRAF6) was reduced by gx‐50 in vitro and in vivo. Interestingly, silencing of TLR4 reduced Aβ‐induced upregulation of IL‐1β and TRAF6 to levels similar to gx‐50 inhibition; moreover, overexpression of TLR4 increased the expression of MyD88 and TRAF6, which was significantly reduced by gx‐50. These findings provide strong evidence that gx‐50 has anti‐inflammatory effects against Aβ‐triggered microglial overactivation via a mechanism that involves the TLR4‐mediated NF‐κBB/MAPK signaling cascade.     

The induced RNA‐binding protein,HuR, targets 3′‐UTR region of IL‐6 mRNA and enhances its stabilization in periodontitis

RNA‐binding proteins (RBPs) regulate mRNA stability by binding to the 3′‐untranslated region (UTR) region of mRNA. Human antigen‐R (HuR), one of the RBPs, is involved in the progression of diseases, such as rheumatoid arthritis, diabetes mellitus and some inflammatory diseases. Interleukin (IL)‐6 is a major inflammatory cytokine regulated by HuR binding to mRNA. Periodontal disease (PD) is also an inflammatory disease caused by elevations in IL‐6 following an infection by periodontopathogenic bacteria. The involvement of HuR in the progression of PD was assessed using in‐vitro and in‐vivo experiments. Immunohistochemistry of inflamed periodontal tissue showed strong staining of HuR in the epithelium and connective tissue. HuR mRNA and protein level was increased following stimulation with Porphyromonas gingivalis (Pg), one of the periodontopathogenic bacteria, lipopolysacchride (LPS)‐derived from Pg (PgLPS) and tumour necrosis factor (TNF)‐α in OBA‐9, an immortalized human gingival epithelial cell. The luciferase activity of 3′‐UTR of IL‐6 mRNA was increased by TNF‐α, Pg and PgLPS in OBA‐9. Luciferase activity was also increased in HuR‐over‐expressing OBA‐9 following a bacterial stimulation. Down‐regulation of HuR by siRNA resulted in a decrease in mRNA expression and production of IL‐6. In contrast, the over‐expression of HuR increased IL‐6 mRNA expression and production in OBA‐9. The HuR inhibitor, quercetin, suppressed Pg‐induced HuR mRNA expression and IL‐6 production in OBA‐9. An oral inoculation with quercetin also inhibited bone resorption in ligature‐induced periodontitis model mice as a result of down‐regulation of IL‐6. These results show that HuR modulates inflammatory responses by regulating IL‐6.     

Characterization of the 18 kDa translocator protein (TSPO) expression in post‐mortem normal and Alzheimer's disease brains

The 18 kDa translocator protein (TSPO) is a widely used target for microglial PET imaging radioligands, but its expression in post‐mortem normal and diseased human brain is not well described. We aimed at characterizing the TSPO expression in human control (CTRL) and Alzheimer's disease (AD) brains. Specifically, we sought to: (1) define the cell type(s) expressing TSPO; (2) compare tspo mRNA and TSPO levels between AD and CTRL brains; (3) correlate TSPO levels with quantitative neuropathological measures of reactive glia and AD neuropathological changes; and (4) investigate the effects of the TSPO rs6971 SNP on tspo mRNA and TSPO levels, glial responses and AD neuropathological changes. We performed quantitative immunohistochemistry and Western blot in post‐mortem brain samples from CTRL and AD subjects, as well as analysis of publicly available mouse and human brain RNA‐Seq datasets. We found that: (1) TSPO is expressed not just in microglia, but also in astrocytes, endothelial cells and vascular smooth muscle cells; (2) there is substantial overlap of tspo mRNA and TSPO levels between AD and CTRL subjects and in TSPO levels between temporal neocortex and white matter in both groups; (3) TSPO cortical burden does not correlate with the burden of activated microglia or reactive astrocytes, Aβ plaques or neurofibrillary tangles, or the cortical thickness; (4) the TSPO rs6971 SNP does not significantly impact tspo mRNA or TSPO levels, the magnitude of glial responses, the cortical thickness, or the burden of AD neuropathological changes. These results could inform ongoing efforts toward the development of reactive glia‐specific PET radioligands.     

