Karlie Jones

Expansion of CUG RNA repeats causes stress and inhibition of translation in myotonic dystrophy 1 (DM1) cells

The purpose of this study was to investigate the role of the mutant CUGn RNA in the induction of stress in type 1 myotonic dystrophy (DM1) cells and in the stressmediated inhibition of protein translation in DM1. To achieve our goals, we performed HPLC‐based purification of stress granules (SGs), immunoanalysis of SGs with stress markers TIA‐1, CUGBP1, and ph‐eIF2, site‐specific mutagenesis, and examinations of RNA‐protein and proteinprotein interactions in myoblasts from control and DM1 patients. The cause‐and‐effect relationships were addressed in stable cells expressing mutant CUG repeats. We found that the mutant CUGn RNA induces formation of SGs through the increase of the double‐stranded RNA‐dependent protein kinase (PKR) and following inactivation of eIF2α, one of the substrates of PKR We show that SGs trap mRNA coding for the DNA repair and remodeling factor MRG15 (MORF4L1), translation of which is regulated by CUGBP1. As the result of the trapping, the levels of MRG15 are reduced in DM1 cells and in CUG‐expressing cells. These data show that CUG repeats cause stress in DM1 through the PKR‐ph‐eIF2α pathway inhibiting translation of certain mRNAs, such as MRG15 mRNA. The repression of protein translation by stress might contribute to the progressive muscle loss in DM1.—Huichalaf, C., Sakai, K., Jin, B., Jones, K., Wang, G.‐L., Schoser, B., Schneider‐Gold, C., Sarkar, P., Pereira‐Smith, O. M., Timchenko, N., Lubov, T. Expansion of CUG RNA repeats causes stress and inhibition of translation in myotonic dystrophy 1 (DM1) cells. FASEB J. 24, 3706–3719 (2010). www.fasebj.org

KCa1.1 Potassium Channels Regulate Key Proinflammatory and Invasive Properties of Fibroblast-like Synoviocytes in Rheumatoid Arthritis

Background: Fibroblast-like synoviocytes participate in the pathogenesis of rheumatoid arthritis. Results: KCa1.1 is the major potassium channel on fibroblast-like synoviocytes from patients with rheumatoid arthritis, and blocking KCa1.1 channels perturbs the function of these cells. Conclusion: KCa1.1 channels play important regulatory roles in the function of fibroblast-like synoviocytes from patients with rheumatoid arthritis. Significance: KCa1.1 channel are potential new therapeutic targets for rheumatoid arthritis. Fibroblast-like synoviocytes (FLS) play important roles in the pathogenesis of rheumatoid arthritis (RA). Potassium channels have regulatory roles in many cell functions. We have identified the calcium- and voltage-gated KCa1.1 channel (BK, Maxi-K, Slo1, KCNMA1) as the major potassium channel expressed at the plasma membrane of FLS isolated from patients with RA (RA-FLS). We further show that blocking this channel perturbs the calcium homeostasis of the cells and inhibits the proliferation, production of VEGF, IL-8, and pro-MMP-2, and migration and invasion of RA-FLS. Our findings indicate a regulatory role of KCa1.1 channels in RA-FLS function and suggest this channel as a potential target for the treatment of RA.

RNA Foci, CUGBP1, and ZNF9 are the primary targets of the mutant CUG and CCUG repeats expanded in myotonic dystrophies type 1 and type 2

Expansions of noncoding CUG and CCUG repeats in myotonic dystrophies type 1 (DM1) and DM2 cause complex molecular pathology, the features of which include accumulation of RNA aggregates and misregulation of the RNA-binding proteins muscleblind-like 1 (MBNL1) and CUG-binding protein 1 (CUGBP1). CCUG repeats also decrease amounts of the nucleic acid binding protein ZNF9. Using tetracycline (Tet)-regulated monoclonal cell models that express CUG and CCUG repeats, we found that low levels of long CUG and CCUG repeats result in nuclear and cytoplasmic RNA aggregation with a simultaneous increase of CUGBP1 and a reduction of ZNF9. Elevation of CUGBP1 and reduction of ZNF9 were also observed before strong aggregation of the mutant CUG/CCUG repeats. Degradation of CUG and CCUG repeats normalizes ZNF9 and CUGBP1 levels. Comparison of short and long CUG and CCUG RNAs showed that great expression of short repeats form foci and alter CUGBP1 and ZNF9; however, long CUG/CCUG repeats misregulate CUGBP1 and ZNF9 much faster than high levels of the short repeats. These data suggest that correction of DM1 and DM2 might be achieved by complete and efficient degradation of CUG and CCUG repeats or by a simultaneous disruption of CUG/CCUG foci and correction of CUGBP1 and ZNF9.