SMARCB1 expression in epithelioid sarcoma is regulated by miR‐206, miR‐381, and miR‐671‐5p on Both mRNA and protein levels

Proximal type epithelioid sarcoma shares similarities with malignant rhabdoid tumor, including the lack of nuclear immunoreactivity of SMARCB1. Biallelic mutation of SMARCB1 has been convincingly established as the cause of loss of protein expression in rhabdoid tumor, but the cause in epithelioid sarcoma remains unknown. In our previous work, we demonstrated that DNA hypermethylation and post‐translational modification mechanisms were not involved. In this current work, we explored the hypothesis that miRNAs regulate SMARCB1 gene expression in epithelioid sarcomas. In silico target prediction analysis revealed eight candidate miRNAs, and quantitative PCR—in 32 formalin‐fixed, paraffin‐embedded tumor samples comprising 30 epithelioid sarcomas and two malignant rhabdoid tumors—demonstrated significant (P < 0.001) overexpression of four miRNAs in epithelioid sarcomas: miR‐206, miR‐381, miR‐671‐5p, and miR‐765. Two human tumors (fibrosarcoma and colon adenocarcinoma) and a normal cell line (human dermal fibroblast) with retained SMARCB1 expression were cultured for miRNA transient transfection (electroporation) experiments. SMARCB1 mRNA expression was analyzed by quantitative real‐time PCR and immunostaining of SMARCB1 was performed to examine the effect of miRNAs transfections on both RNA and protein levels. Only three of the overexpressed miRNAs (miR‐206, miR‐381, and miR‐671‐5p) could silence the SMARCB1 mRNA expression in cell cultures; most effectively miR‐206. Transfection of miR‐206, miR‐381, miR‐671‐5p, and some combination of them also eliminated SMARCB1 nuclear staining, demonstrating a strong effect on not only mRNA but also protein levels. Our results suggest loss of SMARCB1 protein expression in epithelioid sarcoma is due to the epigenetic mechanism of gene silencing by oncomiRs. © 2013 Wiley Periodicals, Inc.     

Inhibition of peroxisomal β-oxidation by thioridazine increases the amount of VLCFAs and Aβ generation in the rat brain

Alzheimer's disease (AD) is characterized by the accumulation of the β-amyloid peptide (Aβ), which is generated from sequential cleavages of the amyloid precursor protein (APP) by β-secretase (BACE1) and γ-secretase. Fatty acid alterations in AD brains have recently received substantial attention. Because increased very long chain fatty acid (VLCFA) levels in AD brains imply that peroxisomal β-oxidation dysfunction may be associated with AD pathogenesis, we investigated the effects of impaired peroxisomal β-oxidation on Aβ generation in vivo and in vitro using thioridazine, a selective peroxisomal β-oxidation inhibitor. Under the experimental conditions, thioridazine caused VLCFA accumulation and increases in Aβ₄₀ content, APP immunoreactivity and APP_751+770 mRNA expressions in the rat cerebral cortex. A correlation analysis showed that the Aβ₄₀ levels were positively correlated with the cortex C_24:0 and C_26:0 levels. Additionally, the primary cerebral cortex neurons treated with this compound showed increases in APP_751+770 mRNA, APP protein, BACE1 mRNA and protein, and secreted Aβ40 levels. This work supports an emerging viewpoint that impaired peroxisomal function may play an important role in the progression of AD pathology.     

The sterol regulatory element‐binding protein 2 is dysregulated by tau alterations in Alzheimer disease

Disturbed neuronal cholesterol homeostasis has been observed in Alzheimer disease (AD) and contributes to the pathogenesis of AD. As the master switch of cholesterol biosynthesis, the sterol regulatory element‐binding protein 2 (SREBP‐2) translocates to the nucleus after cleavage/activation, but its expression and activation have not been studied in AD which is the focus of the current study. We found both a significant decrease in the nuclear translocation of N‐terminal SREBP‐2 accompanied by a significant accumulation of C‐terminal SREBP‐2 in NFT‐containing pyramidal neurons in AD. N‐terminal‐ SREBP‐2 is also found in dystrophic neurites around plaques in AD brain. Western blot confirmed a significantly reduced nuclear translocation of mature SREBP‐2 (mSREBP‐2) in AD brain. Interestingly, reduced nuclear mSREBP‐2 was only found in animal models of tauopathies such as 3XTg AD mice and P301L Tau Tg mice but not in CRND8 APP transgenic mice, suggesting that tau alterations likely are involved in the changes of mSREBP‐2 distribution and activation in AD. Altogether, our study demonstrated disturbed SREBP‐2 signaling in AD and related models, and proved for the first time that tau alterations contribute to disturbed cholesterol homeostasis in AD likely through modulation of nuclear mSREBP‐2 translocation.     