The role of CUGBP1 in age-dependent changes of liver functions

Aging liver is characterized by alterations of liver biology and by a reduction of many functions which are important for the maintenance of body homeostasis. The main dysfunctions include appearance of enlarged hepatocytes, impaired liver regeneration after partial hepatectomy (PH), development of hepatic steatosis, reduction of secretion of proteins and alterations in the hepatic sinusoid. RNA binding proteins are involved in the regulation of gene expression in all tissues including regulation of biological processes in the liver. This review is focused on the role of a conserved, multi-functional RNA-binding protein, CUGBP1, in the development of aging phenotype in the liver. CUGBP1 has been identified as a protein which binds to RNA CUG repeats expanded in Myotonic Dystrophy type 1 (DM1). CUGBP1 is highly expressed in the liver and regulates translation of proteins which are critical for maintenance of liver functions. In livers of young mice, CUGBP1 forms complexes with eukaryotic translation initiation factor eIF2 and supports translation of C/EBPβ and HDAC1 proteins, which are involved in liver growth, differentiation and liver cancer. Aging changes several signaling pathways which lead to the elevation of the CUGBP1-eIF2α complex and to an increase of translation of C/EBPβ and HDAC1. These proteins form multi-protein complexes with additional transcription factors and with chromatin remodeling proteins causing epigenetic alterations of gene expression in livers of old mice. It appears that CUGBP1-mediated translational elevation of HDAC1 is one of the key events in the epigenetic changes in livers of old mice, leading to the development of age-associated dysfunctions of the liver. This review will also discuss a possible role of CUGBP1 in liver dysfunction in patients affected with DM1.

Reduction of toxic RNAs in myotonic dystrophies type 1 and type 2 by the RNA helicase p68/DDX5

Significance Inherited multisystemic diseases, myotonic dystrophies type 1 (DM1) and type 2 (DM2), are caused by long CUG and CCUG RNA repeats. The mutant RNA CCUG repeats should be degraded after intron excision; however, this RNA accumulates in cells, leading to pathology. Although mutant RNAs may be degraded with synthetic oligonucleotides, the identification of a cause of the increased stability of CUG and CCUG RNAs would help to improve the efficiency of their degradation. We found that the reduction of RNA helicase p68 in skeletal muscle biopsies of DM1 and DM2 patients contributes to the delay of degradation of the mutant RNAs. Our work suggests RNA helicase p68 as a therapeutic target in DM, correction of which improves degradation of the mutant RNAs, reducing DM pathology. Myotonic dystrophies type 1 (DM1) and type 2 (DM2) are neuromuscular diseases, caused by accumulation of CUG and CCUG RNAs in toxic aggregates. Here we report that the increased stability of the mutant RNAs in both types of DM is caused by deficiency of RNA helicase p68. We have identified p68 by studying CCUG-binding proteins associated with degradation of the mutant CCUG repeats. Protein levels of p68 are reduced in DM1 and DM2 biopsied skeletal muscle. Delivery of p68 in DM1/2 cells causes degradation of the mutant RNAs, whereas delivery of p68 in skeletal muscle of DM1 mouse model reduces skeletal muscle myopathy and atrophy. Our study shows that correction of p68 may reduce toxicity of the mutant RNAs in DM1 and in DM2.

Smaug/SAMD4A Restores Translational Activity of CUGBP1 and Suppresses CUG-Induced Myopathy

We report the identification and characterization of a previously unknown suppressor of myopathy caused by expansion of CUG repeats, the mutation that triggers Myotonic Dystrophy Type 1 (DM1). We screened a collection of genes encoding RNA–binding proteins as candidates to modify DM1 pathogenesis using a well established Drosophila model of the disease. The screen revealed smaug as a powerful modulator of CUG-induced toxicity. Increasing smaug levels prevents muscle wasting and restores muscle function, while reducing its function exacerbates CUG-induced phenotypes. Using human myoblasts, we show physical interactions between human Smaug (SMAUG1/SMAD4A) and CUGBP1. Increased levels of SMAUG1 correct the abnormally high nuclear accumulation of CUGBP1 in myoblasts from DM1 patients. In addition, augmenting SMAUG1 levels leads to a reduction of inactive CUGBP1-eIF2α translational complexes and to a correction of translation of MRG15, a downstream target of CUGBP1. Therefore, Smaug suppresses CUG-mediated muscle wasting at least in part via restoration of translational activity of CUGBP1.

Graft versus host disease: New insights into A2A receptor agonist therapy

Allogeneic transplantation can cure many disorders, including sickle cell disease, chronic granulomatous disease (CGD), severe combined immunodeficiency (SCID) and many types of cancers. However, there are several associated risks that can result in severe immunological reactions and, in some cases, death. Much of this morbidity is related to graft versus host disease (GVHD) [1]. GVHD is an immune mediated reaction in which donor T cells recognize the host as antigenically foreign, causing donor T cells to expand and attack host tissues. The current method of treating recent transplant patients with immunosuppressants to prevent this reaction has met with only partial success, emphasizing a need for new methods of GVHD treatment and prevention. Recently, a novel strategy has emerged targeting adenosine A2A receptors (A2AR) through the use of adenosine agonists. These agonists have been shown in vitro to increase the TGFβ-induced generation of FoxP3+ regulatory T cells (Tregs) and in vivo to improve weight gain and mortality as well as inhibit the release of pro-inflammatory cytokines in GVHD murine models [2,3]. Positive results involving A2AR agonists in vitro and in vivo are promising, suggesting that A2AR agonists should be a part of the management of clinical GvHD.