MicroRNA‐155 may be involved in the pathogenesis of atopic dermatitis by modulating the differentiation and function of T helper type 17 (Th17) cells

Our aims were to identify the differential expression of microRNA (miR)‐155, as well as to explore the possible regulatory effects of miR‐155 on the differentiation and function of T helper type 17 (Th17) cells in atopic dermatitis (AD). The Th17 cell percentage and expression levels of miR‐155, retinoic acid‐related orphan receptor (ROR)γt, interleukin (IL)‐17 and suppressor of cytokine signalling‐1 (SOCS1) in peripheral CD4⁺ T cells, plasma and skin specimens were detected and compared in AD patients and healthy subjects. A miR‐155 mimic and an inhibitor were transfected separately into AD CD4⁺ T cells to confirm the in‐vivo data. The Th17 cell percentage, miR‐155 expression, RORγt mRNA expression, IL‐17 mRNA expression and plasma concentration were increased significantly in AD patients compared with healthy subjects. Conversely, SOCS1 mRNA expression and plasma concentration were decreased significantly. Similar results were detected in cultured CD4⁺ T cells transfected with the miR‐155 mimic compared with a miR‐155 inhibitor or a negative control. Additionally, there was a sequential decrease in miR‐155 expression, as well as RORγt and IL‐17 mRNA expression, but an increase in SOCS1 mRNA expression, from AD lesional skin and perilesional skin to normal skin. Positive correlations were found between miR‐155 expression and AD severity, Th17 cell percentage, RORγt mRNA expression and IL‐17 mRNA expression and plasma concentration, while negative correlations were observed between miR‐155 expression and SOCS1 mRNA expression and plasma concentration in AD peripheral circulation and skin lesions. In conclusion, miR‐155 is over‐expressed and may be involved in AD pathogenesis by modulating the differentiation and function of Th17 cells.     

B‐cell‐activating factor expressions in salivary epithelial cells after dsRNA virus infection depends on RNA‐activated protein kinase activation

B‐cell‐activating factor (BAFF) plays a key role in promoting activation of autoimmune B cells. This cytokine may be expressed in and secreted by salivary gland epithelial cells (SGEC) after stimulation with type I IFN or viral or synthetic dsRNA. Because this BAFF expression depends only in part on endosomal TLR and type I IFN, we investigated whether other dsRNA sensors could be implicated in BAFF expression. Using human SGEC, we confirmed the partial dependence of BAFF expression on TLR‐3 by replicating the partial inhibition of BAFF expression observed upon endosomal inhibition using TLR‐3 or Toll/IL‐1R domain‐containing protein inducing IFN‐β silencing mRNA, but not with TLR‐7 silencing mRNA. Melanoma differentiation‐associated gene 5 silencing mRNA had no effect on BAFF expression, but retinoic acid‐inducible gene I silencing mRNA had a slight effect observed following infection with dsRNA reovirus‐1. Inhibition of RNA‐activated protein kinase (PKR) by 2‐aminopurine completely abolished both BAFF mRNA and protein production after reovirus‐1 infection and poly(I:C) stimulation through NF‐κB and p38 MAPK pathways, with the latter implicated only after poly(I:C) stimulation. Thus, PKR is the dsRNA sensor implicated in BAFF induction in SGEC after dsRNA stimulation. In autoimmune diseases, PKR may be an interesting target for preventing BAFF following the induction of innate immunity.     