GSK3β is a new therapeutic target for myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1), an incurable, neuromuscular disease, is caused by the expansion of CTG repeats within the 3′ UTR of DMPK on chromosome 19q. In DM1 patients, mutant DMPK transcripts deregulate RNA metabolism by altering CUG RNA-binding proteins. Several approaches have been proposed for DM1 therapy focused on specific degradation of the mutant CUG repeats or on correction of RNA-binding proteins, affected by CUG repeats. One such protein is CUG RNA-binding protein (CUGBP1). The ability of CUGBP1 to increase or inhibit translation depends on phosphorylation at Ser302, which is mediated by cyclin D3-CDK4. The mutant CUG repeats increase the levels of CUGBP1 protein and inhibit Ser302 phosphorylation, leading to the accumulation of CUGBP1 isoforms that repress translation (i.e., CUGBP1REP). Elevation of CUGBP1REP in DM1 is caused by increased GSK3β kinase, which reduces the cyclin D3-CDK4 pathway and subsequent phosphorylation of CUGBP1 at Ser302. In this review, we discuss our recent discovery showing that correction of GSK3β activity in the DM1 mouse model (i.e., HSALR mice) reduces DM1 muscle pathology. These findings demonstrate that GSK3β is a novel therapeutic target for treating DM1.

A Novel Method for Screening Adenosine Receptor Specific Agonists for Use in Adenosine Drug Development

Agonists that target the A1, A2A, A2B and A3 adenosine receptors have potential to be potent treatment options for a number of diseases, including autoimmune diseases, cardiovascular disease and cancer. Because each of these adenosine receptors plays a distinct role throughout the body, obtaining highly specific receptor agonists is essential. Of these receptors, the adenosine A2AR and A2BR share many sequence and structural similarities but highly differ in their responses to inflammatory stimuli. Our laboratory, using a combination of specially developed cell lines and calcium release analysis hardware, has created a new and faster method for determining specificity of synthetic adenosine agonist compounds for the A2A and A2B receptors in human cells. A2A receptor expression was effectively removed from K562 cells, resulting in the development of a distinct null line. Using HIV-lentivector and plasmid DNA transfection, we also developed A2A and A2B receptor over-expressing lines. As adenosine is known to cause changes in intracellular calcium levels upon addition to cell culture, calcium release can be determined in these cell lines upon compound addition, providing a functional readout of receptor activation and allowing us to isolate the most specific adenosine agonist compounds.

Recombinant Pregnancy-Specific Glycoprotein 1 Has a Protective Role in a Murine Model of Acute Graft-versus-Host Disease

Acute graft-versus-host disease (aGVHD) is an immune-mediated reaction that can occur after hematopoietic stem cell transplantation in which donor T cells recognize the host antigens as foreign, destroying host tissues. Establishment of a tolerogenic immune environment while preserving the immune response to infectious agents is required for successful bone marrow transplantation. Pregnancy-specific glycoprotein 1 (PSG1), which is secreted by the human placenta into the maternal circulation throughout pregnancy, likely plays a role in maintaining immunotolerance to prevent rejection of the fetus by the maternal immune system. We have previously shown that PSG1 activates the latent form of transforming growth factor β1 (TGF-β), a cytokine essential for the differentiation of tolerance-inducing CD4+FoxP3+ regulatory T cells (Tregs). Consistent with this observation, treatment of naïve murine T cells with PSG1 resulted in a significant increase in FoxP3+ cells that was blocked by a TGF-β receptor I inhibitor. We also show here that PSG1 can increase the availability of active TGF-β in vivo. As the role of CD4+FoxP3+ cells in the prevention of aGVHD is well established, we tested whether PSG1 has beneficial effects in a murine aGHVD transplantation model. PSG1-treated mice had reduced numbers of tissue-infiltrating inflammatory CD3+ T cells and had increased expression of FoxP3 in T cells compared with vehicle-treated mice. In addition, administration of PSG1 significantly inhibited aGVHD-associated weight loss and mortality. On the other hand, administration of PSG1 was less effective in managing aGVHD in the presence of an alloimmune reaction against a malignancy in a graft-versus-leukemia experimental model. Combined, this data strongly suggests that PSG1 could be a promising treatment option for patients with aGVHD following bone marrow transplantation for a nonmalignant condition, such as an autoimmune disorder or a genetic immunodeficiency.