T‐cell regulation through a basic suppressive mechanism targeting low‐density lipoprotein receptor‐related protein 1

Cell adhesion is generally considered to depend on positive regulation through ligation of integrins and cytokine receptors. However, here we show that T‐cell adhesion, and notably also T‐cell receptor (TCR) ‐induced activation, are subject to constant suppression through shedding of low‐density lipoprotein receptor‐related protein 1 (LRP1). The broad‐spectrum metalloprotease inhibitor GM6001 abrogated shedding, so inducing prominent cell surface expression of LRP1 while enhancing TCR‐induced activation and adhesion to β₁ and β₂ integrin ligands, hence arresting the cells. Integrin ligands also inhibited shedding but the effect was less potent than that of GM6001. Unlike GM6001, integrin ligands also induced cell surface expression of full‐length thrombospondin‐1 (TSP170) and TSP130, which associated with LRP1, and TSP110, which did not associate with LRP1. Cell surface expression of LRP1 and TSP130 were induced exclusively in adhering cells, expression of TSP110 preferentially in non‐adhering cells and expression of TSP170 correlated with T‐cell motility. The pro‐adhesive chemokine CXCL12 also inhibited LRP1 shedding and induced surface expression of TSP170 and TSP130 while inhibiting TSP110. Exogenous TSP‐1 and ligation of CD28 inhibited shedding although less effectively than GM6001, and the inhibition through CD28 was independent of TSP‐1. Small interfering RNA silencing experiments confirmed involvement of LRP1 and TSP‐1 in integrin‐dependent adhesion and TCR‐induced activation. Hence, the poor LRP1 expression in T cells depends on shedding. Integrin ligands and CXCL12 antagonize shedding through a TSP‐1‐dependent pathway and ligation of CD28 antagonizes shedding independent of TSP‐1. The disappearance of LRP1 from the cell surface may provide basic immunosuppression at the T‐cell level.     

XPC branch‐point sequence mutations disrupt U2 snRNP binding,resulting in abnormal pre‐mRNA splicing in xeroderma pigmentosum patients

Mutations in two branch‐point sequences (BPS) in intron 3 of the XPC DNA repair gene affect pre‐mRNA splicing in association with xeroderma pigmentosum (XP) with many skin cancers (XP101TMA) or no skin cancer (XP72TMA), respectively. To investigate the mechanism of these abnormalities we now report that transfection of minigenes with these mutations revealed abnormal XPC pre‐mRNA splicing that mimicked pre‐mRNA splicing in the patients' cells. DNA oligonucleotide‐directed RNase H digestion demonstrated that mutations in these BPS disrupt U2 snRNP–BPS interaction. XP101TMA cells had no detectable XPC protein but XP72TMA had 29% of normal levels. A small amount of XPC protein was detected at sites of localized ultraviolet (UV)‐damaged DNA in XP72TMA cells which then recruited other nucleotide excision repair (NER) proteins. In contrast, XP101TMA cells had no detectable recruitment of XPC or other NER proteins. Post‐UV survival and photoproduct assays revealed greater reduction in DNA repair in XP101TMA cells than in XP72TMA. Thus mutations in XPC BPS resulted in disruption of U2 snRNP‐BPS interaction leading to abnormal pre‐mRNA splicing and reduced XPC protein. At the cellular level these changes were associated with features of reduced DNA repair including diminished NER protein recruitment, reduced post‐UV survival and impaired photoproduct removal. Hum Mutat 30:1–9, 2009. Published 2009 Wiley‐Liss, Inc.     

The ETFDH c.158A>G Variation Disrupts the Balanced Interplay of ESE‐ and ESS‐Binding Proteins thereby Causing Missplicing and Multiple Acyl‐CoA Dehydrogenation Deficiency

Multiple acyl‐CoA dehydrogenation deficiency is a disorder of fatty acid and amino acid oxidation caused by defects of electron transfer flavoprotein (ETF) or its dehydrogenase (ETFDH). A clear relationship between genotype and phenotype makes genotyping of patients important not only diagnostically but also for prognosis and for assessment of treatment. In the present study, we show that a predicted benign ETFDH missense variation (c.158A>G/p.Lys53Arg) in exon 2 causes exon skipping and degradation of ETFDH protein in patient samples. Using splicing reporter minigenes and RNA pull‐down of nuclear proteins, we show that the c.158A>G variation increases the strength of a preexisting exonic splicing silencer (ESS) motif UAGGGA. This ESS motif binds splice inhibitory hnRNP A1, hnRNP A2/B1, and hnRNP H proteins. Binding of these inhibitory proteins prevents binding of the positive splicing regulatory SRSF1 and SRSF5 proteins to nearby and overlapping exonic splicing enhancer elements and this causes exon skipping. We further suggest that binding of hnRNP proteins to UAGGGA is increased by triggering synergistic hnRNP H binding to GGG triplets located upstream and downsteam of the UAGGGA motif. A number of disease‐causing exonic elements that induce exon skipping in other genes have a similar architecture as the one in ETFDH exon 2.     

Role of Cytotoxic Protease Granzyme‐b in Neuronal Degeneration During Human Stroke

Infiltration of leukocytes into post‐ischemic cerebrum is a well‐described phenomenon in stroke injury. Because CD‐8⁺ T‐lymphocytes secrete cytotoxic proteases, including granzyme‐b (Gra‐b) that exacerbates post‐ischemic brain damage, we investigated roles of Gra‐b in human stroke. To study the role of Gra‐b in stroke, ischemic and non‐ischemic tissues (from post‐mortem stroke patients) were analyzed using immunoblotting, co‐immunoprecipitation, terminal deoxy uridine nick end labeling (TUNEL) and Annexin–V immunostaining, and in vitro neuron survival assays. Activated CG‐SH cells and supernatants were used to model leukocyte‐dependent injury. Non‐ischemic brain tissues were used as non‐pathological controls. Non‐activated CG‐SH cells and supernatants were used as controls for in vitro experiments. Human stroke (ischemic) samples contained significantly higher levels of Gra‐b and interferon‐gamma inducible protein‐10 (IP‐10/CXCL10) than non‐ischemic controls. In stroke, poly (ADP‐ribose) polymerase‐1 and heat shock protein‐70 were cleaved to canonical proteolytic “signature” fragments by Gra‐b. Gra‐b was also found to bind to Bid and caspase‐3. Gra‐b also co‐localized with Annexin‐V⁺/TUNEL⁺ in degenerating neurons. Importantly, Gra‐b inhibition protected both normal and ischemia‐reperfused neurons against in vitro neurotoxicity mediated by activated CG‐SH cells and supernatants. These results suggest that increased leukocyte infiltration and elevated Gra‐b levels in the post‐stroke brain can induce contact‐dependent and independent post‐ischemic neuronal death to aggravate stroke injury.     

GRP78 up-regulation is associated with androgen receptor status, Hsp70-Hsp90 client proteins and castrate-resistant prostate cancer

GRP78/BiP is a key member of the molecular chaperone heat shock protein (Hsp) 70 family. It has a critical role in prostate cancer (PC) including Pten loss‐driven carcinogenesis, but the molecular basis of this remains unclear. We investigated the effect of GRP78 and its putative client proteins, including androgen receptor (AR) in clinical PC. Expression of GRP78 and key Hsp70–hsp90 client proteins (HER2, HER3, AR and AKT) were studied in an incidence tissue microarray (TMA) of prostate cancer. The relationship of GRP78 and AR was further tested in in vitro cell models (LNCaP and its derived LNCaP‐CR subclone) and a matched TMA of hormone‐naïve (HNPC) and castrate‐resistant prostate cancer (CRPC). In vitro and in vivo expression of GRP78 and client proteins were assessed by western blotting and immunohistochemistry, respectively, using the weighted histoscore method. Significant co‐expression of GRP78, pAKT, HER2, HER3 and AR was observed in PC. Abnormal AR, GRP78 and pAKT expression have significant impact on patient survival. GRP78 expression in AR⁺ tumours was significantly higher than in AR^? tumours. In keeping with our clinical data, activation of AR by dihydrotestosterone (DHT) potently activated GRP78 expression in both LNCaP and LNCaP‐CR cells. For the first time, using a matched HNPC and CRPC TMA, enhanced cytoplasmic and membranous GRP78 expression was observed in CRPC. Future prospective studies are therefore warranted to validate GRP78 as prognostic marker and therapeutic target, in the context of the AR and pAKT status. In summary, GRP78 is co‐expressed with Hsp70–hsp90 client proteins. Up‐regulated expression of AR and GRP78 expression in untreated prostate cancer predicts a less favourable outcome. This points to the importance of understanding in the molecular interaction among AR, GRP78 and AKT. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.